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What was the outcome of reaction 'Appendicitis'? | Midostaurin after allogeneic stem cell transplant in patients with FLT3-internal tandem duplication-positive acute myeloid leukemia.
We evaluated standard-of-care (SOC) treatment with or without midostaurin to prevent relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) in patients with acute myeloid leukemia (AML) harboring internal tandem duplication (ITD) in FLT3. Adults (aged 18-70 years) who received alloHSCT in first complete remission, had achieved hematologic recovery, and were transfusion independent were randomized to receive SOC with or without midostaurin (50 mg twice daily) continuously in twelve 4-week cycles. The primary endpoint was relapse-free survival (RFS) 18 months post-alloHSCT. Sixty patients were randomized (30/arm); 30 completed all 12 cycles (midostaurin + SOC, n = 16; SOC, n = 14). The estimated 18-month RFS (95% CI) was 89% (69-96%) in the midostaurin arm and 76% (54-88%) in the SOC arm (hazard ratio, 0.46 [95% CI, 0.12-1.86]; P = 0.27); estimated relapse rates were 11% and 24%, respectively. Inhibition of FLT3 phosphorylation to <70% of baseline (achieved by 50% of midostaurin-treated patients) was associated with improved RFS. The most common serious adverse events were diarrhea, nausea, and vomiting. Rates of graft-vs-host disease were similar between both arms (midostaurin + SOC, 70%; SOC, 73%). The addition of midostaurin maintenance therapy following alloHSCT may provide clinical benefit in some patients with FLT3-ITD AML. (ClinicalTrials.gov identifier: NCT01883362).
Introduction
Acute myeloid leukemia (AML), the most common acute leukemia, is difficult to treat and has a poor prognosis, with a 5-year survival of ~25% [1, 2]. Multiple factors, including age, performance status (e.g., Eastern Cooperative Oncology Group), and cytogenetic and molecular features, affect treatment decisions and outcomes [3, 4]. Mutations in fms-like tyrosine kinase 3 (FLT3) are among the most common in AML and confer a poor prognosis with poor overall survival (OS) [5–7]. Consequently, these patients, particularly those with internal tandem duplications (ITDs), historically have more frequent and earlier relapses than patients without FLT3 mutations [7, 8].
Midostaurin, a multikinase inhibitor that targets FLT3 and other kinases, was approved for the treatment of adult patients with newly diagnosed, FLT3-mutated AML when combined with intensive induction and consolidation chemotherapy [9]. Approval was based on the phase 3 RATIFY/CALGB 10603 trial, which demonstrated improved survival with the addition of midostaurin to intensive chemotherapy followed by single-agent maintenance therapy in patients aged <60 years with newly diagnosed, FLT3-mutated AML. The RATIFY trial did not allow patients receiving alloHSCT to continue midostaurin [10].
AlloHSCT in first complete remission (CR1) provides patients with FLT3-ITD-positive AML the highest likelihood of sustained remission [11, 12], but relapse rates remain high [13–15]. The prognosis for patients with FLT3-ITD mutations has been poor following standard alloHSCT, primarily because these patients have a higher risk of relapse than patients with FLT3-ITD-negative AML [14–16].
Post-HSCT maintenance therapy with tyrosine kinase inhibitors (TKIs) may improve outcomes in patients with FLT3-mutated AML. In a phase 2 trial (AMLSG 16-10), midostaurin combined with intensive chemotherapy followed by alloHSCT and single-agent maintenance therapy demonstrated improved rates of event-free survival in patients receiving midostaurin compared with historical controls [17]. In AMLSG 16-10, midostaurin was administered as in RATIFY; however, patients who underwent alloHSCT could resume midostaurin as maintenance therapy post-transplant [10, 17]. Data from phase 1 and 2 trials suggest there may be a benefit with sorafenib, another TKI, as maintenance therapy post-HSCT [18–20]. Results from the phase 2 SORMAIN trial, which evaluated post-alloHSCT maintenance with sorafenib, suggested a benefit with sorafenib versus placebo with a median 2-year relapse-free survival (RFS) rate of 85% (95% CI, 70–93%) vs 53% (95% CI, 37–68%), respectively, (hazard ratio [HR], 0.39 [95% CI, 0.183—0.848]; P = 0.013) [20]. Similarly, quizartinib, a FLT3 TKI, was safely administered after alloHSCT in a phase 1 study [21]. Detailed trials evaluating FLT3 TKIs as maintenance therapy are ongoing [22–25].
Here, we report the results of the RADIUS trial investigating whether the addition of midostaurin to standard-of-care (SOC) treatment post-alloHSCT improves RFS over SOC alone in patients with FLT3-ITD-positive AML.
Patients and methods
Study design
RADIUS (NCT01883362) was a phase 2, randomized, open-label trial of SOC with or without midostaurin in patients (aged 18–70 years) with documented FLT3-ITD-positive AML who had undergone a protocol-specified conditioning regimen before alloHSCT in CR1 (following hematologic recovery, transfusion independence, and controlled graft-vs-host disease [GVHD]). Patients were enrolled after engraftment and randomized 1:1 within 28 to 60 days after alloHSCT to receive SOC ± midostaurin (50 mg twice daily in twelve 4-week cycles). SOC was dictated by the treating physician but excluded alternate TKI therapy. Currently, SOC therapy varies per treating institution in the post-alloHSCT setting. SOC therapy includes anti-infective prophylaxis and treatment as well as GVHD prophylaxis and treatment along with supportive care. Anti-infective and GVHD prophylaxis treatments were based on institutional guidelines.
Patients were assessed for relapse and survival through 24 months post-alloHSCT and/or until the end of the study. Patient visits occurred monthly for 1 year during treatment and every other month during the 24-month follow-up. Adverse events (AEs) were tracked for 30 days after treatment ended and assessed per the Common Terminology Criteria for Adverse Events version 4.0 [26].
The study was performed in accordance with the International Council for Harmonisation Good Clinical Practice guidelines and the principles of the Declaration of Helsinki and was approved by institutional review boards at participating institutions. All patients provided written informed consent.
Endpoints
The primary endpoint was RFS (time from transplant to relapse or death due to disease) 18 months after alloHSCT. Key secondary endpoints were safety, OS (time from transplant to the date of death from any cause), and RFS 24 months after alloHSCT.
Pharmacokinetics and in vivo FLT3 inhibition by FLT3 plasma inhibitory activity (PIA) assay were assessed as preplanned exploratory endpoints (see Supplementary methods). FLT3 inhibition and FLT3 ligand levels were evaluated on the basis of phosphorylated FLT3 (P-FLT3) and FLT3 ligand levels in the plasma [27].
The incidence and severity of GVHD were also exploratory study objectives. The percentage of patients developing acute or chronic GVHD (categorized according to the National Institutes of Health Consensus Development Project Working Group criteria [28]) and grade of GVHD were collected throughout the study by local assessment. GVHD by category and organ class was assessed at each study visit.
Statistical analysis
RADIUS was an exploratory, signal-finding study not powered to detect a statistical difference between treatment arms. A sample size of 60 was calculated to detect a 50% reduction in the risk of relapse with 71% power, assuming a 15% incidence of relapse in the midostaurin arm.
For time-to-event analyses, Kaplan–Meier curves were used to estimate survival distributions. A Cox proportional hazards model was used to estimate the HR and associated 95% CIs.
Results
Patients
Between February 5, 2014, and June 13, 2016, 74 patients were screened and 60 patients (30 per arm) were randomized at 18 sites in the United States and 1 site in Canada (Fig. 1 and Table S1). All patients were in CR1 prior to transplant; 18 patients (30%) received transplant directly following induction, 39 (65%) of patients had received consolidation without additional maintenance, and 3 (5%) of patients had received pretransplant maintenance. All patients had completed a protocol-specified conditioning regimen before alloHSCT (Table S2). Overall, 30 patients completed the per-protocol 12 cycles of therapy (midostaurin + SOC: 16 patients [53%]; SOC: 14 patients [47%]). The number of patients discontinuing early from the study was comparable between arms (midostaurin + SOC, n = 13; SOC, n = 15); however, the reasons for treatment discontinuation differed by arm, with AEs being the most common reason in the midostaurin arm (27% vs 3%) and consent withdrawal being the most common reason in the SOC arm (7% vs 20%). Patients who withdrew from treatment were to return for relapse and follow-up assessments and were not considered withdrawn from the study. Patients who withdrew consent were censored at the time of withdrawal. Patient demographics, baseline characteristics, and transplant characteristics are shown in Table 1. Most patients (midostaurin + SOC, 100%; SOC, 90%) had de novo AML. The 2 arms were balanced with regard to age, sex, and race.Fig. 1 CONSORT diagram.
AE adverse event, alloHSCT allogeneic hematopoietic stem cell transplant, SOC, standard of care. aA single patient might have had >1 reason for screen failure. bEarly termination due to work schedule conflicts. cPatients lost to follow-up (n = 2), early termination due to hospitalization at outside facility (n = 1), and early termination due to large travel distance (n = 1).
Table 1 Baseline patient and transplant characteristics.
Full analysis set Midostaurin + SOC
(n = 30) SOC
(n = 30)
Median age (range), yearsa 48 (20–61) 56 (20–68)
Sex, n (%)b
Male 16 (53) 18 (60)
Female 14 (47) 12 (40)
Race, n (%)c
White 27 (90) 27 (90)
Other 3 (10) 3 (10)
AML status at initial diagnosis, n (%)
De novo 27 (90) 30 (100)
Secondary to AHD 1 (3) 0
Therapy related 2 (7) 0
Median peripheral WBC count (range), × 109/L 48 (<1–278) 55 (<1–344)
Presence of FLT3-TKD mutation
Yes 3 (10) 2 (7)
No 17 (57) 20 (67)
Unknown 10 (33) 8 (27)
Purpose of pre-HSCT treatment, n (%)
Induction 30 (100) 30 (100)
Consolidation 22 (73) 20 (67)
Maintenance 2 (7) 1 (3)
Median time to randomization (range), days 54 (34–61) 54 (30–64)
Donor type, n (%)
Syngeneic 0 1 (3)
Allogeneic, matched relatedd 10 (33) 14 (47)
Allogeneic, matched unrelatedd 20 (67) 15 (50)
Stem cell source, n (%)
Peripheral blood 29 (97) 28 (93)
Bone marrow 1 (3) 2 (7)
AHD antecedent hematologic disorder, AML acute myeloid leukemia, FLT3 fms-like tyrosine kinase 3, HLA human leukocyte antigen, HSCT hematopoietic stem cell transplant, SOC standard of care, TKD tyrosine kinase domain, WBC white blood cell.
aP = 0.14; 2-sample t-test.
bP = 0.60; Fisher exact test.
cP = 0.72; Fisher exact test.
dMatched donors had HLA typing to include an 8/8 or 7/8 allele match rate at HLA-A, -B, -C, and -DRB1. A single mismatch was allowed.
Efficacy
The estimated RFS at 18 months (95% CI) was 89% (69–96%) with midostaurin and 76% (54–88%) with SOC alone (HR, 0.46 [95% CI, 0.12–1.86]; P = 0.27) (Fig. 2A). There were 3 RFS events in the midostaurin arm and 6 RFS events in the SOC arm at 18 months. The predicted relative reduction in the risk of relapse with the addition of midostaurin was 54% at 18 months post-alloHSCT.Fig. 2 Outcomes after alloHSCT.
Kaplan–Meier curves of A RFS by treatment arm at 18 months after undergoing alloHSCT and B OS by treatment arm at 24 months after undergoing alloHSCT. Blue, midostaurin + SOC; red, SOC. Tick marks indicate censoring of data. alloHSCT allogeneic hematopoietic stem cell transplant, HR hazard ratio, OS overall survival, RFS relapse-free survival, SOC standard of care. aMedian RFS was not reached. bLog-rank P value. cMedian OS was not reached.
At 24 months, addition of midostaurin to SOC continued to demonstrate reduced risk of relapse and prolonged survival compared with SOC alone (Figs. 2B and S1). At the time of final analysis (i.e., when all patients who remained on the study had reached 24 months post-alloHSCT), the median RFS and OS were not reached in either treatment arm. There were 4 relapses (13%) in the midostaurin arm vs 5 relapses (17%) in the SOC arm; median time to relapse from transplant was similar across both arms (median [range]; midostaurin + SOC, 323.5 days [69–1028 days]; SOC alone, 323 days [94–456 days]). The estimated 24-month RFS (95% CI) was 85% (64–94%) with midostaurin and 76% (54–88%) with SOC alone (HR, 0.60 [95% CI, 0.17–2.14]; P = 0.4297), and the relative reduction in the risk of relapse with the addition of midostaurin remained high at 40%.
Survival outcomes also improved; the estimated 24-month OS (95% CI) was 85% (65%-94%) with midostaurin and 76% (54%-89%) with SOC alone (HR, 0.58 [95% CI, 0.19–1.79]; P = 0.34), which is a 42% reduction in the risk of death with the addition of midostaurin (albeit not statistically significant). Eight patients died in the SOC arm vs 5 patients in the midostaurin arm; relapse accounted for a similar fraction of deaths in each arm. Details of post-relapse treatment were not captured. A total of 7 patients died due to reasons other than relapse: 5 in the SOC arm and 2 in the midostaurin arm; these patients were censored at the date of death. Non-relapse mortality was due to study indication (n = 2) and 1 instance each of cardiac arrest, GVHD, hepatic failure, cardiopulmonary arrest, and encephalitis infection.
Pharmacokinetics and PIA assay
The pharmacokinetics of midostaurin and its main metabolites (CGP62221 and CGP52421) were evaluated in 29 patients. The mean plasma concentration of midostaurin reached a maximum duringcycle 1 day 15, where as CGP52421 and CGP62221 peaked at cycle 3 day 1; all reached steady-state levels at cycle 4 (Fig. S2).
Among patients who received midostaurin, 28 were evaluable using the PIA assay. The degree of P-FLT3 inhibition was greatest during the first 2 cycles of therapy (Fig. 3A, B). In an exploratory biomarker analysis that assessed the correlation between plasma levels of midostaurin and its primary metabolites with the degree of FLT3 inhibition (i.e., lower levels of P-FLT3), early inhibition of FLT3 correlated inversely with drug levels (Fig. 3B). Peak FLT3 inhibition occurred at cycle 3 day 1; this time point was chosen for the correlative analysis.Fig. 3 Correlation between exploratory biomarker analyses and clinical outcomes.
A Median FLT3 ligand levels and B median P-FLT3 levels relative to baseline and concurrent combined levels of midostaurin and its metabolites in patients who received midostaurin + SOC. Median P-FLT3 levels were 70% of baseline at C3D1. C RFS and D OS at 24 months after alloHSCT in patients who received midostaurin + SOC stratified by P-FLT3 level (<70% vs >70%). C cycle; D day; FLT3, fms-like tyrosine kinase 3; M midostaurin, P-FLT3 phosphorylated FLT3, OS overall survival, RFS relapse-free survival, SOC standard of care. aFor this analysis, RFS was defined as time from transplant to relapse or death from any cause. bLog-rank P value vs SOC (n = 28). cPatients who reached C3D1 and received midostaurin + SOC (n = 28) were stratified according to FLT3 inhibition levels above or below the median (median P-FLT3, 70%). FLT3 inhibition was higher in patients with P-FLT3 levels <70% of baseline. dP-FLT3 > 70% includes patients with missing P-FLT3 at C3D1.
In patients receiving midostaurin (n = 28), the median P-FLT3 level at cycle 3 day 1 was 70% of baseline P-FLT3 levels. Thus, 14 of these patients had more effective inhibition of FLT3 activity (i.e., P-FLT3 levels <70% of baseline) on cycle 3 day 1 with P-FLT3 levels ranging from 20% to 69%. Of these 14 patients, 10 completed all 12 cycles of midostaurin therapy (Fig. S3). Among the remaining 14 patients who had less effective inhibition of FLT3 activity (i.e., P-FLT3 levels >70% of baseline), P-FLT3 was not measured at cycle 3 day 1 in 8 patients (6 were not receiving midostaurin on cycle 3 day 1). Six of 14 patients completed 12 cycles of midostaurin therapy and had P-FLT3 levels ranging from 74% to 100%. These higher P-FLT3 levels indicate less effective FLT3 inhibition, possibly resulting from the biological response of the patient to midostaurin or likely related to patient adherence to midostaurin, indicating the importance of proactive AE management to support patients throughout treatment.
Stratifying patients who received midostaurin by levels of FLT3 inhibition above or below the median revealed an association with clinical outcomes. Higher levels of FLT3 inhibition correlated with prolonged RFS, a reduced risk of relapse (P = 0.06), and significantly improved survival (P = 0.048) (Fig. 3C, D). Patients with less FLT3 inhibition had a similar risk of relapse and survival rate to those observed in patients receiving SOC alone (P = 0.9 and P = 0.92, respectively).
Safety
With midostaurin + SOC and SOC alone, AEs occurred in 100% and 87% of patients, respectively (Table 2). Most AEs in both arms were grade 1/2. The most common AEs were low-grade gastrointestinal AEs (grades 1–3, midostaurin arm vs SOC arm): vomiting (73% vs 23%), nausea (67% vs 27%), and diarrhea (49% vs 23%). Gastrointestinal AEs were more common in the midostaurin arm than in the SOC arm. The most common grade 3/4 laboratory abnormalities, increased alanine aminotransferase, increased aspartate aminotransferase, and decreased neutrophils, occurred in both arms. Serious AEs (Table 3) occurred in 57% of patients with midostaurin and 30% of patients with SOC alone. The most common serious AEs (midostaurin arm vs SOC arm) were diarrhea (13% vs 7%), nausea and vomiting (both, 3% vs 10%), and pyrexia (7% vs 7%).Table 2 Most common AEs (occurring in ≥15% of patients).
AE, n (%) Midostaurin + SOC
(n = 30) SOC
(n = 30)
Any grade Grade ≥ 3 Any grade Grade ≥ 3
Vomiting 7 (23) 1 (3) 22 (73) 2 (7)
Nausea 8 (27) 3 (10) 20 (67) 1 (3)
Diarrhea 7 (23) 1 (3) 12 (40) 3 (10)
Fatigue 9 (30) 0 8 (27) 1 (3)
Peripheral edema 9 (30) 0 8 (27) 0
Headache 7 (23) 0 8 (27) 0
Cough 6 (20) 0 8 (27) 0
ALT increased 7 (23) 4 (13) 6 (20) 3 (10)
Anemia 6 (20) 2 (7) 7 (23) 3 (10)
AST increased 8 (27) 4 (13) 5 (17) 2 (7)
Pruritus 6 (20) 0 7 (23) 3 (10)
Dry eye 6 (20) 0 5 (17) 0
Pyrexia 5 (17) 1 (3) 4 (20) 0
Rash 6 (20) 0 6 (17) 0
Tremor 4 (13) 0 7 (23) 0
Dyspnea 7 (23) 1 (3) 3 (10) 0
Insomnia 6 (20) 0 4 (13) 0
Neutrophil count decreased 3 (10) 2 (7) 7 (23) 4 (13)
Arthralgia 6 (20) 1 (3) 3 (10) 0
Dizziness 6 (20) 0 3 (10) 0
Hypertension 6 (20) 4 (13) 3 (10) 0
Upper respiratory tract infection 6 (20) 0 3 (10) 0
AE adverse event, ALT alanine aminotransferase, AST aspartate aminotransferase, SOC standard of care.
Table 3 Serious AEs occurring in ≥1 of patients overall.
AE, n (%) Midostaurin + SOC
(n = 30) SOC
(n = 30)
Diarrhea 4 (13) 2 (7)
Nausea 1 (3) 3 (10)
Vomiting 1 (3) 3 (10)
Pyrexia 2 (7) 2 (7)
Deep vein thrombosis 1 (3) 2 (7)
Febrile neutropenia 1 (3) 2 (7)
Anemia 2 (7) 1 (3)
Acute kidney injury 0 2 (7)
Abdominal pain 1 (3) 1 (3)
Parainfluenza virus infection 1 (3) 1 (3)
AE adverse event, SOC standard of care.
Median midostaurin exposure was 10.5 months (range, 0.2–11.5 months; defined by time of last midostaurin dose); 16 patients completed all 12 cycles of treatment. The median dose intensity was 93 mg/day (range, 25–100 mg/day). Dose adjustments were required per protocol in 19 patients (63%), most commonly due to AEs (84%). AEs leading to dose adjustment in ≥10% of patients included vomiting (27%), nausea (20%), and aspartate aminotransferase levels increased (10%). One patient was reported to have received a modified dose of midostaurin due to concomitant posaconazole, a cytochrome P450 3A4 inhibitor, per protocol.
AEs resulted in discontinuation from the study in 9 patients: 8 (27%) in the midostaurin arm and 1 (3%) in the SOC arm. The 8 patients in the midostaurin arm who discontinued treatment had 9 events: nausea (n = 3), vomiting (n = 2), liver function test levels increased (n = 2), pulmonary mycosis (n = 1), and pneumonitis (n = 1). The patient in the SOC arm discontinued from the study due to hypoxia. Twelve patients died on study during the follow-up phase (midostaurin + SOC, n = 4; SOC, n = 8). Death due to AML disease progression occurred in 2 patients receiving midostaurin and 4 receiving SOC alone. The addition of midostaurin to SOC did not result in an increase in the severity or rate of acute or chronic GVHD (Table 4). Rates of GVHD, as determined by local assessment, were similar between the midostaurin and SOC arms (overall, 70% vs 73%; acute, 53% vs 50%; and chronic, 37% vs 33%, respectively). Ninety-seven percent of patients received concomitant medication for the management of GVHD, including 28 (93%) in the midostaurin arm and 30 (100%) in the SOC arm. The most common concomitant medications typical of GVHD management were calcineurin inhibitors (85%), glucocorticoids (57%), moderately potent corticosteroids (18%), and selective immunosuppressants (17%) (Table S3).Table 4 Incidence of GVHD.
GVHD, n (%)a Midostaurin + SOC
(n = 30) SOC
(n = 30)
Acute 15 (50) 16 (53)
Grade I 7 (23) 4 (13)
Grade II 8 (27) 10 (33)
Grade III 0 2 (7)
Grade IV 0 0
Chronic 9 (30) 10 (33)
Mild 2 (7) 5 (17)
Moderate 5 (17) 4 (13)
Severe 2 (7) 1 (3)
GVHD graft-vs-host disease, SOC standard of care.
aPatients could be counted in multiple categories.
The most common organ toxicity due to GVHD was localized to the skin and affected 50% of patients in the midostaurin arm and 47% of patients in the SOC arm (Fig. S4). All patients with skin involvement in the midostaurin arm had stage 1 or 2 disease, whereas 2 patients in the SOC arm experienced stage 3 disease. Neither arm reported stage 4 organ involvement. Upper gastrointestinal toxicity was similar in both groups and did not exceed stage 1. Lower gastrointestinal toxicity was reported only in patients in the SOC arm and was primarily stage 1.
Discussion
This is the first randomized study of midostaurin as maintenance therapy after alloHSCT. We show that for patients with FLT3-ITD-positive AML in CR1, a defined course of up to 12 months of maintenance therapy with midostaurin was safely added to SOC after recovery from alloHSCT and improved RFS at 18 months after alloHSCT by 13% (over SOC alone). Although the study was not powered to detect a treatment difference, there was a trend toward benefit with midostaurin for all efficacy endpoints evaluated.
The survival outcomes in all participants in this study were better than anticipated for this high-risk leukemia population. Historically, the expected 2-year OS with SOC was closer to 60% compared with 76% observed in this study [15]. The stringent enrollment criteria, including recovery of counts (i.e., absolute neutrophil count >1000/μL and platelet count ≥20,000/μL without platelet transfusion) by day 42, ability to start treatment by day 60 post- alloHSCT, and no active, advanced, acute GVHD, may have contributed to the survival outcomes observed for all participants in this study. Moreover, the median time from the date of alloHSCT to initiation of study drug for both arms was 54 days; patients who had morphological relapse before that date were ineligible. Consistently, factors related to these inclusion/exclusion criteria, such as unacceptable test procedure results (8%) and unacceptable medical history/concomitant diagnosis (4%), were common reasons for screen failure, though the overall rate of screen failures (14 of 74 patients screened [19%]) was relatively low. Censoring of patients at the date of death due to non-relapse mortality may also have contributed to survival rates, particularly given the small patient population in this study. Similarly, patients were not stratified by European LeukemiaNet or National Comprehensive Cancer Network molecular risk classification due to the size of the study; thus, enrollment of patients with favorable molecular risk factors may also have affected the survival rates observed.
Correlative analysis suggests that patients who tolerated midostaurin and remained on therapy, as demonstrated by relatively higher levels of P-FLT3 inhibition, may have sustained benefit and long-term outcomes. The PIA assay allows for an indirect measurement of the phosphorylation of FLT3. P-FLT3 inhibition to <70% of baseline was achieved by 50% of patients receiving midostaurin and was associated with improved RFS and OS, indicating that inhibiting FLT3, even modestly, can have clinical benefit. Treatment adherence was not uniform in all patients receiving midostaurin, possibly due to tolerability (e.g., gastrointestinal toxicity). Prophylactic support, including antiemetics, in the management of gastrointestinal toxicities was crucial in keeping patients on therapy to provide the clinical benefit suggested by these data. Thus, increases in gastrointestinal toxicities were primarily low grade and manageable, consistent with other reports with single-agent midostaurin [29, 30]. Addition of midostaurin to SOC did not increase rates or severity of GVHD. Although the PIA assay is not used in clinical practice, FLT3 inhibition measured by this assay has tightly correlated with clinical activity across a broad array of FLT3 inhibitors [27, 31–34]. The results from the exploratory analysis in this study suggest that midostaurin therapy after alloHSCT may provide high levels of FLT3 inhibition in the long term in patients who remain on treatment, though further validation is required.
These data are consistent with the safety profile of midostaurin in patients with FLT3-ITD AML. In line with the AMLSG 16-10 study [17], the median time of midostaurin exposure during maintenance was similar (9 months in AMLSG 16-10 and 10 months in RADIUS); discontinuation due to toxicity was more common in AMLSG 16-10 (55%) than in RADIUS (27%), which may be explained by the stringent inclusion criteria of RADIUS. However, both studies demonstrated the safety and feasibility of midostaurin maintenance therapy.
Post-alloHSCT maintenance therapy with FLT3 TKIs, including midostaurin, is a viable treatment for reducing the risk of relapse in patients with FLT3-ITD AML. We anticipate that this study will provide a landmark for future studies, as the population had no pretransplant TKI exposure. These results complement those of the AMLSG 16-10 trial, which demonstrated improved event-free survival for patients with FLT3-ITD AML who received pretransplant midostaurin and began midostaurin within 100 days post-transplant compared with patients who only received pretransplant midostaurin [17]. Evidence from the present study and AMLSG 16-10 suggest that midostaurin maintenance therapy may be most appropriate for patients aged 18–70 years with FLT3-ITD AML who have undergone alloHSCT in CR1 and can begin midostaurin therapy quickly (within 100 days, ideally <60 days).
With the approval of midostaurin as up-front therapy for FLT3-ITD AML, new trials are emerging to better clarify the role of post-transplant TKI therapy in patients with deeper molecular remission, such as the large, phase 3, multinational, randomized trial assessing gilteritinib vs placebo as post-transplant adjuvant therapy for patients with FLT3-ITD AML in CR1 (BMT-CTN 1506; NCT02997202). As available treatment options increase, more detailed scrutiny of the risk-benefit profiles of these targeted agents is likely to be required.
With a post-transplant 2-year OS of ~80%, this study highlights the impact of recent advances in the management of FLT3-ITD AML on survival outcomes. Because FLT3-mutated AML has a higher risk of relapse than FLT3-mutation-negative AML, the addition of midostaurin maintenance therapy post-HSCT may be a viable option to reduce the risk of relapse in some patients after alloHSCT. These results provide evidence of clinical benefit and an estimate of treatment effect that could inform larger-scale studies in the future.
Supplementary information
Supplemental Material
Supplementary information
The online version of this article (10.1038/s41409-020-01153-1) contains supplementary material, which is available to authorized users.
Acknowledgements
The authors would like to thank the patients and the investigators who participated in the RADIUS study. Medical editorial assistance was provided by JoAnna Anderson, Ph.D., and Amy Ghiretti, Ph.D., of ArticulateScience LLC, and was supported by Novartis Pharmaceuticals Corporation. This study was funded by Novartis Pharmaceuticals Corporation.
Compliance with ethical standards
Conflict of interest
RTM discloses honoraria from Novartis, Incyte, Juno Therapeutics, and Kite Therapeutics; Board of Directors membership at Novartis Pharmaceuticals Corporation; consultancies with Incyte and Juno Therapeutics; and patents and royalties from Athersys, Inc; as an OHSU employee who provided and received payment for consultancy services to Novartis Pharmaceuticals Corporation, this potential conflict of interest has been reviewed and managed by OHSU. ML discloses consultancy with Novartis Pharmaceuticals Corporation, Astellas, and Daiichi Sankyo; research funding from Novartis Pharmaceuticals Corporation, Astellas, and Fujifilm; and honoraria from Novartis Pharmaceuticals Corporation. MMP discloses advisory board membership with Stemline. BLS discloses consultancy with Acceleron, Incyte, Agios, Celgene, and Alexion and research funding from Novartis Pharmaceuticals Corporation and Celgene. SRM has nothing to disclose. AD discloses consultancies with Kite Therapeutics and Novartis Pharmaceuticals Corporation. SDR has nothing to disclose. DDHK discloses consultancies with Novartis Pharmaceuticals Corporation, Bristol-Meyers Squibb, Paladin, and Pfizer and honoraria and research funding from Novartis Pharmaceuticals Corporation and Bristol-Meyers Squibb. DH and TR have nothing to disclose. KH discloses former employment with Novartis Pharmaceuticals Corporation and current employment with Regeneron Pharmaceuticals, Inc. GB and DP disclose employment with Novartis Pharmaceuticals Corporation. PR discloses former employment with Novartis Pharmaceuticals Corporation and current employment with Target CW. HFF discloses honoraria from Pfizer and Sanofi and speakers’ bureau membership with Sanofi.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Recovered | ReactionOutcome | CC BY | 33288862 | 18,658,791 | 2021-05 |
What was the outcome of reaction 'Nausea'? | Midostaurin after allogeneic stem cell transplant in patients with FLT3-internal tandem duplication-positive acute myeloid leukemia.
We evaluated standard-of-care (SOC) treatment with or without midostaurin to prevent relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) in patients with acute myeloid leukemia (AML) harboring internal tandem duplication (ITD) in FLT3. Adults (aged 18-70 years) who received alloHSCT in first complete remission, had achieved hematologic recovery, and were transfusion independent were randomized to receive SOC with or without midostaurin (50 mg twice daily) continuously in twelve 4-week cycles. The primary endpoint was relapse-free survival (RFS) 18 months post-alloHSCT. Sixty patients were randomized (30/arm); 30 completed all 12 cycles (midostaurin + SOC, n = 16; SOC, n = 14). The estimated 18-month RFS (95% CI) was 89% (69-96%) in the midostaurin arm and 76% (54-88%) in the SOC arm (hazard ratio, 0.46 [95% CI, 0.12-1.86]; P = 0.27); estimated relapse rates were 11% and 24%, respectively. Inhibition of FLT3 phosphorylation to <70% of baseline (achieved by 50% of midostaurin-treated patients) was associated with improved RFS. The most common serious adverse events were diarrhea, nausea, and vomiting. Rates of graft-vs-host disease were similar between both arms (midostaurin + SOC, 70%; SOC, 73%). The addition of midostaurin maintenance therapy following alloHSCT may provide clinical benefit in some patients with FLT3-ITD AML. (ClinicalTrials.gov identifier: NCT01883362).
Introduction
Acute myeloid leukemia (AML), the most common acute leukemia, is difficult to treat and has a poor prognosis, with a 5-year survival of ~25% [1, 2]. Multiple factors, including age, performance status (e.g., Eastern Cooperative Oncology Group), and cytogenetic and molecular features, affect treatment decisions and outcomes [3, 4]. Mutations in fms-like tyrosine kinase 3 (FLT3) are among the most common in AML and confer a poor prognosis with poor overall survival (OS) [5–7]. Consequently, these patients, particularly those with internal tandem duplications (ITDs), historically have more frequent and earlier relapses than patients without FLT3 mutations [7, 8].
Midostaurin, a multikinase inhibitor that targets FLT3 and other kinases, was approved for the treatment of adult patients with newly diagnosed, FLT3-mutated AML when combined with intensive induction and consolidation chemotherapy [9]. Approval was based on the phase 3 RATIFY/CALGB 10603 trial, which demonstrated improved survival with the addition of midostaurin to intensive chemotherapy followed by single-agent maintenance therapy in patients aged <60 years with newly diagnosed, FLT3-mutated AML. The RATIFY trial did not allow patients receiving alloHSCT to continue midostaurin [10].
AlloHSCT in first complete remission (CR1) provides patients with FLT3-ITD-positive AML the highest likelihood of sustained remission [11, 12], but relapse rates remain high [13–15]. The prognosis for patients with FLT3-ITD mutations has been poor following standard alloHSCT, primarily because these patients have a higher risk of relapse than patients with FLT3-ITD-negative AML [14–16].
Post-HSCT maintenance therapy with tyrosine kinase inhibitors (TKIs) may improve outcomes in patients with FLT3-mutated AML. In a phase 2 trial (AMLSG 16-10), midostaurin combined with intensive chemotherapy followed by alloHSCT and single-agent maintenance therapy demonstrated improved rates of event-free survival in patients receiving midostaurin compared with historical controls [17]. In AMLSG 16-10, midostaurin was administered as in RATIFY; however, patients who underwent alloHSCT could resume midostaurin as maintenance therapy post-transplant [10, 17]. Data from phase 1 and 2 trials suggest there may be a benefit with sorafenib, another TKI, as maintenance therapy post-HSCT [18–20]. Results from the phase 2 SORMAIN trial, which evaluated post-alloHSCT maintenance with sorafenib, suggested a benefit with sorafenib versus placebo with a median 2-year relapse-free survival (RFS) rate of 85% (95% CI, 70–93%) vs 53% (95% CI, 37–68%), respectively, (hazard ratio [HR], 0.39 [95% CI, 0.183—0.848]; P = 0.013) [20]. Similarly, quizartinib, a FLT3 TKI, was safely administered after alloHSCT in a phase 1 study [21]. Detailed trials evaluating FLT3 TKIs as maintenance therapy are ongoing [22–25].
Here, we report the results of the RADIUS trial investigating whether the addition of midostaurin to standard-of-care (SOC) treatment post-alloHSCT improves RFS over SOC alone in patients with FLT3-ITD-positive AML.
Patients and methods
Study design
RADIUS (NCT01883362) was a phase 2, randomized, open-label trial of SOC with or without midostaurin in patients (aged 18–70 years) with documented FLT3-ITD-positive AML who had undergone a protocol-specified conditioning regimen before alloHSCT in CR1 (following hematologic recovery, transfusion independence, and controlled graft-vs-host disease [GVHD]). Patients were enrolled after engraftment and randomized 1:1 within 28 to 60 days after alloHSCT to receive SOC ± midostaurin (50 mg twice daily in twelve 4-week cycles). SOC was dictated by the treating physician but excluded alternate TKI therapy. Currently, SOC therapy varies per treating institution in the post-alloHSCT setting. SOC therapy includes anti-infective prophylaxis and treatment as well as GVHD prophylaxis and treatment along with supportive care. Anti-infective and GVHD prophylaxis treatments were based on institutional guidelines.
Patients were assessed for relapse and survival through 24 months post-alloHSCT and/or until the end of the study. Patient visits occurred monthly for 1 year during treatment and every other month during the 24-month follow-up. Adverse events (AEs) were tracked for 30 days after treatment ended and assessed per the Common Terminology Criteria for Adverse Events version 4.0 [26].
The study was performed in accordance with the International Council for Harmonisation Good Clinical Practice guidelines and the principles of the Declaration of Helsinki and was approved by institutional review boards at participating institutions. All patients provided written informed consent.
Endpoints
The primary endpoint was RFS (time from transplant to relapse or death due to disease) 18 months after alloHSCT. Key secondary endpoints were safety, OS (time from transplant to the date of death from any cause), and RFS 24 months after alloHSCT.
Pharmacokinetics and in vivo FLT3 inhibition by FLT3 plasma inhibitory activity (PIA) assay were assessed as preplanned exploratory endpoints (see Supplementary methods). FLT3 inhibition and FLT3 ligand levels were evaluated on the basis of phosphorylated FLT3 (P-FLT3) and FLT3 ligand levels in the plasma [27].
The incidence and severity of GVHD were also exploratory study objectives. The percentage of patients developing acute or chronic GVHD (categorized according to the National Institutes of Health Consensus Development Project Working Group criteria [28]) and grade of GVHD were collected throughout the study by local assessment. GVHD by category and organ class was assessed at each study visit.
Statistical analysis
RADIUS was an exploratory, signal-finding study not powered to detect a statistical difference between treatment arms. A sample size of 60 was calculated to detect a 50% reduction in the risk of relapse with 71% power, assuming a 15% incidence of relapse in the midostaurin arm.
For time-to-event analyses, Kaplan–Meier curves were used to estimate survival distributions. A Cox proportional hazards model was used to estimate the HR and associated 95% CIs.
Results
Patients
Between February 5, 2014, and June 13, 2016, 74 patients were screened and 60 patients (30 per arm) were randomized at 18 sites in the United States and 1 site in Canada (Fig. 1 and Table S1). All patients were in CR1 prior to transplant; 18 patients (30%) received transplant directly following induction, 39 (65%) of patients had received consolidation without additional maintenance, and 3 (5%) of patients had received pretransplant maintenance. All patients had completed a protocol-specified conditioning regimen before alloHSCT (Table S2). Overall, 30 patients completed the per-protocol 12 cycles of therapy (midostaurin + SOC: 16 patients [53%]; SOC: 14 patients [47%]). The number of patients discontinuing early from the study was comparable between arms (midostaurin + SOC, n = 13; SOC, n = 15); however, the reasons for treatment discontinuation differed by arm, with AEs being the most common reason in the midostaurin arm (27% vs 3%) and consent withdrawal being the most common reason in the SOC arm (7% vs 20%). Patients who withdrew from treatment were to return for relapse and follow-up assessments and were not considered withdrawn from the study. Patients who withdrew consent were censored at the time of withdrawal. Patient demographics, baseline characteristics, and transplant characteristics are shown in Table 1. Most patients (midostaurin + SOC, 100%; SOC, 90%) had de novo AML. The 2 arms were balanced with regard to age, sex, and race.Fig. 1 CONSORT diagram.
AE adverse event, alloHSCT allogeneic hematopoietic stem cell transplant, SOC, standard of care. aA single patient might have had >1 reason for screen failure. bEarly termination due to work schedule conflicts. cPatients lost to follow-up (n = 2), early termination due to hospitalization at outside facility (n = 1), and early termination due to large travel distance (n = 1).
Table 1 Baseline patient and transplant characteristics.
Full analysis set Midostaurin + SOC
(n = 30) SOC
(n = 30)
Median age (range), yearsa 48 (20–61) 56 (20–68)
Sex, n (%)b
Male 16 (53) 18 (60)
Female 14 (47) 12 (40)
Race, n (%)c
White 27 (90) 27 (90)
Other 3 (10) 3 (10)
AML status at initial diagnosis, n (%)
De novo 27 (90) 30 (100)
Secondary to AHD 1 (3) 0
Therapy related 2 (7) 0
Median peripheral WBC count (range), × 109/L 48 (<1–278) 55 (<1–344)
Presence of FLT3-TKD mutation
Yes 3 (10) 2 (7)
No 17 (57) 20 (67)
Unknown 10 (33) 8 (27)
Purpose of pre-HSCT treatment, n (%)
Induction 30 (100) 30 (100)
Consolidation 22 (73) 20 (67)
Maintenance 2 (7) 1 (3)
Median time to randomization (range), days 54 (34–61) 54 (30–64)
Donor type, n (%)
Syngeneic 0 1 (3)
Allogeneic, matched relatedd 10 (33) 14 (47)
Allogeneic, matched unrelatedd 20 (67) 15 (50)
Stem cell source, n (%)
Peripheral blood 29 (97) 28 (93)
Bone marrow 1 (3) 2 (7)
AHD antecedent hematologic disorder, AML acute myeloid leukemia, FLT3 fms-like tyrosine kinase 3, HLA human leukocyte antigen, HSCT hematopoietic stem cell transplant, SOC standard of care, TKD tyrosine kinase domain, WBC white blood cell.
aP = 0.14; 2-sample t-test.
bP = 0.60; Fisher exact test.
cP = 0.72; Fisher exact test.
dMatched donors had HLA typing to include an 8/8 or 7/8 allele match rate at HLA-A, -B, -C, and -DRB1. A single mismatch was allowed.
Efficacy
The estimated RFS at 18 months (95% CI) was 89% (69–96%) with midostaurin and 76% (54–88%) with SOC alone (HR, 0.46 [95% CI, 0.12–1.86]; P = 0.27) (Fig. 2A). There were 3 RFS events in the midostaurin arm and 6 RFS events in the SOC arm at 18 months. The predicted relative reduction in the risk of relapse with the addition of midostaurin was 54% at 18 months post-alloHSCT.Fig. 2 Outcomes after alloHSCT.
Kaplan–Meier curves of A RFS by treatment arm at 18 months after undergoing alloHSCT and B OS by treatment arm at 24 months after undergoing alloHSCT. Blue, midostaurin + SOC; red, SOC. Tick marks indicate censoring of data. alloHSCT allogeneic hematopoietic stem cell transplant, HR hazard ratio, OS overall survival, RFS relapse-free survival, SOC standard of care. aMedian RFS was not reached. bLog-rank P value. cMedian OS was not reached.
At 24 months, addition of midostaurin to SOC continued to demonstrate reduced risk of relapse and prolonged survival compared with SOC alone (Figs. 2B and S1). At the time of final analysis (i.e., when all patients who remained on the study had reached 24 months post-alloHSCT), the median RFS and OS were not reached in either treatment arm. There were 4 relapses (13%) in the midostaurin arm vs 5 relapses (17%) in the SOC arm; median time to relapse from transplant was similar across both arms (median [range]; midostaurin + SOC, 323.5 days [69–1028 days]; SOC alone, 323 days [94–456 days]). The estimated 24-month RFS (95% CI) was 85% (64–94%) with midostaurin and 76% (54–88%) with SOC alone (HR, 0.60 [95% CI, 0.17–2.14]; P = 0.4297), and the relative reduction in the risk of relapse with the addition of midostaurin remained high at 40%.
Survival outcomes also improved; the estimated 24-month OS (95% CI) was 85% (65%-94%) with midostaurin and 76% (54%-89%) with SOC alone (HR, 0.58 [95% CI, 0.19–1.79]; P = 0.34), which is a 42% reduction in the risk of death with the addition of midostaurin (albeit not statistically significant). Eight patients died in the SOC arm vs 5 patients in the midostaurin arm; relapse accounted for a similar fraction of deaths in each arm. Details of post-relapse treatment were not captured. A total of 7 patients died due to reasons other than relapse: 5 in the SOC arm and 2 in the midostaurin arm; these patients were censored at the date of death. Non-relapse mortality was due to study indication (n = 2) and 1 instance each of cardiac arrest, GVHD, hepatic failure, cardiopulmonary arrest, and encephalitis infection.
Pharmacokinetics and PIA assay
The pharmacokinetics of midostaurin and its main metabolites (CGP62221 and CGP52421) were evaluated in 29 patients. The mean plasma concentration of midostaurin reached a maximum duringcycle 1 day 15, where as CGP52421 and CGP62221 peaked at cycle 3 day 1; all reached steady-state levels at cycle 4 (Fig. S2).
Among patients who received midostaurin, 28 were evaluable using the PIA assay. The degree of P-FLT3 inhibition was greatest during the first 2 cycles of therapy (Fig. 3A, B). In an exploratory biomarker analysis that assessed the correlation between plasma levels of midostaurin and its primary metabolites with the degree of FLT3 inhibition (i.e., lower levels of P-FLT3), early inhibition of FLT3 correlated inversely with drug levels (Fig. 3B). Peak FLT3 inhibition occurred at cycle 3 day 1; this time point was chosen for the correlative analysis.Fig. 3 Correlation between exploratory biomarker analyses and clinical outcomes.
A Median FLT3 ligand levels and B median P-FLT3 levels relative to baseline and concurrent combined levels of midostaurin and its metabolites in patients who received midostaurin + SOC. Median P-FLT3 levels were 70% of baseline at C3D1. C RFS and D OS at 24 months after alloHSCT in patients who received midostaurin + SOC stratified by P-FLT3 level (<70% vs >70%). C cycle; D day; FLT3, fms-like tyrosine kinase 3; M midostaurin, P-FLT3 phosphorylated FLT3, OS overall survival, RFS relapse-free survival, SOC standard of care. aFor this analysis, RFS was defined as time from transplant to relapse or death from any cause. bLog-rank P value vs SOC (n = 28). cPatients who reached C3D1 and received midostaurin + SOC (n = 28) were stratified according to FLT3 inhibition levels above or below the median (median P-FLT3, 70%). FLT3 inhibition was higher in patients with P-FLT3 levels <70% of baseline. dP-FLT3 > 70% includes patients with missing P-FLT3 at C3D1.
In patients receiving midostaurin (n = 28), the median P-FLT3 level at cycle 3 day 1 was 70% of baseline P-FLT3 levels. Thus, 14 of these patients had more effective inhibition of FLT3 activity (i.e., P-FLT3 levels <70% of baseline) on cycle 3 day 1 with P-FLT3 levels ranging from 20% to 69%. Of these 14 patients, 10 completed all 12 cycles of midostaurin therapy (Fig. S3). Among the remaining 14 patients who had less effective inhibition of FLT3 activity (i.e., P-FLT3 levels >70% of baseline), P-FLT3 was not measured at cycle 3 day 1 in 8 patients (6 were not receiving midostaurin on cycle 3 day 1). Six of 14 patients completed 12 cycles of midostaurin therapy and had P-FLT3 levels ranging from 74% to 100%. These higher P-FLT3 levels indicate less effective FLT3 inhibition, possibly resulting from the biological response of the patient to midostaurin or likely related to patient adherence to midostaurin, indicating the importance of proactive AE management to support patients throughout treatment.
Stratifying patients who received midostaurin by levels of FLT3 inhibition above or below the median revealed an association with clinical outcomes. Higher levels of FLT3 inhibition correlated with prolonged RFS, a reduced risk of relapse (P = 0.06), and significantly improved survival (P = 0.048) (Fig. 3C, D). Patients with less FLT3 inhibition had a similar risk of relapse and survival rate to those observed in patients receiving SOC alone (P = 0.9 and P = 0.92, respectively).
Safety
With midostaurin + SOC and SOC alone, AEs occurred in 100% and 87% of patients, respectively (Table 2). Most AEs in both arms were grade 1/2. The most common AEs were low-grade gastrointestinal AEs (grades 1–3, midostaurin arm vs SOC arm): vomiting (73% vs 23%), nausea (67% vs 27%), and diarrhea (49% vs 23%). Gastrointestinal AEs were more common in the midostaurin arm than in the SOC arm. The most common grade 3/4 laboratory abnormalities, increased alanine aminotransferase, increased aspartate aminotransferase, and decreased neutrophils, occurred in both arms. Serious AEs (Table 3) occurred in 57% of patients with midostaurin and 30% of patients with SOC alone. The most common serious AEs (midostaurin arm vs SOC arm) were diarrhea (13% vs 7%), nausea and vomiting (both, 3% vs 10%), and pyrexia (7% vs 7%).Table 2 Most common AEs (occurring in ≥15% of patients).
AE, n (%) Midostaurin + SOC
(n = 30) SOC
(n = 30)
Any grade Grade ≥ 3 Any grade Grade ≥ 3
Vomiting 7 (23) 1 (3) 22 (73) 2 (7)
Nausea 8 (27) 3 (10) 20 (67) 1 (3)
Diarrhea 7 (23) 1 (3) 12 (40) 3 (10)
Fatigue 9 (30) 0 8 (27) 1 (3)
Peripheral edema 9 (30) 0 8 (27) 0
Headache 7 (23) 0 8 (27) 0
Cough 6 (20) 0 8 (27) 0
ALT increased 7 (23) 4 (13) 6 (20) 3 (10)
Anemia 6 (20) 2 (7) 7 (23) 3 (10)
AST increased 8 (27) 4 (13) 5 (17) 2 (7)
Pruritus 6 (20) 0 7 (23) 3 (10)
Dry eye 6 (20) 0 5 (17) 0
Pyrexia 5 (17) 1 (3) 4 (20) 0
Rash 6 (20) 0 6 (17) 0
Tremor 4 (13) 0 7 (23) 0
Dyspnea 7 (23) 1 (3) 3 (10) 0
Insomnia 6 (20) 0 4 (13) 0
Neutrophil count decreased 3 (10) 2 (7) 7 (23) 4 (13)
Arthralgia 6 (20) 1 (3) 3 (10) 0
Dizziness 6 (20) 0 3 (10) 0
Hypertension 6 (20) 4 (13) 3 (10) 0
Upper respiratory tract infection 6 (20) 0 3 (10) 0
AE adverse event, ALT alanine aminotransferase, AST aspartate aminotransferase, SOC standard of care.
Table 3 Serious AEs occurring in ≥1 of patients overall.
AE, n (%) Midostaurin + SOC
(n = 30) SOC
(n = 30)
Diarrhea 4 (13) 2 (7)
Nausea 1 (3) 3 (10)
Vomiting 1 (3) 3 (10)
Pyrexia 2 (7) 2 (7)
Deep vein thrombosis 1 (3) 2 (7)
Febrile neutropenia 1 (3) 2 (7)
Anemia 2 (7) 1 (3)
Acute kidney injury 0 2 (7)
Abdominal pain 1 (3) 1 (3)
Parainfluenza virus infection 1 (3) 1 (3)
AE adverse event, SOC standard of care.
Median midostaurin exposure was 10.5 months (range, 0.2–11.5 months; defined by time of last midostaurin dose); 16 patients completed all 12 cycles of treatment. The median dose intensity was 93 mg/day (range, 25–100 mg/day). Dose adjustments were required per protocol in 19 patients (63%), most commonly due to AEs (84%). AEs leading to dose adjustment in ≥10% of patients included vomiting (27%), nausea (20%), and aspartate aminotransferase levels increased (10%). One patient was reported to have received a modified dose of midostaurin due to concomitant posaconazole, a cytochrome P450 3A4 inhibitor, per protocol.
AEs resulted in discontinuation from the study in 9 patients: 8 (27%) in the midostaurin arm and 1 (3%) in the SOC arm. The 8 patients in the midostaurin arm who discontinued treatment had 9 events: nausea (n = 3), vomiting (n = 2), liver function test levels increased (n = 2), pulmonary mycosis (n = 1), and pneumonitis (n = 1). The patient in the SOC arm discontinued from the study due to hypoxia. Twelve patients died on study during the follow-up phase (midostaurin + SOC, n = 4; SOC, n = 8). Death due to AML disease progression occurred in 2 patients receiving midostaurin and 4 receiving SOC alone. The addition of midostaurin to SOC did not result in an increase in the severity or rate of acute or chronic GVHD (Table 4). Rates of GVHD, as determined by local assessment, were similar between the midostaurin and SOC arms (overall, 70% vs 73%; acute, 53% vs 50%; and chronic, 37% vs 33%, respectively). Ninety-seven percent of patients received concomitant medication for the management of GVHD, including 28 (93%) in the midostaurin arm and 30 (100%) in the SOC arm. The most common concomitant medications typical of GVHD management were calcineurin inhibitors (85%), glucocorticoids (57%), moderately potent corticosteroids (18%), and selective immunosuppressants (17%) (Table S3).Table 4 Incidence of GVHD.
GVHD, n (%)a Midostaurin + SOC
(n = 30) SOC
(n = 30)
Acute 15 (50) 16 (53)
Grade I 7 (23) 4 (13)
Grade II 8 (27) 10 (33)
Grade III 0 2 (7)
Grade IV 0 0
Chronic 9 (30) 10 (33)
Mild 2 (7) 5 (17)
Moderate 5 (17) 4 (13)
Severe 2 (7) 1 (3)
GVHD graft-vs-host disease, SOC standard of care.
aPatients could be counted in multiple categories.
The most common organ toxicity due to GVHD was localized to the skin and affected 50% of patients in the midostaurin arm and 47% of patients in the SOC arm (Fig. S4). All patients with skin involvement in the midostaurin arm had stage 1 or 2 disease, whereas 2 patients in the SOC arm experienced stage 3 disease. Neither arm reported stage 4 organ involvement. Upper gastrointestinal toxicity was similar in both groups and did not exceed stage 1. Lower gastrointestinal toxicity was reported only in patients in the SOC arm and was primarily stage 1.
Discussion
This is the first randomized study of midostaurin as maintenance therapy after alloHSCT. We show that for patients with FLT3-ITD-positive AML in CR1, a defined course of up to 12 months of maintenance therapy with midostaurin was safely added to SOC after recovery from alloHSCT and improved RFS at 18 months after alloHSCT by 13% (over SOC alone). Although the study was not powered to detect a treatment difference, there was a trend toward benefit with midostaurin for all efficacy endpoints evaluated.
The survival outcomes in all participants in this study were better than anticipated for this high-risk leukemia population. Historically, the expected 2-year OS with SOC was closer to 60% compared with 76% observed in this study [15]. The stringent enrollment criteria, including recovery of counts (i.e., absolute neutrophil count >1000/μL and platelet count ≥20,000/μL without platelet transfusion) by day 42, ability to start treatment by day 60 post- alloHSCT, and no active, advanced, acute GVHD, may have contributed to the survival outcomes observed for all participants in this study. Moreover, the median time from the date of alloHSCT to initiation of study drug for both arms was 54 days; patients who had morphological relapse before that date were ineligible. Consistently, factors related to these inclusion/exclusion criteria, such as unacceptable test procedure results (8%) and unacceptable medical history/concomitant diagnosis (4%), were common reasons for screen failure, though the overall rate of screen failures (14 of 74 patients screened [19%]) was relatively low. Censoring of patients at the date of death due to non-relapse mortality may also have contributed to survival rates, particularly given the small patient population in this study. Similarly, patients were not stratified by European LeukemiaNet or National Comprehensive Cancer Network molecular risk classification due to the size of the study; thus, enrollment of patients with favorable molecular risk factors may also have affected the survival rates observed.
Correlative analysis suggests that patients who tolerated midostaurin and remained on therapy, as demonstrated by relatively higher levels of P-FLT3 inhibition, may have sustained benefit and long-term outcomes. The PIA assay allows for an indirect measurement of the phosphorylation of FLT3. P-FLT3 inhibition to <70% of baseline was achieved by 50% of patients receiving midostaurin and was associated with improved RFS and OS, indicating that inhibiting FLT3, even modestly, can have clinical benefit. Treatment adherence was not uniform in all patients receiving midostaurin, possibly due to tolerability (e.g., gastrointestinal toxicity). Prophylactic support, including antiemetics, in the management of gastrointestinal toxicities was crucial in keeping patients on therapy to provide the clinical benefit suggested by these data. Thus, increases in gastrointestinal toxicities were primarily low grade and manageable, consistent with other reports with single-agent midostaurin [29, 30]. Addition of midostaurin to SOC did not increase rates or severity of GVHD. Although the PIA assay is not used in clinical practice, FLT3 inhibition measured by this assay has tightly correlated with clinical activity across a broad array of FLT3 inhibitors [27, 31–34]. The results from the exploratory analysis in this study suggest that midostaurin therapy after alloHSCT may provide high levels of FLT3 inhibition in the long term in patients who remain on treatment, though further validation is required.
These data are consistent with the safety profile of midostaurin in patients with FLT3-ITD AML. In line with the AMLSG 16-10 study [17], the median time of midostaurin exposure during maintenance was similar (9 months in AMLSG 16-10 and 10 months in RADIUS); discontinuation due to toxicity was more common in AMLSG 16-10 (55%) than in RADIUS (27%), which may be explained by the stringent inclusion criteria of RADIUS. However, both studies demonstrated the safety and feasibility of midostaurin maintenance therapy.
Post-alloHSCT maintenance therapy with FLT3 TKIs, including midostaurin, is a viable treatment for reducing the risk of relapse in patients with FLT3-ITD AML. We anticipate that this study will provide a landmark for future studies, as the population had no pretransplant TKI exposure. These results complement those of the AMLSG 16-10 trial, which demonstrated improved event-free survival for patients with FLT3-ITD AML who received pretransplant midostaurin and began midostaurin within 100 days post-transplant compared with patients who only received pretransplant midostaurin [17]. Evidence from the present study and AMLSG 16-10 suggest that midostaurin maintenance therapy may be most appropriate for patients aged 18–70 years with FLT3-ITD AML who have undergone alloHSCT in CR1 and can begin midostaurin therapy quickly (within 100 days, ideally <60 days).
With the approval of midostaurin as up-front therapy for FLT3-ITD AML, new trials are emerging to better clarify the role of post-transplant TKI therapy in patients with deeper molecular remission, such as the large, phase 3, multinational, randomized trial assessing gilteritinib vs placebo as post-transplant adjuvant therapy for patients with FLT3-ITD AML in CR1 (BMT-CTN 1506; NCT02997202). As available treatment options increase, more detailed scrutiny of the risk-benefit profiles of these targeted agents is likely to be required.
With a post-transplant 2-year OS of ~80%, this study highlights the impact of recent advances in the management of FLT3-ITD AML on survival outcomes. Because FLT3-mutated AML has a higher risk of relapse than FLT3-mutation-negative AML, the addition of midostaurin maintenance therapy post-HSCT may be a viable option to reduce the risk of relapse in some patients after alloHSCT. These results provide evidence of clinical benefit and an estimate of treatment effect that could inform larger-scale studies in the future.
Supplementary information
Supplemental Material
Supplementary information
The online version of this article (10.1038/s41409-020-01153-1) contains supplementary material, which is available to authorized users.
Acknowledgements
The authors would like to thank the patients and the investigators who participated in the RADIUS study. Medical editorial assistance was provided by JoAnna Anderson, Ph.D., and Amy Ghiretti, Ph.D., of ArticulateScience LLC, and was supported by Novartis Pharmaceuticals Corporation. This study was funded by Novartis Pharmaceuticals Corporation.
Compliance with ethical standards
Conflict of interest
RTM discloses honoraria from Novartis, Incyte, Juno Therapeutics, and Kite Therapeutics; Board of Directors membership at Novartis Pharmaceuticals Corporation; consultancies with Incyte and Juno Therapeutics; and patents and royalties from Athersys, Inc; as an OHSU employee who provided and received payment for consultancy services to Novartis Pharmaceuticals Corporation, this potential conflict of interest has been reviewed and managed by OHSU. ML discloses consultancy with Novartis Pharmaceuticals Corporation, Astellas, and Daiichi Sankyo; research funding from Novartis Pharmaceuticals Corporation, Astellas, and Fujifilm; and honoraria from Novartis Pharmaceuticals Corporation. MMP discloses advisory board membership with Stemline. BLS discloses consultancy with Acceleron, Incyte, Agios, Celgene, and Alexion and research funding from Novartis Pharmaceuticals Corporation and Celgene. SRM has nothing to disclose. AD discloses consultancies with Kite Therapeutics and Novartis Pharmaceuticals Corporation. SDR has nothing to disclose. DDHK discloses consultancies with Novartis Pharmaceuticals Corporation, Bristol-Meyers Squibb, Paladin, and Pfizer and honoraria and research funding from Novartis Pharmaceuticals Corporation and Bristol-Meyers Squibb. DH and TR have nothing to disclose. KH discloses former employment with Novartis Pharmaceuticals Corporation and current employment with Regeneron Pharmaceuticals, Inc. GB and DP disclose employment with Novartis Pharmaceuticals Corporation. PR discloses former employment with Novartis Pharmaceuticals Corporation and current employment with Target CW. HFF discloses honoraria from Pfizer and Sanofi and speakers’ bureau membership with Sanofi.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Recovered | ReactionOutcome | CC BY | 33288862 | 18,658,791 | 2021-05 |
What was the outcome of reaction 'Vomiting'? | Midostaurin after allogeneic stem cell transplant in patients with FLT3-internal tandem duplication-positive acute myeloid leukemia.
We evaluated standard-of-care (SOC) treatment with or without midostaurin to prevent relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) in patients with acute myeloid leukemia (AML) harboring internal tandem duplication (ITD) in FLT3. Adults (aged 18-70 years) who received alloHSCT in first complete remission, had achieved hematologic recovery, and were transfusion independent were randomized to receive SOC with or without midostaurin (50 mg twice daily) continuously in twelve 4-week cycles. The primary endpoint was relapse-free survival (RFS) 18 months post-alloHSCT. Sixty patients were randomized (30/arm); 30 completed all 12 cycles (midostaurin + SOC, n = 16; SOC, n = 14). The estimated 18-month RFS (95% CI) was 89% (69-96%) in the midostaurin arm and 76% (54-88%) in the SOC arm (hazard ratio, 0.46 [95% CI, 0.12-1.86]; P = 0.27); estimated relapse rates were 11% and 24%, respectively. Inhibition of FLT3 phosphorylation to <70% of baseline (achieved by 50% of midostaurin-treated patients) was associated with improved RFS. The most common serious adverse events were diarrhea, nausea, and vomiting. Rates of graft-vs-host disease were similar between both arms (midostaurin + SOC, 70%; SOC, 73%). The addition of midostaurin maintenance therapy following alloHSCT may provide clinical benefit in some patients with FLT3-ITD AML. (ClinicalTrials.gov identifier: NCT01883362).
Introduction
Acute myeloid leukemia (AML), the most common acute leukemia, is difficult to treat and has a poor prognosis, with a 5-year survival of ~25% [1, 2]. Multiple factors, including age, performance status (e.g., Eastern Cooperative Oncology Group), and cytogenetic and molecular features, affect treatment decisions and outcomes [3, 4]. Mutations in fms-like tyrosine kinase 3 (FLT3) are among the most common in AML and confer a poor prognosis with poor overall survival (OS) [5–7]. Consequently, these patients, particularly those with internal tandem duplications (ITDs), historically have more frequent and earlier relapses than patients without FLT3 mutations [7, 8].
Midostaurin, a multikinase inhibitor that targets FLT3 and other kinases, was approved for the treatment of adult patients with newly diagnosed, FLT3-mutated AML when combined with intensive induction and consolidation chemotherapy [9]. Approval was based on the phase 3 RATIFY/CALGB 10603 trial, which demonstrated improved survival with the addition of midostaurin to intensive chemotherapy followed by single-agent maintenance therapy in patients aged <60 years with newly diagnosed, FLT3-mutated AML. The RATIFY trial did not allow patients receiving alloHSCT to continue midostaurin [10].
AlloHSCT in first complete remission (CR1) provides patients with FLT3-ITD-positive AML the highest likelihood of sustained remission [11, 12], but relapse rates remain high [13–15]. The prognosis for patients with FLT3-ITD mutations has been poor following standard alloHSCT, primarily because these patients have a higher risk of relapse than patients with FLT3-ITD-negative AML [14–16].
Post-HSCT maintenance therapy with tyrosine kinase inhibitors (TKIs) may improve outcomes in patients with FLT3-mutated AML. In a phase 2 trial (AMLSG 16-10), midostaurin combined with intensive chemotherapy followed by alloHSCT and single-agent maintenance therapy demonstrated improved rates of event-free survival in patients receiving midostaurin compared with historical controls [17]. In AMLSG 16-10, midostaurin was administered as in RATIFY; however, patients who underwent alloHSCT could resume midostaurin as maintenance therapy post-transplant [10, 17]. Data from phase 1 and 2 trials suggest there may be a benefit with sorafenib, another TKI, as maintenance therapy post-HSCT [18–20]. Results from the phase 2 SORMAIN trial, which evaluated post-alloHSCT maintenance with sorafenib, suggested a benefit with sorafenib versus placebo with a median 2-year relapse-free survival (RFS) rate of 85% (95% CI, 70–93%) vs 53% (95% CI, 37–68%), respectively, (hazard ratio [HR], 0.39 [95% CI, 0.183—0.848]; P = 0.013) [20]. Similarly, quizartinib, a FLT3 TKI, was safely administered after alloHSCT in a phase 1 study [21]. Detailed trials evaluating FLT3 TKIs as maintenance therapy are ongoing [22–25].
Here, we report the results of the RADIUS trial investigating whether the addition of midostaurin to standard-of-care (SOC) treatment post-alloHSCT improves RFS over SOC alone in patients with FLT3-ITD-positive AML.
Patients and methods
Study design
RADIUS (NCT01883362) was a phase 2, randomized, open-label trial of SOC with or without midostaurin in patients (aged 18–70 years) with documented FLT3-ITD-positive AML who had undergone a protocol-specified conditioning regimen before alloHSCT in CR1 (following hematologic recovery, transfusion independence, and controlled graft-vs-host disease [GVHD]). Patients were enrolled after engraftment and randomized 1:1 within 28 to 60 days after alloHSCT to receive SOC ± midostaurin (50 mg twice daily in twelve 4-week cycles). SOC was dictated by the treating physician but excluded alternate TKI therapy. Currently, SOC therapy varies per treating institution in the post-alloHSCT setting. SOC therapy includes anti-infective prophylaxis and treatment as well as GVHD prophylaxis and treatment along with supportive care. Anti-infective and GVHD prophylaxis treatments were based on institutional guidelines.
Patients were assessed for relapse and survival through 24 months post-alloHSCT and/or until the end of the study. Patient visits occurred monthly for 1 year during treatment and every other month during the 24-month follow-up. Adverse events (AEs) were tracked for 30 days after treatment ended and assessed per the Common Terminology Criteria for Adverse Events version 4.0 [26].
The study was performed in accordance with the International Council for Harmonisation Good Clinical Practice guidelines and the principles of the Declaration of Helsinki and was approved by institutional review boards at participating institutions. All patients provided written informed consent.
Endpoints
The primary endpoint was RFS (time from transplant to relapse or death due to disease) 18 months after alloHSCT. Key secondary endpoints were safety, OS (time from transplant to the date of death from any cause), and RFS 24 months after alloHSCT.
Pharmacokinetics and in vivo FLT3 inhibition by FLT3 plasma inhibitory activity (PIA) assay were assessed as preplanned exploratory endpoints (see Supplementary methods). FLT3 inhibition and FLT3 ligand levels were evaluated on the basis of phosphorylated FLT3 (P-FLT3) and FLT3 ligand levels in the plasma [27].
The incidence and severity of GVHD were also exploratory study objectives. The percentage of patients developing acute or chronic GVHD (categorized according to the National Institutes of Health Consensus Development Project Working Group criteria [28]) and grade of GVHD were collected throughout the study by local assessment. GVHD by category and organ class was assessed at each study visit.
Statistical analysis
RADIUS was an exploratory, signal-finding study not powered to detect a statistical difference between treatment arms. A sample size of 60 was calculated to detect a 50% reduction in the risk of relapse with 71% power, assuming a 15% incidence of relapse in the midostaurin arm.
For time-to-event analyses, Kaplan–Meier curves were used to estimate survival distributions. A Cox proportional hazards model was used to estimate the HR and associated 95% CIs.
Results
Patients
Between February 5, 2014, and June 13, 2016, 74 patients were screened and 60 patients (30 per arm) were randomized at 18 sites in the United States and 1 site in Canada (Fig. 1 and Table S1). All patients were in CR1 prior to transplant; 18 patients (30%) received transplant directly following induction, 39 (65%) of patients had received consolidation without additional maintenance, and 3 (5%) of patients had received pretransplant maintenance. All patients had completed a protocol-specified conditioning regimen before alloHSCT (Table S2). Overall, 30 patients completed the per-protocol 12 cycles of therapy (midostaurin + SOC: 16 patients [53%]; SOC: 14 patients [47%]). The number of patients discontinuing early from the study was comparable between arms (midostaurin + SOC, n = 13; SOC, n = 15); however, the reasons for treatment discontinuation differed by arm, with AEs being the most common reason in the midostaurin arm (27% vs 3%) and consent withdrawal being the most common reason in the SOC arm (7% vs 20%). Patients who withdrew from treatment were to return for relapse and follow-up assessments and were not considered withdrawn from the study. Patients who withdrew consent were censored at the time of withdrawal. Patient demographics, baseline characteristics, and transplant characteristics are shown in Table 1. Most patients (midostaurin + SOC, 100%; SOC, 90%) had de novo AML. The 2 arms were balanced with regard to age, sex, and race.Fig. 1 CONSORT diagram.
AE adverse event, alloHSCT allogeneic hematopoietic stem cell transplant, SOC, standard of care. aA single patient might have had >1 reason for screen failure. bEarly termination due to work schedule conflicts. cPatients lost to follow-up (n = 2), early termination due to hospitalization at outside facility (n = 1), and early termination due to large travel distance (n = 1).
Table 1 Baseline patient and transplant characteristics.
Full analysis set Midostaurin + SOC
(n = 30) SOC
(n = 30)
Median age (range), yearsa 48 (20–61) 56 (20–68)
Sex, n (%)b
Male 16 (53) 18 (60)
Female 14 (47) 12 (40)
Race, n (%)c
White 27 (90) 27 (90)
Other 3 (10) 3 (10)
AML status at initial diagnosis, n (%)
De novo 27 (90) 30 (100)
Secondary to AHD 1 (3) 0
Therapy related 2 (7) 0
Median peripheral WBC count (range), × 109/L 48 (<1–278) 55 (<1–344)
Presence of FLT3-TKD mutation
Yes 3 (10) 2 (7)
No 17 (57) 20 (67)
Unknown 10 (33) 8 (27)
Purpose of pre-HSCT treatment, n (%)
Induction 30 (100) 30 (100)
Consolidation 22 (73) 20 (67)
Maintenance 2 (7) 1 (3)
Median time to randomization (range), days 54 (34–61) 54 (30–64)
Donor type, n (%)
Syngeneic 0 1 (3)
Allogeneic, matched relatedd 10 (33) 14 (47)
Allogeneic, matched unrelatedd 20 (67) 15 (50)
Stem cell source, n (%)
Peripheral blood 29 (97) 28 (93)
Bone marrow 1 (3) 2 (7)
AHD antecedent hematologic disorder, AML acute myeloid leukemia, FLT3 fms-like tyrosine kinase 3, HLA human leukocyte antigen, HSCT hematopoietic stem cell transplant, SOC standard of care, TKD tyrosine kinase domain, WBC white blood cell.
aP = 0.14; 2-sample t-test.
bP = 0.60; Fisher exact test.
cP = 0.72; Fisher exact test.
dMatched donors had HLA typing to include an 8/8 or 7/8 allele match rate at HLA-A, -B, -C, and -DRB1. A single mismatch was allowed.
Efficacy
The estimated RFS at 18 months (95% CI) was 89% (69–96%) with midostaurin and 76% (54–88%) with SOC alone (HR, 0.46 [95% CI, 0.12–1.86]; P = 0.27) (Fig. 2A). There were 3 RFS events in the midostaurin arm and 6 RFS events in the SOC arm at 18 months. The predicted relative reduction in the risk of relapse with the addition of midostaurin was 54% at 18 months post-alloHSCT.Fig. 2 Outcomes after alloHSCT.
Kaplan–Meier curves of A RFS by treatment arm at 18 months after undergoing alloHSCT and B OS by treatment arm at 24 months after undergoing alloHSCT. Blue, midostaurin + SOC; red, SOC. Tick marks indicate censoring of data. alloHSCT allogeneic hematopoietic stem cell transplant, HR hazard ratio, OS overall survival, RFS relapse-free survival, SOC standard of care. aMedian RFS was not reached. bLog-rank P value. cMedian OS was not reached.
At 24 months, addition of midostaurin to SOC continued to demonstrate reduced risk of relapse and prolonged survival compared with SOC alone (Figs. 2B and S1). At the time of final analysis (i.e., when all patients who remained on the study had reached 24 months post-alloHSCT), the median RFS and OS were not reached in either treatment arm. There were 4 relapses (13%) in the midostaurin arm vs 5 relapses (17%) in the SOC arm; median time to relapse from transplant was similar across both arms (median [range]; midostaurin + SOC, 323.5 days [69–1028 days]; SOC alone, 323 days [94–456 days]). The estimated 24-month RFS (95% CI) was 85% (64–94%) with midostaurin and 76% (54–88%) with SOC alone (HR, 0.60 [95% CI, 0.17–2.14]; P = 0.4297), and the relative reduction in the risk of relapse with the addition of midostaurin remained high at 40%.
Survival outcomes also improved; the estimated 24-month OS (95% CI) was 85% (65%-94%) with midostaurin and 76% (54%-89%) with SOC alone (HR, 0.58 [95% CI, 0.19–1.79]; P = 0.34), which is a 42% reduction in the risk of death with the addition of midostaurin (albeit not statistically significant). Eight patients died in the SOC arm vs 5 patients in the midostaurin arm; relapse accounted for a similar fraction of deaths in each arm. Details of post-relapse treatment were not captured. A total of 7 patients died due to reasons other than relapse: 5 in the SOC arm and 2 in the midostaurin arm; these patients were censored at the date of death. Non-relapse mortality was due to study indication (n = 2) and 1 instance each of cardiac arrest, GVHD, hepatic failure, cardiopulmonary arrest, and encephalitis infection.
Pharmacokinetics and PIA assay
The pharmacokinetics of midostaurin and its main metabolites (CGP62221 and CGP52421) were evaluated in 29 patients. The mean plasma concentration of midostaurin reached a maximum duringcycle 1 day 15, where as CGP52421 and CGP62221 peaked at cycle 3 day 1; all reached steady-state levels at cycle 4 (Fig. S2).
Among patients who received midostaurin, 28 were evaluable using the PIA assay. The degree of P-FLT3 inhibition was greatest during the first 2 cycles of therapy (Fig. 3A, B). In an exploratory biomarker analysis that assessed the correlation between plasma levels of midostaurin and its primary metabolites with the degree of FLT3 inhibition (i.e., lower levels of P-FLT3), early inhibition of FLT3 correlated inversely with drug levels (Fig. 3B). Peak FLT3 inhibition occurred at cycle 3 day 1; this time point was chosen for the correlative analysis.Fig. 3 Correlation between exploratory biomarker analyses and clinical outcomes.
A Median FLT3 ligand levels and B median P-FLT3 levels relative to baseline and concurrent combined levels of midostaurin and its metabolites in patients who received midostaurin + SOC. Median P-FLT3 levels were 70% of baseline at C3D1. C RFS and D OS at 24 months after alloHSCT in patients who received midostaurin + SOC stratified by P-FLT3 level (<70% vs >70%). C cycle; D day; FLT3, fms-like tyrosine kinase 3; M midostaurin, P-FLT3 phosphorylated FLT3, OS overall survival, RFS relapse-free survival, SOC standard of care. aFor this analysis, RFS was defined as time from transplant to relapse or death from any cause. bLog-rank P value vs SOC (n = 28). cPatients who reached C3D1 and received midostaurin + SOC (n = 28) were stratified according to FLT3 inhibition levels above or below the median (median P-FLT3, 70%). FLT3 inhibition was higher in patients with P-FLT3 levels <70% of baseline. dP-FLT3 > 70% includes patients with missing P-FLT3 at C3D1.
In patients receiving midostaurin (n = 28), the median P-FLT3 level at cycle 3 day 1 was 70% of baseline P-FLT3 levels. Thus, 14 of these patients had more effective inhibition of FLT3 activity (i.e., P-FLT3 levels <70% of baseline) on cycle 3 day 1 with P-FLT3 levels ranging from 20% to 69%. Of these 14 patients, 10 completed all 12 cycles of midostaurin therapy (Fig. S3). Among the remaining 14 patients who had less effective inhibition of FLT3 activity (i.e., P-FLT3 levels >70% of baseline), P-FLT3 was not measured at cycle 3 day 1 in 8 patients (6 were not receiving midostaurin on cycle 3 day 1). Six of 14 patients completed 12 cycles of midostaurin therapy and had P-FLT3 levels ranging from 74% to 100%. These higher P-FLT3 levels indicate less effective FLT3 inhibition, possibly resulting from the biological response of the patient to midostaurin or likely related to patient adherence to midostaurin, indicating the importance of proactive AE management to support patients throughout treatment.
Stratifying patients who received midostaurin by levels of FLT3 inhibition above or below the median revealed an association with clinical outcomes. Higher levels of FLT3 inhibition correlated with prolonged RFS, a reduced risk of relapse (P = 0.06), and significantly improved survival (P = 0.048) (Fig. 3C, D). Patients with less FLT3 inhibition had a similar risk of relapse and survival rate to those observed in patients receiving SOC alone (P = 0.9 and P = 0.92, respectively).
Safety
With midostaurin + SOC and SOC alone, AEs occurred in 100% and 87% of patients, respectively (Table 2). Most AEs in both arms were grade 1/2. The most common AEs were low-grade gastrointestinal AEs (grades 1–3, midostaurin arm vs SOC arm): vomiting (73% vs 23%), nausea (67% vs 27%), and diarrhea (49% vs 23%). Gastrointestinal AEs were more common in the midostaurin arm than in the SOC arm. The most common grade 3/4 laboratory abnormalities, increased alanine aminotransferase, increased aspartate aminotransferase, and decreased neutrophils, occurred in both arms. Serious AEs (Table 3) occurred in 57% of patients with midostaurin and 30% of patients with SOC alone. The most common serious AEs (midostaurin arm vs SOC arm) were diarrhea (13% vs 7%), nausea and vomiting (both, 3% vs 10%), and pyrexia (7% vs 7%).Table 2 Most common AEs (occurring in ≥15% of patients).
AE, n (%) Midostaurin + SOC
(n = 30) SOC
(n = 30)
Any grade Grade ≥ 3 Any grade Grade ≥ 3
Vomiting 7 (23) 1 (3) 22 (73) 2 (7)
Nausea 8 (27) 3 (10) 20 (67) 1 (3)
Diarrhea 7 (23) 1 (3) 12 (40) 3 (10)
Fatigue 9 (30) 0 8 (27) 1 (3)
Peripheral edema 9 (30) 0 8 (27) 0
Headache 7 (23) 0 8 (27) 0
Cough 6 (20) 0 8 (27) 0
ALT increased 7 (23) 4 (13) 6 (20) 3 (10)
Anemia 6 (20) 2 (7) 7 (23) 3 (10)
AST increased 8 (27) 4 (13) 5 (17) 2 (7)
Pruritus 6 (20) 0 7 (23) 3 (10)
Dry eye 6 (20) 0 5 (17) 0
Pyrexia 5 (17) 1 (3) 4 (20) 0
Rash 6 (20) 0 6 (17) 0
Tremor 4 (13) 0 7 (23) 0
Dyspnea 7 (23) 1 (3) 3 (10) 0
Insomnia 6 (20) 0 4 (13) 0
Neutrophil count decreased 3 (10) 2 (7) 7 (23) 4 (13)
Arthralgia 6 (20) 1 (3) 3 (10) 0
Dizziness 6 (20) 0 3 (10) 0
Hypertension 6 (20) 4 (13) 3 (10) 0
Upper respiratory tract infection 6 (20) 0 3 (10) 0
AE adverse event, ALT alanine aminotransferase, AST aspartate aminotransferase, SOC standard of care.
Table 3 Serious AEs occurring in ≥1 of patients overall.
AE, n (%) Midostaurin + SOC
(n = 30) SOC
(n = 30)
Diarrhea 4 (13) 2 (7)
Nausea 1 (3) 3 (10)
Vomiting 1 (3) 3 (10)
Pyrexia 2 (7) 2 (7)
Deep vein thrombosis 1 (3) 2 (7)
Febrile neutropenia 1 (3) 2 (7)
Anemia 2 (7) 1 (3)
Acute kidney injury 0 2 (7)
Abdominal pain 1 (3) 1 (3)
Parainfluenza virus infection 1 (3) 1 (3)
AE adverse event, SOC standard of care.
Median midostaurin exposure was 10.5 months (range, 0.2–11.5 months; defined by time of last midostaurin dose); 16 patients completed all 12 cycles of treatment. The median dose intensity was 93 mg/day (range, 25–100 mg/day). Dose adjustments were required per protocol in 19 patients (63%), most commonly due to AEs (84%). AEs leading to dose adjustment in ≥10% of patients included vomiting (27%), nausea (20%), and aspartate aminotransferase levels increased (10%). One patient was reported to have received a modified dose of midostaurin due to concomitant posaconazole, a cytochrome P450 3A4 inhibitor, per protocol.
AEs resulted in discontinuation from the study in 9 patients: 8 (27%) in the midostaurin arm and 1 (3%) in the SOC arm. The 8 patients in the midostaurin arm who discontinued treatment had 9 events: nausea (n = 3), vomiting (n = 2), liver function test levels increased (n = 2), pulmonary mycosis (n = 1), and pneumonitis (n = 1). The patient in the SOC arm discontinued from the study due to hypoxia. Twelve patients died on study during the follow-up phase (midostaurin + SOC, n = 4; SOC, n = 8). Death due to AML disease progression occurred in 2 patients receiving midostaurin and 4 receiving SOC alone. The addition of midostaurin to SOC did not result in an increase in the severity or rate of acute or chronic GVHD (Table 4). Rates of GVHD, as determined by local assessment, were similar between the midostaurin and SOC arms (overall, 70% vs 73%; acute, 53% vs 50%; and chronic, 37% vs 33%, respectively). Ninety-seven percent of patients received concomitant medication for the management of GVHD, including 28 (93%) in the midostaurin arm and 30 (100%) in the SOC arm. The most common concomitant medications typical of GVHD management were calcineurin inhibitors (85%), glucocorticoids (57%), moderately potent corticosteroids (18%), and selective immunosuppressants (17%) (Table S3).Table 4 Incidence of GVHD.
GVHD, n (%)a Midostaurin + SOC
(n = 30) SOC
(n = 30)
Acute 15 (50) 16 (53)
Grade I 7 (23) 4 (13)
Grade II 8 (27) 10 (33)
Grade III 0 2 (7)
Grade IV 0 0
Chronic 9 (30) 10 (33)
Mild 2 (7) 5 (17)
Moderate 5 (17) 4 (13)
Severe 2 (7) 1 (3)
GVHD graft-vs-host disease, SOC standard of care.
aPatients could be counted in multiple categories.
The most common organ toxicity due to GVHD was localized to the skin and affected 50% of patients in the midostaurin arm and 47% of patients in the SOC arm (Fig. S4). All patients with skin involvement in the midostaurin arm had stage 1 or 2 disease, whereas 2 patients in the SOC arm experienced stage 3 disease. Neither arm reported stage 4 organ involvement. Upper gastrointestinal toxicity was similar in both groups and did not exceed stage 1. Lower gastrointestinal toxicity was reported only in patients in the SOC arm and was primarily stage 1.
Discussion
This is the first randomized study of midostaurin as maintenance therapy after alloHSCT. We show that for patients with FLT3-ITD-positive AML in CR1, a defined course of up to 12 months of maintenance therapy with midostaurin was safely added to SOC after recovery from alloHSCT and improved RFS at 18 months after alloHSCT by 13% (over SOC alone). Although the study was not powered to detect a treatment difference, there was a trend toward benefit with midostaurin for all efficacy endpoints evaluated.
The survival outcomes in all participants in this study were better than anticipated for this high-risk leukemia population. Historically, the expected 2-year OS with SOC was closer to 60% compared with 76% observed in this study [15]. The stringent enrollment criteria, including recovery of counts (i.e., absolute neutrophil count >1000/μL and platelet count ≥20,000/μL without platelet transfusion) by day 42, ability to start treatment by day 60 post- alloHSCT, and no active, advanced, acute GVHD, may have contributed to the survival outcomes observed for all participants in this study. Moreover, the median time from the date of alloHSCT to initiation of study drug for both arms was 54 days; patients who had morphological relapse before that date were ineligible. Consistently, factors related to these inclusion/exclusion criteria, such as unacceptable test procedure results (8%) and unacceptable medical history/concomitant diagnosis (4%), were common reasons for screen failure, though the overall rate of screen failures (14 of 74 patients screened [19%]) was relatively low. Censoring of patients at the date of death due to non-relapse mortality may also have contributed to survival rates, particularly given the small patient population in this study. Similarly, patients were not stratified by European LeukemiaNet or National Comprehensive Cancer Network molecular risk classification due to the size of the study; thus, enrollment of patients with favorable molecular risk factors may also have affected the survival rates observed.
Correlative analysis suggests that patients who tolerated midostaurin and remained on therapy, as demonstrated by relatively higher levels of P-FLT3 inhibition, may have sustained benefit and long-term outcomes. The PIA assay allows for an indirect measurement of the phosphorylation of FLT3. P-FLT3 inhibition to <70% of baseline was achieved by 50% of patients receiving midostaurin and was associated with improved RFS and OS, indicating that inhibiting FLT3, even modestly, can have clinical benefit. Treatment adherence was not uniform in all patients receiving midostaurin, possibly due to tolerability (e.g., gastrointestinal toxicity). Prophylactic support, including antiemetics, in the management of gastrointestinal toxicities was crucial in keeping patients on therapy to provide the clinical benefit suggested by these data. Thus, increases in gastrointestinal toxicities were primarily low grade and manageable, consistent with other reports with single-agent midostaurin [29, 30]. Addition of midostaurin to SOC did not increase rates or severity of GVHD. Although the PIA assay is not used in clinical practice, FLT3 inhibition measured by this assay has tightly correlated with clinical activity across a broad array of FLT3 inhibitors [27, 31–34]. The results from the exploratory analysis in this study suggest that midostaurin therapy after alloHSCT may provide high levels of FLT3 inhibition in the long term in patients who remain on treatment, though further validation is required.
These data are consistent with the safety profile of midostaurin in patients with FLT3-ITD AML. In line with the AMLSG 16-10 study [17], the median time of midostaurin exposure during maintenance was similar (9 months in AMLSG 16-10 and 10 months in RADIUS); discontinuation due to toxicity was more common in AMLSG 16-10 (55%) than in RADIUS (27%), which may be explained by the stringent inclusion criteria of RADIUS. However, both studies demonstrated the safety and feasibility of midostaurin maintenance therapy.
Post-alloHSCT maintenance therapy with FLT3 TKIs, including midostaurin, is a viable treatment for reducing the risk of relapse in patients with FLT3-ITD AML. We anticipate that this study will provide a landmark for future studies, as the population had no pretransplant TKI exposure. These results complement those of the AMLSG 16-10 trial, which demonstrated improved event-free survival for patients with FLT3-ITD AML who received pretransplant midostaurin and began midostaurin within 100 days post-transplant compared with patients who only received pretransplant midostaurin [17]. Evidence from the present study and AMLSG 16-10 suggest that midostaurin maintenance therapy may be most appropriate for patients aged 18–70 years with FLT3-ITD AML who have undergone alloHSCT in CR1 and can begin midostaurin therapy quickly (within 100 days, ideally <60 days).
With the approval of midostaurin as up-front therapy for FLT3-ITD AML, new trials are emerging to better clarify the role of post-transplant TKI therapy in patients with deeper molecular remission, such as the large, phase 3, multinational, randomized trial assessing gilteritinib vs placebo as post-transplant adjuvant therapy for patients with FLT3-ITD AML in CR1 (BMT-CTN 1506; NCT02997202). As available treatment options increase, more detailed scrutiny of the risk-benefit profiles of these targeted agents is likely to be required.
With a post-transplant 2-year OS of ~80%, this study highlights the impact of recent advances in the management of FLT3-ITD AML on survival outcomes. Because FLT3-mutated AML has a higher risk of relapse than FLT3-mutation-negative AML, the addition of midostaurin maintenance therapy post-HSCT may be a viable option to reduce the risk of relapse in some patients after alloHSCT. These results provide evidence of clinical benefit and an estimate of treatment effect that could inform larger-scale studies in the future.
Supplementary information
Supplemental Material
Supplementary information
The online version of this article (10.1038/s41409-020-01153-1) contains supplementary material, which is available to authorized users.
Acknowledgements
The authors would like to thank the patients and the investigators who participated in the RADIUS study. Medical editorial assistance was provided by JoAnna Anderson, Ph.D., and Amy Ghiretti, Ph.D., of ArticulateScience LLC, and was supported by Novartis Pharmaceuticals Corporation. This study was funded by Novartis Pharmaceuticals Corporation.
Compliance with ethical standards
Conflict of interest
RTM discloses honoraria from Novartis, Incyte, Juno Therapeutics, and Kite Therapeutics; Board of Directors membership at Novartis Pharmaceuticals Corporation; consultancies with Incyte and Juno Therapeutics; and patents and royalties from Athersys, Inc; as an OHSU employee who provided and received payment for consultancy services to Novartis Pharmaceuticals Corporation, this potential conflict of interest has been reviewed and managed by OHSU. ML discloses consultancy with Novartis Pharmaceuticals Corporation, Astellas, and Daiichi Sankyo; research funding from Novartis Pharmaceuticals Corporation, Astellas, and Fujifilm; and honoraria from Novartis Pharmaceuticals Corporation. MMP discloses advisory board membership with Stemline. BLS discloses consultancy with Acceleron, Incyte, Agios, Celgene, and Alexion and research funding from Novartis Pharmaceuticals Corporation and Celgene. SRM has nothing to disclose. AD discloses consultancies with Kite Therapeutics and Novartis Pharmaceuticals Corporation. SDR has nothing to disclose. DDHK discloses consultancies with Novartis Pharmaceuticals Corporation, Bristol-Meyers Squibb, Paladin, and Pfizer and honoraria and research funding from Novartis Pharmaceuticals Corporation and Bristol-Meyers Squibb. DH and TR have nothing to disclose. KH discloses former employment with Novartis Pharmaceuticals Corporation and current employment with Regeneron Pharmaceuticals, Inc. GB and DP disclose employment with Novartis Pharmaceuticals Corporation. PR discloses former employment with Novartis Pharmaceuticals Corporation and current employment with Target CW. HFF discloses honoraria from Pfizer and Sanofi and speakers’ bureau membership with Sanofi.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Recovered | ReactionOutcome | CC BY | 33288862 | 18,658,791 | 2021-05 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective for unapproved indication'. | Fatal pulmonary fibrosis complicating COVID-19 infection in preexistent emphysema.
Only a few earlier clinical radiologic reports exist describing post-COVID-19 pulmonary fibrosis. We report a case of 74-year-old woman referred with dizziness and hypoxemic respiratory failure with chest high resolution computer tomography (HRCT) showing ground glass opacities and emphysema. The patient was tested for Sars-CoV-2 and resulted positive, she was treated with medical therapy and supported with mechanical ventilation. Despite initial clinical and radiological improvements, subsequently the respiratory failure worsened as ground glass opacities evolved, with the appearance of combined pulmonary fibrosis and emphysema and the patient eventually died. Development of pulmonary fibrosis after SARS-CoV-2 infection and the overlap with preexistent emphysema could be a fatal complication.
Background
Sars-CoV-2 is an RNA virus of the beta-coronavirus family identified in December 2019 in Wuhan and it is known to cause COVID-19 disease, which could include interstitial pneumonia with evolution into acute respiratory distress syndrome in some individuals. World Health Organization (WHO) has classified SARS-CoV-2 as a pandemic. According to WHO data, SARS-CoV-2 has infected, as of November 8, 2020, more than 49 million people worldwide and has caused over 1 million confirmed deaths. The virus mainly affects men with a history of smoking and comorbidities including diabetes mellitus, arterial hypertension, obesity, heart disease, and chronic lung disease [1].TheSARS-CoV-2 virus has an incubation period between 3 and 7 days and about 80% of patients have a mild infection or are asymptomatic, 15% have mild respiratory failure, and 5% require noninvasive or invasive mechanical ventilation. As of yet, the long-term sequelae of COVID-19 pneumonia are unknown. We report a case of Sars-CoV-2infection with severe pulmonary involvement in preexisting emphysema.
Case report
In March 2020, a 74-year-old woman was admitted to the emergency department for dizziness. Her past medical history was significant for emphysematous chronic obstructive pulmonary disease with smoking habit, severe chronic ischemic heart disease, and hypercholesterolemia. The patient presented with hypoxemic respiratory failure with SpO2 82% and 24/min respiratory frequency in room air and spontaneous breathing. Oxygen therapy with a Venturi mask was started. The electrocardiogram was negative for ischemia and body temperature was 37.6°C. The laboratory tests revealed lymphopenia and C-reactive protein increase. Chest HRCT was performed showing centrilobular emphysema and 2 ground glass opacity (GGO) areas at the ventral segment of the superior right lobe and at the lateral segment of the middle right lobe (Fig. 1A). The patient was tested for Coronavirus-19 with real-time polymerase chain reaction and resulted positive. She was admitted to the medicine department where therapy with hydroxychloroquine and azithromycin was started. After 7 days from admission the patient deteriorated and was admitted to the intensive care unit and then to our “Covid-19 Sub-ICU” where noninvasive mechanical ventilation (NIMV) was started. Intubation was not indicated because of her severe cardiopulmonary comorbidities. She received 8 mg/kg single dose of tocilizumab and started methylprednisolone 1 mg/kg and enoxaparin 100 U/kg 2 times a day. After 48 hours from tocilizumab administration respiratory failure improved. At 20 days from admission a HRCT (not showed) was repeated and revealed regression of GGO. Arterial blood gasses showed improvement of hypoxemia with arterial oxygen partial pressure/fraction of inspired oxygen (P/F) ratio of 176 mm Hg, allowing the discontinuation of NIMV. High flow oxygen was still required. Clinical conditions deteriorated again a few days later with increasing respiratory frequency and worsening of hypoxemia. A new chest HRCT (Fig. 1B) scan showed increasing basal nonspecific GGOs with sign of loss of volume and traction bronchiectasis. The situation was complicated by a gram-positive sepsis and the patient was treated successfully with linezolid. Subsequently respiratory conditions worsened and NIMV was started again. At 55 days from admission arterial blood gasses showed very severe hypoxemic respiratory failure with P/F ratio less than 40 mm Hg. A last chest HRCT (Fig. 1C) scan revealed radiological progression of pulmonary fibrosis at inferior lobes associated with global architecture distortion. Two months from admission the patient died due to severe hypoxemic respiratory failure.Fig. 1 Lung HRCT of the patient with axial images and multiplanar reconstructions. (A) First CT. Bilateral centrilobular emphysema more prominent in the upper right lobe with minimal GGO. (B) Second CT (2 weeks later from the first HRCT). New bilateral peripheral mild ground glass opacities in all lobes. (C) Last CT (7 weeks later from the first CT). More extension of GGO but also evidence of volume loss, distortion of the architecture and traction bronchiectasis due to fibrosis, the latter more evident in the lower lobes. Enlargement of centrilobular emphysema more evident in the upper lobes. The radiological appearance is globally similar to the combined pulmonary fibrosis and emphysema syndrome. GGO, ground glass opacity; HRCT, high resolution computer tomography.
Fig 1
Discussion
Evolution of SARS-CoV-19 related pneumonia is still under debate. In the usual course of mild lung involvement, the resolution of consolidations and crazy-paving pattern requires 14 to 30 days after the onset of the initial symptoms. Ground glass opacities are the main findings in the follow up and can be found whereas consolidations were previously observed [2,3]. On the other hand more severe cases may result in persistent lung alterations. Fourteen patients in a Korean observational study [4] showed fibrotic signs due to the SARS-CoV-19 pneumonia, and most frequent alterations reported after discharge were irregular interface and parenchymal bands. In a recent long-term study [5] on the lung consequences of 2003 SARS outbreak, Zhang et al found 31% of patients have radiologic abnormalities at CT scans across 15 years of follow up. Lung abnormalities reported were GGO or cord-like consolidations. Clinical course of our case study was initially quite usual for SARS-Cov-19 with only small GGOs at admission and subsequently development of progressive hypoxemic respiratory failure as the ground glass involvement progressed. We observed initial recovery from the acute phase and a slight improvement in respiratory failure after tocilizumab administration while HRCT scan showed resolution of GGO. After a month from the onset of symptoms a respiratory failure with extremely severe hypoxemia was still present (P/F <40 mm Hg). As the respiratory conditions further worsened the chest HRCT scan showed initial signs of loss of volume associated with progressive lung fibrosis; despite the severity of the condition inflammatory markers were reduced, suggesting a low activity of the SARS-CoV-19-related disease. HRCT scans never showed a clear pattern of ARDS, instead a slow but progressive fibrotic process which after 2 months, unlike the cases cited above, lead to the radiological alterations resembling a pattern of combined pulmonary fibrosis and emphysema. Our findings agree with the considerations suggested by Spagnolo et al[6] of potential development of severe pulmonary fibrosis after SARS-CoV-19 infection.
Conclusion
In our case study, a lung fibrosis overlapping a preexistent emphysema could be a potential lethal complication of COVID-19. Further studies are required to assess risk of development of subacute pulmonary fibrosis in patients with SARS-CoV-19 related pneumonia.
Patient Consent statement
The authors declare that they have acquired the patient's verbal informed consent for publication of her case; unfortunately she was unable to express written consent due to the severity of the disease.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declarations of competing interest: None. | AZITHROMYCIN ANHYDROUS, HYDROXYCHLOROQUINE | DrugsGivenReaction | CC BY-NC-ND | 33288987 | 19,138,872 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'. | Fatal pulmonary fibrosis complicating COVID-19 infection in preexistent emphysema.
Only a few earlier clinical radiologic reports exist describing post-COVID-19 pulmonary fibrosis. We report a case of 74-year-old woman referred with dizziness and hypoxemic respiratory failure with chest high resolution computer tomography (HRCT) showing ground glass opacities and emphysema. The patient was tested for Sars-CoV-2 and resulted positive, she was treated with medical therapy and supported with mechanical ventilation. Despite initial clinical and radiological improvements, subsequently the respiratory failure worsened as ground glass opacities evolved, with the appearance of combined pulmonary fibrosis and emphysema and the patient eventually died. Development of pulmonary fibrosis after SARS-CoV-2 infection and the overlap with preexistent emphysema could be a fatal complication.
Background
Sars-CoV-2 is an RNA virus of the beta-coronavirus family identified in December 2019 in Wuhan and it is known to cause COVID-19 disease, which could include interstitial pneumonia with evolution into acute respiratory distress syndrome in some individuals. World Health Organization (WHO) has classified SARS-CoV-2 as a pandemic. According to WHO data, SARS-CoV-2 has infected, as of November 8, 2020, more than 49 million people worldwide and has caused over 1 million confirmed deaths. The virus mainly affects men with a history of smoking and comorbidities including diabetes mellitus, arterial hypertension, obesity, heart disease, and chronic lung disease [1].TheSARS-CoV-2 virus has an incubation period between 3 and 7 days and about 80% of patients have a mild infection or are asymptomatic, 15% have mild respiratory failure, and 5% require noninvasive or invasive mechanical ventilation. As of yet, the long-term sequelae of COVID-19 pneumonia are unknown. We report a case of Sars-CoV-2infection with severe pulmonary involvement in preexisting emphysema.
Case report
In March 2020, a 74-year-old woman was admitted to the emergency department for dizziness. Her past medical history was significant for emphysematous chronic obstructive pulmonary disease with smoking habit, severe chronic ischemic heart disease, and hypercholesterolemia. The patient presented with hypoxemic respiratory failure with SpO2 82% and 24/min respiratory frequency in room air and spontaneous breathing. Oxygen therapy with a Venturi mask was started. The electrocardiogram was negative for ischemia and body temperature was 37.6°C. The laboratory tests revealed lymphopenia and C-reactive protein increase. Chest HRCT was performed showing centrilobular emphysema and 2 ground glass opacity (GGO) areas at the ventral segment of the superior right lobe and at the lateral segment of the middle right lobe (Fig. 1A). The patient was tested for Coronavirus-19 with real-time polymerase chain reaction and resulted positive. She was admitted to the medicine department where therapy with hydroxychloroquine and azithromycin was started. After 7 days from admission the patient deteriorated and was admitted to the intensive care unit and then to our “Covid-19 Sub-ICU” where noninvasive mechanical ventilation (NIMV) was started. Intubation was not indicated because of her severe cardiopulmonary comorbidities. She received 8 mg/kg single dose of tocilizumab and started methylprednisolone 1 mg/kg and enoxaparin 100 U/kg 2 times a day. After 48 hours from tocilizumab administration respiratory failure improved. At 20 days from admission a HRCT (not showed) was repeated and revealed regression of GGO. Arterial blood gasses showed improvement of hypoxemia with arterial oxygen partial pressure/fraction of inspired oxygen (P/F) ratio of 176 mm Hg, allowing the discontinuation of NIMV. High flow oxygen was still required. Clinical conditions deteriorated again a few days later with increasing respiratory frequency and worsening of hypoxemia. A new chest HRCT (Fig. 1B) scan showed increasing basal nonspecific GGOs with sign of loss of volume and traction bronchiectasis. The situation was complicated by a gram-positive sepsis and the patient was treated successfully with linezolid. Subsequently respiratory conditions worsened and NIMV was started again. At 55 days from admission arterial blood gasses showed very severe hypoxemic respiratory failure with P/F ratio less than 40 mm Hg. A last chest HRCT (Fig. 1C) scan revealed radiological progression of pulmonary fibrosis at inferior lobes associated with global architecture distortion. Two months from admission the patient died due to severe hypoxemic respiratory failure.Fig. 1 Lung HRCT of the patient with axial images and multiplanar reconstructions. (A) First CT. Bilateral centrilobular emphysema more prominent in the upper right lobe with minimal GGO. (B) Second CT (2 weeks later from the first HRCT). New bilateral peripheral mild ground glass opacities in all lobes. (C) Last CT (7 weeks later from the first CT). More extension of GGO but also evidence of volume loss, distortion of the architecture and traction bronchiectasis due to fibrosis, the latter more evident in the lower lobes. Enlargement of centrilobular emphysema more evident in the upper lobes. The radiological appearance is globally similar to the combined pulmonary fibrosis and emphysema syndrome. GGO, ground glass opacity; HRCT, high resolution computer tomography.
Fig 1
Discussion
Evolution of SARS-CoV-19 related pneumonia is still under debate. In the usual course of mild lung involvement, the resolution of consolidations and crazy-paving pattern requires 14 to 30 days after the onset of the initial symptoms. Ground glass opacities are the main findings in the follow up and can be found whereas consolidations were previously observed [2,3]. On the other hand more severe cases may result in persistent lung alterations. Fourteen patients in a Korean observational study [4] showed fibrotic signs due to the SARS-CoV-19 pneumonia, and most frequent alterations reported after discharge were irregular interface and parenchymal bands. In a recent long-term study [5] on the lung consequences of 2003 SARS outbreak, Zhang et al found 31% of patients have radiologic abnormalities at CT scans across 15 years of follow up. Lung abnormalities reported were GGO or cord-like consolidations. Clinical course of our case study was initially quite usual for SARS-Cov-19 with only small GGOs at admission and subsequently development of progressive hypoxemic respiratory failure as the ground glass involvement progressed. We observed initial recovery from the acute phase and a slight improvement in respiratory failure after tocilizumab administration while HRCT scan showed resolution of GGO. After a month from the onset of symptoms a respiratory failure with extremely severe hypoxemia was still present (P/F <40 mm Hg). As the respiratory conditions further worsened the chest HRCT scan showed initial signs of loss of volume associated with progressive lung fibrosis; despite the severity of the condition inflammatory markers were reduced, suggesting a low activity of the SARS-CoV-19-related disease. HRCT scans never showed a clear pattern of ARDS, instead a slow but progressive fibrotic process which after 2 months, unlike the cases cited above, lead to the radiological alterations resembling a pattern of combined pulmonary fibrosis and emphysema. Our findings agree with the considerations suggested by Spagnolo et al[6] of potential development of severe pulmonary fibrosis after SARS-CoV-19 infection.
Conclusion
In our case study, a lung fibrosis overlapping a preexistent emphysema could be a potential lethal complication of COVID-19. Further studies are required to assess risk of development of subacute pulmonary fibrosis in patients with SARS-CoV-19 related pneumonia.
Patient Consent statement
The authors declare that they have acquired the patient's verbal informed consent for publication of her case; unfortunately she was unable to express written consent due to the severity of the disease.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declarations of competing interest: None. | AZITHROMYCIN ANHYDROUS, HYDROXYCHLOROQUINE | DrugsGivenReaction | CC BY-NC-ND | 33288987 | 19,138,872 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cerebrovascular accident'. | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | CABOZANTINIB, CINACALCET HYDROCHLORIDE, DENOSUMAB, FUROSEMIDE, METHYLPREDNISOLONE, NIVOLUMAB, PAZOPANIB, PREDNISONE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug effective for unapproved indication'. | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | CABOZANTINIB, CINACALCET HYDROCHLORIDE, DENOSUMAB, FUROSEMIDE, METHYLPREDNISOLONE, NIVOLUMAB, PAZOPANIB, PREDNISONE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hypercalcaemia'. | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | CABOZANTINIB, CINACALCET HYDROCHLORIDE, DENOSUMAB, FUROSEMIDE, METHYLPREDNISOLONE, NIVOLUMAB, PAZOPANIB, PREDNISONE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Metastatic renal cell carcinoma'. | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | CABOZANTINIB, CINACALCET HYDROCHLORIDE, DENOSUMAB, FUROSEMIDE, METHYLPREDNISOLONE, NIVOLUMAB, PAZOPANIB, PREDNISONE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'. | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | CABOZANTINIB, CINACALCET HYDROCHLORIDE, DENOSUMAB, FUROSEMIDE, METHYLPREDNISOLONE, NIVOLUMAB, PAZOPANIB, PREDNISONE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Therapy non-responder'. | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | CABOZANTINIB, CINACALCET HYDROCHLORIDE, DENOSUMAB, FUROSEMIDE, METHYLPREDNISOLONE, NIVOLUMAB, PAZOPANIB, PREDNISONE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the administration route of drug 'CINACALCET HYDROCHLORIDE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the administration route of drug 'DENOSUMAB'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Subcutaneous | DrugAdministrationRoute | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the administration route of drug 'FUROSEMIDE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the administration route of drug 'METHYLPREDNISOLONE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the administration route of drug 'SODIUM CHLORIDE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the dosage of drug 'DENOSUMAB'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | 120 MILLIGRAM, Q4WK | DrugDosageText | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the dosage of drug 'FUROSEMIDE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | 40 MILLIGRAM | DrugDosageText | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the dosage of drug 'METHYLPREDNISOLONE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | 40 MILLIGRAM, ONE TIME DOSE | DrugDosageText | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the dosage of drug 'SODIUM CHLORIDE'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | 1 LITER | DrugDosageText | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the outcome of reaction 'Hypercalcaemia'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Recovered | ReactionOutcome | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
What was the outcome of reaction 'Metastatic renal cell carcinoma'? | Treatment of malignancy-associated hypercalcemia with cinacalcet: a paradigm shift.
Palliation of symptoms related to malignancy-associated hypercalcemia (MAH) is essential and clinically meaningful for patients, given the continued poor prognosis, with high morbidity and mortality associated with this disease process. Historically, agents have been temporizing, having no impact on patient morbidity nor survival. We suggest that cinacalcet can be an efficacious agent to be taken orally, reducing patients' time in the hospital/clinic settings. It is well-tolerated and maintains serum calcium levels in the normal range, while targeted cancer treatments can be employed. This has a direct, major impact on morbidity. Maintaining eucalcemia can increase quality of life, while allowing targeted therapies time to improve survival. Given that our case (and others) showed calcium reduction in MAH, there is promising evidence that cinacalcet can be more widely employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Though the exact mechanism of action for cinacalcet's reduction in calcium in this setting is not currently known, we can still afford patients the possible benefit from it.
Introduction
Malignancy-associated hypercalcemia (MAH) has long been described in medical literature and has posed a therapeutic conundrum. Over decades, this form of hypercalcemia has eluded conventional therapies, in that, it responds only temporarily and often is refractory. Clinically, for the patient it negatively impacts quality of life, and patients can succumb to hypercalcemic crisis. Indeed, MAH not uncommonly, constitutes a metabolic oncologic emergency (1, 2).
Malignancy-associated hypercalcemia is the second most common cause of hypercalcemia in the general population and the most common cause of hypercalcemia among patients in the inpatient setting. Incidence has been reported at 15 cases per 100,000 annually, and approximately 20–30% of patients with cancer develop MAH (3). The clinical symptomatology of hypercalcemia depends on the degree of elevation of calcium. The patient may be asymptomatic, has few constitutional symptoms, or may develop neurovascular symptoms resulting in a state of metabolic emergency (1).
Survival
Historically, once MAH presents, up to 50% of patients die in an average of 30 days, and up to 75% die within 3 months (4, 5). It has been suggested that therapy for hypercalcemia is interim, with no effect on survival; this has been observed over time (4, 6). Despite advances in therapeutics, survival after diagnosis of MAH has not changed over the decades. In the 1980s, patients with bone metastases from breast cancer were observed to survive about 3 months after the onset of hypercalcemia (7). Median survival in patients with squamous cell carcinoma and hypercalcemia was 17–64 days (8, 9). In a series of patients with parathyroid hormone-related peptide (PTH-RP) mediated hypercalcemia associated with solid organ malignancy, the median survival was 52 days (10). A 2017 study revealed similar survival rates with the cohort having median survival of 40 days (11). Neither degree of elevation of hypercalcemia nor degree of elevation of PTH-RP has shown an associated change in survival (10). This recapitulates early studies showing that the absolute level of calcium is not a good prognosticator, but the mere presence of hypercalcemia portends poor prognosis (6).
Survival may be impacted by controlling the calcium level, to the extent that patients whose calcium is normal or near-normal are not succumbing to hypercalcemia-related complications (e.g. cardiac arrhythmias) as a cause of death. It is thought that controlling calcium can increase quality of life, reduce morbidity, and give time for targeted cancer therapy to be implemented (12). Ramos et al. showed that after MAH was diagnosed, there was a lengthened survival in those patients whose calcium normalized and were subsequently able to receive chemotherapy (11). Nonetheless, their study confirmed that for patients developing MAH, there remains dismal prognosis. Specifically looking at effects on morbidity and mortality, bisphosphonate therapy has brought about no change in these parameters (13). Ling et al. confirm this, observing that patients died within 2 months, while some who received bisphosphonate died within 3 months of developing hypercalcemia (14). They noted that tumor type, time from tumor diagnosis to hypercalcemia, nor level of serum calcium impacted survival. It has also been observed that there is no difference in survival in patients treated with different anti-hypercalcemic agents (5).
Historic and current observations continue to confirm that MAH portends a poor prognosis (8). In fact, a bedside prognostic score has been developed and used in studies evaluating hypercalcemia as an independent prognostic factor (9, 15). Certainly, newer targeted anti-cancer therapies may extend overall survival in cancer patients and can lengthen progression time to malignancy-associated complications such as bone metastases and/or hypercalcemia. There are currently no studies describing the impact of newer, targeted anti-cancer therapies and their impact on MAH and survival. Is it possible that if hypercalcemia is normalized, patients can experience fewer morbidities (those that relate to hypercalcemia) and have extended survival simply because they can continue with targeted anti-cancer therapies?
Historical perspective of classification and pathophysiology
In 1941, Albright proposed that tumors be tested for parathyroid hormone (PTH), as it seemed a hormone causing PTH-like effects were produced from tumors (16). Since this hormone early on was thought to be PTH, the process was termed ectopic PTH syndrome. Still in the 1970s, more studies showed that tumors can secrete a hormone other than PTH which exerts PTH-like effects (17, 18). Though this PTH-like substance remained elusive for decades, it had been concluded that the prior known ‘ectopic PTH syndrome’ was very rare (<1% of cases), as most cases of MAH had no detectable PTH (3, 19, 20). As these cases continued to be described, the term ‘pseudo-hyperparathyroidism’ was given in lieu of ectopic PTH syndrome. To describe the process more accurately, more than 30 years after Albright’s supposition, the term ‘humoral hypercalcemia of malignancy’ (HHM) was proposed (21).
Researchers postulated that there were many factors that drive MAH, including bone resorption by local tumor growth, substances causing bone resorption, and renal effects of PTH-like factors (22, 23, 24). Previously, it was estimated that PTH-like factors were produced by at least 75–80% of solid tumors associated with hypercalcemia (23); the current estimate remains at -80% (3).
Current perspective of classification and pathophysiology
Various pathophysiologic mechanisms have been found to be responsible for MAH. Overall, general mechanisms are osteolytic and humoral (Table 1). Mechanisms within these two main states are further considered briefly.
Table 1 General mechanisms of malignancy-associated hypercalcemia.
Osteolytic Humoral
↑ Bone resorption ↑ PTH-RP
Local destruction by metastasis ↑ PTH
Humoral factors ↑ 1,25(OH)2D3
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Humoral hypercalcemia of malignancy (HHM)
Most cases of MAH are driven by means which are humoral (3). The mechanism is most frequently via tumor secretion of PTH-RP, and/or other humoral factors. Most often, it is observed in cancers involving solid tumors (without bone metastases), but it can manifest in a variety of cancers.
Another mechanism that can drive HHM is the elevation of 1,25-dihydroxy vitamin D (1,25(OH)2D3), leading to increased absorption of calcium. This is mainly seen in hematologic cancers like lymphomas, and it has been reported in ovarian dysgerminomas (3, 25, 26, 27).
True ectopic PTH secretion by tumors is the least common mechanism to drive HHM; there have been cases reported in neuroendocrine tumors (3, 20).
Specifically speaking to cases of HHM driven by PTH-RP, it was first commonly observed in cancers involving solid tumors but without bone metastases. Bone metastases had long been described in breast cancer, yet without production of PTH-RP. However, HHM has been described coincident with bone metastases, and a PTH-like peptide was identified in breast cancer cells in (28, 29, 30). Furthermore, the first report of expression of the PTH-RP gene and the production of PTH-RP has been documented in multiple myeloma with marked elevation of serum calcium, evidence that a humoral component can also contribute to the skeletal complications and hypercalcemia in myeloma (31). Of note, patients with normocalcemic states have been found to have tumors expressing PTH-RP, suggesting that levels in circulation may not have been high enough to achieve and maintain a hypercalcemic state (32). There can be overlap in the way tumor activity results in a hypercalcemic state (Fig. 1).
Figure 1 Intersecting and independent etiologies of HHM. Parathyroid hormone (PTH); parathyroid hormone-related peptide (PTH-RP). 1,25-dihydroxy vitamin D (1,25(OH)2D3).
Osteolytic
Other factors that can drive MAH are osteolytic. Osteoclast-mediated destruction and osteosclerosis due to impaired/increased osteoblastic activity are the predominant forces contributing to the formation of bone lesions. Hypercalcemia can develop when the predominant force is osteoclastic, and hypocalcemia can develop due to calcium sequestration when the driving force is osteoblastic. Although cancers can exhibit predominantly increased resorption or formation of bone, a mixed picture is not uncommonly observed (33, 34, 35). Increased resorption and impaired formation are driven by local factors and humoral tumor factors produced by the tumor. Bone metastases themselves ultimately can destroy bone locally and exert mass effect. Thus, another mechanism for MAH is explained by local osteolytic effects resulting in hypercalcemia, seen mainly in cancers with significant skeletal lysis and/or increased resorption like breast cancer and multiple myeloma, respectively.
PTH-RP in perspective
Parathyroid hormone-related peptide is in many tissues and is involved in normal physiology (36, 37). In normal states, PTH-RP is not elevated. In a pathologic state like HHM, PTH-RP is produced and secreted in excess, therefore, it was proposed that PTH-RP could serve as a tumor marker (38).
Before its actual identification, this PTH-like protein from tumor extracts was described as having multiple times the biologic activity of PTH, being a different form of PTH, and working in concert with other substances resulting in hypercalcemia (17, 39). In the 1980s, parathyroid hormone-like proteins identified in breast (30) and lung cancers displayed homology to PTH, yet with greater biologic activity (40, 41). This increased effect on bone and renal activity can explain the development of hypercalcemia above the threshold of the body’s capability to maintain normal calcium homeostasis and can account for the relative severity and acuity of MAH compared with PTH-mediated hypercalcemia. Researchers reported a PTH-like protein that can stimulate adenylate cyclase in the renal cortices (30, 42) and promote calcium retention consistent with the clinical manifestations of HHM, pointing to the kidney as a major therapeutic target for this disease state (42).
Historically, the PTH-RP assays were developed and used in labs for research purposes. Currently, commercial labs have developed and offer PTH-RP testing, though there is currently great need for standardization and improvement in specificity, sensitivity, and analytic precision due to the various isoforms of the molecule (43).
Homology of PTH to PTH-RP as well as their genetic homology
Parathyroid hormone-related protein purified from lung and breast cancer cell lines was cloned; an amino acid sequence with homology to human PTH was observed (30, 40, 41), explaining its PTH-like effects. Considering the homology of PTH and PTH-RP, it was inferred that there was homology in the genes encoding them (40). In 1989, the human PTH-RP gene was characterized (44), structurally confirming the relatedness of the PTH-RP and PTH genes (chromosome 12 and 11, respectively) and showing that three distinct PTH-like proteins are products of the PTH-RP gene. Knowing the structural and genetic similarities of PTH and PTH-RP, it comes as no surprise that there are similarities and overlap in their functional activities relating to calcium homeostasis.
The type 1 parathyroid hormone receptor (PTH1R)
Based on review of prior and ongoing studies, it was surmised in 1989 that the hormone driving MAH acted on PTH target cells at the PTH receptor (19). It is now known that PTH and PTH-RP share the PTH1R to evoke their physiologic actions. After a very elegant literature review discussing the interaction and contribution of PTH1R and the calcium-sensing receptor (CaSR) signaling pathway to the development and perpetuation of breast cancer bone metastases, Yang suggested that future therapeutic modalities target those agents that can influence PTH-RP, the PTH1R, and CaSR signaling pathways (45).
The calcium-sensing receptor
The CaSR on the surface of the parathyroid gland chief cell is the principal regulator of PTH synthesis, secretion, and gene expression by mediating the inhibitory action of calcium (36). In the calcitonin-secreting C-cells of the thyroid, it mediates the stimulatory action of high calcium on calcitonin secretion. Cinacalcet is a calcimimetic that directly lowers PTH levels by increasing the sensitivity of the CaSR to extracellular calcium. In 1998, the first therapeutic use of this novel agent was described in a patient with parathyroid carcinoma and hypercalcemia (46) resulting in a reduction in calcium and PTH levels. Despite disease progression resulting in PTH increases, calcium remained stable with various dosage adjustments. It has been suggested that cinacalcet may potentially be useful in cancers with ectopic production of PTH (20, 47). Review of studies up to 2001, suggested a physiologic relationship between the CaSR and the secretion of PTH-RP (37); a relationship on which to focus future therapy.
Pharmacotherapy for MAH
Reducing tumor burden, can reduce or control calcium at least temporarily (17). This can be by surgical or chemotherapeutic means. Targeted cancer treatment, when successful, can slow progression to a state of hypercalcemia.
Certainly, reducing exogenous influences on calcium burden are paramount. This can be achieved by removing calcium supplements orally, parenterally, and in dialysate. Low calcium or calcium-free dialysate is effective in hypercalcemic crisis when initial treatments fail, or in the setting of fluid overload or renal failure (48). Discontinuation of agents that raise serum calcium (e.g. thiazides or lithium) reduces calcium burden otherwise imposed by the hypercalcemic state. Avoiding immobility and volume depletion and employing volume expansion with isotonic saline where necessary is helpful. Hydration and diuresis with a loop diuretic, directly increasing calcium excretion, have been used to lower serum calcium. However, this is not a safe option in all patients, and it can lead to dehydration with rebound hypercalcemia.
It was thought that long- term management of MAH needed to focus on development of agents targeting bone resorption (39). Some early agents employed to lower calcium were found to be unsafe, are no longer in use, and will not be discussed. For 30 years, bisphosphonates were the focus of studies and were the mainstay of therapy for MAH. In 1977 etidronate was the first diphosphate used to treat hypercalcemia. It slowed bone resorption, thereby affecting calcium metabolism to reduce serum levels. Working similarly was pamidronate, which was approved 14 years later (1991); pamidronate became the first bisphosphonate specifically indicated for treatment of MAH. The next bisphosphonate approved for MAH was zolendronate (2001). These agents are dosed intravenously (IV) in clinic or hospital settings. It can take a few days to see a reduction in calcium levels, and this reduction is temporary.
Denosumab came to market in 2010 as the first novel agent in 30 years targeted at inhibiting bone resorption. It is a human MAB that binds to and inhibits the receptor activator of nuclear factor kappa-B ligand (RANKL), the primary mediator of bone resorption, via activation of osteoclasts. Employing denosumab, Hu et al. observed a 70% response rate (response = calcium level <2.8 mmol/L) for patients with MAH, and the median duration of response was 9 days (49). The longest duration was 104 days. It is promising that this agent can, in some cases, bring about a longer period of lowered calcium levels.
Glucocorticoids can be effective in cases of HHM where overproduction of 1,25(OH)2D3 predominantly drives hypercalcemia. Calcitonin lowers blood calcium by promoting calcium incorporation into bone, however, the effects are minimal and transient.
Historically, the only treatment for hypercalcemia in patients with renal failure was dialysis (50). Currently, denosumab can be used without need for dosage adjustment in renal failure. Cinacalcet, though not indicated for treatment of MAH, can safely reduce calcium levels in renal failure or renal-compromised patients. Therefore, safety in this population is established.
Cinacalcet was approved for use in 2004 and is indicated for patients with secondary hyperparathyroidism with chronic kidney disease on dialysis, hypercalcemia in patients with parathyroid carcinoma, and severe hypercalcemia in patients with primary hyperparathyroidism who are unable to undergo parathyroidectomy. Considering the shared homology of PTH and PTH-RP and given cinacalcet’s current role in controlling PTH-mediated hypercalcemia, Can there be a key role for cinacalcet in treating other hypercalcemic states, especially those driven by PTH-RP? It had been suggested that MAH refractory to bisphosphonate therapy can be treated with denosumab (51). It is now proposed that cinacalcet can be used as adjunctive therapy in HHM (and possibly other forms of MAH) successfully and safely over the long-term.
Cases of cinacalcet-treated MAH
The Netherlands
One of the first cases using cinacalcet in MAH was described in 2012 by Bech (52) and colleagues. In this case, efficacy of cinacalcet as a suppressor of PTH-RP production was explored. A 57 -year-old male with stage cT4N3M1b squamous cell lung carcinoma developed severe, recurrent MAH. On presentation, the patient had symptomatic hypercalcemia with the following laboratory values: PTH <1.0 pmol/L (1.3–6.8 pmol/L), PTH-RP 5.8 pmol/L or 55 ng/L (<0.6 pmol/L or 6 ng/L), and calcium 4.5 mmol/L (routine clinical chemistry assays Roche Diagnostics). The patient was administered normal saline, calcitonin, and pamidronate over 2 weeks. These measures achieved a calcium of 2.8 mmol/L which increased to 4.4 mmol/L after 2 weeks. For the next 5 days, normal saline was resumed along with calcitonin and a single dose of zolendronate. Nonetheless, the calcium and PTH-RP were 3.5 mmol/L and 13.3 pmol/L (125 ng/L), respectively.
At this point, with the patient’s consent, cinacalcet was started and continued for 15 days while chemotherapy with carboplatin and gemcitabine was initiated. During this first cycle, the calcium dropped to a hypocalcemic level, and PTH-RP came down. Cinacalcet was discontinued, bringing about a rise in PTH from undetectable to 5.1 pmol/L with a normalization of serum calcium. There were three more cycles of combination chemotherapy without cinacalcet. After the fourth cycle, the calcium rose to 3.5 mmol/L. The patient was hospitalized, and cinacalcet was started along with hydration and a dose of zolendronate. Calcium improved to 3.0 mmol/L, and the patient was discharged on the cinacalcet. Hospitalization was required after 9 days, and a dose of zolendronate was given. Due to disease progression, the patient succumbed to his illness after 2 weeks. It was concluded that about 71% of the variance in serum calcium correlated with PTH-RP levels and that PTH-RP reduction may be a result of cinacalcet use.
United States of America
Sternlicht & Glezerman report a case of metastatic renal cell carcinoma in 2013 (53). Laboratory reference ranges provided are PTH-RP 14–27 pg/mL (14–27 ng/L) and PTH 12–88 pg/mL (1.3–9.3 pmol/L). After bisphosphonate and denosumab therapy, the calcium was 14.2 mg/dL (3.6 mmol/L), PTH 10 pg/mL (1.1 pmol/L), and PTH-RP 114 pg/mL (114 ng/L). Cinacalcet was started and titrated, and at 10 weeks calcium improved to 10.1 mg/dL (2.5 mmol/L) with PTH-RP 159 pg/mL (159 ng/L). Their theory is that cinacalcet may have a role in the treatment of MAH.
New Zealand
A case presented by abstract at the Endocrine Society’s 97th Annual Meeting by Whitfield and Carroll (54) describes a 54- year-old female diagnosed with inoperable gastroenteropancreatic neuroendocrine tumor (GEP-NET). The tumor was treated with octreotide. Within 1 year, the calcium rose to 3.0 mmol/L (2.2–2.6 mmol/L) with PTH <0.6 pmol/L (1.5–6.0 pmol/L) and PTH-RP 3.3 pmol/L or 31 ng/L (0.0–1.5 pmol/L or 0–14 ng/L). Tumor embolization failed, and funded sunitinib therapy was unavailable. Three weekly infusions of zolendronate and normal saline failed to control calcium and its symptoms, therefore cinacalcet was initiated and titrated. The calcium improved to 2.9 mmol/L within 1 month and remained 2.5–2.9 mmol/L for 18 months (all the while patient remained on octreotide). The observation was that cinacalcet may be a useful therapeutic option for MAH.
Belgium
Another case of a neuroendocrine (NET) tumor with hypercalcemia has been described by Valdes-Socin and colleagues in 2017 (55). A 52- year-old male presented with an unresectable, well-differentiated, metastatic pancreatic NET. Laboratory reference ranges provided are calcium 2.2–2.6 mmol/L and PTH 12–58 pg/mL (1.3–6.2 pmol/L).
Calcium was 3.5 mmol/L with PTH <4 pg/mL (0.4 pmol/L); PTH-RP could not be measured. Several cycles of streptozotocin-adriamycin and FOLFOX (folinate, fluorouracil, oxaliplatin) were given. While the PTH level remained low at 19 pg/mL (2.0 pmol/L), the tumor mass and calcium level (2.6 mmol/L) improved. After 3 months, the calcium and PTH were 2.9 mmol/L and <2 pg/mL (0.2 pmol/L), respectively. Octreotide was given without clinical impact. Calcium had risen to 3.1 mmol/L and was refractory to saline fluids, diuretics, recombinant calcitonin, and zolendronate. Compassionate treatment with cinacalcet was initiated. Calcium levels responded down to 2.8 then 2.6 mmol/L over 3 months. Shortly thereafter, sunitinib was introduced. After 1 month of combined sunitinib-cinacalcet therapy, the calcium fell into the hypocalcemic range at 2.1 mmol/L with PTH 78 pg/mL (8.3 pmol/L). Cinacalcet was discontinued; sunitinib treatment was continued for 4 years with normal calcium levels.
The authors conclude that cinacalcet lowered calcium and improved clinical condition and that sunitinib contributed to lowering calcium.
Greece
Asonitis and colleagues (56) presented a case of a 69-year-old female with a 6-year history of infiltrating ductal and lobular mammary carcinoma with bone metastases. The patient received zolendronate and radioactive samarium due to thoracic, lumbar spine, and pelvic lesions. Of note, the zolendronate was given for bone metastases, not hypercalcemia, and the last dose had been given 2 years prior to presentation with hypercalcemia. Laboratory reference ranges provided are calcium 8.6–10.2 mg/dL (2.3–2.6 mmol/L) and PTH 8–76 pg/mL (8–76 ng/L).
At presentation, the calcium level was 15.2 mg/dL (3.8 mmol/L) with PTH 6.5 pg/mL (0.6 pmol/L). The PTH-RP could not be measured. Treatment consisted of normal saline, furosemide, and zolendronate. On day 2, the calcium was 12.9 mg/dL (3.2 mmol/L), and calcitonin and hydrocortisone were administered. On day 5, the calcium was 10.4 mg/dL (2.6 mmol/L), and the patient was discharged on methylprednisolone, furosemide, reduced calcium intake, and increased water intake. Five days later, denosumab was added due to a calcium level of 13.6 mg/dL (3.4 mmol/L). After 3 weeks, cinacalcet was added to the regimen, since the calcium plateaued at 13.3 mg/dL (3.3 mmol/L). By 2 weeks, the calcium level improved to 11.7 mg/dL (2.9 mmol/L), and the cinacalcet was titrated. At this point the denosumab was administered monthly. The calcium was normal (9.6 mg/dL (2.4 mmol/L)) after 3 weeks and remained normal for 1.5 months. To confirm efficacy, cinacalcet was held, resulting in a rise of calcium by 1.7 mg/dL (0.4 mmol/L). In total, the patient benefitted from stable calcium levels for 11 months with cinacalcet. The authors suggest that cinacalcet can be an effective therapeutic option for MAH.
United States of America
Recently, authors report a case of an 81 -year-old female suffering from non-small cell lung cancer (NSCLC) and recurrent bladder cancer with HHM refractory to traditional therapy (57). Laboratory reference ranges provided are calcium 8.5–10.1 mg/dL (2.1–2.5 mmol/L), PTH 18–85 pg/mL (1.9–9.0 pmol/L), and PTH-RP 0-2 pmol/L (<19 ng/L).
The NSCLC was showing progression, so nivolumab was started. Five weeks later the calcium started to rise (10.6 mg/dL (2.7 mmol/L)). Thereafter, due to progressive clinical deterioration, she was hospitalized with calcium 12.7 mg/dL (3.8 mmol/L), PTH <6 pg/mL (<0.7 pmol/L), and PTH-RP 3.3 pmol/L (31 ng/L). Treatment consisted of pamidronate and fluids. After 4 days, the calcium was 8.2 mg/dL (2.1 mmol/L). She was readmitted due to symptoms with calcium 11.1 md/dL (2.8 mmol/L), PTH 5.8 pg/mL (0.6 pmol/L), and PTH-RP 42 pmol/L (396 ng/L). Treatment consisted of zolendronate and fluids. Within 2 days the calcium was 8.7 mg/dL (2.2 pmol/L) with a rise to 10.1 mg/dL (2.5 mmol/L) in 3 days. Denosumab was given, but readmission was required in 3 days with a calcium of 11.1 mg/dL (2.8 mmol/L). After zolendronate and two doses of calcitonin were given, the calcium was 9.0 mg/dL (2.3 mmol/L). Cinacalcet was initiated and titrated. For nearly 2 months on cinacalcet monotherapy, she had no more hypercalcemia despite rises in the PTH-RP 143–>194 pmol/L (1,348–>1,829 ng/L). Nivolumab was discontinued due to disease progression, and the patient died in hospice care without further laboratory studies.
Our case (United States of America)
We now present a case of HHM treated successfully with cinacalcet. Success being defined as normalization of calcium levels over many months without need for clinic or hospital administration of IV nor s.c. agent and no emergency department visits nor hospital admissions for hypercalcemia urgency or crisis.
Performing labs and reference ranges are provided as follows: Calcium 2.1–2.7 mmol/L, Orlando VA Health Care System, Orlando, Florida, USA; 1,25(OH)2 D3 43–173 pmol/L Quest Diagnostics, chromatography/mass spectrometry, Chantilly, Virginia, USA; 25 hydroxy vitamin D (25 (OH) D3) 75–250 nmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH-RP 14–27 ng/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA; PTH 1.5–6.8 pmol/L Quest Diagnostics, immunoassay, Chantilly, Virginia, USA. Adjusted calcium level was determined using the following equation: ((4-albumin) × 0.8) + serum calcium. All calcium levels referenced below are adjusted serum levels, as the patient’s albumin was low.
A 71-year-old male had a past medical history significant for Von Hippel-Lindau syndrome and metastatic renal cell carcinoma (RCC). The RCC was found to have metastasized (16 years after initial nephrectomy) as evidenced by pulmonary masses, a large pancreatic mass replacing the tail, a right parotid mass, osseous lesions, and numerous hyperdense left renal lesions. Treatment with pazopanib was initiated shortly thereafter. The patient developed MAH 6 months into therapy. The calcium was 3.1 mmol/L with PTH 0.6 pmol/L, and 25 (OH) D3 142 nmol/L, therefore, MAH was presumed. The hypercalcemia responded to zolendronate 4 mg IV on two separate occasions over 11 months (calcium levels normal or slightly elevated) while the patient was able to receive targeted cancer therapy, with a change from pazopanib to nivolumab.
Upon its return, the hypercalcemia at 3.0 mmol/L was refractory to three doses of denosumab 120 mg SC over 4 weeks. Nivolumab was discontinued due to kidney injury, and prednisone was started. At the time of his consultation with our Endocrinology service, the patient presented with a calcium of 3.7 mmol/L, PTH of 0.2 pmol/L, PTH-RP 47 ng/L, 1,25(OH)2 D3 238 pmol/L, and 25 (OH) D3 102 nmol/L. The patient received IV hydration 3 L over 6 h and IV methylprednisolone 40 mg once; he had just received the latest denosumab dose. Day 2, the patient received furosemide 40 mg IV and 1 L normal saline IV and was started on cinacalcet 30 mg by mouth (PO) daily. Four days later, the calcium improved to 3.3 mmol/L, and the cinacalcet was increased to 60 mg PO daily. One week after cinacalcet dose escalation, the calcium was 2.8 mmol/L. Due to the very favorable response and uncertainty as to whether this continued dose would incite hypocalcemia, the cinacalcet was reduced back to 30 mg PO daily. Seven days later the calcium had risen to 3.3 mmol/L; the cinacalcet was again increased to 60 mg PO daily. At this time targeted therapy with cabozantanib was started and was given off and on for 10 months. It had been placed on hold for various medical reasons. The calcium level remained normal for 3 months at which time it dropped to low normal at 2.1 mmol/L. Rather than de-escalating the cinacalcet dose by 50%, the dose was simply reduced to 45 mg PO daily. The calcium remained in the normal range for the next 9 months (with a goal to keep the calcium at the upper limits of normal, so as not to incite hypocalcemia), and the PTH normalized to 1.9 pmol/L. During this time the 1,25(OH)2 D3 normalized and then rose slightly above normal again.
In his 10th month of treatment with cinacalcet, the patient suffered an acute stroke and was hospitalized. During that time, his cinacalcet treatment was interrupted. Resultantly, his calcium rose to 3.6 mmol/L. Cinacalcet was resumed at 90 mg PO daily, and denosumab 120 mg SC was given. By 10 days, the calcium improved to 3.0 mmol/L, and another dose of denosumab 120 mg SC was given. The calcium normalized in 1 week and remained normal with a normal PTH on cinacalcet monotherapy until he succumbed to his disease 17 days later (Fig. 2).
Figure 2 Parathyroid hormone (PTH). The dash line represents calcium response, and the bar denotes change in PTH.
It should be noted that the patient was started on prednisone for chronic kidney inflammation while on nivolumab. It was given off and on prior to and during the course of cinacalcet treatment. Considering the amount of time that the patient was on a stable dose of cinacalcet with normal calcium levels, it is our thought that the prednisone was not significantly influencing calcium levels. Furthermore, while targeted anti-tumor therapies had been on hold, the cinacalcet was, nonetheless, able to maintain normal calcium levels. While the PTH-RP came down to 29 ng/L, it was not profoundly elevated at any given time, and its improvement was only very slight. Therefore, it is postulated that for a given level of PTH-RP, there is not a correlation with the severity of hypercalcemia nor the cinacalcet dose required to achieve normocalcemia (Fig. 3). Changes in 25(OH) D3 were not noteworthy, while there was slight reduction in 1,25(OH)2 D3 (Table 2).
Figure 3 Parathyroid hormone-related peptide (PTH-RP). The dash line represents calcium response, and the bar denotes change in PTH-RP.
Table 2 Effects of cinacalcet treatment on pertinent biochemical parameters.
Parameters (normal range) Day 0 initiated cinacalcet 30 mg/day Day 4 ↑ cinacalcet 60 mg/day Day 11 ↓ cinacalcet 30 mg/day Day 18 ↑ cinacalcet 60 mg/day Day 110 ↓ cinacalcet 45 mg/day Day 260 stable cinacalcet 45 mg/day Day 305 stable cinacalcet 45 mg/day Day 335a restart cinacalcet 90 mg/day + denosumab Day 349b stable cinacalcet 90 mg/day
Calcium (2.1–2.7 mmol/L) 3.6 3.3 2.8 3.3 2.1 2.4 2.6 3.6 2.6
PTH (1.5–6.8 pmol/L) 0.2 – 0.3 – – 1.9 – – –
PTH-RP (14–27 ng/L) – – 47 – 29 32 – – –
25 (OH) D3 (75–250 nmol/L) 102 – – – 72 96 – – –
1,25(OH)2 D3 (43–173 pmol/L) 238 – – – 216 178 – – –
aPatient was hospitalized for a stroke from day 306 to 334 and was off cinacalcet during this period. Cinacalcet was restarted along with one dose of s.c. denosumab 120 mg, bPatient deceased 11 days (day 360) after last lab draw.
1, 25(OH)2 D3, 1, 25-dihydroxy vitamin D; 25(OH) D3, 25 hydroxy vitamin D; PTH, parathyroid hormone; PTH-RP, parathyroid hormone-related peptide.
Discussion
Our patient acquired HHM that was refractory to bisphosphonate and denosumab therapy. As a result of treatment with cinacalcet, there was reduction in and normalization of calcium. As noted above, other cases show cinacalcet’s usefulness in the treatment of HHM. Given that the patients in these cases received multiple therapeutic agents to reduce calcium, it can be difficult to differentiate effects due to cinacalcet and those due to other agents. However, when hypercalcemia is refractory to all conventional modalities yet responds to the addition of cinacalcet, it follows that cinacalcet can serve as adjunctive therapy.
It is well described that the CaSR of the parafollicular C cells of the thyroid modulates calcitonin release in response to hypercalcemia (3). It is possible that this action could be a mechanism by which cinacalcet lowers calcium in HHM; Colloton describes reduction of PTH-RP-mediated calcium levels (accompanied by rise in calcitonin levels) with cinacalcet therapy (58). In our case, the PTH-RP levels did not show significant change, though the calcium showed dramatic response. Certainly, the CaSR’s influence on renal calcium disposition and osteoblast and osteoclast function can play a role in cinacalcet’s calcium lowering ability.
The patient in our case benefited from a eucalcemic state for nearly 1 year until he succumbed to his disease. It was observed that calcium levels start to respond to cinacalcet in 1 week with normalization of calcium by 2 weeks. While considering each of the cases reviewed here, it is important to note that each patient has variations in calcium homeostasis and in the disease states inciting the MAH and will thus respond differently even to the same cinacalcet dose. Great care should be taken in the monitoring and dosage adjustment of cinacalcet. It is proposed that a temporary drug holiday or a reduction in dose in the setting of hypocalcemia would be preferable to drug discontinuation. This reduces the chance of returning to a hypercalcemic state or a hypercalcemic urgency. Lab draws were more frequent with initiation of cinacalcet, for example within 1 week for the first draw and weekly draws until calcium levels are stable on a given dose. For our case there were a couple of instances of 3–4 weeks between blood draws, since the calcium was quite stable.
Reducing morbidity from MAH is important to patients in terms of their symptomatology, but it is equally important in terms of their required clinic visits and hospitalizations. While on oral cinacalcet monotherapy for his HHM, our patient remained eucalcemic, and no longer required clinic visits or hospitalizations specifically for treatment of hypercalcemia. Patients have many clinic encounters and hospitalizations resulting from disease treatment and progression of their primary disease; it follows that reducing the need for these encounters by controlling MAH becomes very meaningful to them.
Early on it was suggested that debulking tumor would favorably impact hypercalcemia regardless of the biochemical factors involved, because a debulked tumor could portend reduction of biochemical factors driving hypercalcemia (59). It follows that PTH-RP could be reduced with physical debulking or with targeted tumor therapy. Interestingly, our patient’s PTH-RP levels came down only slightly, with cinacalcet therapy; the significance of this is unknown. Even with only minimal reductions of PTH-RP and progression of cancer until the time of death, cinacalcet was able to achieve a eucalcemic state.
Conclusion
Even as recent as 2014, it has been suggested that palliation of symptoms related to MAH is essential and clinically meaningful for patients, given the continued poor prognosis and high morbidity and mortality associated with MAH (49). Historically, agents have been temporizing and have not impacted patient survival. The ideal agent for long-term treatment of MAH that was hoped for in the early 1980s was an oral agent which maintains the serum calcium in the normal or near normal range (39). We suggest that cinacalcet can be that oral agent, reducing patients’ time in the hospital and clinic settings. It is well-tolerated and can maintain calcium levels in the normal range. This has a direct, major impact on morbidity. Treatment of MAH to this level of success can increase patient quality of life while targeted cancer therapies can work to improve survival. So far, this is the only agent to treat MAH suggested to favorably impact quality of life. Studies are needed to determine the possible impact of the achievement of eucalcemia on survival with MAH. While it is true that not all patients may respond, depending on the aggressiveness of the late stages of cancer, especially where death is imminent, it seems worthwhile to afford the possible benefit.
Cinacalcet is approved for secondary hyperparathyroidism, parathyroid carcinoma-associated hypercalcemia, and severe hypercalcemia associated with primary hyperparathyroidism. The use of cinacalcet is novel in the treatment of MAH/HHM; the case presented here responded successfully to this therapy (reduction of calcium levels to normal). First line agents for MAH historically have been IV or SC, and no agent had been uniformly safe and effective over a long period of time (23, 39). It is proposed here that oral cinacalcet can favorably influence calcium homeostasis safely over an extended period of time in the setting of HHM as adjunctive therapy or (in some cases) monotherapy. Given that there is often a humoral component to osteolytic MAH, it is postulated that cinacalcet could benefit patients regardless of the predominating etiology of MAH in any given case.
Goals of future therapeutic modalities
Prior to identifying PTH-RP or its receptor, it was postulated that blocking the humoral substance driving the hypercalcemia would be a possible therapeutic option (17). Recognizing the need to target renal resorption of calcium, it was suggested that drugs are needed to inhibit PTH or PTH-RP action or production, or that antibodies are needed to inhibit PTH-RP (19, 53, 60). Further research elucidating this interplay is warranted.
Given that these case reports showed improvement of calcium in MAH, there is promising evidence that cinacalcet can be employed in this setting. Future consideration should be given to studies addressing the efficacy of cinacalcet in calcium normalization, improvement of quality of life, and impact on survival in patients with MAH. Even though the exact mechanism of action for cinacalcet’s reduction in calcium in this setting is not entirely elucidated, we can still afford patients the possible benefit from it.
Declaration of interest
The published viewpoints are those of the individual authors and do not represent the official stance or statements of the respective academic and/or governmental agencies with which the authors are affiliated.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
S O’Callaghan conceived of the idea and subject matter for this review article. S O’Callaghan and H Yau were responsible for the care of the patient presented in the case along with the acquisition, analysis, and interpretation of data. Both authors contributed to the drafting and revising of the manuscript critically for important intellectual content. | Fatal | ReactionOutcome | CC BY-NC-ND | 33289687 | 19,258,843 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Exposure during pregnancy'. | Coombs-negative haemolytic anaemia in pregnancy: A case report.
We present a rare case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman in secondary care. There were no known underlying medical or obstetric risk factors for haemolytic anaemia. Following extensive investigation and a therapeutic trial of oral corticosteroids, a diagnosis was made. Autoimmune haemolytic anaemia is potentially fatal, and prompt diagnosis with haematology input is essential to ensure maternal and fetal safety in pregnancy and the puerperium. With only a small number of cases of Coombs-negative autoimmune haemolytic anaemia reported in the literature, we present this rare case for discussion. We highlight the importance of thorough investigation of refractory anaemia in pregnancy and consider the associated challenges.
1 Introduction
Anaemia, predominantly iron deficiency anaemia, affects up to 30–40% of pregnant women [1]. Anaemia in pregnancy can be associated with an increased risk of maternal death. In one paper a haemoglobin level of less than 89 g/L was associated with the highest risk [2]. Autoimmune haemolytic anaemia (AIHA) has an incidence of approximately 0.83 in 100,0003 in the general population. It is rare in pregnancy, affecting as little as 1 in 140,000 pregnancies [3]. In most cases the diagnosis is straightforward when there is the combination of anaemia, reticulocytosis, a high LDH, low or undetectable haptoglobin and a positive direct Coombs test (DCT). Only 5–10% of all cases of AIHA are Coombs negative. We report a case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman who presented at 16 weeks of gestation with shortness of breath and epigastric pain.
2 Case report
A 41-year-old woman, gravida 5, para 2, presented with epigastric pain and shortness of breath at 16 weeks of gestation. Her BMI was 33.8 kg/m2. She was rhesus positive and had a venous thromboembolic (VTE) score of 2 (moderate), not requiring antenatal thromboprophylaxis. Her booking haemoglobin level was 141 g/L. She had had an emergency Caesarean section 16 years previously for a brow presentation, followed by a successful vaginal birth after caesarean (VBAC) 3 years later. Her medical history includes idiopathic intracranial hypertension with no treatment, previous large loop excision of the transformation zone (LLETZ) of the cervix for an abnormal cervical cytology, and previous left nephrectomy as she was an organ donor. There were no obstetric or haematological complications in any previous pregnancies.
In the index pregnancy the patient was taking 400μg folic acid; there was no other drug history to note. Antenatally she was commenced on 150 mg aspirin, as she was high risk for developing pre-eclampsia (age, > 10-year pregnancy interval). A glucose tolerance test was arranged as she was at high risk for developing gestational diabetes (age, BMI). Cervical length scan screening was arranged in view of previous LLETZ. Serial growth scans were planned for 30,34 and 38 weeks of gestation (for maternal age). There was no relevant family history of note.
On first presentation at 16 weeks of gestation she complained of shortness of breath and epigastric pain. Initial blood results revealed a haemoglobin level of 79 g/L, with a raised bilirubin level of 23 umol/L, raised reticulocyte count of 5% and undetectable haptoglobin. Ferritin, B12 and folate levels were also normal. All other blood results were within normal range for gestation and common causes of abdominal pain in pregnancy were excluded, such as urinary tract infection, pancreatitis and appendicitis. In view of the raised bilirubin level, a referral was made to haematology. An ultrasound scan of the abdomen revealed a normal liver, no gall stones, no evidence of splenic or hepatic venous thrombosis, but evidence of splenomegaly, with the spleen measuring 16 cm. There was no evidence of preceding infection. Serology and polymerase chain reaction (PCR) were negative for cytomegalovirus, Epstein Barr virus and toxoplasmosis. A connective tissue screen was also negative. The haematology team further investigated the cause of her severe refractory anaemia. The blood test results during the course of investigations can be seen in Fig. 1; the patient had raised LDH and persistent anaemia. Bilirubin and reticulocyte count remained raised throughout the antenatal period. Direct Coombs tests were repeatedly negative using anti-IgG and complement antisera and remained negative when repeated with a polyspecific anti-immunoglobulin anti-sera. The findings were in keeping with the rare diagnosis of Coombs-negative haemolytic anaemia. The patient was discharged and had haematology day-unit follow-up, having her first blood transfusion on at 17 + 1/40 weeks of gestation, after tests revealed a haemoglobin level of 66 g/L (Fig. 1). During the course of her investigations, she received a total of five blood transfusions antenatally. Screening for paroxysmal nocturnal haemoglobinuria (PNH) was negative, urine haemosiderin negative with no evidence of PNH clone on flow cytometry. Blood results as seen in Fig. 1 revealed ongoing haemolysis.Fig. 1 Blood results.
Fig. 1
The patient was commenced on 20 mg prednisolone as per haematology plan at 22 + 6/40 weeks of gestation, with resolution of symptoms seen within days. As seen in Fig. 1, haemoglobin and LDH improved following the initiation the therapeutic oral corticosteroid therapy. Reticulocyte count and bilirubin also returned to normal within three days of treatment. A satisfactory Hb was maintained when the prednisolone was reduced to 10 mg daily. Subsequently, this patient went on to develop gestational diabetes, potentially secondary to steroid treatment, with additional underlying risk factors such as age and BMI. She was managed by the obstetric diabetes team.
Antenatally, fetal wellbeing was monitored. A growth scan performed at 28 weeks of gestation showed normal growth on the 90th centile, normal liquor volume and end diastolic flow/umbilical artery doppler. A plan for delivery was made, for an induction of labour at 37 weeks, and the patient had weekly haematology follow-up.
A live female infant was born by normal vaginal delivery at 37+ 3 weeks of gestation. Blood results for the neonate where all normal: Hb 193 g/L, WCC 10.8 × 10^9/L, Plat 386 × 10^9/L, Cord bilirubin 41 umol/L, DCT - negative. Postnatally the patient was weaned off oral corticosteroid treatment, and maintained a normal haemaglobin level of 144 g/L. Follow-up tests also revealed normal bilirubin and reticulocyte count. The potential for recurrence of this condition is unknown, due to its rarity. The patient was counselled regarding postnatal contraception.
3 Discussion
AIHA is caused by a host immune system creating immunoglobulin auto-antibodies against red cell membrane antigens, leading to their premature destruction by the spleen and reticuloendothelial system. The DCT uses an antibody against immunoglobulins to detect these on the red cell surface but when the level of antibody coating is very low, the test may be negative, as in this case [4]. The autoimmune mechanism of haemolysis in this case is confirmed by the prompt response to immunosuppression with cortiosteroids.
As IgG antibodies can cross the placenta, the fetus is subsequently at risk of developing haemolytic disease of the newborn (HDN). Testing of umbilical cord DCT, haemoglobin and bilirubin are required, and monitoring for symptoms essential [5]. Treatment recommendations for maternal autoimmune haemolysis are initially oral corticosteroids with IV immunoglobulins in refractory patients. Occasionally, additional immunosuppression with azathioprine or rituximab is required and rarely splenectomy may be indicated.
4 Conclusion
Conditions such as haemolytic anaemia can be fatal and require multidisciplinary input to ensure safe and effective treatment of the patient. With no preceding history or risk factors, this is a rare case of acquired auto-immune haemolytic anaemia in pregnancy. The case highlights the importance of thorough investigation for what may appear to be simple anaemia in pregnancy, especially when refractory to treatment.
Contributors
Dr Holly George drafted the paper and performed the literature search and review.
Dr. E Haslett contributed to revision of the paper.
Dr. M Macheta provided haematology expertise and contributed to revision of the paper.
Conflict of Interest
The authors declare that they have no conflict of interest regarding the publication of this case report.
Funding
No funding from an external source supported the publication of this case report.
Patient consent
Obtained.
Provenance and peer review
This case report was peer reviewed. | ASPIRIN, FOLIC ACID, PREDNISOLONE | DrugsGivenReaction | CC BY-NC-ND | 33294391 | 18,792,533 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Maternal exposure during pregnancy'. | Coombs-negative haemolytic anaemia in pregnancy: A case report.
We present a rare case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman in secondary care. There were no known underlying medical or obstetric risk factors for haemolytic anaemia. Following extensive investigation and a therapeutic trial of oral corticosteroids, a diagnosis was made. Autoimmune haemolytic anaemia is potentially fatal, and prompt diagnosis with haematology input is essential to ensure maternal and fetal safety in pregnancy and the puerperium. With only a small number of cases of Coombs-negative autoimmune haemolytic anaemia reported in the literature, we present this rare case for discussion. We highlight the importance of thorough investigation of refractory anaemia in pregnancy and consider the associated challenges.
1 Introduction
Anaemia, predominantly iron deficiency anaemia, affects up to 30–40% of pregnant women [1]. Anaemia in pregnancy can be associated with an increased risk of maternal death. In one paper a haemoglobin level of less than 89 g/L was associated with the highest risk [2]. Autoimmune haemolytic anaemia (AIHA) has an incidence of approximately 0.83 in 100,0003 in the general population. It is rare in pregnancy, affecting as little as 1 in 140,000 pregnancies [3]. In most cases the diagnosis is straightforward when there is the combination of anaemia, reticulocytosis, a high LDH, low or undetectable haptoglobin and a positive direct Coombs test (DCT). Only 5–10% of all cases of AIHA are Coombs negative. We report a case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman who presented at 16 weeks of gestation with shortness of breath and epigastric pain.
2 Case report
A 41-year-old woman, gravida 5, para 2, presented with epigastric pain and shortness of breath at 16 weeks of gestation. Her BMI was 33.8 kg/m2. She was rhesus positive and had a venous thromboembolic (VTE) score of 2 (moderate), not requiring antenatal thromboprophylaxis. Her booking haemoglobin level was 141 g/L. She had had an emergency Caesarean section 16 years previously for a brow presentation, followed by a successful vaginal birth after caesarean (VBAC) 3 years later. Her medical history includes idiopathic intracranial hypertension with no treatment, previous large loop excision of the transformation zone (LLETZ) of the cervix for an abnormal cervical cytology, and previous left nephrectomy as she was an organ donor. There were no obstetric or haematological complications in any previous pregnancies.
In the index pregnancy the patient was taking 400μg folic acid; there was no other drug history to note. Antenatally she was commenced on 150 mg aspirin, as she was high risk for developing pre-eclampsia (age, > 10-year pregnancy interval). A glucose tolerance test was arranged as she was at high risk for developing gestational diabetes (age, BMI). Cervical length scan screening was arranged in view of previous LLETZ. Serial growth scans were planned for 30,34 and 38 weeks of gestation (for maternal age). There was no relevant family history of note.
On first presentation at 16 weeks of gestation she complained of shortness of breath and epigastric pain. Initial blood results revealed a haemoglobin level of 79 g/L, with a raised bilirubin level of 23 umol/L, raised reticulocyte count of 5% and undetectable haptoglobin. Ferritin, B12 and folate levels were also normal. All other blood results were within normal range for gestation and common causes of abdominal pain in pregnancy were excluded, such as urinary tract infection, pancreatitis and appendicitis. In view of the raised bilirubin level, a referral was made to haematology. An ultrasound scan of the abdomen revealed a normal liver, no gall stones, no evidence of splenic or hepatic venous thrombosis, but evidence of splenomegaly, with the spleen measuring 16 cm. There was no evidence of preceding infection. Serology and polymerase chain reaction (PCR) were negative for cytomegalovirus, Epstein Barr virus and toxoplasmosis. A connective tissue screen was also negative. The haematology team further investigated the cause of her severe refractory anaemia. The blood test results during the course of investigations can be seen in Fig. 1; the patient had raised LDH and persistent anaemia. Bilirubin and reticulocyte count remained raised throughout the antenatal period. Direct Coombs tests were repeatedly negative using anti-IgG and complement antisera and remained negative when repeated with a polyspecific anti-immunoglobulin anti-sera. The findings were in keeping with the rare diagnosis of Coombs-negative haemolytic anaemia. The patient was discharged and had haematology day-unit follow-up, having her first blood transfusion on at 17 + 1/40 weeks of gestation, after tests revealed a haemoglobin level of 66 g/L (Fig. 1). During the course of her investigations, she received a total of five blood transfusions antenatally. Screening for paroxysmal nocturnal haemoglobinuria (PNH) was negative, urine haemosiderin negative with no evidence of PNH clone on flow cytometry. Blood results as seen in Fig. 1 revealed ongoing haemolysis.Fig. 1 Blood results.
Fig. 1
The patient was commenced on 20 mg prednisolone as per haematology plan at 22 + 6/40 weeks of gestation, with resolution of symptoms seen within days. As seen in Fig. 1, haemoglobin and LDH improved following the initiation the therapeutic oral corticosteroid therapy. Reticulocyte count and bilirubin also returned to normal within three days of treatment. A satisfactory Hb was maintained when the prednisolone was reduced to 10 mg daily. Subsequently, this patient went on to develop gestational diabetes, potentially secondary to steroid treatment, with additional underlying risk factors such as age and BMI. She was managed by the obstetric diabetes team.
Antenatally, fetal wellbeing was monitored. A growth scan performed at 28 weeks of gestation showed normal growth on the 90th centile, normal liquor volume and end diastolic flow/umbilical artery doppler. A plan for delivery was made, for an induction of labour at 37 weeks, and the patient had weekly haematology follow-up.
A live female infant was born by normal vaginal delivery at 37+ 3 weeks of gestation. Blood results for the neonate where all normal: Hb 193 g/L, WCC 10.8 × 10^9/L, Plat 386 × 10^9/L, Cord bilirubin 41 umol/L, DCT - negative. Postnatally the patient was weaned off oral corticosteroid treatment, and maintained a normal haemaglobin level of 144 g/L. Follow-up tests also revealed normal bilirubin and reticulocyte count. The potential for recurrence of this condition is unknown, due to its rarity. The patient was counselled regarding postnatal contraception.
3 Discussion
AIHA is caused by a host immune system creating immunoglobulin auto-antibodies against red cell membrane antigens, leading to their premature destruction by the spleen and reticuloendothelial system. The DCT uses an antibody against immunoglobulins to detect these on the red cell surface but when the level of antibody coating is very low, the test may be negative, as in this case [4]. The autoimmune mechanism of haemolysis in this case is confirmed by the prompt response to immunosuppression with cortiosteroids.
As IgG antibodies can cross the placenta, the fetus is subsequently at risk of developing haemolytic disease of the newborn (HDN). Testing of umbilical cord DCT, haemoglobin and bilirubin are required, and monitoring for symptoms essential [5]. Treatment recommendations for maternal autoimmune haemolysis are initially oral corticosteroids with IV immunoglobulins in refractory patients. Occasionally, additional immunosuppression with azathioprine or rituximab is required and rarely splenectomy may be indicated.
4 Conclusion
Conditions such as haemolytic anaemia can be fatal and require multidisciplinary input to ensure safe and effective treatment of the patient. With no preceding history or risk factors, this is a rare case of acquired auto-immune haemolytic anaemia in pregnancy. The case highlights the importance of thorough investigation for what may appear to be simple anaemia in pregnancy, especially when refractory to treatment.
Contributors
Dr Holly George drafted the paper and performed the literature search and review.
Dr. E Haslett contributed to revision of the paper.
Dr. M Macheta provided haematology expertise and contributed to revision of the paper.
Conflict of Interest
The authors declare that they have no conflict of interest regarding the publication of this case report.
Funding
No funding from an external source supported the publication of this case report.
Patient consent
Obtained.
Provenance and peer review
This case report was peer reviewed. | ASPIRIN, DEXTROSE, FERRIC CARBOXYMALTOSE, FOLIC ACID, PREDNISOLONE | DrugsGivenReaction | CC BY-NC-ND | 33294391 | 18,802,221 | 2021-01 |
What was the administration route of drug 'FERRIC CARBOXYMALTOSE'? | Coombs-negative haemolytic anaemia in pregnancy: A case report.
We present a rare case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman in secondary care. There were no known underlying medical or obstetric risk factors for haemolytic anaemia. Following extensive investigation and a therapeutic trial of oral corticosteroids, a diagnosis was made. Autoimmune haemolytic anaemia is potentially fatal, and prompt diagnosis with haematology input is essential to ensure maternal and fetal safety in pregnancy and the puerperium. With only a small number of cases of Coombs-negative autoimmune haemolytic anaemia reported in the literature, we present this rare case for discussion. We highlight the importance of thorough investigation of refractory anaemia in pregnancy and consider the associated challenges.
1 Introduction
Anaemia, predominantly iron deficiency anaemia, affects up to 30–40% of pregnant women [1]. Anaemia in pregnancy can be associated with an increased risk of maternal death. In one paper a haemoglobin level of less than 89 g/L was associated with the highest risk [2]. Autoimmune haemolytic anaemia (AIHA) has an incidence of approximately 0.83 in 100,0003 in the general population. It is rare in pregnancy, affecting as little as 1 in 140,000 pregnancies [3]. In most cases the diagnosis is straightforward when there is the combination of anaemia, reticulocytosis, a high LDH, low or undetectable haptoglobin and a positive direct Coombs test (DCT). Only 5–10% of all cases of AIHA are Coombs negative. We report a case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman who presented at 16 weeks of gestation with shortness of breath and epigastric pain.
2 Case report
A 41-year-old woman, gravida 5, para 2, presented with epigastric pain and shortness of breath at 16 weeks of gestation. Her BMI was 33.8 kg/m2. She was rhesus positive and had a venous thromboembolic (VTE) score of 2 (moderate), not requiring antenatal thromboprophylaxis. Her booking haemoglobin level was 141 g/L. She had had an emergency Caesarean section 16 years previously for a brow presentation, followed by a successful vaginal birth after caesarean (VBAC) 3 years later. Her medical history includes idiopathic intracranial hypertension with no treatment, previous large loop excision of the transformation zone (LLETZ) of the cervix for an abnormal cervical cytology, and previous left nephrectomy as she was an organ donor. There were no obstetric or haematological complications in any previous pregnancies.
In the index pregnancy the patient was taking 400μg folic acid; there was no other drug history to note. Antenatally she was commenced on 150 mg aspirin, as she was high risk for developing pre-eclampsia (age, > 10-year pregnancy interval). A glucose tolerance test was arranged as she was at high risk for developing gestational diabetes (age, BMI). Cervical length scan screening was arranged in view of previous LLETZ. Serial growth scans were planned for 30,34 and 38 weeks of gestation (for maternal age). There was no relevant family history of note.
On first presentation at 16 weeks of gestation she complained of shortness of breath and epigastric pain. Initial blood results revealed a haemoglobin level of 79 g/L, with a raised bilirubin level of 23 umol/L, raised reticulocyte count of 5% and undetectable haptoglobin. Ferritin, B12 and folate levels were also normal. All other blood results were within normal range for gestation and common causes of abdominal pain in pregnancy were excluded, such as urinary tract infection, pancreatitis and appendicitis. In view of the raised bilirubin level, a referral was made to haematology. An ultrasound scan of the abdomen revealed a normal liver, no gall stones, no evidence of splenic or hepatic venous thrombosis, but evidence of splenomegaly, with the spleen measuring 16 cm. There was no evidence of preceding infection. Serology and polymerase chain reaction (PCR) were negative for cytomegalovirus, Epstein Barr virus and toxoplasmosis. A connective tissue screen was also negative. The haematology team further investigated the cause of her severe refractory anaemia. The blood test results during the course of investigations can be seen in Fig. 1; the patient had raised LDH and persistent anaemia. Bilirubin and reticulocyte count remained raised throughout the antenatal period. Direct Coombs tests were repeatedly negative using anti-IgG and complement antisera and remained negative when repeated with a polyspecific anti-immunoglobulin anti-sera. The findings were in keeping with the rare diagnosis of Coombs-negative haemolytic anaemia. The patient was discharged and had haematology day-unit follow-up, having her first blood transfusion on at 17 + 1/40 weeks of gestation, after tests revealed a haemoglobin level of 66 g/L (Fig. 1). During the course of her investigations, she received a total of five blood transfusions antenatally. Screening for paroxysmal nocturnal haemoglobinuria (PNH) was negative, urine haemosiderin negative with no evidence of PNH clone on flow cytometry. Blood results as seen in Fig. 1 revealed ongoing haemolysis.Fig. 1 Blood results.
Fig. 1
The patient was commenced on 20 mg prednisolone as per haematology plan at 22 + 6/40 weeks of gestation, with resolution of symptoms seen within days. As seen in Fig. 1, haemoglobin and LDH improved following the initiation the therapeutic oral corticosteroid therapy. Reticulocyte count and bilirubin also returned to normal within three days of treatment. A satisfactory Hb was maintained when the prednisolone was reduced to 10 mg daily. Subsequently, this patient went on to develop gestational diabetes, potentially secondary to steroid treatment, with additional underlying risk factors such as age and BMI. She was managed by the obstetric diabetes team.
Antenatally, fetal wellbeing was monitored. A growth scan performed at 28 weeks of gestation showed normal growth on the 90th centile, normal liquor volume and end diastolic flow/umbilical artery doppler. A plan for delivery was made, for an induction of labour at 37 weeks, and the patient had weekly haematology follow-up.
A live female infant was born by normal vaginal delivery at 37+ 3 weeks of gestation. Blood results for the neonate where all normal: Hb 193 g/L, WCC 10.8 × 10^9/L, Plat 386 × 10^9/L, Cord bilirubin 41 umol/L, DCT - negative. Postnatally the patient was weaned off oral corticosteroid treatment, and maintained a normal haemaglobin level of 144 g/L. Follow-up tests also revealed normal bilirubin and reticulocyte count. The potential for recurrence of this condition is unknown, due to its rarity. The patient was counselled regarding postnatal contraception.
3 Discussion
AIHA is caused by a host immune system creating immunoglobulin auto-antibodies against red cell membrane antigens, leading to their premature destruction by the spleen and reticuloendothelial system. The DCT uses an antibody against immunoglobulins to detect these on the red cell surface but when the level of antibody coating is very low, the test may be negative, as in this case [4]. The autoimmune mechanism of haemolysis in this case is confirmed by the prompt response to immunosuppression with cortiosteroids.
As IgG antibodies can cross the placenta, the fetus is subsequently at risk of developing haemolytic disease of the newborn (HDN). Testing of umbilical cord DCT, haemoglobin and bilirubin are required, and monitoring for symptoms essential [5]. Treatment recommendations for maternal autoimmune haemolysis are initially oral corticosteroids with IV immunoglobulins in refractory patients. Occasionally, additional immunosuppression with azathioprine or rituximab is required and rarely splenectomy may be indicated.
4 Conclusion
Conditions such as haemolytic anaemia can be fatal and require multidisciplinary input to ensure safe and effective treatment of the patient. With no preceding history or risk factors, this is a rare case of acquired auto-immune haemolytic anaemia in pregnancy. The case highlights the importance of thorough investigation for what may appear to be simple anaemia in pregnancy, especially when refractory to treatment.
Contributors
Dr Holly George drafted the paper and performed the literature search and review.
Dr. E Haslett contributed to revision of the paper.
Dr. M Macheta provided haematology expertise and contributed to revision of the paper.
Conflict of Interest
The authors declare that they have no conflict of interest regarding the publication of this case report.
Funding
No funding from an external source supported the publication of this case report.
Patient consent
Obtained.
Provenance and peer review
This case report was peer reviewed. | Other | DrugAdministrationRoute | CC BY-NC-ND | 33294391 | 18,802,221 | 2021-01 |
What was the administration route of drug 'PREDNISOLONE'? | Coombs-negative haemolytic anaemia in pregnancy: A case report.
We present a rare case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman in secondary care. There were no known underlying medical or obstetric risk factors for haemolytic anaemia. Following extensive investigation and a therapeutic trial of oral corticosteroids, a diagnosis was made. Autoimmune haemolytic anaemia is potentially fatal, and prompt diagnosis with haematology input is essential to ensure maternal and fetal safety in pregnancy and the puerperium. With only a small number of cases of Coombs-negative autoimmune haemolytic anaemia reported in the literature, we present this rare case for discussion. We highlight the importance of thorough investigation of refractory anaemia in pregnancy and consider the associated challenges.
1 Introduction
Anaemia, predominantly iron deficiency anaemia, affects up to 30–40% of pregnant women [1]. Anaemia in pregnancy can be associated with an increased risk of maternal death. In one paper a haemoglobin level of less than 89 g/L was associated with the highest risk [2]. Autoimmune haemolytic anaemia (AIHA) has an incidence of approximately 0.83 in 100,0003 in the general population. It is rare in pregnancy, affecting as little as 1 in 140,000 pregnancies [3]. In most cases the diagnosis is straightforward when there is the combination of anaemia, reticulocytosis, a high LDH, low or undetectable haptoglobin and a positive direct Coombs test (DCT). Only 5–10% of all cases of AIHA are Coombs negative. We report a case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman who presented at 16 weeks of gestation with shortness of breath and epigastric pain.
2 Case report
A 41-year-old woman, gravida 5, para 2, presented with epigastric pain and shortness of breath at 16 weeks of gestation. Her BMI was 33.8 kg/m2. She was rhesus positive and had a venous thromboembolic (VTE) score of 2 (moderate), not requiring antenatal thromboprophylaxis. Her booking haemoglobin level was 141 g/L. She had had an emergency Caesarean section 16 years previously for a brow presentation, followed by a successful vaginal birth after caesarean (VBAC) 3 years later. Her medical history includes idiopathic intracranial hypertension with no treatment, previous large loop excision of the transformation zone (LLETZ) of the cervix for an abnormal cervical cytology, and previous left nephrectomy as she was an organ donor. There were no obstetric or haematological complications in any previous pregnancies.
In the index pregnancy the patient was taking 400μg folic acid; there was no other drug history to note. Antenatally she was commenced on 150 mg aspirin, as she was high risk for developing pre-eclampsia (age, > 10-year pregnancy interval). A glucose tolerance test was arranged as she was at high risk for developing gestational diabetes (age, BMI). Cervical length scan screening was arranged in view of previous LLETZ. Serial growth scans were planned for 30,34 and 38 weeks of gestation (for maternal age). There was no relevant family history of note.
On first presentation at 16 weeks of gestation she complained of shortness of breath and epigastric pain. Initial blood results revealed a haemoglobin level of 79 g/L, with a raised bilirubin level of 23 umol/L, raised reticulocyte count of 5% and undetectable haptoglobin. Ferritin, B12 and folate levels were also normal. All other blood results were within normal range for gestation and common causes of abdominal pain in pregnancy were excluded, such as urinary tract infection, pancreatitis and appendicitis. In view of the raised bilirubin level, a referral was made to haematology. An ultrasound scan of the abdomen revealed a normal liver, no gall stones, no evidence of splenic or hepatic venous thrombosis, but evidence of splenomegaly, with the spleen measuring 16 cm. There was no evidence of preceding infection. Serology and polymerase chain reaction (PCR) were negative for cytomegalovirus, Epstein Barr virus and toxoplasmosis. A connective tissue screen was also negative. The haematology team further investigated the cause of her severe refractory anaemia. The blood test results during the course of investigations can be seen in Fig. 1; the patient had raised LDH and persistent anaemia. Bilirubin and reticulocyte count remained raised throughout the antenatal period. Direct Coombs tests were repeatedly negative using anti-IgG and complement antisera and remained negative when repeated with a polyspecific anti-immunoglobulin anti-sera. The findings were in keeping with the rare diagnosis of Coombs-negative haemolytic anaemia. The patient was discharged and had haematology day-unit follow-up, having her first blood transfusion on at 17 + 1/40 weeks of gestation, after tests revealed a haemoglobin level of 66 g/L (Fig. 1). During the course of her investigations, she received a total of five blood transfusions antenatally. Screening for paroxysmal nocturnal haemoglobinuria (PNH) was negative, urine haemosiderin negative with no evidence of PNH clone on flow cytometry. Blood results as seen in Fig. 1 revealed ongoing haemolysis.Fig. 1 Blood results.
Fig. 1
The patient was commenced on 20 mg prednisolone as per haematology plan at 22 + 6/40 weeks of gestation, with resolution of symptoms seen within days. As seen in Fig. 1, haemoglobin and LDH improved following the initiation the therapeutic oral corticosteroid therapy. Reticulocyte count and bilirubin also returned to normal within three days of treatment. A satisfactory Hb was maintained when the prednisolone was reduced to 10 mg daily. Subsequently, this patient went on to develop gestational diabetes, potentially secondary to steroid treatment, with additional underlying risk factors such as age and BMI. She was managed by the obstetric diabetes team.
Antenatally, fetal wellbeing was monitored. A growth scan performed at 28 weeks of gestation showed normal growth on the 90th centile, normal liquor volume and end diastolic flow/umbilical artery doppler. A plan for delivery was made, for an induction of labour at 37 weeks, and the patient had weekly haematology follow-up.
A live female infant was born by normal vaginal delivery at 37+ 3 weeks of gestation. Blood results for the neonate where all normal: Hb 193 g/L, WCC 10.8 × 10^9/L, Plat 386 × 10^9/L, Cord bilirubin 41 umol/L, DCT - negative. Postnatally the patient was weaned off oral corticosteroid treatment, and maintained a normal haemaglobin level of 144 g/L. Follow-up tests also revealed normal bilirubin and reticulocyte count. The potential for recurrence of this condition is unknown, due to its rarity. The patient was counselled regarding postnatal contraception.
3 Discussion
AIHA is caused by a host immune system creating immunoglobulin auto-antibodies against red cell membrane antigens, leading to their premature destruction by the spleen and reticuloendothelial system. The DCT uses an antibody against immunoglobulins to detect these on the red cell surface but when the level of antibody coating is very low, the test may be negative, as in this case [4]. The autoimmune mechanism of haemolysis in this case is confirmed by the prompt response to immunosuppression with cortiosteroids.
As IgG antibodies can cross the placenta, the fetus is subsequently at risk of developing haemolytic disease of the newborn (HDN). Testing of umbilical cord DCT, haemoglobin and bilirubin are required, and monitoring for symptoms essential [5]. Treatment recommendations for maternal autoimmune haemolysis are initially oral corticosteroids with IV immunoglobulins in refractory patients. Occasionally, additional immunosuppression with azathioprine or rituximab is required and rarely splenectomy may be indicated.
4 Conclusion
Conditions such as haemolytic anaemia can be fatal and require multidisciplinary input to ensure safe and effective treatment of the patient. With no preceding history or risk factors, this is a rare case of acquired auto-immune haemolytic anaemia in pregnancy. The case highlights the importance of thorough investigation for what may appear to be simple anaemia in pregnancy, especially when refractory to treatment.
Contributors
Dr Holly George drafted the paper and performed the literature search and review.
Dr. E Haslett contributed to revision of the paper.
Dr. M Macheta provided haematology expertise and contributed to revision of the paper.
Conflict of Interest
The authors declare that they have no conflict of interest regarding the publication of this case report.
Funding
No funding from an external source supported the publication of this case report.
Patient consent
Obtained.
Provenance and peer review
This case report was peer reviewed. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33294391 | 18,802,221 | 2021-01 |
What was the outcome of reaction 'Gestational diabetes'? | Coombs-negative haemolytic anaemia in pregnancy: A case report.
We present a rare case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman in secondary care. There were no known underlying medical or obstetric risk factors for haemolytic anaemia. Following extensive investigation and a therapeutic trial of oral corticosteroids, a diagnosis was made. Autoimmune haemolytic anaemia is potentially fatal, and prompt diagnosis with haematology input is essential to ensure maternal and fetal safety in pregnancy and the puerperium. With only a small number of cases of Coombs-negative autoimmune haemolytic anaemia reported in the literature, we present this rare case for discussion. We highlight the importance of thorough investigation of refractory anaemia in pregnancy and consider the associated challenges.
1 Introduction
Anaemia, predominantly iron deficiency anaemia, affects up to 30–40% of pregnant women [1]. Anaemia in pregnancy can be associated with an increased risk of maternal death. In one paper a haemoglobin level of less than 89 g/L was associated with the highest risk [2]. Autoimmune haemolytic anaemia (AIHA) has an incidence of approximately 0.83 in 100,0003 in the general population. It is rare in pregnancy, affecting as little as 1 in 140,000 pregnancies [3]. In most cases the diagnosis is straightforward when there is the combination of anaemia, reticulocytosis, a high LDH, low or undetectable haptoglobin and a positive direct Coombs test (DCT). Only 5–10% of all cases of AIHA are Coombs negative. We report a case of Coombs-negative autoimmune haemolytic anaemia in a multiparous woman who presented at 16 weeks of gestation with shortness of breath and epigastric pain.
2 Case report
A 41-year-old woman, gravida 5, para 2, presented with epigastric pain and shortness of breath at 16 weeks of gestation. Her BMI was 33.8 kg/m2. She was rhesus positive and had a venous thromboembolic (VTE) score of 2 (moderate), not requiring antenatal thromboprophylaxis. Her booking haemoglobin level was 141 g/L. She had had an emergency Caesarean section 16 years previously for a brow presentation, followed by a successful vaginal birth after caesarean (VBAC) 3 years later. Her medical history includes idiopathic intracranial hypertension with no treatment, previous large loop excision of the transformation zone (LLETZ) of the cervix for an abnormal cervical cytology, and previous left nephrectomy as she was an organ donor. There were no obstetric or haematological complications in any previous pregnancies.
In the index pregnancy the patient was taking 400μg folic acid; there was no other drug history to note. Antenatally she was commenced on 150 mg aspirin, as she was high risk for developing pre-eclampsia (age, > 10-year pregnancy interval). A glucose tolerance test was arranged as she was at high risk for developing gestational diabetes (age, BMI). Cervical length scan screening was arranged in view of previous LLETZ. Serial growth scans were planned for 30,34 and 38 weeks of gestation (for maternal age). There was no relevant family history of note.
On first presentation at 16 weeks of gestation she complained of shortness of breath and epigastric pain. Initial blood results revealed a haemoglobin level of 79 g/L, with a raised bilirubin level of 23 umol/L, raised reticulocyte count of 5% and undetectable haptoglobin. Ferritin, B12 and folate levels were also normal. All other blood results were within normal range for gestation and common causes of abdominal pain in pregnancy were excluded, such as urinary tract infection, pancreatitis and appendicitis. In view of the raised bilirubin level, a referral was made to haematology. An ultrasound scan of the abdomen revealed a normal liver, no gall stones, no evidence of splenic or hepatic venous thrombosis, but evidence of splenomegaly, with the spleen measuring 16 cm. There was no evidence of preceding infection. Serology and polymerase chain reaction (PCR) were negative for cytomegalovirus, Epstein Barr virus and toxoplasmosis. A connective tissue screen was also negative. The haematology team further investigated the cause of her severe refractory anaemia. The blood test results during the course of investigations can be seen in Fig. 1; the patient had raised LDH and persistent anaemia. Bilirubin and reticulocyte count remained raised throughout the antenatal period. Direct Coombs tests were repeatedly negative using anti-IgG and complement antisera and remained negative when repeated with a polyspecific anti-immunoglobulin anti-sera. The findings were in keeping with the rare diagnosis of Coombs-negative haemolytic anaemia. The patient was discharged and had haematology day-unit follow-up, having her first blood transfusion on at 17 + 1/40 weeks of gestation, after tests revealed a haemoglobin level of 66 g/L (Fig. 1). During the course of her investigations, she received a total of five blood transfusions antenatally. Screening for paroxysmal nocturnal haemoglobinuria (PNH) was negative, urine haemosiderin negative with no evidence of PNH clone on flow cytometry. Blood results as seen in Fig. 1 revealed ongoing haemolysis.Fig. 1 Blood results.
Fig. 1
The patient was commenced on 20 mg prednisolone as per haematology plan at 22 + 6/40 weeks of gestation, with resolution of symptoms seen within days. As seen in Fig. 1, haemoglobin and LDH improved following the initiation the therapeutic oral corticosteroid therapy. Reticulocyte count and bilirubin also returned to normal within three days of treatment. A satisfactory Hb was maintained when the prednisolone was reduced to 10 mg daily. Subsequently, this patient went on to develop gestational diabetes, potentially secondary to steroid treatment, with additional underlying risk factors such as age and BMI. She was managed by the obstetric diabetes team.
Antenatally, fetal wellbeing was monitored. A growth scan performed at 28 weeks of gestation showed normal growth on the 90th centile, normal liquor volume and end diastolic flow/umbilical artery doppler. A plan for delivery was made, for an induction of labour at 37 weeks, and the patient had weekly haematology follow-up.
A live female infant was born by normal vaginal delivery at 37+ 3 weeks of gestation. Blood results for the neonate where all normal: Hb 193 g/L, WCC 10.8 × 10^9/L, Plat 386 × 10^9/L, Cord bilirubin 41 umol/L, DCT - negative. Postnatally the patient was weaned off oral corticosteroid treatment, and maintained a normal haemaglobin level of 144 g/L. Follow-up tests also revealed normal bilirubin and reticulocyte count. The potential for recurrence of this condition is unknown, due to its rarity. The patient was counselled regarding postnatal contraception.
3 Discussion
AIHA is caused by a host immune system creating immunoglobulin auto-antibodies against red cell membrane antigens, leading to their premature destruction by the spleen and reticuloendothelial system. The DCT uses an antibody against immunoglobulins to detect these on the red cell surface but when the level of antibody coating is very low, the test may be negative, as in this case [4]. The autoimmune mechanism of haemolysis in this case is confirmed by the prompt response to immunosuppression with cortiosteroids.
As IgG antibodies can cross the placenta, the fetus is subsequently at risk of developing haemolytic disease of the newborn (HDN). Testing of umbilical cord DCT, haemoglobin and bilirubin are required, and monitoring for symptoms essential [5]. Treatment recommendations for maternal autoimmune haemolysis are initially oral corticosteroids with IV immunoglobulins in refractory patients. Occasionally, additional immunosuppression with azathioprine or rituximab is required and rarely splenectomy may be indicated.
4 Conclusion
Conditions such as haemolytic anaemia can be fatal and require multidisciplinary input to ensure safe and effective treatment of the patient. With no preceding history or risk factors, this is a rare case of acquired auto-immune haemolytic anaemia in pregnancy. The case highlights the importance of thorough investigation for what may appear to be simple anaemia in pregnancy, especially when refractory to treatment.
Contributors
Dr Holly George drafted the paper and performed the literature search and review.
Dr. E Haslett contributed to revision of the paper.
Dr. M Macheta provided haematology expertise and contributed to revision of the paper.
Conflict of Interest
The authors declare that they have no conflict of interest regarding the publication of this case report.
Funding
No funding from an external source supported the publication of this case report.
Patient consent
Obtained.
Provenance and peer review
This case report was peer reviewed. | Recovering | ReactionOutcome | CC BY-NC-ND | 33294391 | 18,802,221 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Blood testosterone decreased'. | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | LEUPROLIDE ACETATE | DrugsGivenReaction | CC BY | 33294761 | 18,656,149 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'. | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | ABIRATERONE ACETATE, BICALUTAMIDE, DEGARELIX, LEUPROLIDE ACETATE, PREDNISONE | DrugsGivenReaction | CC BY | 33294761 | 18,916,525 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Prostate cancer'. | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | ABIRATERONE ACETATE, BICALUTAMIDE, DEGARELIX, LEUPROLIDE ACETATE, PREDNISONE | DrugsGivenReaction | CC BY | 33294761 | 18,916,525 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Prostatic specific antigen abnormal'. | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | LEUPROLIDE ACETATE | DrugsGivenReaction | CC BY | 33294761 | 18,656,149 | 2021-01-01 |
What was the outcome of reaction 'Adrenogenital syndrome'? | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | Recovered | ReactionOutcome | CC BY | 33294761 | 18,916,528 | 2021-01-01 |
What was the outcome of reaction 'Blood testosterone decreased'? | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | Recovering | ReactionOutcome | CC BY | 33294761 | 18,656,149 | 2021-01-01 |
What was the outcome of reaction 'Blood testosterone increased'? | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | Recovered | ReactionOutcome | CC BY | 33294761 | 18,916,528 | 2021-01-01 |
What was the outcome of reaction 'Prostate cancer'? | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | Not recovered | ReactionOutcome | CC BY | 33294761 | 18,916,525 | 2021-01-01 |
What was the outcome of reaction 'Prostatic specific antigen abnormal'? | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | Recovering | ReactionOutcome | CC BY | 33294761 | 18,656,149 | 2021-01-01 |
What was the outcome of reaction 'Prostatic specific antigen increased'? | Congenital Adrenal Hyperplasia Causing Poor Response to Androgen Deprivation Therapy in Prostate Cancer.
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
Androgen deprivation therapy (ADT) is a component of the standard treatment for men with regionally localized high-risk or metastatic prostate cancer [1]. ADT is associated with delayed disease progression and survival benefit [2-4].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase enzyme deficiency is an autosomal recessive disorder affecting the adrenal cortex. Impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol leads to impaired cortisol synthesis. The lack of negative feedback in the hypothalamic-pituitary-adrenal axis due to cortisol deficiency leads to increased adrenocorticotropic hormone (ACTH) levels, which in turn results in stimulation and hyperplasia of the adrenal cortex. The diversion of cortisol precursors to adrenal androgens can cause virilization in affected girls [5, 6]. The severity of the disease correlates closely with the degree of enzyme dysfunction. Simple virilizing form of CAH usually has approximately 1% to 2% of preserved 21-hydroxylase enzyme function and without newborn screening programs can be unrecognized until rapid growth and accelerated skeletal maturation is observed in later childhood, leading to compromised adult stature. Nonclassic (late-onset) CAH (NCCAH) is a less severe form of the disorder with about 5% to 20% of 21-hydroxylase enzyme activity. The degree of hyperandrogenemia is more moderate than that seen in patients with classic CAH. Although usually asymptomatic, NCCAH can present later in life with signs of androgen excess. Androgen excess in males can lead to premature puberty, acne, advanced bone age, short stature, and infertility.
Although the prevalence of classic CAH is rare, with a worldwide incidence of 1 in 14 000 to 18 000 births, NCCAH is one of the most common autosomal recessive diseases reported in 1 in 1000 individuals in the general White population [7], with even higher prevalence among certain ethnic groups [8, 9].
We describe 2 cases of patients diagnosed with CAH following inadequate suppression of testosterone with ADT for prostate cancer.
Case Presentation
Patient 1
We present a 73-year-old man with prostate adenocarcinoma (pT2CN0M0; Gleason score 4 + 5 = 9) who underwent radical prostatectomy on initial diagnosis. He was later treated with ADT following local recurrence of the tumor with involvement of pelvic lymph nodes. Despite treatment with leuprolide, a gonadotropin-releasing hormone (GnRH) analog, he continued to have an elevated testosterone level of 294.4 ng/dL and an elevated prostate-specific antigen (PSA) level of 17.7 ng/mL. He was also noted to have incidental adrenal hyperplasia on a computed tomography (CT) imaging performed for prostate cancer staging (Fig. 1). He was referred to the Endocrinology Clinic at Orlando VA Medical Center for evaluation of persistent elevation of testosterone despite treatment with leuprolide. Given concerns about overproduction of adrenal androgens, the levels of 17-hydroxyprogesterone and dehydroepiandrosterone sulfate (DHEA-S) were measured and noted to be elevated at 10917 ng/dL (reference range, 28-250 ng/dL) and 378 mcg/dL (reference range, 5-253 mcg/dL), respectively. Consequently, an ACTH stimulation test showed significant elevation in 17-hydroxyprogesterone at baseline (19 910 ng/dL) with increase to greater than 20 000 ng/dL at 30 and 60 minutes (Table 1). The cortisol level, however, remained unchanged at 5 mcg/dL at 30 and 60 minutes, consistent with clinically occult adrenal insufficiency. Genetic testing showed biallelic mutations of the CYP21A2 gene. The first mutation was located in intron 2 (c.293-13A/C > G), usually associated with simple virilizing or salt-wasting phenotypes of classic CAH. The second mutation was detected in the I172N sequence (c.518T > A), commonly associated with the simple virilizing phenotype. The patient had no salt-wasting features. He reported a history of infertility but no premature puberty or short stature. Based on the clinical and biochemical findings as well as genotype-phenotype association, he was diagnosed with simple virilizing CAH. The patient was treated with dexamethasone (1 mg daily) and had marked decrease in adrenal androgens, testosterone, and PSA levels (Fig. 2). He was later switched to a maintenance dose of prednisone (3 mg daily). A year after initiation of glucocorticoid therapy, he continued to have adequate control of his prostate cancer with no signs of biochemical or radiographic progression.
Figure 1. Cross-sectional abdominal computed tomography shows bilateral adrenal nodular hyperplasia (arrows) in Patient 1 with simple virilizing congenital adrenal hyperplasia.
Table 1. ACTH stimulation test results in patient 1 with simple virilizing congenital adrenal hyperplasia
Test Baseline 30 min after ACTH stimulation 60 min after ACTH stimulation Reference range
17-hydroxypregnenolone 1101 1701 1998 < 700 ng/dL
DHEA 449 169 1384 147-1760 mcg/dL
Progesterone 8.0 22.3 17.7 < 0.4 ng/mL
17-hydroxyprogesterone 19 910 > 20 000 > 20 000 28-250 ng/dL
Androstenedione 1659 1631 1782 23-125 ng/dL
Deoxycorticosterone < 16 < 16 < 16 < 15 ng/dL
11-deoxycortisol 62 53 54 < 110 ng/dL
Testosterone 284 281 302 190-928 ng/dL
Cortisol 5.2 4.8 5.3 2.5-22.0 ug/dL
Abbreviations: ACTH, adrenocorticotropin; DHEA, dehydroepiandrosterone.
Figure 2. Downtrend of testosterone and prostate-specific antigen (PSA) levels following initiation of glucocorticoid therapy in patient 1 with simple virilizing congenital adrenal hyperplasia.
Patient 2
The second patient is an 82-year-old man with prostate adenocarcinoma (T1cNxMx, Gleason score 4 + 3 = 7) diagnosed 5 years before presentation. He initially pursued active surveillance. On surveillance, his PSA reached 14.2 ng/mL with a testosterone level of 239 ng/dL. He was treated with stereotactic body radiation therapy to the prostate in combination with leuprolide as ADT as definitive therapy in the absence of radiographic evidence of metastatic disease. However, his testosterone level remained inappropriately elevated at 87 ng/dL despite treatment with leuprolide (goal testosterone < 5 mg/dL). Consequently, the androgen receptor inhibitor bicalutamide (50 mg daily) was added to his treatment with ongoing ADT. He received 7 months of treatment with leuprolide and 4 months of combined androgen signaling inhibition with leuprolide and bicalutamide. His PSA nadir was 0.29 ng/mL, but his testosterone level remained relatively unchanged at 88.4 ng/dL. After completion of treatment with combined androgen signaling inhibition, his PSA rose to 10.86 ng/dL and testosterone to 218 ng/dL. He underwent a positron emission tomography/CT scan (PET/CT), which showed bilateral posterior iliac, right sacral, thoracic, and lumbar spine metastases as well as incidental bilateral nodular adrenal enlargement (Fig. 3). ADT was resumed with the GnRH antagonist degarelix. However, there was no improvement in his PSA (11.8 ng/mL), and testosterone remained well above castrate level (119 ng/dL). He was referred to the Endocrinology Clinic at UT Southwestern Medical Center for evaluation of adrenal androgen overproduction. A review of the patient’s history revealed premature puberty, short stature, and infertility. Further laboratory workup revealed elevated 17-hydroxyprogesterone at 4910 ng/dL (reference range, < 200 ng/dL) and DHEA-S at 312 mcg/dL (reference, < 16.2 mcg/dL) (Table 2). Based on the clinical, radiographic, and biochemical findings, the patient was diagnosed with NCCAH. He was started on treatment with the CYP17A1 inhibitor, abiraterone acetate (1000 mg daily) in combination with glucocorticoid replacement with prednisone (2.5 mg twice daily). His testosterone decreased to undetectable levels and his PSA declined to 0.41 ng/mL. One year later, an F18-fluciclovine PET/CT demonstrated interval resolution of previously seen fluciclovine-avid bone lesions, representing response to treatment. Unfortunately, his most recent evaluation showed signs of cancer progression with 2 new bone metastases in the right seventh and ninth ribs despite treatment with leuprolide, degarelix, abiraterone acetate, and prednisone, consistent with treatment failure.
Table 2. Laboratory evaluation before and after treatment of CAH in patient 2 with nonclassic congenital adrenal hyperplasia
Test Pre treatment Post treatment Reference range
17-hydroxyprogesterone 4900 < 200 ng/dL
Androstenedione 317 40-180 ng/dL
ACTH 39 pg/mL 2.2 and 13.3 pmol/L
Cortisol 5.6 2.5-22.0 µg/dL
FSH < 1.0 2-7 mIU/mL
LH < 1.0 1.24-7.8 IU/L
DHEA-sulfate 312 < 16.2 mcg/dL
Aldosterone 4.0 2-9 ng/dL
Renin 3.5 2.5-45.1 pg/mL
Testosterone 117.6 < 5.0 Goal < 5.0 ng/dL
PSA 12.9 0.41 Goal < 5.0 ng/mL
Abbreviations: ACTH, adrenocorticotropin; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PSA, prostate-specific antigen.
Figure 3. Computed tomography identified enlarged (A) right adrenal gland and (B) left adrenal gland on the axial images (arrows) in patient 2 with nonclassic congenital adrenal hyperplasia.
Discussion
Our 2 patients were diagnosed with CAH at an advanced age as a result of inadequate response to ADT for prostate cancer. Based on their clinical history and biochemical findings, these patients were diagnosed with simple virilizing and NCCAH, respectively.
Clinical manifestations of CAH range from mild to severe, depending on the degree of 21-hydroxylase deficiency. Males with the classic simple virilizing form typically present with early virilization (pubic hair, growth spurt, adult body odor) at age 2 to 4 years. Although CAH is one of the most common inborn endocrine disorders, the diagnosis can be missed or delayed because of subtle clinical presentation, lack of clinical suspicion, and/or awareness of the diagnosis.
Several previous observations demonstrated that diagnosis of CAH is established in fewer males compared to females, with even more pronounced discrepancy in simple virilizing patients [10-13]. In a retrospective study of 484 patients with classic forms of CAH, males were diagnosed significantly later than females with both forms (salt wasting: 26 vs 13 days [median], P < .001; simple virilizing: 5.0 vs 2.8 years, P = .03) [11]. Estimated 2 to 2.5 salt-wasting and up to 5 simple virilizing patients remain undiagnosed out of 40 expected CAH patients per year in the countries investigated in the study [11]. Clinical detection and treatment of CAH in our 2 patients were insufficient because of absent newborn screening at the time of birth of both patients and lack of clinical suspicion in the setting-provided manifestations. Newborn screening for CAH as well as greater awareness of the medical community should improve the efficacy of CAH detection and management.
Genetic testing for patient 1 revealed heterozygous, missense mutations of I172N and intron 2G (I2G) parts of the CYP21A2 gene. According to a study of 1507 families with CAH, the frequency of I172N and I2G mutations are 8.2% and 22.9%, respectively [14]. The biallelic I2G/I172N mutation genotype is most prevalent in patients of European ethnicity (30/50 cases) and is predominantly associated with the simple virilizing phenotype (36/50 cases). Salt-wasting (13/50 cases) and nonclassic types (1/50 cases) were less common. These mutations result in reduced 21-hydroxylase enzyme activity to about 2% [14]. Based on the genotype-phenotype correlation, patient 1 likely had classic virilizing phenotype. He did not come to medical attention until later in his life and even then biochemical workup, rather than clinical history and clinical manifestations, prompted further workup and diagnosis. Although delineating between whether this patient has classic virilizing vs nonclassic phenotypes is inconsequential in this case, genetic testing does enrich our collective understanding of CAH. The utility of genetic testing is more paramount in younger patients for purposes of genetic counseling, fertility considerations, and for establishing diagnosis in equivocal cases [15, 16].
Nonclassic or late-onset 21-hydroxylase deficiency may present as early pubarche in school-age children, hirsutism and menstrual irregularity in young women, or there may be no symptoms. Accordingly, patient 2 with NCCAH reported early puberty and short stature. His baseline 17-hydroxyprogesterone level was moderately elevated. The diagnosis of NCCAH was made based on biochemical and clinical findings. The patient received prostate cancer treatment with combined androgen suppression with GnRH-targeted therapy plus the CYP17A1 inhibitor abiraterone in combination with prednisone. A similar case of persistent testosterone elevation despite ADT and also surgical castration was previously reported in the literature. That patient was ultimately diagnosed with NCCAH and successfully treated with hydrocortisone and prednisolone, resulting in the target castration serum testosterone level [17]. It is worth noting that our patients were born before newborn screening for CAH was introduced. Nowadays, most classic CAH cases are detected shortly after birth owing to newborn screening measures.
ADT with GnRH agonists or antagonists, or surgical castration, is recommended by the National Comprehensive Cancer Network and American Society of Clinical Oncology for treating patients with advanced prostate cancer [18]. Testosterone and PSA should both be monitored to assess for the effectiveness of ADT to suppress androgen production and cancer growth, respectively. Inadequate testosterone suppression by ADT impairs anticancer efficacy and warrants further evaluation. Several causes of persistent testosterone elevation have been described and should be considered prior to changing the treatment course. When testosterone remains elevated above castrate level (> 50 ng/dL), the first step is to repeat the test to account for possible laboratory error. Although chemiluminescent immunoassay is accurate at high levels of testosterone, it is less reliable at lower levels (< 50 ng/dL). Therefore, liquid chromatography–tandem mass spectroscopy is preferred in patients on ADT [19]. It is also recommended that the same laboratory and assay be used for testosterone monitoring to improve consistency and comparability of results [20]. The timing of the laboratory test is also important. A surge in testosterone is expected following the first injection of GnRH agonist due to temporary stimulation of the GnRH receptor. Subsequently, downregulation of testosterone production by the testicles occurs and the levels decline. Some patients may experience similar testosterone surges with readministration of GnRH agonists even after multiple injections (acute-on-chronic effect). Testosterone levels may also trend up at any point during treatment, a phenomenon known as “testosterone escape” or “breakthrough response” [18]. Therefore, it is important to take into consideration anticipated surges when interpreting the testosterone levels to avoid unwarranted changes in treatment regimen [18].
Another potential pitfall is incorrect preparation and administration of the medication, which can impair the efficacy of the medication. This can be addressed by switching the injection site and reviewing the injection procedure with the nursing staff [20]. Biodegradable lactic acid polymer microcapsules present in leuprolide injections can induce granulomatous skin reactions at the drug injection site. This has been proposed as another possible cause of hormonal escape [21]. Unfortunately, there is no treatment or preventive measure for injection-site reactions and alternative agents should be considered [22].
An uncommon case of gonadotropin-producing pituitary adenoma has been described as a cause of sustained testosterone production despite therapy with a GnRH agonist [23]. Insufficient response to GnRH agonists and higher prostate cancer mortality have also been correlated with obesity, but the mechanism is not clear [18, 24]. Molecular mechanisms involving the expression, splicing, and posttranslational modifications of the androgen receptor have been associated with resistance to ADT [25].
It is established that medical or surgical castration does not completely eliminate androgen levels and that intratumoral and adrenal androgens remain detectable [25]. While testosterone is the main circulating androgen, adrenal androgens like androstenedione and DHEA-S are also important contributors to androgen homeostasis [18]. Adrenal androgen levels are reduced by only 60% during ADT [25]. Moreover, virilizing adrenal syndromes can amplify the effects of adrenal androgens even further. Therefore, it is important to evaluate for undiagnosed adrenal etiologies of hyperandrogenemia. Based on our literature review, 2 additional cases of patients with prostate cancer with inadequate testosterone suppression despite ADT were attributed to underlying CAH [17, 26].
In patients with CAH, glucocorticoid therapy reinstates the negative feedback mechanism in the hypothalamic-pituitary-adrenal axis. The decrease in ACTH level leads to rapid decline in androstenedione and testosterone production [17]. The decrease in testosterone levels was accompanied by remarkable improvement in PSA levels in both cases presented here, though glucocorticoid therapy was used in combination with abiraterone in the treatment of the second patient. Abiraterone, a CYP17A1 inhibitor, can be used to inhibit adrenal androgen production in castration-resistant prostate cancer. By blocking CYP17A1 enzyme, abiraterone also inhibits cortisol production and can lead to mineralocorticoid excess. Therefore, abiraterone is used in conjunction with a physiologic dose of glucocorticoids to replace cortisol deficiency and prevent further escalation of ACTH stimulation and mineralocorticoid toxicity [17, 27, 28].
In conclusion, it is important to monitor serum testosterone levels in patients receiving ADT for prostate cancer and to evaluate for virilizing disorders such as milder forms of CAH in patients who show inadequate decline in androgen levels despite ADT.
Acknowledgments
Author Contributions: All authors made substantial contributions through drafting of the manuscript or revisions, and all authors read and approved the final manuscript.
Abbreviations
ACTH adrenocorticotropic hormone
ADT androgen deprivation therapy
CAH congenital adrenal hyperplasia
CT computed tomography
DHEA-S dehydroepiandrosterone sulfate
FSH follicle-stimulating hormone
GnRH gonadotropin-releasing hormone
I2G intron 2G
NCCAH nonclassic congenital adrenal hyperplasia
LH luteinizing hormone
LHRH luteinizing hormone-releasing hormone
PET/CT positron emission tomography/computed tomography
PSA prostate-specific antigen
Additional Information
Disclosure Summary: O.H. reports research collaboration with Mayo Clinic and advisory board participation with Corcept Therapeutics and Pfizer outside the submitted work. The remaining authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. | Recovered | ReactionOutcome | CC BY | 33294761 | 18,916,528 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hyperglycaemia'. | Pasireotide: A Novel Treatment for Tumor-Induced Hypoglycemia Due to Insulinoma and Non-Islet Cell Tumor Hypoglycemia.
Tumor-induced hypoglycemia is a serious disorder most commonly caused by insulinoma or non-islet cell tumor hypoglycemia (NICTH). The hypoglycemia can be severe and refractory to conventional therapy, leading to significant morbidity and mortality. The objective of this work is to describe a series of challenging cases in which refractory, tumor-induced hypoglycemia was shown to respond to the use of pasireotide, a second-generation somatostatin receptor ligand. We describe the clinical and biochemical features of 3 patients with tumor-induced hypoglycemia due to an occult insulinoma, malignant insulinoma, and non-islet cell tumor hypoglycemia. In these 3 individuals, the hypoglycemia remained refractory to guideline-recommended medical therapy, such as diazoxide, nonpasireotide somatostatin analogues, and glucocorticoids. Pasireotide was substituted to attenuate the refractory hypoglycemia for each patient. The addition of pasireotide led to prompt improvement in the frequency and severity of hypoglycemic episodes for each tumor-induced hypoglycemia patient. We demonstrate the successful treatment of 3 individuals with refractory, tumor-induced hypoglycemia with pasireotide. We offer the first reported use of pasireotide for the successful treatment of nonmalignant insulinoma and non-islet cell tumor hypoglycemia.
Hypoglycemia in individuals without diabetes is uncommon and warrants further investigation if the Whipple triad is fulfilled. Insulinoma and non-islet cell tumor hypoglycemia (NICTH) are rare causes of hypoglycemia. They may present with refractory hypoglycemia, and in severe cases, can cause irreversible neurocognitive impairment and death. The medical management of refractory hypoglycemia is challenging because there are limited therapeutic options available with unpredictable response and significant adverse effects [1].
The most common cause of hypoglycemia due to endogenous hyperinsulinism is insulinoma, which is generally single and benign [2]. Malignant insulinomas are rare, comprising only 5.8% of all insulinomas [3]. Malignant insulinomas have a poor prognosis, with a 10-year survival of less than 20%, and present with distant metastatic involvement, predominately to the liver and regional lymph nodes [4]. The definitive treatment of solitary insulinomas is surgical, though medical therapy has a role in patients who are poor surgical candidates or who decide against surgery. In cases of malignant insulinomas, however, surgery is not curative; thus, medical therapy has an important role in controlling symptomatic hypoglycemia and reducing tumor burden. Medications indicated for alleviating hypoglycemia in malignant insulinoma include diazoxide, glucocorticoids, and somatostatin analogues, which have shown variable responses [1, 5].
NICTH is a rare paraneoplastic syndrome associated with tumors of epithelial and mesenchymal origin. It is the second-most common cause of tumor-induced hypoglycemia after insulinoma and is most commonly seen in hepatocellular and adrenocortical carcinoma [6]. NICTH is caused by tumor overexpression of insulin-like growth factor (IGF)-2: both mature IGF-2 and incompletely processed “big IGF-2,” which promotes hypoglycemia due to insulin-like effects [7]. NICTH should be considered in the presence of hypoinsulinemic hypoglycemia with low growth hormone (GH) and IGF-1 levels, and an IGF-2:IGF-1 ratio greater than 10. There are no commercially available assays for big IGF-2 [8]. Definitive treatment of NICTH involves complete tumor resection. If resection is not feasible, local antitumor therapies are generally pursued, with a trial of glucocorticoids and/or recombinant human GH for refractory hypoglycemia [9].
We report 3 cases of refractory hypoglycemia due to occult insulinoma, malignant insulinoma, and NICTH that were successfully treated with pasireotide, a second-generation somatostatin receptor ligand. Although not formally approved for use in hypoglycemia, pasireotide has unique features that make it an appealing choice for refractory, tumor-induced hypoglycemia.
Case 1
Pasireotide for the Treatment of Insulinoma in a Poor Surgical Candidate
An 80-year-old woman was referred to our endocrine clinic for recurrent hypoglycemic episodes. She reported a 9-year history of spells of dizziness, tremors, and diaphoresis that improved with eating peanut butter. When symptomatic, the blood glucose levels measured in her assisted living facility were approximately 50 mg/dL. These episodes occurred both in the fasting and postprandial states and were increasing in frequency. Additionally, the patient had gained 12 pounds in the last 6 months.
The patient was admitted to the hospital for a 72-hour fast, which revealed endogenous hyperinsulinism. When the patient’s blood glucose measured 43 mg/dL, she had an insulin level of 47 (normal, 2.0-19.6 µIU/mL), C-peptide of 8.2 (normal, 0.80-3.85 ng/mL), proinsulin level of 27.9 (normal, ≤ 18.8 pmol/L), and negative sulfonylurea screen and insulin antibody. Computed tomography (CT) of the abdomen and pelvis showed a subtle 3-mm nodular focus of arterial enhancement within the pancreatic head, suspicious for a small insulinoma, but endoscopic ultrasound failed to localize the tumor. 68Ga DOTATATE positron emission tomography/CT did not reveal any abnormal uptake concerning for neuroendocrine tumor, and a selective arterial calcium stimulation test failed to localize the patient’s insulinoma as well.
The patient opted for medical management of her insulinoma as surgery was considered high risk because of her age and significant cardiovascular history. She could not tolerate diazoxide because of edema and was transitioned to short-acting octreotide without improvement in her hypoglycemic episodes. The patient was switched to octreotide long-acting release (LAR) 20 mg every 4 weeks, which was continued for 3 months. During this time, she required 2 hospitalizations for severe hypoglycemia.
Given the patient’s inadequate response to octreotide LAR, pasireotide LAR 40 mg intramuscularly (IM) every 4 weeks was initiated, which completely resolved the hypoglycemic episodes within 1 month, with average blood glucose of 200 mg/dL. However, owing to some postural lightheadedness, her dose was decreased to 20 mg IM every 4 weeks. Approximately 1 year later, the patient developed persistent hyperglycemia, with a mean blood glucose of 180 mg/dL and glycated hemoglobin (HbA1c) of 8% (64 mmol/mol). At this time, definitive treatment of her insulinoma was discussed (eg, repeat selective arterial calcium stimulation test followed by surgery), but the patient decided to continue medical management citing concerns with COVID-19 infection risk with an elective radiologic procedure followed by hospitalization for tumor resection. As such, the patient’s pasireotide dose was lowered to 10 mg every 4 weeks, and she has maintained glucose levels in the 90- to 120-mg/dL range without any further episodes of hypoglycemia or hyperglycemia.
Case 2
Pasireotide for the Treatment of Malignant Insulinoma
We have published the preliminary details of this case previously [10]. A 53-year-old woman presented with a 2-year history of recurrent hypoglycemia that fulfilled the Whipple triad. Within the first hour of a 72-hour fast, she was found to have a blood glucose of 53 mg/dL, insulin level of 87 mIU/mL (normal, 2.6-24.9 µIU/mL), C-peptide of 13.2 ng/mL (normal, 1.1-4.4ng/mL), and proinsulin of 2822 pmol/L (normal, 3-20 pmol/L), confirming endogenous hyperinsulinism. She was found to have a 3.6 × 2.8 × 2.4-cm pancreatic tail mass with innumerable masses in the liver measuring up to 10 cm. Core biopsy of the hepatic lesions revealed a well-differentiated neuroendocrine tumor (World Health Organization grade 2, Ki-67 4%), and she was diagnosed with stage IV malignant insulinoma with liver metastases.
Consensus from our institutional tumor board was to manage the patient medically given her extensive tumor burden. She was initiated on diazoxide and octreotide LAR 30 mg IM every 4 weeks but continued to have persistent hypoglycemia (30% of recorded glucose levels were < 70 mg/dL and 5% < 55 mg/dL) on her Dexcom G4 Platinum continuous glucose monitor (CGM) (Fig. 1A). As such, she was switched to pasireotide LAR 60 mg IM every 4 weeks with marked improvement in her hypoglycemic episodes within 1 month: Only 3% of recorded readings were below 70 mg/dL with no serious hypoglycemia (≤ 55 mg/dL), with mean sensor glucose of 129 mg/dL (Fig. 1B).
Figure 1. Dexcom G4 Platinum continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 30% to 3% of recorded values, respectively.
The following month, the patient started chemotherapy with capecitabine and temozolomide followed by transarterial chemoembolization of her hepatic metastases. Two months after chemoembolization, the patient developed hyperglycemia, so pasireotide was discontinued. Subsequently, at her 3-month follow-up, the patient was no longer experiencing any hypoglycemic episodes and her average blood glucose was 122 mg/dL. She was started on lanreotide 120 mg IM every 4 weeks for antineoplastic effect and continued on the single chemotherapeutic agent capecitabine by the oncology service.
Two years later, the patient developed type 2 diabetes mellitus with an HbA1c of 9.2% (75 mmol/mol). She was started on empagliflozin and 4 months later, her HbA1c improved to 7.5% (53 mmol/mol). The patient has no evidence of tumor progression 4 years from her initial diagnosis while on capecitabine plus lanreotide therapy, with stable radiographic appearance of her hepatic metastases.
Case 3
Pasireotide for the Treatment of Non-Islet Cell Tumor Hypoglycemia
A 72-year-old man with history of cirrhosis from hepatitis C and recently diagnosed hepatocellular carcinoma (HCC) presented to our institution with refractory hypoglycemia. The patient’s HCC was diagnosed 2 months prior to admission in the setting of severe hypoglycemia and seizure. The patient noted lightheadedness and diaphoresis occurring at 2-hour intervals during the day and night with fingerstick glucose readings of approximately 40 mg/dL during these episodes. His symptoms resolved on consuming carbohydrates. The patient was not taking any medications associated with glucose-lowering and denied alcohol consumption. He had normal renal and hepatic function. A download of his Dexcom G6 CGM revealed that 48% of his recorded blood glucose readings were below 70 mg/dL with multiple episodes (28%) of severe nocturnal hypoglycemia with blood glucose below 54 mg/dL (Fig. 2A).
Figure 2. Dexcom G6 continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 48% to 8% of recorded values, respectively.
A 72-hour fast was initiated, and within 1 hour, the patient had a blood glucose measurement of 30 mg/dL, with an insulin level of 2.2 mcIU/mL (normal, 2.6-24.9 mcIU/mL), C-peptide of 0.17 ng/mL (normal, 0.80-3.85 ng/mL), β-hydroxybutyrate less than 0.1 mmol/L (normal, 0.0-0.3 mmol/L), and proinsulin less than 0.4 pmol/L (normal, ≤ 18.8pmol/L). The patient had a negative insulin antibody (< 0.4 U/mL) and negative serum hypoglycemic agent screen. Further evaluation of his noninsulin-mediated hypoglycemia excluded adrenal insufficiency via cosyntropin stimulation testing. His GH level was 0.06 ng/mL (normal, 0.01-0.97 ng/mL), IGF-1 was less than 10 ng/mL (normal, 32-200 ng/ml), and IGF-2 level was 780 ng/mL (normal, 333-967 ng/mL). Owing to the patient’s hypoinsulinemic hypoglycemia with low IGF-1 level and an IGF-2:IGF-1 ratio greater than 10, the patient was diagnosed with NICTH.
CT abdomen/pelvis revealed a cirrhotic liver with a 7-cm lesion involving the right hepatic lobe; biopsy confirmed the diagnosis of well-differentiated hepatocellular carcinoma. The tumor was unresectable because of portal vein thrombosis, so the patient was started on an immunotherapy clinical trial with pembrolizumab and bavituximab. He was not a candidate for yttrium-90 radioembolization because of an anterioportal shunt that could not be adequately embolized, so he received palliative external beam radiotherapy. However, the patient continued to experience refractory hypoglycemic episodes, which necessitated hourly waking by his family to encourage him to eat carbohydrates, despite the addition of prednisone 30 mg daily, 1 month after the diagnosis of HCC. He was readmitted to the hospital for glucose optimization.
During hospitalization, the patient required a titratable dextrose 50% infusion, 37.5 g of dextrose gel orally every 3 hours, and frequent meals for management of his hypoglycemia. He was started on combination medical therapy with diazoxide, prednisolone 20 mg twice daily, and short-acting octreotide 100 mg subcutaneously every 8 hours. Despite that, the patient remained in the hospital for 3 weeks, as he promptly developed hypoglycemia when the dextrose infusion was discontinued. Pasireotide LAR was ordered for the patient but could not be given inpatient, so he was discharged to a long-term assisted care facility while remaining on the dextrose infusion.
Shortly after discharge, the patient was started on pasireotide 40 mg IM every 4 weeks. Within 1 week, he was weaned off the dextrose infusion. Three weeks later, at his endocrine follow-up appointment, his Dexcom G6 CGM data revealed markedly improved glycemic control, with a mean sensor glucose of 121 mg/dL, 8% of readings below 70 mg/dL, and only 2% of readings below 54 mg/dL (Fig. 2B). Unfortunately, the patient’s HCC progressed despite the use of sorafenib and later nivolumab, and he died of shock and decompensated liver failure 5 months after his initial HCC diagnosis.
Discussion
Medications that are currently approved for refractory, tumor-induced hypoglycemia are generally of limited efficacy and tolerability [5]. In the cases described here, each patient with tumor-induced hypoglycemia was started on guideline-recommended medications for the treatment of hypoglycemia (ie, diazoxide, glucocorticoids, and/or octreotide) without attenuation of their refractory hypoglycemia. Then, pasireotide was substituted with marked improvement of hypoglycemia in all cases within 1 month of administration.
Pasireotide, like other somatostatin analogues, exerts its biologic effect by binding to somatostatin receptors (SSTRs). There are 5 somatostatin receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) distributed heterogeneously throughout the body. Once stimulated, somatostatin is a potent inhibitor of endocrine and exocrine hormonal release in humans [11]. Inhibition of insulin and glucagon secretion is primarily mediated by SSTR2, whereas insulin secretion is predominantly mediated via SSTR5 [12]. Neuroendocrine tumors largely express somatostatin receptors, with SSTR2 and SSTR5 expression shown in 70% of insulinomas [13]. Pasireotide, a second-generation SSTR ligand, has a 30- to 40-fold higher binding affinity for SSTR5 than the first-generation SSTR ligand octreotide, which accounts for the former’s advantage in treating hypoglycemia [14]. The hyperglycemia effect of pasireotide is related to a decrease in insulin secretion and incretin hormone response without change to hepatic or peripheral insulin sensitivity [15]. Although octreotide has been shown to improve hypoglycemia in two-thirds of insulinoma patients in one study [16], there are insufficient data for the use of pasireotide in insulinoma patients to calculate a treatment response. However, pasireotide induces a potent hyperglycemic effect in its currently indicated uses for Cushing disease and acromegaly: Seventy-three percent of Cushing disease patients [17] and 57% of acromegaly patients [18] developed hyperglycemia-related adverse events while on pasireotide. Pasireotide-induced hyperglycemia has been shown to respond to vildagliptin and liraglutide therapy [19].
Additionally, pasireotide has been shown to have antiproliferative effects comparable to the more conventionally used somatostatin analogues octreotide and lanreotide in the treatment of advanced neuroendocrine tumors [20].
Treatment of NICTH involves tumor resection or the use of alternate antitumor modalities when resection is not possible. While the use of pasireotide in the management of NICTH has not been previously described, high-dosed octreotide given as continuous infusion following uptake of 111In-labeled octreotide by a solitary fibrous, pleural tumor was not effective in suppressing big IGF-2 production or improving hypoglycemia [21]. In a separate case, the mechanism of refractory hypoglycemia due to NICTH from an intra-abdominal hemagiopericytoma was attributed to muscle tissue uptake of glucose mediated by IGF-2; during somatostatin treatment, big IGF-2 levels decreased modestly but could not adequately control hypoglycemia without the simultaneous infusion of exogenous glucose [22]. The effect of pasireotide therapy on big IGF-2 levels has not been described previously, though a potent lowering of big IGF-2 levels, potentially through SSTR5 interaction, could account for the hyperglycemic effect noted in our patient with hepatocellular carcinoma. Hepatocellular carcinoma tumor cells have been shown previously to express a high proportion of SSTR5 [23]. However, the treatment of advanced hepatocellular carcinoma with somatostatin analogues has shown variable response [24]. A recent phase 2 trial of pasireotide LAR in patients with unresectable HCC showed limited antitumor benefit; however, 30% of participants developed grade 3 hyperglycemia, and 5% of participants developed grade 4 hyperglycemia [25].
Despite the presumed advantage of pasireotide in treating tumor-induced hypoglycemia, only a single case, besides our own, has been published using pasireotide for the treatment of malignant insulinoma. Tirosh et al found that pasireotide LAR was more effective in treating refractory hypoglycemia compared with lanreotide in a patient with malignant insulinoma with hepatic metastases [26]. This manuscript adds to the literature by outlining 3 cases of refractory, tumor-induced hypoglycemia—due to an occult insulinoma, malignant insulinoma, and NICTH from HCC—that all failed to respond to conventional medical therapy, and for which pasireotide caused rapid, dramatic improvement in glucose levels (resolving the hypoglycemia completely in the first 2 cases). Two of our cases—pasireotide for treating hypoglycemia in occult insulinoma and NICTH—are not previously reported in the literature.
In conclusion, we recommend that pasireotide be considered for the treatment of tumor-induced hypoglycemia in 3 settings. First, in a patient with insulinoma who is either not a surgical candidate or who decides against surgery, and in whom hypoglycemia persists despite conventional medical therapy. Second, in the patient with NICTH for whom tumor resection is not possible and adjunctive medical therapy is unhelpful. Third, during the COVID-19 pandemic, there is added concern on the part of providers and patients, both from an infectious disease and resource use standpoint, in terms of admitting tumor-induced hypoglycemia patients for the purposes of diagnosis, localization, and/or surgical management. In such case, there may be a role for temporary pasireotide therapy as a “bridging” technique until definitive diagnostic and therapeutic strategies can be implemented. In summary, given its potent antihypoglycemic and antitumor properties, pasireotide is a reasonable choice for the treatment of tumor-induced hypoglycemia, though additional investigation is warranted.
Abbreviations
COVID-19 2019 novel coronavirus
GH growth hormone
HbA1c glycated hemoglobin
HCC hepatocellular carcinoma
IGF insulin-like growth factor
LAR long-acting release
NICTH non-islet cell tumor hypoglycemia
SSTR somatostatin receptor
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | CAPECITABINE, PASIREOTIDE, TEMOZOLOMIDE | DrugsGivenReaction | CC BY-NC-ND | 33294765 | 20,443,328 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Product use in unapproved indication'. | Pasireotide: A Novel Treatment for Tumor-Induced Hypoglycemia Due to Insulinoma and Non-Islet Cell Tumor Hypoglycemia.
Tumor-induced hypoglycemia is a serious disorder most commonly caused by insulinoma or non-islet cell tumor hypoglycemia (NICTH). The hypoglycemia can be severe and refractory to conventional therapy, leading to significant morbidity and mortality. The objective of this work is to describe a series of challenging cases in which refractory, tumor-induced hypoglycemia was shown to respond to the use of pasireotide, a second-generation somatostatin receptor ligand. We describe the clinical and biochemical features of 3 patients with tumor-induced hypoglycemia due to an occult insulinoma, malignant insulinoma, and non-islet cell tumor hypoglycemia. In these 3 individuals, the hypoglycemia remained refractory to guideline-recommended medical therapy, such as diazoxide, nonpasireotide somatostatin analogues, and glucocorticoids. Pasireotide was substituted to attenuate the refractory hypoglycemia for each patient. The addition of pasireotide led to prompt improvement in the frequency and severity of hypoglycemic episodes for each tumor-induced hypoglycemia patient. We demonstrate the successful treatment of 3 individuals with refractory, tumor-induced hypoglycemia with pasireotide. We offer the first reported use of pasireotide for the successful treatment of nonmalignant insulinoma and non-islet cell tumor hypoglycemia.
Hypoglycemia in individuals without diabetes is uncommon and warrants further investigation if the Whipple triad is fulfilled. Insulinoma and non-islet cell tumor hypoglycemia (NICTH) are rare causes of hypoglycemia. They may present with refractory hypoglycemia, and in severe cases, can cause irreversible neurocognitive impairment and death. The medical management of refractory hypoglycemia is challenging because there are limited therapeutic options available with unpredictable response and significant adverse effects [1].
The most common cause of hypoglycemia due to endogenous hyperinsulinism is insulinoma, which is generally single and benign [2]. Malignant insulinomas are rare, comprising only 5.8% of all insulinomas [3]. Malignant insulinomas have a poor prognosis, with a 10-year survival of less than 20%, and present with distant metastatic involvement, predominately to the liver and regional lymph nodes [4]. The definitive treatment of solitary insulinomas is surgical, though medical therapy has a role in patients who are poor surgical candidates or who decide against surgery. In cases of malignant insulinomas, however, surgery is not curative; thus, medical therapy has an important role in controlling symptomatic hypoglycemia and reducing tumor burden. Medications indicated for alleviating hypoglycemia in malignant insulinoma include diazoxide, glucocorticoids, and somatostatin analogues, which have shown variable responses [1, 5].
NICTH is a rare paraneoplastic syndrome associated with tumors of epithelial and mesenchymal origin. It is the second-most common cause of tumor-induced hypoglycemia after insulinoma and is most commonly seen in hepatocellular and adrenocortical carcinoma [6]. NICTH is caused by tumor overexpression of insulin-like growth factor (IGF)-2: both mature IGF-2 and incompletely processed “big IGF-2,” which promotes hypoglycemia due to insulin-like effects [7]. NICTH should be considered in the presence of hypoinsulinemic hypoglycemia with low growth hormone (GH) and IGF-1 levels, and an IGF-2:IGF-1 ratio greater than 10. There are no commercially available assays for big IGF-2 [8]. Definitive treatment of NICTH involves complete tumor resection. If resection is not feasible, local antitumor therapies are generally pursued, with a trial of glucocorticoids and/or recombinant human GH for refractory hypoglycemia [9].
We report 3 cases of refractory hypoglycemia due to occult insulinoma, malignant insulinoma, and NICTH that were successfully treated with pasireotide, a second-generation somatostatin receptor ligand. Although not formally approved for use in hypoglycemia, pasireotide has unique features that make it an appealing choice for refractory, tumor-induced hypoglycemia.
Case 1
Pasireotide for the Treatment of Insulinoma in a Poor Surgical Candidate
An 80-year-old woman was referred to our endocrine clinic for recurrent hypoglycemic episodes. She reported a 9-year history of spells of dizziness, tremors, and diaphoresis that improved with eating peanut butter. When symptomatic, the blood glucose levels measured in her assisted living facility were approximately 50 mg/dL. These episodes occurred both in the fasting and postprandial states and were increasing in frequency. Additionally, the patient had gained 12 pounds in the last 6 months.
The patient was admitted to the hospital for a 72-hour fast, which revealed endogenous hyperinsulinism. When the patient’s blood glucose measured 43 mg/dL, she had an insulin level of 47 (normal, 2.0-19.6 µIU/mL), C-peptide of 8.2 (normal, 0.80-3.85 ng/mL), proinsulin level of 27.9 (normal, ≤ 18.8 pmol/L), and negative sulfonylurea screen and insulin antibody. Computed tomography (CT) of the abdomen and pelvis showed a subtle 3-mm nodular focus of arterial enhancement within the pancreatic head, suspicious for a small insulinoma, but endoscopic ultrasound failed to localize the tumor. 68Ga DOTATATE positron emission tomography/CT did not reveal any abnormal uptake concerning for neuroendocrine tumor, and a selective arterial calcium stimulation test failed to localize the patient’s insulinoma as well.
The patient opted for medical management of her insulinoma as surgery was considered high risk because of her age and significant cardiovascular history. She could not tolerate diazoxide because of edema and was transitioned to short-acting octreotide without improvement in her hypoglycemic episodes. The patient was switched to octreotide long-acting release (LAR) 20 mg every 4 weeks, which was continued for 3 months. During this time, she required 2 hospitalizations for severe hypoglycemia.
Given the patient’s inadequate response to octreotide LAR, pasireotide LAR 40 mg intramuscularly (IM) every 4 weeks was initiated, which completely resolved the hypoglycemic episodes within 1 month, with average blood glucose of 200 mg/dL. However, owing to some postural lightheadedness, her dose was decreased to 20 mg IM every 4 weeks. Approximately 1 year later, the patient developed persistent hyperglycemia, with a mean blood glucose of 180 mg/dL and glycated hemoglobin (HbA1c) of 8% (64 mmol/mol). At this time, definitive treatment of her insulinoma was discussed (eg, repeat selective arterial calcium stimulation test followed by surgery), but the patient decided to continue medical management citing concerns with COVID-19 infection risk with an elective radiologic procedure followed by hospitalization for tumor resection. As such, the patient’s pasireotide dose was lowered to 10 mg every 4 weeks, and she has maintained glucose levels in the 90- to 120-mg/dL range without any further episodes of hypoglycemia or hyperglycemia.
Case 2
Pasireotide for the Treatment of Malignant Insulinoma
We have published the preliminary details of this case previously [10]. A 53-year-old woman presented with a 2-year history of recurrent hypoglycemia that fulfilled the Whipple triad. Within the first hour of a 72-hour fast, she was found to have a blood glucose of 53 mg/dL, insulin level of 87 mIU/mL (normal, 2.6-24.9 µIU/mL), C-peptide of 13.2 ng/mL (normal, 1.1-4.4ng/mL), and proinsulin of 2822 pmol/L (normal, 3-20 pmol/L), confirming endogenous hyperinsulinism. She was found to have a 3.6 × 2.8 × 2.4-cm pancreatic tail mass with innumerable masses in the liver measuring up to 10 cm. Core biopsy of the hepatic lesions revealed a well-differentiated neuroendocrine tumor (World Health Organization grade 2, Ki-67 4%), and she was diagnosed with stage IV malignant insulinoma with liver metastases.
Consensus from our institutional tumor board was to manage the patient medically given her extensive tumor burden. She was initiated on diazoxide and octreotide LAR 30 mg IM every 4 weeks but continued to have persistent hypoglycemia (30% of recorded glucose levels were < 70 mg/dL and 5% < 55 mg/dL) on her Dexcom G4 Platinum continuous glucose monitor (CGM) (Fig. 1A). As such, she was switched to pasireotide LAR 60 mg IM every 4 weeks with marked improvement in her hypoglycemic episodes within 1 month: Only 3% of recorded readings were below 70 mg/dL with no serious hypoglycemia (≤ 55 mg/dL), with mean sensor glucose of 129 mg/dL (Fig. 1B).
Figure 1. Dexcom G4 Platinum continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 30% to 3% of recorded values, respectively.
The following month, the patient started chemotherapy with capecitabine and temozolomide followed by transarterial chemoembolization of her hepatic metastases. Two months after chemoembolization, the patient developed hyperglycemia, so pasireotide was discontinued. Subsequently, at her 3-month follow-up, the patient was no longer experiencing any hypoglycemic episodes and her average blood glucose was 122 mg/dL. She was started on lanreotide 120 mg IM every 4 weeks for antineoplastic effect and continued on the single chemotherapeutic agent capecitabine by the oncology service.
Two years later, the patient developed type 2 diabetes mellitus with an HbA1c of 9.2% (75 mmol/mol). She was started on empagliflozin and 4 months later, her HbA1c improved to 7.5% (53 mmol/mol). The patient has no evidence of tumor progression 4 years from her initial diagnosis while on capecitabine plus lanreotide therapy, with stable radiographic appearance of her hepatic metastases.
Case 3
Pasireotide for the Treatment of Non-Islet Cell Tumor Hypoglycemia
A 72-year-old man with history of cirrhosis from hepatitis C and recently diagnosed hepatocellular carcinoma (HCC) presented to our institution with refractory hypoglycemia. The patient’s HCC was diagnosed 2 months prior to admission in the setting of severe hypoglycemia and seizure. The patient noted lightheadedness and diaphoresis occurring at 2-hour intervals during the day and night with fingerstick glucose readings of approximately 40 mg/dL during these episodes. His symptoms resolved on consuming carbohydrates. The patient was not taking any medications associated with glucose-lowering and denied alcohol consumption. He had normal renal and hepatic function. A download of his Dexcom G6 CGM revealed that 48% of his recorded blood glucose readings were below 70 mg/dL with multiple episodes (28%) of severe nocturnal hypoglycemia with blood glucose below 54 mg/dL (Fig. 2A).
Figure 2. Dexcom G6 continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 48% to 8% of recorded values, respectively.
A 72-hour fast was initiated, and within 1 hour, the patient had a blood glucose measurement of 30 mg/dL, with an insulin level of 2.2 mcIU/mL (normal, 2.6-24.9 mcIU/mL), C-peptide of 0.17 ng/mL (normal, 0.80-3.85 ng/mL), β-hydroxybutyrate less than 0.1 mmol/L (normal, 0.0-0.3 mmol/L), and proinsulin less than 0.4 pmol/L (normal, ≤ 18.8pmol/L). The patient had a negative insulin antibody (< 0.4 U/mL) and negative serum hypoglycemic agent screen. Further evaluation of his noninsulin-mediated hypoglycemia excluded adrenal insufficiency via cosyntropin stimulation testing. His GH level was 0.06 ng/mL (normal, 0.01-0.97 ng/mL), IGF-1 was less than 10 ng/mL (normal, 32-200 ng/ml), and IGF-2 level was 780 ng/mL (normal, 333-967 ng/mL). Owing to the patient’s hypoinsulinemic hypoglycemia with low IGF-1 level and an IGF-2:IGF-1 ratio greater than 10, the patient was diagnosed with NICTH.
CT abdomen/pelvis revealed a cirrhotic liver with a 7-cm lesion involving the right hepatic lobe; biopsy confirmed the diagnosis of well-differentiated hepatocellular carcinoma. The tumor was unresectable because of portal vein thrombosis, so the patient was started on an immunotherapy clinical trial with pembrolizumab and bavituximab. He was not a candidate for yttrium-90 radioembolization because of an anterioportal shunt that could not be adequately embolized, so he received palliative external beam radiotherapy. However, the patient continued to experience refractory hypoglycemic episodes, which necessitated hourly waking by his family to encourage him to eat carbohydrates, despite the addition of prednisone 30 mg daily, 1 month after the diagnosis of HCC. He was readmitted to the hospital for glucose optimization.
During hospitalization, the patient required a titratable dextrose 50% infusion, 37.5 g of dextrose gel orally every 3 hours, and frequent meals for management of his hypoglycemia. He was started on combination medical therapy with diazoxide, prednisolone 20 mg twice daily, and short-acting octreotide 100 mg subcutaneously every 8 hours. Despite that, the patient remained in the hospital for 3 weeks, as he promptly developed hypoglycemia when the dextrose infusion was discontinued. Pasireotide LAR was ordered for the patient but could not be given inpatient, so he was discharged to a long-term assisted care facility while remaining on the dextrose infusion.
Shortly after discharge, the patient was started on pasireotide 40 mg IM every 4 weeks. Within 1 week, he was weaned off the dextrose infusion. Three weeks later, at his endocrine follow-up appointment, his Dexcom G6 CGM data revealed markedly improved glycemic control, with a mean sensor glucose of 121 mg/dL, 8% of readings below 70 mg/dL, and only 2% of readings below 54 mg/dL (Fig. 2B). Unfortunately, the patient’s HCC progressed despite the use of sorafenib and later nivolumab, and he died of shock and decompensated liver failure 5 months after his initial HCC diagnosis.
Discussion
Medications that are currently approved for refractory, tumor-induced hypoglycemia are generally of limited efficacy and tolerability [5]. In the cases described here, each patient with tumor-induced hypoglycemia was started on guideline-recommended medications for the treatment of hypoglycemia (ie, diazoxide, glucocorticoids, and/or octreotide) without attenuation of their refractory hypoglycemia. Then, pasireotide was substituted with marked improvement of hypoglycemia in all cases within 1 month of administration.
Pasireotide, like other somatostatin analogues, exerts its biologic effect by binding to somatostatin receptors (SSTRs). There are 5 somatostatin receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) distributed heterogeneously throughout the body. Once stimulated, somatostatin is a potent inhibitor of endocrine and exocrine hormonal release in humans [11]. Inhibition of insulin and glucagon secretion is primarily mediated by SSTR2, whereas insulin secretion is predominantly mediated via SSTR5 [12]. Neuroendocrine tumors largely express somatostatin receptors, with SSTR2 and SSTR5 expression shown in 70% of insulinomas [13]. Pasireotide, a second-generation SSTR ligand, has a 30- to 40-fold higher binding affinity for SSTR5 than the first-generation SSTR ligand octreotide, which accounts for the former’s advantage in treating hypoglycemia [14]. The hyperglycemia effect of pasireotide is related to a decrease in insulin secretion and incretin hormone response without change to hepatic or peripheral insulin sensitivity [15]. Although octreotide has been shown to improve hypoglycemia in two-thirds of insulinoma patients in one study [16], there are insufficient data for the use of pasireotide in insulinoma patients to calculate a treatment response. However, pasireotide induces a potent hyperglycemic effect in its currently indicated uses for Cushing disease and acromegaly: Seventy-three percent of Cushing disease patients [17] and 57% of acromegaly patients [18] developed hyperglycemia-related adverse events while on pasireotide. Pasireotide-induced hyperglycemia has been shown to respond to vildagliptin and liraglutide therapy [19].
Additionally, pasireotide has been shown to have antiproliferative effects comparable to the more conventionally used somatostatin analogues octreotide and lanreotide in the treatment of advanced neuroendocrine tumors [20].
Treatment of NICTH involves tumor resection or the use of alternate antitumor modalities when resection is not possible. While the use of pasireotide in the management of NICTH has not been previously described, high-dosed octreotide given as continuous infusion following uptake of 111In-labeled octreotide by a solitary fibrous, pleural tumor was not effective in suppressing big IGF-2 production or improving hypoglycemia [21]. In a separate case, the mechanism of refractory hypoglycemia due to NICTH from an intra-abdominal hemagiopericytoma was attributed to muscle tissue uptake of glucose mediated by IGF-2; during somatostatin treatment, big IGF-2 levels decreased modestly but could not adequately control hypoglycemia without the simultaneous infusion of exogenous glucose [22]. The effect of pasireotide therapy on big IGF-2 levels has not been described previously, though a potent lowering of big IGF-2 levels, potentially through SSTR5 interaction, could account for the hyperglycemic effect noted in our patient with hepatocellular carcinoma. Hepatocellular carcinoma tumor cells have been shown previously to express a high proportion of SSTR5 [23]. However, the treatment of advanced hepatocellular carcinoma with somatostatin analogues has shown variable response [24]. A recent phase 2 trial of pasireotide LAR in patients with unresectable HCC showed limited antitumor benefit; however, 30% of participants developed grade 3 hyperglycemia, and 5% of participants developed grade 4 hyperglycemia [25].
Despite the presumed advantage of pasireotide in treating tumor-induced hypoglycemia, only a single case, besides our own, has been published using pasireotide for the treatment of malignant insulinoma. Tirosh et al found that pasireotide LAR was more effective in treating refractory hypoglycemia compared with lanreotide in a patient with malignant insulinoma with hepatic metastases [26]. This manuscript adds to the literature by outlining 3 cases of refractory, tumor-induced hypoglycemia—due to an occult insulinoma, malignant insulinoma, and NICTH from HCC—that all failed to respond to conventional medical therapy, and for which pasireotide caused rapid, dramatic improvement in glucose levels (resolving the hypoglycemia completely in the first 2 cases). Two of our cases—pasireotide for treating hypoglycemia in occult insulinoma and NICTH—are not previously reported in the literature.
In conclusion, we recommend that pasireotide be considered for the treatment of tumor-induced hypoglycemia in 3 settings. First, in a patient with insulinoma who is either not a surgical candidate or who decides against surgery, and in whom hypoglycemia persists despite conventional medical therapy. Second, in the patient with NICTH for whom tumor resection is not possible and adjunctive medical therapy is unhelpful. Third, during the COVID-19 pandemic, there is added concern on the part of providers and patients, both from an infectious disease and resource use standpoint, in terms of admitting tumor-induced hypoglycemia patients for the purposes of diagnosis, localization, and/or surgical management. In such case, there may be a role for temporary pasireotide therapy as a “bridging” technique until definitive diagnostic and therapeutic strategies can be implemented. In summary, given its potent antihypoglycemic and antitumor properties, pasireotide is a reasonable choice for the treatment of tumor-induced hypoglycemia, though additional investigation is warranted.
Abbreviations
COVID-19 2019 novel coronavirus
GH growth hormone
HbA1c glycated hemoglobin
HCC hepatocellular carcinoma
IGF insulin-like growth factor
LAR long-acting release
NICTH non-islet cell tumor hypoglycemia
SSTR somatostatin receptor
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | CAPECITABINE, PASIREOTIDE, TEMOZOLOMIDE | DrugsGivenReaction | CC BY-NC-ND | 33294765 | 20,443,328 | 2021-01-01 |
What was the administration route of drug 'PASIREOTIDE'? | Pasireotide: A Novel Treatment for Tumor-Induced Hypoglycemia Due to Insulinoma and Non-Islet Cell Tumor Hypoglycemia.
Tumor-induced hypoglycemia is a serious disorder most commonly caused by insulinoma or non-islet cell tumor hypoglycemia (NICTH). The hypoglycemia can be severe and refractory to conventional therapy, leading to significant morbidity and mortality. The objective of this work is to describe a series of challenging cases in which refractory, tumor-induced hypoglycemia was shown to respond to the use of pasireotide, a second-generation somatostatin receptor ligand. We describe the clinical and biochemical features of 3 patients with tumor-induced hypoglycemia due to an occult insulinoma, malignant insulinoma, and non-islet cell tumor hypoglycemia. In these 3 individuals, the hypoglycemia remained refractory to guideline-recommended medical therapy, such as diazoxide, nonpasireotide somatostatin analogues, and glucocorticoids. Pasireotide was substituted to attenuate the refractory hypoglycemia for each patient. The addition of pasireotide led to prompt improvement in the frequency and severity of hypoglycemic episodes for each tumor-induced hypoglycemia patient. We demonstrate the successful treatment of 3 individuals with refractory, tumor-induced hypoglycemia with pasireotide. We offer the first reported use of pasireotide for the successful treatment of nonmalignant insulinoma and non-islet cell tumor hypoglycemia.
Hypoglycemia in individuals without diabetes is uncommon and warrants further investigation if the Whipple triad is fulfilled. Insulinoma and non-islet cell tumor hypoglycemia (NICTH) are rare causes of hypoglycemia. They may present with refractory hypoglycemia, and in severe cases, can cause irreversible neurocognitive impairment and death. The medical management of refractory hypoglycemia is challenging because there are limited therapeutic options available with unpredictable response and significant adverse effects [1].
The most common cause of hypoglycemia due to endogenous hyperinsulinism is insulinoma, which is generally single and benign [2]. Malignant insulinomas are rare, comprising only 5.8% of all insulinomas [3]. Malignant insulinomas have a poor prognosis, with a 10-year survival of less than 20%, and present with distant metastatic involvement, predominately to the liver and regional lymph nodes [4]. The definitive treatment of solitary insulinomas is surgical, though medical therapy has a role in patients who are poor surgical candidates or who decide against surgery. In cases of malignant insulinomas, however, surgery is not curative; thus, medical therapy has an important role in controlling symptomatic hypoglycemia and reducing tumor burden. Medications indicated for alleviating hypoglycemia in malignant insulinoma include diazoxide, glucocorticoids, and somatostatin analogues, which have shown variable responses [1, 5].
NICTH is a rare paraneoplastic syndrome associated with tumors of epithelial and mesenchymal origin. It is the second-most common cause of tumor-induced hypoglycemia after insulinoma and is most commonly seen in hepatocellular and adrenocortical carcinoma [6]. NICTH is caused by tumor overexpression of insulin-like growth factor (IGF)-2: both mature IGF-2 and incompletely processed “big IGF-2,” which promotes hypoglycemia due to insulin-like effects [7]. NICTH should be considered in the presence of hypoinsulinemic hypoglycemia with low growth hormone (GH) and IGF-1 levels, and an IGF-2:IGF-1 ratio greater than 10. There are no commercially available assays for big IGF-2 [8]. Definitive treatment of NICTH involves complete tumor resection. If resection is not feasible, local antitumor therapies are generally pursued, with a trial of glucocorticoids and/or recombinant human GH for refractory hypoglycemia [9].
We report 3 cases of refractory hypoglycemia due to occult insulinoma, malignant insulinoma, and NICTH that were successfully treated with pasireotide, a second-generation somatostatin receptor ligand. Although not formally approved for use in hypoglycemia, pasireotide has unique features that make it an appealing choice for refractory, tumor-induced hypoglycemia.
Case 1
Pasireotide for the Treatment of Insulinoma in a Poor Surgical Candidate
An 80-year-old woman was referred to our endocrine clinic for recurrent hypoglycemic episodes. She reported a 9-year history of spells of dizziness, tremors, and diaphoresis that improved with eating peanut butter. When symptomatic, the blood glucose levels measured in her assisted living facility were approximately 50 mg/dL. These episodes occurred both in the fasting and postprandial states and were increasing in frequency. Additionally, the patient had gained 12 pounds in the last 6 months.
The patient was admitted to the hospital for a 72-hour fast, which revealed endogenous hyperinsulinism. When the patient’s blood glucose measured 43 mg/dL, she had an insulin level of 47 (normal, 2.0-19.6 µIU/mL), C-peptide of 8.2 (normal, 0.80-3.85 ng/mL), proinsulin level of 27.9 (normal, ≤ 18.8 pmol/L), and negative sulfonylurea screen and insulin antibody. Computed tomography (CT) of the abdomen and pelvis showed a subtle 3-mm nodular focus of arterial enhancement within the pancreatic head, suspicious for a small insulinoma, but endoscopic ultrasound failed to localize the tumor. 68Ga DOTATATE positron emission tomography/CT did not reveal any abnormal uptake concerning for neuroendocrine tumor, and a selective arterial calcium stimulation test failed to localize the patient’s insulinoma as well.
The patient opted for medical management of her insulinoma as surgery was considered high risk because of her age and significant cardiovascular history. She could not tolerate diazoxide because of edema and was transitioned to short-acting octreotide without improvement in her hypoglycemic episodes. The patient was switched to octreotide long-acting release (LAR) 20 mg every 4 weeks, which was continued for 3 months. During this time, she required 2 hospitalizations for severe hypoglycemia.
Given the patient’s inadequate response to octreotide LAR, pasireotide LAR 40 mg intramuscularly (IM) every 4 weeks was initiated, which completely resolved the hypoglycemic episodes within 1 month, with average blood glucose of 200 mg/dL. However, owing to some postural lightheadedness, her dose was decreased to 20 mg IM every 4 weeks. Approximately 1 year later, the patient developed persistent hyperglycemia, with a mean blood glucose of 180 mg/dL and glycated hemoglobin (HbA1c) of 8% (64 mmol/mol). At this time, definitive treatment of her insulinoma was discussed (eg, repeat selective arterial calcium stimulation test followed by surgery), but the patient decided to continue medical management citing concerns with COVID-19 infection risk with an elective radiologic procedure followed by hospitalization for tumor resection. As such, the patient’s pasireotide dose was lowered to 10 mg every 4 weeks, and she has maintained glucose levels in the 90- to 120-mg/dL range without any further episodes of hypoglycemia or hyperglycemia.
Case 2
Pasireotide for the Treatment of Malignant Insulinoma
We have published the preliminary details of this case previously [10]. A 53-year-old woman presented with a 2-year history of recurrent hypoglycemia that fulfilled the Whipple triad. Within the first hour of a 72-hour fast, she was found to have a blood glucose of 53 mg/dL, insulin level of 87 mIU/mL (normal, 2.6-24.9 µIU/mL), C-peptide of 13.2 ng/mL (normal, 1.1-4.4ng/mL), and proinsulin of 2822 pmol/L (normal, 3-20 pmol/L), confirming endogenous hyperinsulinism. She was found to have a 3.6 × 2.8 × 2.4-cm pancreatic tail mass with innumerable masses in the liver measuring up to 10 cm. Core biopsy of the hepatic lesions revealed a well-differentiated neuroendocrine tumor (World Health Organization grade 2, Ki-67 4%), and she was diagnosed with stage IV malignant insulinoma with liver metastases.
Consensus from our institutional tumor board was to manage the patient medically given her extensive tumor burden. She was initiated on diazoxide and octreotide LAR 30 mg IM every 4 weeks but continued to have persistent hypoglycemia (30% of recorded glucose levels were < 70 mg/dL and 5% < 55 mg/dL) on her Dexcom G4 Platinum continuous glucose monitor (CGM) (Fig. 1A). As such, she was switched to pasireotide LAR 60 mg IM every 4 weeks with marked improvement in her hypoglycemic episodes within 1 month: Only 3% of recorded readings were below 70 mg/dL with no serious hypoglycemia (≤ 55 mg/dL), with mean sensor glucose of 129 mg/dL (Fig. 1B).
Figure 1. Dexcom G4 Platinum continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 30% to 3% of recorded values, respectively.
The following month, the patient started chemotherapy with capecitabine and temozolomide followed by transarterial chemoembolization of her hepatic metastases. Two months after chemoembolization, the patient developed hyperglycemia, so pasireotide was discontinued. Subsequently, at her 3-month follow-up, the patient was no longer experiencing any hypoglycemic episodes and her average blood glucose was 122 mg/dL. She was started on lanreotide 120 mg IM every 4 weeks for antineoplastic effect and continued on the single chemotherapeutic agent capecitabine by the oncology service.
Two years later, the patient developed type 2 diabetes mellitus with an HbA1c of 9.2% (75 mmol/mol). She was started on empagliflozin and 4 months later, her HbA1c improved to 7.5% (53 mmol/mol). The patient has no evidence of tumor progression 4 years from her initial diagnosis while on capecitabine plus lanreotide therapy, with stable radiographic appearance of her hepatic metastases.
Case 3
Pasireotide for the Treatment of Non-Islet Cell Tumor Hypoglycemia
A 72-year-old man with history of cirrhosis from hepatitis C and recently diagnosed hepatocellular carcinoma (HCC) presented to our institution with refractory hypoglycemia. The patient’s HCC was diagnosed 2 months prior to admission in the setting of severe hypoglycemia and seizure. The patient noted lightheadedness and diaphoresis occurring at 2-hour intervals during the day and night with fingerstick glucose readings of approximately 40 mg/dL during these episodes. His symptoms resolved on consuming carbohydrates. The patient was not taking any medications associated with glucose-lowering and denied alcohol consumption. He had normal renal and hepatic function. A download of his Dexcom G6 CGM revealed that 48% of his recorded blood glucose readings were below 70 mg/dL with multiple episodes (28%) of severe nocturnal hypoglycemia with blood glucose below 54 mg/dL (Fig. 2A).
Figure 2. Dexcom G6 continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 48% to 8% of recorded values, respectively.
A 72-hour fast was initiated, and within 1 hour, the patient had a blood glucose measurement of 30 mg/dL, with an insulin level of 2.2 mcIU/mL (normal, 2.6-24.9 mcIU/mL), C-peptide of 0.17 ng/mL (normal, 0.80-3.85 ng/mL), β-hydroxybutyrate less than 0.1 mmol/L (normal, 0.0-0.3 mmol/L), and proinsulin less than 0.4 pmol/L (normal, ≤ 18.8pmol/L). The patient had a negative insulin antibody (< 0.4 U/mL) and negative serum hypoglycemic agent screen. Further evaluation of his noninsulin-mediated hypoglycemia excluded adrenal insufficiency via cosyntropin stimulation testing. His GH level was 0.06 ng/mL (normal, 0.01-0.97 ng/mL), IGF-1 was less than 10 ng/mL (normal, 32-200 ng/ml), and IGF-2 level was 780 ng/mL (normal, 333-967 ng/mL). Owing to the patient’s hypoinsulinemic hypoglycemia with low IGF-1 level and an IGF-2:IGF-1 ratio greater than 10, the patient was diagnosed with NICTH.
CT abdomen/pelvis revealed a cirrhotic liver with a 7-cm lesion involving the right hepatic lobe; biopsy confirmed the diagnosis of well-differentiated hepatocellular carcinoma. The tumor was unresectable because of portal vein thrombosis, so the patient was started on an immunotherapy clinical trial with pembrolizumab and bavituximab. He was not a candidate for yttrium-90 radioembolization because of an anterioportal shunt that could not be adequately embolized, so he received palliative external beam radiotherapy. However, the patient continued to experience refractory hypoglycemic episodes, which necessitated hourly waking by his family to encourage him to eat carbohydrates, despite the addition of prednisone 30 mg daily, 1 month after the diagnosis of HCC. He was readmitted to the hospital for glucose optimization.
During hospitalization, the patient required a titratable dextrose 50% infusion, 37.5 g of dextrose gel orally every 3 hours, and frequent meals for management of his hypoglycemia. He was started on combination medical therapy with diazoxide, prednisolone 20 mg twice daily, and short-acting octreotide 100 mg subcutaneously every 8 hours. Despite that, the patient remained in the hospital for 3 weeks, as he promptly developed hypoglycemia when the dextrose infusion was discontinued. Pasireotide LAR was ordered for the patient but could not be given inpatient, so he was discharged to a long-term assisted care facility while remaining on the dextrose infusion.
Shortly after discharge, the patient was started on pasireotide 40 mg IM every 4 weeks. Within 1 week, he was weaned off the dextrose infusion. Three weeks later, at his endocrine follow-up appointment, his Dexcom G6 CGM data revealed markedly improved glycemic control, with a mean sensor glucose of 121 mg/dL, 8% of readings below 70 mg/dL, and only 2% of readings below 54 mg/dL (Fig. 2B). Unfortunately, the patient’s HCC progressed despite the use of sorafenib and later nivolumab, and he died of shock and decompensated liver failure 5 months after his initial HCC diagnosis.
Discussion
Medications that are currently approved for refractory, tumor-induced hypoglycemia are generally of limited efficacy and tolerability [5]. In the cases described here, each patient with tumor-induced hypoglycemia was started on guideline-recommended medications for the treatment of hypoglycemia (ie, diazoxide, glucocorticoids, and/or octreotide) without attenuation of their refractory hypoglycemia. Then, pasireotide was substituted with marked improvement of hypoglycemia in all cases within 1 month of administration.
Pasireotide, like other somatostatin analogues, exerts its biologic effect by binding to somatostatin receptors (SSTRs). There are 5 somatostatin receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) distributed heterogeneously throughout the body. Once stimulated, somatostatin is a potent inhibitor of endocrine and exocrine hormonal release in humans [11]. Inhibition of insulin and glucagon secretion is primarily mediated by SSTR2, whereas insulin secretion is predominantly mediated via SSTR5 [12]. Neuroendocrine tumors largely express somatostatin receptors, with SSTR2 and SSTR5 expression shown in 70% of insulinomas [13]. Pasireotide, a second-generation SSTR ligand, has a 30- to 40-fold higher binding affinity for SSTR5 than the first-generation SSTR ligand octreotide, which accounts for the former’s advantage in treating hypoglycemia [14]. The hyperglycemia effect of pasireotide is related to a decrease in insulin secretion and incretin hormone response without change to hepatic or peripheral insulin sensitivity [15]. Although octreotide has been shown to improve hypoglycemia in two-thirds of insulinoma patients in one study [16], there are insufficient data for the use of pasireotide in insulinoma patients to calculate a treatment response. However, pasireotide induces a potent hyperglycemic effect in its currently indicated uses for Cushing disease and acromegaly: Seventy-three percent of Cushing disease patients [17] and 57% of acromegaly patients [18] developed hyperglycemia-related adverse events while on pasireotide. Pasireotide-induced hyperglycemia has been shown to respond to vildagliptin and liraglutide therapy [19].
Additionally, pasireotide has been shown to have antiproliferative effects comparable to the more conventionally used somatostatin analogues octreotide and lanreotide in the treatment of advanced neuroendocrine tumors [20].
Treatment of NICTH involves tumor resection or the use of alternate antitumor modalities when resection is not possible. While the use of pasireotide in the management of NICTH has not been previously described, high-dosed octreotide given as continuous infusion following uptake of 111In-labeled octreotide by a solitary fibrous, pleural tumor was not effective in suppressing big IGF-2 production or improving hypoglycemia [21]. In a separate case, the mechanism of refractory hypoglycemia due to NICTH from an intra-abdominal hemagiopericytoma was attributed to muscle tissue uptake of glucose mediated by IGF-2; during somatostatin treatment, big IGF-2 levels decreased modestly but could not adequately control hypoglycemia without the simultaneous infusion of exogenous glucose [22]. The effect of pasireotide therapy on big IGF-2 levels has not been described previously, though a potent lowering of big IGF-2 levels, potentially through SSTR5 interaction, could account for the hyperglycemic effect noted in our patient with hepatocellular carcinoma. Hepatocellular carcinoma tumor cells have been shown previously to express a high proportion of SSTR5 [23]. However, the treatment of advanced hepatocellular carcinoma with somatostatin analogues has shown variable response [24]. A recent phase 2 trial of pasireotide LAR in patients with unresectable HCC showed limited antitumor benefit; however, 30% of participants developed grade 3 hyperglycemia, and 5% of participants developed grade 4 hyperglycemia [25].
Despite the presumed advantage of pasireotide in treating tumor-induced hypoglycemia, only a single case, besides our own, has been published using pasireotide for the treatment of malignant insulinoma. Tirosh et al found that pasireotide LAR was more effective in treating refractory hypoglycemia compared with lanreotide in a patient with malignant insulinoma with hepatic metastases [26]. This manuscript adds to the literature by outlining 3 cases of refractory, tumor-induced hypoglycemia—due to an occult insulinoma, malignant insulinoma, and NICTH from HCC—that all failed to respond to conventional medical therapy, and for which pasireotide caused rapid, dramatic improvement in glucose levels (resolving the hypoglycemia completely in the first 2 cases). Two of our cases—pasireotide for treating hypoglycemia in occult insulinoma and NICTH—are not previously reported in the literature.
In conclusion, we recommend that pasireotide be considered for the treatment of tumor-induced hypoglycemia in 3 settings. First, in a patient with insulinoma who is either not a surgical candidate or who decides against surgery, and in whom hypoglycemia persists despite conventional medical therapy. Second, in the patient with NICTH for whom tumor resection is not possible and adjunctive medical therapy is unhelpful. Third, during the COVID-19 pandemic, there is added concern on the part of providers and patients, both from an infectious disease and resource use standpoint, in terms of admitting tumor-induced hypoglycemia patients for the purposes of diagnosis, localization, and/or surgical management. In such case, there may be a role for temporary pasireotide therapy as a “bridging” technique until definitive diagnostic and therapeutic strategies can be implemented. In summary, given its potent antihypoglycemic and antitumor properties, pasireotide is a reasonable choice for the treatment of tumor-induced hypoglycemia, though additional investigation is warranted.
Abbreviations
COVID-19 2019 novel coronavirus
GH growth hormone
HbA1c glycated hemoglobin
HCC hepatocellular carcinoma
IGF insulin-like growth factor
LAR long-acting release
NICTH non-islet cell tumor hypoglycemia
SSTR somatostatin receptor
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Intramuscular | DrugAdministrationRoute | CC BY-NC-ND | 33294765 | 20,443,328 | 2021-01-01 |
What was the outcome of reaction 'Hyperglycaemia'? | Pasireotide: A Novel Treatment for Tumor-Induced Hypoglycemia Due to Insulinoma and Non-Islet Cell Tumor Hypoglycemia.
Tumor-induced hypoglycemia is a serious disorder most commonly caused by insulinoma or non-islet cell tumor hypoglycemia (NICTH). The hypoglycemia can be severe and refractory to conventional therapy, leading to significant morbidity and mortality. The objective of this work is to describe a series of challenging cases in which refractory, tumor-induced hypoglycemia was shown to respond to the use of pasireotide, a second-generation somatostatin receptor ligand. We describe the clinical and biochemical features of 3 patients with tumor-induced hypoglycemia due to an occult insulinoma, malignant insulinoma, and non-islet cell tumor hypoglycemia. In these 3 individuals, the hypoglycemia remained refractory to guideline-recommended medical therapy, such as diazoxide, nonpasireotide somatostatin analogues, and glucocorticoids. Pasireotide was substituted to attenuate the refractory hypoglycemia for each patient. The addition of pasireotide led to prompt improvement in the frequency and severity of hypoglycemic episodes for each tumor-induced hypoglycemia patient. We demonstrate the successful treatment of 3 individuals with refractory, tumor-induced hypoglycemia with pasireotide. We offer the first reported use of pasireotide for the successful treatment of nonmalignant insulinoma and non-islet cell tumor hypoglycemia.
Hypoglycemia in individuals without diabetes is uncommon and warrants further investigation if the Whipple triad is fulfilled. Insulinoma and non-islet cell tumor hypoglycemia (NICTH) are rare causes of hypoglycemia. They may present with refractory hypoglycemia, and in severe cases, can cause irreversible neurocognitive impairment and death. The medical management of refractory hypoglycemia is challenging because there are limited therapeutic options available with unpredictable response and significant adverse effects [1].
The most common cause of hypoglycemia due to endogenous hyperinsulinism is insulinoma, which is generally single and benign [2]. Malignant insulinomas are rare, comprising only 5.8% of all insulinomas [3]. Malignant insulinomas have a poor prognosis, with a 10-year survival of less than 20%, and present with distant metastatic involvement, predominately to the liver and regional lymph nodes [4]. The definitive treatment of solitary insulinomas is surgical, though medical therapy has a role in patients who are poor surgical candidates or who decide against surgery. In cases of malignant insulinomas, however, surgery is not curative; thus, medical therapy has an important role in controlling symptomatic hypoglycemia and reducing tumor burden. Medications indicated for alleviating hypoglycemia in malignant insulinoma include diazoxide, glucocorticoids, and somatostatin analogues, which have shown variable responses [1, 5].
NICTH is a rare paraneoplastic syndrome associated with tumors of epithelial and mesenchymal origin. It is the second-most common cause of tumor-induced hypoglycemia after insulinoma and is most commonly seen in hepatocellular and adrenocortical carcinoma [6]. NICTH is caused by tumor overexpression of insulin-like growth factor (IGF)-2: both mature IGF-2 and incompletely processed “big IGF-2,” which promotes hypoglycemia due to insulin-like effects [7]. NICTH should be considered in the presence of hypoinsulinemic hypoglycemia with low growth hormone (GH) and IGF-1 levels, and an IGF-2:IGF-1 ratio greater than 10. There are no commercially available assays for big IGF-2 [8]. Definitive treatment of NICTH involves complete tumor resection. If resection is not feasible, local antitumor therapies are generally pursued, with a trial of glucocorticoids and/or recombinant human GH for refractory hypoglycemia [9].
We report 3 cases of refractory hypoglycemia due to occult insulinoma, malignant insulinoma, and NICTH that were successfully treated with pasireotide, a second-generation somatostatin receptor ligand. Although not formally approved for use in hypoglycemia, pasireotide has unique features that make it an appealing choice for refractory, tumor-induced hypoglycemia.
Case 1
Pasireotide for the Treatment of Insulinoma in a Poor Surgical Candidate
An 80-year-old woman was referred to our endocrine clinic for recurrent hypoglycemic episodes. She reported a 9-year history of spells of dizziness, tremors, and diaphoresis that improved with eating peanut butter. When symptomatic, the blood glucose levels measured in her assisted living facility were approximately 50 mg/dL. These episodes occurred both in the fasting and postprandial states and were increasing in frequency. Additionally, the patient had gained 12 pounds in the last 6 months.
The patient was admitted to the hospital for a 72-hour fast, which revealed endogenous hyperinsulinism. When the patient’s blood glucose measured 43 mg/dL, she had an insulin level of 47 (normal, 2.0-19.6 µIU/mL), C-peptide of 8.2 (normal, 0.80-3.85 ng/mL), proinsulin level of 27.9 (normal, ≤ 18.8 pmol/L), and negative sulfonylurea screen and insulin antibody. Computed tomography (CT) of the abdomen and pelvis showed a subtle 3-mm nodular focus of arterial enhancement within the pancreatic head, suspicious for a small insulinoma, but endoscopic ultrasound failed to localize the tumor. 68Ga DOTATATE positron emission tomography/CT did not reveal any abnormal uptake concerning for neuroendocrine tumor, and a selective arterial calcium stimulation test failed to localize the patient’s insulinoma as well.
The patient opted for medical management of her insulinoma as surgery was considered high risk because of her age and significant cardiovascular history. She could not tolerate diazoxide because of edema and was transitioned to short-acting octreotide without improvement in her hypoglycemic episodes. The patient was switched to octreotide long-acting release (LAR) 20 mg every 4 weeks, which was continued for 3 months. During this time, she required 2 hospitalizations for severe hypoglycemia.
Given the patient’s inadequate response to octreotide LAR, pasireotide LAR 40 mg intramuscularly (IM) every 4 weeks was initiated, which completely resolved the hypoglycemic episodes within 1 month, with average blood glucose of 200 mg/dL. However, owing to some postural lightheadedness, her dose was decreased to 20 mg IM every 4 weeks. Approximately 1 year later, the patient developed persistent hyperglycemia, with a mean blood glucose of 180 mg/dL and glycated hemoglobin (HbA1c) of 8% (64 mmol/mol). At this time, definitive treatment of her insulinoma was discussed (eg, repeat selective arterial calcium stimulation test followed by surgery), but the patient decided to continue medical management citing concerns with COVID-19 infection risk with an elective radiologic procedure followed by hospitalization for tumor resection. As such, the patient’s pasireotide dose was lowered to 10 mg every 4 weeks, and she has maintained glucose levels in the 90- to 120-mg/dL range without any further episodes of hypoglycemia or hyperglycemia.
Case 2
Pasireotide for the Treatment of Malignant Insulinoma
We have published the preliminary details of this case previously [10]. A 53-year-old woman presented with a 2-year history of recurrent hypoglycemia that fulfilled the Whipple triad. Within the first hour of a 72-hour fast, she was found to have a blood glucose of 53 mg/dL, insulin level of 87 mIU/mL (normal, 2.6-24.9 µIU/mL), C-peptide of 13.2 ng/mL (normal, 1.1-4.4ng/mL), and proinsulin of 2822 pmol/L (normal, 3-20 pmol/L), confirming endogenous hyperinsulinism. She was found to have a 3.6 × 2.8 × 2.4-cm pancreatic tail mass with innumerable masses in the liver measuring up to 10 cm. Core biopsy of the hepatic lesions revealed a well-differentiated neuroendocrine tumor (World Health Organization grade 2, Ki-67 4%), and she was diagnosed with stage IV malignant insulinoma with liver metastases.
Consensus from our institutional tumor board was to manage the patient medically given her extensive tumor burden. She was initiated on diazoxide and octreotide LAR 30 mg IM every 4 weeks but continued to have persistent hypoglycemia (30% of recorded glucose levels were < 70 mg/dL and 5% < 55 mg/dL) on her Dexcom G4 Platinum continuous glucose monitor (CGM) (Fig. 1A). As such, she was switched to pasireotide LAR 60 mg IM every 4 weeks with marked improvement in her hypoglycemic episodes within 1 month: Only 3% of recorded readings were below 70 mg/dL with no serious hypoglycemia (≤ 55 mg/dL), with mean sensor glucose of 129 mg/dL (Fig. 1B).
Figure 1. Dexcom G4 Platinum continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 30% to 3% of recorded values, respectively.
The following month, the patient started chemotherapy with capecitabine and temozolomide followed by transarterial chemoembolization of her hepatic metastases. Two months after chemoembolization, the patient developed hyperglycemia, so pasireotide was discontinued. Subsequently, at her 3-month follow-up, the patient was no longer experiencing any hypoglycemic episodes and her average blood glucose was 122 mg/dL. She was started on lanreotide 120 mg IM every 4 weeks for antineoplastic effect and continued on the single chemotherapeutic agent capecitabine by the oncology service.
Two years later, the patient developed type 2 diabetes mellitus with an HbA1c of 9.2% (75 mmol/mol). She was started on empagliflozin and 4 months later, her HbA1c improved to 7.5% (53 mmol/mol). The patient has no evidence of tumor progression 4 years from her initial diagnosis while on capecitabine plus lanreotide therapy, with stable radiographic appearance of her hepatic metastases.
Case 3
Pasireotide for the Treatment of Non-Islet Cell Tumor Hypoglycemia
A 72-year-old man with history of cirrhosis from hepatitis C and recently diagnosed hepatocellular carcinoma (HCC) presented to our institution with refractory hypoglycemia. The patient’s HCC was diagnosed 2 months prior to admission in the setting of severe hypoglycemia and seizure. The patient noted lightheadedness and diaphoresis occurring at 2-hour intervals during the day and night with fingerstick glucose readings of approximately 40 mg/dL during these episodes. His symptoms resolved on consuming carbohydrates. The patient was not taking any medications associated with glucose-lowering and denied alcohol consumption. He had normal renal and hepatic function. A download of his Dexcom G6 CGM revealed that 48% of his recorded blood glucose readings were below 70 mg/dL with multiple episodes (28%) of severe nocturnal hypoglycemia with blood glucose below 54 mg/dL (Fig. 2A).
Figure 2. Dexcom G6 continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 48% to 8% of recorded values, respectively.
A 72-hour fast was initiated, and within 1 hour, the patient had a blood glucose measurement of 30 mg/dL, with an insulin level of 2.2 mcIU/mL (normal, 2.6-24.9 mcIU/mL), C-peptide of 0.17 ng/mL (normal, 0.80-3.85 ng/mL), β-hydroxybutyrate less than 0.1 mmol/L (normal, 0.0-0.3 mmol/L), and proinsulin less than 0.4 pmol/L (normal, ≤ 18.8pmol/L). The patient had a negative insulin antibody (< 0.4 U/mL) and negative serum hypoglycemic agent screen. Further evaluation of his noninsulin-mediated hypoglycemia excluded adrenal insufficiency via cosyntropin stimulation testing. His GH level was 0.06 ng/mL (normal, 0.01-0.97 ng/mL), IGF-1 was less than 10 ng/mL (normal, 32-200 ng/ml), and IGF-2 level was 780 ng/mL (normal, 333-967 ng/mL). Owing to the patient’s hypoinsulinemic hypoglycemia with low IGF-1 level and an IGF-2:IGF-1 ratio greater than 10, the patient was diagnosed with NICTH.
CT abdomen/pelvis revealed a cirrhotic liver with a 7-cm lesion involving the right hepatic lobe; biopsy confirmed the diagnosis of well-differentiated hepatocellular carcinoma. The tumor was unresectable because of portal vein thrombosis, so the patient was started on an immunotherapy clinical trial with pembrolizumab and bavituximab. He was not a candidate for yttrium-90 radioembolization because of an anterioportal shunt that could not be adequately embolized, so he received palliative external beam radiotherapy. However, the patient continued to experience refractory hypoglycemic episodes, which necessitated hourly waking by his family to encourage him to eat carbohydrates, despite the addition of prednisone 30 mg daily, 1 month after the diagnosis of HCC. He was readmitted to the hospital for glucose optimization.
During hospitalization, the patient required a titratable dextrose 50% infusion, 37.5 g of dextrose gel orally every 3 hours, and frequent meals for management of his hypoglycemia. He was started on combination medical therapy with diazoxide, prednisolone 20 mg twice daily, and short-acting octreotide 100 mg subcutaneously every 8 hours. Despite that, the patient remained in the hospital for 3 weeks, as he promptly developed hypoglycemia when the dextrose infusion was discontinued. Pasireotide LAR was ordered for the patient but could not be given inpatient, so he was discharged to a long-term assisted care facility while remaining on the dextrose infusion.
Shortly after discharge, the patient was started on pasireotide 40 mg IM every 4 weeks. Within 1 week, he was weaned off the dextrose infusion. Three weeks later, at his endocrine follow-up appointment, his Dexcom G6 CGM data revealed markedly improved glycemic control, with a mean sensor glucose of 121 mg/dL, 8% of readings below 70 mg/dL, and only 2% of readings below 54 mg/dL (Fig. 2B). Unfortunately, the patient’s HCC progressed despite the use of sorafenib and later nivolumab, and he died of shock and decompensated liver failure 5 months after his initial HCC diagnosis.
Discussion
Medications that are currently approved for refractory, tumor-induced hypoglycemia are generally of limited efficacy and tolerability [5]. In the cases described here, each patient with tumor-induced hypoglycemia was started on guideline-recommended medications for the treatment of hypoglycemia (ie, diazoxide, glucocorticoids, and/or octreotide) without attenuation of their refractory hypoglycemia. Then, pasireotide was substituted with marked improvement of hypoglycemia in all cases within 1 month of administration.
Pasireotide, like other somatostatin analogues, exerts its biologic effect by binding to somatostatin receptors (SSTRs). There are 5 somatostatin receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) distributed heterogeneously throughout the body. Once stimulated, somatostatin is a potent inhibitor of endocrine and exocrine hormonal release in humans [11]. Inhibition of insulin and glucagon secretion is primarily mediated by SSTR2, whereas insulin secretion is predominantly mediated via SSTR5 [12]. Neuroendocrine tumors largely express somatostatin receptors, with SSTR2 and SSTR5 expression shown in 70% of insulinomas [13]. Pasireotide, a second-generation SSTR ligand, has a 30- to 40-fold higher binding affinity for SSTR5 than the first-generation SSTR ligand octreotide, which accounts for the former’s advantage in treating hypoglycemia [14]. The hyperglycemia effect of pasireotide is related to a decrease in insulin secretion and incretin hormone response without change to hepatic or peripheral insulin sensitivity [15]. Although octreotide has been shown to improve hypoglycemia in two-thirds of insulinoma patients in one study [16], there are insufficient data for the use of pasireotide in insulinoma patients to calculate a treatment response. However, pasireotide induces a potent hyperglycemic effect in its currently indicated uses for Cushing disease and acromegaly: Seventy-three percent of Cushing disease patients [17] and 57% of acromegaly patients [18] developed hyperglycemia-related adverse events while on pasireotide. Pasireotide-induced hyperglycemia has been shown to respond to vildagliptin and liraglutide therapy [19].
Additionally, pasireotide has been shown to have antiproliferative effects comparable to the more conventionally used somatostatin analogues octreotide and lanreotide in the treatment of advanced neuroendocrine tumors [20].
Treatment of NICTH involves tumor resection or the use of alternate antitumor modalities when resection is not possible. While the use of pasireotide in the management of NICTH has not been previously described, high-dosed octreotide given as continuous infusion following uptake of 111In-labeled octreotide by a solitary fibrous, pleural tumor was not effective in suppressing big IGF-2 production or improving hypoglycemia [21]. In a separate case, the mechanism of refractory hypoglycemia due to NICTH from an intra-abdominal hemagiopericytoma was attributed to muscle tissue uptake of glucose mediated by IGF-2; during somatostatin treatment, big IGF-2 levels decreased modestly but could not adequately control hypoglycemia without the simultaneous infusion of exogenous glucose [22]. The effect of pasireotide therapy on big IGF-2 levels has not been described previously, though a potent lowering of big IGF-2 levels, potentially through SSTR5 interaction, could account for the hyperglycemic effect noted in our patient with hepatocellular carcinoma. Hepatocellular carcinoma tumor cells have been shown previously to express a high proportion of SSTR5 [23]. However, the treatment of advanced hepatocellular carcinoma with somatostatin analogues has shown variable response [24]. A recent phase 2 trial of pasireotide LAR in patients with unresectable HCC showed limited antitumor benefit; however, 30% of participants developed grade 3 hyperglycemia, and 5% of participants developed grade 4 hyperglycemia [25].
Despite the presumed advantage of pasireotide in treating tumor-induced hypoglycemia, only a single case, besides our own, has been published using pasireotide for the treatment of malignant insulinoma. Tirosh et al found that pasireotide LAR was more effective in treating refractory hypoglycemia compared with lanreotide in a patient with malignant insulinoma with hepatic metastases [26]. This manuscript adds to the literature by outlining 3 cases of refractory, tumor-induced hypoglycemia—due to an occult insulinoma, malignant insulinoma, and NICTH from HCC—that all failed to respond to conventional medical therapy, and for which pasireotide caused rapid, dramatic improvement in glucose levels (resolving the hypoglycemia completely in the first 2 cases). Two of our cases—pasireotide for treating hypoglycemia in occult insulinoma and NICTH—are not previously reported in the literature.
In conclusion, we recommend that pasireotide be considered for the treatment of tumor-induced hypoglycemia in 3 settings. First, in a patient with insulinoma who is either not a surgical candidate or who decides against surgery, and in whom hypoglycemia persists despite conventional medical therapy. Second, in the patient with NICTH for whom tumor resection is not possible and adjunctive medical therapy is unhelpful. Third, during the COVID-19 pandemic, there is added concern on the part of providers and patients, both from an infectious disease and resource use standpoint, in terms of admitting tumor-induced hypoglycemia patients for the purposes of diagnosis, localization, and/or surgical management. In such case, there may be a role for temporary pasireotide therapy as a “bridging” technique until definitive diagnostic and therapeutic strategies can be implemented. In summary, given its potent antihypoglycemic and antitumor properties, pasireotide is a reasonable choice for the treatment of tumor-induced hypoglycemia, though additional investigation is warranted.
Abbreviations
COVID-19 2019 novel coronavirus
GH growth hormone
HbA1c glycated hemoglobin
HCC hepatocellular carcinoma
IGF insulin-like growth factor
LAR long-acting release
NICTH non-islet cell tumor hypoglycemia
SSTR somatostatin receptor
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Recovered | ReactionOutcome | CC BY-NC-ND | 33294765 | 20,443,328 | 2021-01-01 |
What was the outcome of reaction 'Product use in unapproved indication'? | Pasireotide: A Novel Treatment for Tumor-Induced Hypoglycemia Due to Insulinoma and Non-Islet Cell Tumor Hypoglycemia.
Tumor-induced hypoglycemia is a serious disorder most commonly caused by insulinoma or non-islet cell tumor hypoglycemia (NICTH). The hypoglycemia can be severe and refractory to conventional therapy, leading to significant morbidity and mortality. The objective of this work is to describe a series of challenging cases in which refractory, tumor-induced hypoglycemia was shown to respond to the use of pasireotide, a second-generation somatostatin receptor ligand. We describe the clinical and biochemical features of 3 patients with tumor-induced hypoglycemia due to an occult insulinoma, malignant insulinoma, and non-islet cell tumor hypoglycemia. In these 3 individuals, the hypoglycemia remained refractory to guideline-recommended medical therapy, such as diazoxide, nonpasireotide somatostatin analogues, and glucocorticoids. Pasireotide was substituted to attenuate the refractory hypoglycemia for each patient. The addition of pasireotide led to prompt improvement in the frequency and severity of hypoglycemic episodes for each tumor-induced hypoglycemia patient. We demonstrate the successful treatment of 3 individuals with refractory, tumor-induced hypoglycemia with pasireotide. We offer the first reported use of pasireotide for the successful treatment of nonmalignant insulinoma and non-islet cell tumor hypoglycemia.
Hypoglycemia in individuals without diabetes is uncommon and warrants further investigation if the Whipple triad is fulfilled. Insulinoma and non-islet cell tumor hypoglycemia (NICTH) are rare causes of hypoglycemia. They may present with refractory hypoglycemia, and in severe cases, can cause irreversible neurocognitive impairment and death. The medical management of refractory hypoglycemia is challenging because there are limited therapeutic options available with unpredictable response and significant adverse effects [1].
The most common cause of hypoglycemia due to endogenous hyperinsulinism is insulinoma, which is generally single and benign [2]. Malignant insulinomas are rare, comprising only 5.8% of all insulinomas [3]. Malignant insulinomas have a poor prognosis, with a 10-year survival of less than 20%, and present with distant metastatic involvement, predominately to the liver and regional lymph nodes [4]. The definitive treatment of solitary insulinomas is surgical, though medical therapy has a role in patients who are poor surgical candidates or who decide against surgery. In cases of malignant insulinomas, however, surgery is not curative; thus, medical therapy has an important role in controlling symptomatic hypoglycemia and reducing tumor burden. Medications indicated for alleviating hypoglycemia in malignant insulinoma include diazoxide, glucocorticoids, and somatostatin analogues, which have shown variable responses [1, 5].
NICTH is a rare paraneoplastic syndrome associated with tumors of epithelial and mesenchymal origin. It is the second-most common cause of tumor-induced hypoglycemia after insulinoma and is most commonly seen in hepatocellular and adrenocortical carcinoma [6]. NICTH is caused by tumor overexpression of insulin-like growth factor (IGF)-2: both mature IGF-2 and incompletely processed “big IGF-2,” which promotes hypoglycemia due to insulin-like effects [7]. NICTH should be considered in the presence of hypoinsulinemic hypoglycemia with low growth hormone (GH) and IGF-1 levels, and an IGF-2:IGF-1 ratio greater than 10. There are no commercially available assays for big IGF-2 [8]. Definitive treatment of NICTH involves complete tumor resection. If resection is not feasible, local antitumor therapies are generally pursued, with a trial of glucocorticoids and/or recombinant human GH for refractory hypoglycemia [9].
We report 3 cases of refractory hypoglycemia due to occult insulinoma, malignant insulinoma, and NICTH that were successfully treated with pasireotide, a second-generation somatostatin receptor ligand. Although not formally approved for use in hypoglycemia, pasireotide has unique features that make it an appealing choice for refractory, tumor-induced hypoglycemia.
Case 1
Pasireotide for the Treatment of Insulinoma in a Poor Surgical Candidate
An 80-year-old woman was referred to our endocrine clinic for recurrent hypoglycemic episodes. She reported a 9-year history of spells of dizziness, tremors, and diaphoresis that improved with eating peanut butter. When symptomatic, the blood glucose levels measured in her assisted living facility were approximately 50 mg/dL. These episodes occurred both in the fasting and postprandial states and were increasing in frequency. Additionally, the patient had gained 12 pounds in the last 6 months.
The patient was admitted to the hospital for a 72-hour fast, which revealed endogenous hyperinsulinism. When the patient’s blood glucose measured 43 mg/dL, she had an insulin level of 47 (normal, 2.0-19.6 µIU/mL), C-peptide of 8.2 (normal, 0.80-3.85 ng/mL), proinsulin level of 27.9 (normal, ≤ 18.8 pmol/L), and negative sulfonylurea screen and insulin antibody. Computed tomography (CT) of the abdomen and pelvis showed a subtle 3-mm nodular focus of arterial enhancement within the pancreatic head, suspicious for a small insulinoma, but endoscopic ultrasound failed to localize the tumor. 68Ga DOTATATE positron emission tomography/CT did not reveal any abnormal uptake concerning for neuroendocrine tumor, and a selective arterial calcium stimulation test failed to localize the patient’s insulinoma as well.
The patient opted for medical management of her insulinoma as surgery was considered high risk because of her age and significant cardiovascular history. She could not tolerate diazoxide because of edema and was transitioned to short-acting octreotide without improvement in her hypoglycemic episodes. The patient was switched to octreotide long-acting release (LAR) 20 mg every 4 weeks, which was continued for 3 months. During this time, she required 2 hospitalizations for severe hypoglycemia.
Given the patient’s inadequate response to octreotide LAR, pasireotide LAR 40 mg intramuscularly (IM) every 4 weeks was initiated, which completely resolved the hypoglycemic episodes within 1 month, with average blood glucose of 200 mg/dL. However, owing to some postural lightheadedness, her dose was decreased to 20 mg IM every 4 weeks. Approximately 1 year later, the patient developed persistent hyperglycemia, with a mean blood glucose of 180 mg/dL and glycated hemoglobin (HbA1c) of 8% (64 mmol/mol). At this time, definitive treatment of her insulinoma was discussed (eg, repeat selective arterial calcium stimulation test followed by surgery), but the patient decided to continue medical management citing concerns with COVID-19 infection risk with an elective radiologic procedure followed by hospitalization for tumor resection. As such, the patient’s pasireotide dose was lowered to 10 mg every 4 weeks, and she has maintained glucose levels in the 90- to 120-mg/dL range without any further episodes of hypoglycemia or hyperglycemia.
Case 2
Pasireotide for the Treatment of Malignant Insulinoma
We have published the preliminary details of this case previously [10]. A 53-year-old woman presented with a 2-year history of recurrent hypoglycemia that fulfilled the Whipple triad. Within the first hour of a 72-hour fast, she was found to have a blood glucose of 53 mg/dL, insulin level of 87 mIU/mL (normal, 2.6-24.9 µIU/mL), C-peptide of 13.2 ng/mL (normal, 1.1-4.4ng/mL), and proinsulin of 2822 pmol/L (normal, 3-20 pmol/L), confirming endogenous hyperinsulinism. She was found to have a 3.6 × 2.8 × 2.4-cm pancreatic tail mass with innumerable masses in the liver measuring up to 10 cm. Core biopsy of the hepatic lesions revealed a well-differentiated neuroendocrine tumor (World Health Organization grade 2, Ki-67 4%), and she was diagnosed with stage IV malignant insulinoma with liver metastases.
Consensus from our institutional tumor board was to manage the patient medically given her extensive tumor burden. She was initiated on diazoxide and octreotide LAR 30 mg IM every 4 weeks but continued to have persistent hypoglycemia (30% of recorded glucose levels were < 70 mg/dL and 5% < 55 mg/dL) on her Dexcom G4 Platinum continuous glucose monitor (CGM) (Fig. 1A). As such, she was switched to pasireotide LAR 60 mg IM every 4 weeks with marked improvement in her hypoglycemic episodes within 1 month: Only 3% of recorded readings were below 70 mg/dL with no serious hypoglycemia (≤ 55 mg/dL), with mean sensor glucose of 129 mg/dL (Fig. 1B).
Figure 1. Dexcom G4 Platinum continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 30% to 3% of recorded values, respectively.
The following month, the patient started chemotherapy with capecitabine and temozolomide followed by transarterial chemoembolization of her hepatic metastases. Two months after chemoembolization, the patient developed hyperglycemia, so pasireotide was discontinued. Subsequently, at her 3-month follow-up, the patient was no longer experiencing any hypoglycemic episodes and her average blood glucose was 122 mg/dL. She was started on lanreotide 120 mg IM every 4 weeks for antineoplastic effect and continued on the single chemotherapeutic agent capecitabine by the oncology service.
Two years later, the patient developed type 2 diabetes mellitus with an HbA1c of 9.2% (75 mmol/mol). She was started on empagliflozin and 4 months later, her HbA1c improved to 7.5% (53 mmol/mol). The patient has no evidence of tumor progression 4 years from her initial diagnosis while on capecitabine plus lanreotide therapy, with stable radiographic appearance of her hepatic metastases.
Case 3
Pasireotide for the Treatment of Non-Islet Cell Tumor Hypoglycemia
A 72-year-old man with history of cirrhosis from hepatitis C and recently diagnosed hepatocellular carcinoma (HCC) presented to our institution with refractory hypoglycemia. The patient’s HCC was diagnosed 2 months prior to admission in the setting of severe hypoglycemia and seizure. The patient noted lightheadedness and diaphoresis occurring at 2-hour intervals during the day and night with fingerstick glucose readings of approximately 40 mg/dL during these episodes. His symptoms resolved on consuming carbohydrates. The patient was not taking any medications associated with glucose-lowering and denied alcohol consumption. He had normal renal and hepatic function. A download of his Dexcom G6 CGM revealed that 48% of his recorded blood glucose readings were below 70 mg/dL with multiple episodes (28%) of severe nocturnal hypoglycemia with blood glucose below 54 mg/dL (Fig. 2A).
Figure 2. Dexcom G6 continuous glucose monitor tracings A, before, and B, after pasireotide addition, showing a reduction in glucose readings of less than 70 mg/dL from 48% to 8% of recorded values, respectively.
A 72-hour fast was initiated, and within 1 hour, the patient had a blood glucose measurement of 30 mg/dL, with an insulin level of 2.2 mcIU/mL (normal, 2.6-24.9 mcIU/mL), C-peptide of 0.17 ng/mL (normal, 0.80-3.85 ng/mL), β-hydroxybutyrate less than 0.1 mmol/L (normal, 0.0-0.3 mmol/L), and proinsulin less than 0.4 pmol/L (normal, ≤ 18.8pmol/L). The patient had a negative insulin antibody (< 0.4 U/mL) and negative serum hypoglycemic agent screen. Further evaluation of his noninsulin-mediated hypoglycemia excluded adrenal insufficiency via cosyntropin stimulation testing. His GH level was 0.06 ng/mL (normal, 0.01-0.97 ng/mL), IGF-1 was less than 10 ng/mL (normal, 32-200 ng/ml), and IGF-2 level was 780 ng/mL (normal, 333-967 ng/mL). Owing to the patient’s hypoinsulinemic hypoglycemia with low IGF-1 level and an IGF-2:IGF-1 ratio greater than 10, the patient was diagnosed with NICTH.
CT abdomen/pelvis revealed a cirrhotic liver with a 7-cm lesion involving the right hepatic lobe; biopsy confirmed the diagnosis of well-differentiated hepatocellular carcinoma. The tumor was unresectable because of portal vein thrombosis, so the patient was started on an immunotherapy clinical trial with pembrolizumab and bavituximab. He was not a candidate for yttrium-90 radioembolization because of an anterioportal shunt that could not be adequately embolized, so he received palliative external beam radiotherapy. However, the patient continued to experience refractory hypoglycemic episodes, which necessitated hourly waking by his family to encourage him to eat carbohydrates, despite the addition of prednisone 30 mg daily, 1 month after the diagnosis of HCC. He was readmitted to the hospital for glucose optimization.
During hospitalization, the patient required a titratable dextrose 50% infusion, 37.5 g of dextrose gel orally every 3 hours, and frequent meals for management of his hypoglycemia. He was started on combination medical therapy with diazoxide, prednisolone 20 mg twice daily, and short-acting octreotide 100 mg subcutaneously every 8 hours. Despite that, the patient remained in the hospital for 3 weeks, as he promptly developed hypoglycemia when the dextrose infusion was discontinued. Pasireotide LAR was ordered for the patient but could not be given inpatient, so he was discharged to a long-term assisted care facility while remaining on the dextrose infusion.
Shortly after discharge, the patient was started on pasireotide 40 mg IM every 4 weeks. Within 1 week, he was weaned off the dextrose infusion. Three weeks later, at his endocrine follow-up appointment, his Dexcom G6 CGM data revealed markedly improved glycemic control, with a mean sensor glucose of 121 mg/dL, 8% of readings below 70 mg/dL, and only 2% of readings below 54 mg/dL (Fig. 2B). Unfortunately, the patient’s HCC progressed despite the use of sorafenib and later nivolumab, and he died of shock and decompensated liver failure 5 months after his initial HCC diagnosis.
Discussion
Medications that are currently approved for refractory, tumor-induced hypoglycemia are generally of limited efficacy and tolerability [5]. In the cases described here, each patient with tumor-induced hypoglycemia was started on guideline-recommended medications for the treatment of hypoglycemia (ie, diazoxide, glucocorticoids, and/or octreotide) without attenuation of their refractory hypoglycemia. Then, pasireotide was substituted with marked improvement of hypoglycemia in all cases within 1 month of administration.
Pasireotide, like other somatostatin analogues, exerts its biologic effect by binding to somatostatin receptors (SSTRs). There are 5 somatostatin receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) distributed heterogeneously throughout the body. Once stimulated, somatostatin is a potent inhibitor of endocrine and exocrine hormonal release in humans [11]. Inhibition of insulin and glucagon secretion is primarily mediated by SSTR2, whereas insulin secretion is predominantly mediated via SSTR5 [12]. Neuroendocrine tumors largely express somatostatin receptors, with SSTR2 and SSTR5 expression shown in 70% of insulinomas [13]. Pasireotide, a second-generation SSTR ligand, has a 30- to 40-fold higher binding affinity for SSTR5 than the first-generation SSTR ligand octreotide, which accounts for the former’s advantage in treating hypoglycemia [14]. The hyperglycemia effect of pasireotide is related to a decrease in insulin secretion and incretin hormone response without change to hepatic or peripheral insulin sensitivity [15]. Although octreotide has been shown to improve hypoglycemia in two-thirds of insulinoma patients in one study [16], there are insufficient data for the use of pasireotide in insulinoma patients to calculate a treatment response. However, pasireotide induces a potent hyperglycemic effect in its currently indicated uses for Cushing disease and acromegaly: Seventy-three percent of Cushing disease patients [17] and 57% of acromegaly patients [18] developed hyperglycemia-related adverse events while on pasireotide. Pasireotide-induced hyperglycemia has been shown to respond to vildagliptin and liraglutide therapy [19].
Additionally, pasireotide has been shown to have antiproliferative effects comparable to the more conventionally used somatostatin analogues octreotide and lanreotide in the treatment of advanced neuroendocrine tumors [20].
Treatment of NICTH involves tumor resection or the use of alternate antitumor modalities when resection is not possible. While the use of pasireotide in the management of NICTH has not been previously described, high-dosed octreotide given as continuous infusion following uptake of 111In-labeled octreotide by a solitary fibrous, pleural tumor was not effective in suppressing big IGF-2 production or improving hypoglycemia [21]. In a separate case, the mechanism of refractory hypoglycemia due to NICTH from an intra-abdominal hemagiopericytoma was attributed to muscle tissue uptake of glucose mediated by IGF-2; during somatostatin treatment, big IGF-2 levels decreased modestly but could not adequately control hypoglycemia without the simultaneous infusion of exogenous glucose [22]. The effect of pasireotide therapy on big IGF-2 levels has not been described previously, though a potent lowering of big IGF-2 levels, potentially through SSTR5 interaction, could account for the hyperglycemic effect noted in our patient with hepatocellular carcinoma. Hepatocellular carcinoma tumor cells have been shown previously to express a high proportion of SSTR5 [23]. However, the treatment of advanced hepatocellular carcinoma with somatostatin analogues has shown variable response [24]. A recent phase 2 trial of pasireotide LAR in patients with unresectable HCC showed limited antitumor benefit; however, 30% of participants developed grade 3 hyperglycemia, and 5% of participants developed grade 4 hyperglycemia [25].
Despite the presumed advantage of pasireotide in treating tumor-induced hypoglycemia, only a single case, besides our own, has been published using pasireotide for the treatment of malignant insulinoma. Tirosh et al found that pasireotide LAR was more effective in treating refractory hypoglycemia compared with lanreotide in a patient with malignant insulinoma with hepatic metastases [26]. This manuscript adds to the literature by outlining 3 cases of refractory, tumor-induced hypoglycemia—due to an occult insulinoma, malignant insulinoma, and NICTH from HCC—that all failed to respond to conventional medical therapy, and for which pasireotide caused rapid, dramatic improvement in glucose levels (resolving the hypoglycemia completely in the first 2 cases). Two of our cases—pasireotide for treating hypoglycemia in occult insulinoma and NICTH—are not previously reported in the literature.
In conclusion, we recommend that pasireotide be considered for the treatment of tumor-induced hypoglycemia in 3 settings. First, in a patient with insulinoma who is either not a surgical candidate or who decides against surgery, and in whom hypoglycemia persists despite conventional medical therapy. Second, in the patient with NICTH for whom tumor resection is not possible and adjunctive medical therapy is unhelpful. Third, during the COVID-19 pandemic, there is added concern on the part of providers and patients, both from an infectious disease and resource use standpoint, in terms of admitting tumor-induced hypoglycemia patients for the purposes of diagnosis, localization, and/or surgical management. In such case, there may be a role for temporary pasireotide therapy as a “bridging” technique until definitive diagnostic and therapeutic strategies can be implemented. In summary, given its potent antihypoglycemic and antitumor properties, pasireotide is a reasonable choice for the treatment of tumor-induced hypoglycemia, though additional investigation is warranted.
Abbreviations
COVID-19 2019 novel coronavirus
GH growth hormone
HbA1c glycated hemoglobin
HCC hepatocellular carcinoma
IGF insulin-like growth factor
LAR long-acting release
NICTH non-islet cell tumor hypoglycemia
SSTR somatostatin receptor
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Recovered | ReactionOutcome | CC BY-NC-ND | 33294765 | 20,443,328 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Disease progression'. | Molecularly guided treatment of metastatic parotid gland carcinoma in adults.
BACKGROUND
Advanced therapy-refractory parotid gland carcinomas have a poor prognosis with limited therapy options. We used molecular profiling to offer molecular guided therapies to patients with advanced metastatic parotid gland malignancies.
METHODS
In this retrospective analysis we describe the molecular profiling of ten patients diagnosed with therapy-refractory metastatic parotid gland malignancies.
RESULTS
We identified seven genetic aberrations in five patients: two mutations in CDKN2A and one mutation in APC, ATM, TP53, SMARCB1 and FGFR1, respectively. No mutations were detected in five patients. The IHC demonstrated frequent expressions of EGFR and p‑mTOR, as well as PTEN in eight patients. For four fifths (n = 8) of the patients, a targeted therapy was suggested. Eventually, three patients received the targeted therapy recommendation and one patient achieved stable disease for 14 months.
CONCLUSIONS
A total of eight therapy recommendations were provided. Based on our observations, molecular-guided therapies may be a feasible treatment approach for this rare disease entity.
Introduction
Salivary gland carcinomas (SGC) comprise rare heterogeneous malignancies that account for only 5% of all head and neck cancers. The SGCs are classified into 24 subtypes according to the World Health Organization (WHO) definition. Likewise, the tumor biology and prognosis of SGCs markedly differ between histological types [1–4]. Among these glands, most malignancies occur in the parotid gland. The parotid gland carcinoma (PGC) is a relatively rare cancer, making up only 0.3% of all cancers combined [5]. The PGC with distant metastases, mainly in the lungs and bones, has a dismal median survival prognosis of 7.3 months despite therapeutic efforts [6].
The mainstay of treatment is complete surgical resection followed by postoperative radiotherapy (depending on the subtype and risk features). In surgical interventions, complete excision of the PGC is carried out with preservation of the functioning facial nerve, provided there is no tumor invasion. Systemic chemotherapy is generally indicated for patients with recurrent and/or metastatic PGC [5, 7–9].
The most common histological subtype in primary PGC is mucoepidermoid carcinoma (MEC) [10, 11]. Given the rarity of this disease, there are, apart from parotidectomy and radiotherapy, few well-established therapy standards for how to treat patients with progressive stage IV PGC [7].
There has recently been an effort to individualize therapy options in cancer diseases. In some instances, tailored therapy attempts with immunotherapeutics or tyrosine kinase inhibitors are used, e.g., trastuzumab in HER2-positive breast cancer or gastric cancer, imatinib in Philadelphia chromosome-positive chronic myeloid leukemia (Ph + CML), BRAF-directed therapy with vemurafenib or dabrafenib/trametinib in melanoma [12–14].
Emerging novel agents, such as the profiling of tumor molecular alterations and mutations as well as the identification of druggable targets and the ground-breaking pilot trial by von Hoff et al. have ushered in a new era of medicine; this approach has received many titles, such as individualized, stratified, tailored, or precision cancer medicine [15]. The main rationale of PCM is to match a therapeutic agent to its corresponding target for precise tailored therapy fitting a specific patient, aiming to achieve a deep durable and sustainable response without damaging healthy cells and tissues. This matches the tailored “therapeutic dress” to the patient [16].
We conducted a retrospective subgroup analysis of our precision molecular register, exclusively focusing on patients with progressive PGC with no available standard treatment options. These patients had been enrolled and whose tumors had been profiled in our special PCM platform. We sought to map the molecular profiles of advanced, relapsed and therapy-refractory PGC to evaluate whether there are any aberrations that can be targeted by a tailored therapy.
Material and methods
Ethics, consent and permission
The study was conducted in accordance with the International Conference on Harmonization E6 requirements for good clinical practice and with the ethical principles outlined in the Declaration of Helsinki. All patients had to provide written informed consent before inclusion in our PCM platform. Furthermore, the institutional ethics committee has also approved this subanalysis (Nr. 1039/2017).
Patients and design of the precision medicine platform
Patients with PGC who had progressed through all standard treatment options were eligible for inclusion in our platform for precision medicine, provided archival tissue samples were available. Patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Our platform for precision medicine is not a clinical trial, but intends to provide the possibility of a targeted therapy to patients where no active anti-tumor treatment is available.
Tissue samples
Formalin-fixed, paraffin-embedded tissue from patients with advanced PGC that were refractory to all available standard treatment lines were sent to or retrieved from the archive of the Department of Pathology.
Cancer gene panel sequencing
DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany) and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA). The panel includes driver mutations, oncogenes, tumor suppressor genes, and gene fusions. See supplementary information for complete list of the gene panel. The Oncomine Comprehensive Assay v3 was optimized for sequencing on an Ion Personal Genome Machine System (Thermo Fisher Scientific). The generated sequencing data were afterwards analyzed with the help of the Ion Reporter Software (Thermo Scientific Fisher). We referred to BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM and 1000 genomes for variant calling and classification. The variants were classified according to a five-tier system comprised of the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. This classification was based on the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.
Immunohistochemistry
The IHC was performed using 2‑μm-thin tissue sections read by a Ventana Benchmark Ultra stainer (Ventana, Tucson, AZ, USA). The following antibodies were applied: anaplastic lymphoma kinase (ALK, clone 1A4; Zytomed, Berlin, Germany), CD20 (clone L26; Dako Omnis from Agilent Technologies, Santa Clara, CA, USA), CD30 (clone BerH2; Agilent Technologies, Vienna, Austria), epidermal growth factor receptor (EGFR, clone 3C6; Ventana), estrogen receptor (clone SP1; Ventana), human epidermal growth factor receptor 2 (HER2, clone 4B5; Ventana), HER3 (clone SP71; Abcam, Cambridge, UK), C‑kit receptor (KIT, clone 9.7; Ventana), MET (clone SP44; Ventana), NTRK (clone EPR17341, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR, clone 49F9; Cell Signaling Technology, Danvers, MA, USA), platelet-derived growth factor alpha (PDGFRA, rabbit polyclonal; Thermo Fisher Scientific), PDGFRB (clone 28E1, Cell Signaling Technology), programmed death-ligand 1 (PD-L1, clone E1L3N; Cell Signaling Technology), progesterone receptor (clone 1E2; Ventana), phosphatase and tensin homolog (PTEN, clone Y184; Abcam) and ROS1 (clone D4D6; Cell Signaling Technology).
To assess the immunostaining intensity for the antigens EGFR, p-mTOR, PDGFRA, PDGFRB and PTEN, a combinative semiquantitative score for immunohistochemistry was used. The immunostaining intensity was graded from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong). To calculate the score, the intensity grade was multiplied by the percentage of corresponding positive cells: (maximum 300) = (% negative × 0) + (% weak × 1) + (% moderate × 2) + (% strong × 3).
The immunohistochemical staining intensity for HER2 was scored from 0 to 3+ (0 = negative, 1+ = negative, 2+ = positive, 3+ = positive) pursuant to the scoring guidelines of the Dako HercepTestR from the company Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA). In the case of HER2 2+, a further test with HER2 in situ hybridization was performed to verify amplification of the HER2 gene.
Estrogen receptor and progesterone receptor staining were graded according to the Allred scoring system [17] from 0 to 8 and MET staining was scored from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong).
For PD-L1, the tumor proportion score was calculated, which is the percentage of viable malignant cells showing membrane staining.
Staining for ALK, CD30, CD20 and ROS1 was classified as positive or negative based on the percentage of reactive tumor cells but without graduation of the staining intensity. In ALK or ROS1 positive cases, the presence of a possible gene translocation was evaluated by fluorescence in situ hybridization (FISH).
All antibodies used in this study were validated and approved at the clinical institute of pathology and are used in routine IHC staining for clinical purposes. The antibodies have been validated, by proper positive and negative tissue controls and by non-IHC methods, such as immunoblotting and flow cytometry, to detect the respective epitope of the antigens. For the control, the use of the antibodies was optimized in terms of intensity, concentration, signal/noise ratio, incubation times and blocking. The negative control was conducted by omitting the primary antibody and by substitution of isotype-specific antibody and serum at the exact same dilution and laboratory conditions as the primary antibody to preclude unspecific binding.
For the positive control, the antibodies were shown not to cross-react with closely related molecules of the target epitope.
Fluorescence in situ hybridization
The FISH was performed with 4‑μm-thick formalin-fixed, paraffin-embedded tissue sections. The following FISH probes were employed: ALK (2p23.1; Abbott, Abbott Park, IL, USA), RET (10q11; Kreatech, Berlin, Germany), PTEN (10q23.31)/centromere 10, and ROS1 (ZytoVision, Bremerhaven, Germany), 200 cell nuclei per tumor were evaluated. The cut-off level for an aberrant ALK, RET, and ROS1 FISH was ≥15% of cells with a split-apart signal. The PTEN FISH was considered positive for PTEN gene loss with ≥30% of cells with only one or no PTEN signals. A chromosome 10 centromere FISH probe served as a control for ploidy of chromosome 10.
Multidisciplinary boards (molecular tumor boards for PCM)
After thorough examination of the molecular profile of each tumor sample by a qualified and competent molecular pathologist, the results and findings were reviewed in multidisciplinary tumor boards (MTB) that were held every other week. Members of the board included molecular pathologists, radiologists, clinical oncologists, biostatisticians, and basic scientists. The MTB recommended the targeted therapy based on the specific molecular profile of each patient. The targeted therapies included tyrosine kinase inhibitors, checkpoint inhibitors (e.g. anti-PD-L1 monoclonal antibodies), and growth factor receptor antibodies with or without endocrine therapy. The treatment recommendations by the MTB were prioritized depending on the level of evidence from high to low according to phase III to phase I trials.
If more than one druggable molecular aberration was identified, the MTB recommended a therapy regimen to target as many molecular aberrations as possible, with special consideration given to the toxicity profile of each antitumor agent and their potential interactions. Since all patients were given all available standard treatment options for their cancer disease prior to their inclusion in our PCM platform, nearly all targeted agents were suggested as off-label use. If the tumor profile and the clinical characteristics of a patient met the requirements of a clinical trial for targeted therapies that was conducted in our cancer center, patients were preferentially asked if they wanted to participate in this trial.
Descriptive statistics
For data description, we used measures of central tendency including the mean and median. We also used the method of frequency distribution to delineate the characteristics of the PGC patients.
Results
All ten patients diagnosed with progressive primary PGC were included in this analysis from our platform for precision medicine that has so far profiled over 600 patients with various advanced solid tumors. All PGC patients were Europeans. Five men and five women were diagnosed with five different histological subtypes of primary PGC. The subtypes were acinic cell carcinoma (n = 1), adenocarcinoma NOS (n = 3), adenoid cystic carcinoma (n = 3), carcinoma ex pleomorphic adenoma (n = 1), and primary squamous cell carcinoma (n = 2). The primary tumor location was the right side in six patients (60%) and the left side in four patients (40%).
At the time of molecular profiling, all patients had an advanced, therapy-refractory and relapsed PGC in stage IV with distant metastases, mainly in the bones and lungs. The whole cohort had undergone parotidectomy and radiation therapy. Four patients had also received prior chemotherapy: two patients were treated with carboplatin and paclitaxel, one patient received cisplatin and cetuximab, and another patient was given a CAP regimen consisting of cyclophosphamid, doxorubicin (trade name Adriamycin) and a platinum-based agent (usually cisplatin).
The median age at the time of initial diagnosis was 59.5 years, ranging from 27 to 82 years, and the median age at the time of molecular profiling was 63 years, ranging from 37 to 83 years (Table 1).Table 1 Patient characteristics (N = 10)
Patient characteristics Number
Median age at first diagnosis (years) 59.5
Median age at molecular profiling (years) 63
Men 5
Women 5
Histological subtypes of parotid gland carcinoma 5
Caucasian 10
Relapsed disease 10
Stage IV 10
Parotid gland carcinoma on the right side 6
Parotid gland carcinoma on the left side 4
Therapy recommendations 8
Of the ten tissue samples, five were from metastatic sites and five from the primary site.
In total, we identified seven molecular aberrations in five patients: two mutations in CDKN2A and one mutation in each of APC, ATM, TP53, SMARCB1, and FGFR1. No mutations were detected in five patients.
Expression of EGFR, p-mTOR and PTEN was detected by IHC in eight patients. The EGFR median score was 120, and 3 patients had a high EGFR score of between 200 and 300. The expression of p-mTOR was lower with a median score of 70, and 2 patients had a high p-mTOR score of between 200 and 300. Expression of MET and PDGFRA was detectable in six and five samples, respectively. MET expression was weak in four patients and moderate in one patient. One sample exhibited a strong MET expression. Less common expressions were observed for KIT and AR which were observed in three and two patients, respectively. The KIT expression was found to be weak in two samples and moderate in one sample, AR was moderately expressed in both patients with adenocarcinoma.
IHC and FISH were not performed in one patient due to insufficient tumor material.
For eight of the ten patients, a targeted therapy was suggested based on their individual molecular profile (Table 1). Androgen deprivation therapy (ADT), crizotinib, and cetuximab each were offered in two cases and imatinib and sunitinib were proposed in one case. We refer here to Tables 2 and 3 for the rationale of the therapy suggestions.Table 2 Rational for therapy recommendations
Therapeutic agent (trade name) Targets Overview of current FDA approval in different entities Overview of current EMA approval in different entities
Cetuximab (Erbitux)
(n = 2)
EGFR CRC, HNSCC CRC, HNSCC
Crizotinib (Xalkori)
(n = 2)
ALK, ROS1
MET overexpression
ALK or ROS1 positive NSCLC ALK or ROS1 positive NSCLC
Imatinib (Gleevec)
(n = 1)
PDGFR, KIT, Bcr/Abl Ph + CML, KIT + GIST, MDS/MPD associated with PDGFR, Ph + ALL Ph + CML, KIT+ GIST, MDS/MPD associated with PDGFR, Ph + ALL
Sunitinib (Sutent)
(n = 1)
PDGFR, KIT, VEGFR, RET, FLT3 RCC, PDAC, GIST RCC, PDAC, GIST
ABL Abelson murine leukemia viral oncogene homolog 1, ALK Anaplastic lymphoma kinase, ALL acute lymphatic leukemia, BCR breakpoint cluster region, CML chronic myleloid leukemia, CRC colorectal cancer, EGFR epidermal growth factor receptor, EMA European Medicines Agency, FDA Food and Drug Administration, FLT3 fms like tyrosine kinase 3, GIST gastrointestinal stromal tumor, HNSCC Head and neck squamous cell carcinoma, MDS/MPD myelodysplastic syndrome/ myeloproliferative disorder, NSCLC Non-small cell lung carcinoma, PDAC pancreatic ductal adenocarcinoma, PDGFR platelet derived growth factor receptor, Ph+ Philadelphia chromosome positive, p‑mTOR phosphorylated mammalian target of rapamycin, RCC renal cell carcinoma, RET rearranged during transfection, TP53 tumor protein 53, VEGFR vascular endothelial growth factor
Table 3 Detailed characteristics of the PGC patients (n = 10)
Patient number, gender and age Histological subtype and Stage and side Site of metastasis Tissue tested Detected mutations by NGS IHC Therapy recommendation
1
Female
61 years
Acinic cell carcinoma IV°
Right
Lung Metastatic No mutation detected Not done (due to insufficient tissue material) No recommendation
2
Male
83 years
Adenocarcinoma IV°
Right
Liver lung Metastatic
(liver)
No mutation detected EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 3+,
AR 2+
Androgen deprivation therapy
3
Female
37 years
Adenocarcinoma IV°
Right
Bone Metastatic No mutation detected EGFR 2+,
KIT 2+,
PTEN 2+,
p‑mTOR 3+
No recommendation
4
Male
71 years
Adenocarcinoma IV°
Left
Bone Primary No mutation detected EGFR 3+,
PTEN 1+,
p‑mTOR 1+,
AR 2+
Androgen deprivation therapy
5
Male
46 years
Adenoid cystic carcinoma IV°
Left
Lung Metastatic No mutation detected KIT 1+,
MET 3+,
PDGFR 1+,
PTEN 1+,
p‑mTOR 2+
Crizotinib
6
Female
56 years
Adenoid cystic carcinoma IV°
Right
Lung Primary ATM: exon 32
c.C9142G (p.Leu3048Val)
EGFR 3+,
MET 1+,
p‑mTOR 1+,
PTEN 1+
Cetuximab
7
Male
47 years
Adenoid cystic carcinoma IV°
Right
Lung Primary APC: exon 16
c.T3920A (p.I1307K)
KIT 1+,
EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 1+
Imatinib
8
Male
65 years
Carcinoma ex pleomorphic adenoma IV°
Right
Lung,
Brain
Metastatic
(lung)
TP53 (exon 7):
c.C742T
(p.R248W)
EGFR 1+,
MET 1+,
PDGFRA 2+,
PTEN 1+,
p‑mTOR 1+
Sunitinib
9
Female
67 years
Primary squamous cell carcinoma IV°
Left
Bone,
lung
Primary CDKN2A (exon 2): c.151_155delGTCT (p.V51 fs);
FGFR1 (exon 5):
c.478_480delGAT (p.Asp.160del);
SMARCB1 (exon 9):
c.G1130A (p.R3677H)
EGFR 3+,
PTEN 2+
Cetuximab
10
Female
72 years
Primary squamous cell carcinoma IV°
Left
Lung Primary CDKN2A (exon 2):
c.C341T,
(p.P114L)
EGFR 1+,
MET 3+,
PDGFRA 1+,
p‑mTOR 1+
Crizotinib
Values in parentheses indicate the immunohistochemical score that was calculated as mentioned in the “Materials and methods” section
APC adenomatous polyposis coli, AR androgen receptor, CDKN2A cyclin-dependent kinase inhibitor 2A, EGFR epidermal growth factor receptor, FiSH fluorescence in situ hybridization, PDGFR platelet derived growth factor receptor, p‑mTOR phosphorylated mammalian target of rapamycin, PTEN phosphatase and tensin homolog, TP53 tumor protein 53, IHC immunohistochemistry, NGS next-generation sequencing
The median turnaround time from the initiation of molecular profiling to therapy initiation was 43 days.
Eventually, three patients received the targeted therapy. One male patient with an adenocarcinoma was administered bicalutamide as ADT but died because of disease progression before restaging was performed. The second patient with a carcinoma ex pleomorphic adenoma received sunitinib 50 mg orally once daily combined with docetaxel every third week but did not respond to this therapy regimen and experienced progressive disease. The third patient had an adenoid cystic carcinoma and was given imatinib 400 mg orally once daily. He achieved a stable disease for 14 months and tolerated the therapy without any treatment-related adverse events.
Discussion
To our knowledge, this is the first study of individual genomic alterations that have been translated into concrete tailored therapy recommendations in a group of patients with exclusively recurrent, progressive, and therapy-refractory PGC in stage IV in a real-world setting. None of these patients had the histological subtype mucoepidermoid carcinoma (MEC); instead, they had rarer subtypes, making this subgroup analysis even more valuable and unique.
In this retrospective single center subgroup analysis, we exclusively present the molecular profile of all ten patients with PGC. Their disease was relapsed, therapy-refractory and advanced. Tumor tissue was obtained from all patients and characterized regarding molecular profiles. Subsequently, the genomic information of the patients was discussed in a multidisciplinary tumor board (MTB) for PCM to evaluate the possibility of a genomic-based therapy concept that is independent of the tumor’s histological classification (tissue-agnostic drugs).
Tumor samples harbored mutations in APC, ATM, CDKN2A, FGFR1, SMARCB1, and TP53. The IHC revealed expressions of EGFR and p-mTOR as well as PTEN in eight patients.
Therapeutic options recommended were ADT, cetuximab, crizotinib, sunitinib, and imatinib. Two patients with AR expression were offered ADT to control the disease. Two patients with strong MET expression were suggested crizotinib as a tailored therapy. For two patients with high EGFR expression cetuximab was recommended. Imatinib was considered in one patient due to expression of KIT and PDGFRA. Sunitinib was proposed to one patient because of PDGFRA overexpression. A treatment recommendation was derived for eight patients from the MTB. The drugs were carefully selected for an individualized treatment with special respect to the patient’s clinical and treatment history and concomitant therapies and comorbidities. Interestingly, all these recommendations were based on the protein expressions obtained by immunohistochemistry. Thus, our analysis underscores the clinical relevance of immunohistochemistry in precision medicine.
Eventually, three patients received the targeted therapy. One patient died before restaging was performed. The second patient received sunitinib and did not respond. Imatinib was applied to the third patient who experienced a stable disease for 14 months. Although this analysis showed that PCM is implementable in daily clinical routine, only one patient had a clinical benefit from this therapy approach. One reason may be the turnaround time: a shorter turnaround time may help to start the targeted therapy earlier and to control the cancer disease. Liquid biopsy may be a viable option to reduce the turnaround time, to monitor the disease and to assess the therapy response. Another reason may be the complexity of PGC. The major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguishes over 20 types of malignant salivary gland tumors [4].
The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance.
The observed genomic aberrations and overexpression of AR, KIT, and EGFR, PTEN, p-mTOR, and PDGFRA in PGC in this analysis are in keeping with previous studies [18–33]. The rationale for the therapy recommendation with ADT was corroborated by a study by Boon et al. They studied the application of ADT in 35 patients with androgen receptor-positive advanced salivary duct carcinoma, which lead to a median overall survival (OS) of 17 months versus 5 months in 43 patients receiving best supportive care [34].
The overexpression of MET was seen in all three patients with adenoid cystic carcinoma and is in line with other studies [35]; however, to our knowledge, this is the first report of an overexpression also in carcinoma ex pleomorphic adenoma and primary squamous cell carcinoma. Crizotinib was offered as a molecularly driven treatment approach. Its clinical efficacy in salivary gland cancers has not yet been described in clinical trials.
Only one study has used molecular profiling to offer an individualized therapy in patients with metastatic salivary gland adenoid cystic carcinoma (ACC). They enrolled a limited 14 patients, of whom 11 actually received the recommended treatment. The investigators reported the clinical benefit of molecularly guided treatment [36]. Imatinib and sunitinib are tyrosine kinase inhibitors that were offered, each in one case, as an alternative therapy in the case of overexpression in PDGFRA/B or KIT. The data pertaining to the use of imatinib in salivary gland cancer are contradictory and unclear. According to two phase II trials that applied imatinib in patients with KIT-positive adenoid cystic cancers of salivary glands, imatinib was not of significant clinical benefit and the best observed response was a stable disease (SD).
As a limitation, however, it should be noted that only ACC was studied, and PDGFR expression of the tumor tissue was not evaluated [37, 38]. In contrast, another phase II trial tested imatinib in 15 patients with ACC of salivary glands and concluded that imatinib was of clinical benefit because it achieved a partial response (PR) in two patients and a SD in five other patients [39]. Likewise, in another study, imatinib achieved significant regression of initially unresectable ACC of salivary glands in two patients, making them eligible for a salvage resection [40].
Similar to imatinib, sunitinib was also tested in a phase II trial in 13 patients with ACC of salivary glands and 11 of these achieved stable disease; however, the investigators did not test the patients’ tumors for KIT or PDGFR expression [41]. Dasatinib is another tyrosine kinase inhibitor that was investigated in a phase II trial for patients with recurrent or metastatic KIT expressing ACC and for nonadenoid cystic malignant salivary tumors. It achieved only one PR in a patient with ACC and the experimental treatment demonstrated no activity in non-ACC salivary gland cancer [42].
In another phase II trial, axitinib was applied in 33 patients with unresectable ACC. Ho et al. reported that axitinib achieved a PR in 3 patients and a SD in 25 patients. The median progression-free survival (PFS) was 5.7 months [43]. Overexpression of EGFR was often observed in salivary gland cancers and provides a solid and sound rationale for the administration of cetuximab [26, 27]. Its clinical efficacy was examined by Locati et al. in 2009 in salivary gland carcinomas, and they reported a clinical benefit rate of 50% [44].
Notably, we identified p-mTOR overexpression in eight patients; however, we did not consider p-mTOR inhibition with everolimus because of the low evidence for clinical efficacy.
Despite great research efforts and the investigated agents in PGC and other salivary gland cancers, progressive recurrent PGC has a dismal prognosis, and because of the rarity of the disease, well-established therapeutic options are scarce.
Great strides in the in-depth analysis of the vast genetic and epigenetic landscape of salivary gland cancers have been made in recent years; however, the PCM approach remains in its infancy when it comes to implementing novel individualized therapeutic strategies and concepts for this malignancy [33, 45, 46].
It is challenging to classify and prioritize the plethora of reported genetic alterations and epigenetic changes to identify actionable targets and to choose adequate tailored therapeutic measures. Thus, the roles of most identified alterations are undefined regarding pathogenesis, therapeutic consequences, and implications [47].
Another major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguished over 20 types of malignant salivary gland tumors [4]. The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance. In light of the complexity of PGC, molecularly driven clinical trials in PCM have to be designed as basket and umbrella trials that take into account the diversity of this malignancy for a better outcome.
Conclusion
This analysis clearly shows that molecular profiling from tumor samples of patients with advanced, heavily pretreated and therapy-refractory PGC in stage IV is feasible and results in meaningful and rational therapy recommendations and strategies; however, the complex tumor biology, heterogeneity, and extreme rarity remain unique challenges for the management of PGC and need to be addressed by further studies seeking a better understanding of this malignancy. In rare diseases such as PGC where randomized trials cannot be performed easily, molecularly driven treatment approaches and strategies may be particularly useful tools and viable options.
Abbreviations
ADTAndrogen deprivation therapy
AKTAlpha serine/threonine-protein kinase
ALKAnaplastic lymphoma kinase
ALLAcute lymphoblastic leukemia
APCAdenomatous polyposis coli
ARAndrogen receptor
ATMAtaxia telangiectasia mutated
BAP1BRCA1 associated protein‑1
BRAFB‑rapidly accelerated fibrosarcoma
BRCABreast cancer 1
CDKN2ACyclin-dependent kinase inhibitor 2A
CMLChronic myeloid leukemia
CRCColorectal cancer
DCRDisease control rate
ECOGEastern Cooperative Oncology Group
EGFREpidermal growth factor receptor
EMAEuropean Medicines Agency
FDAFood and Drug Administration
FGFRFibroblast growth factor receptor
FiSHFluorescence in situ hybridization
FLT3Fms like tyrosine kinase 3
GISTGastrointestinal stromal tumor
HER2Human epidermal growth factor receptor 2
HNSCCHead and neck squamous cell carcinoma
HLHodgkin lymphoma
IDHIsocitrate dehydrogenase 1
IHCImmunohistochemistry
lossPTENLoss of phosphatase and tensin homolog
MDS/MPDMyelodysplastic syndrome/myeloproliferative disorder
MECMucoepidermoid carcinoma
MTBMultidisciplinary tumor board
mTORMammalian target of rapamycin
NF1Neurofibromin 1
NOSNot otherwise specified
NSCLCNon-small cell lung carcinoma
PARPPoly [ADP-ribose] polymerase 1
PCMPrecision cancer medicine
PDACPancreatic ductal adenocarcinoma
PDGFRPlatelet-derived growth factor receptor
PD-L1Programmed death-ligand 1
PGCParotid gland carcinoma
Ph+Philadelphia chromosome positive
PIK3CBPhosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit beta
p-mTORphosphorylated Mammalian target of rapamycin
PRPartial response
PTENPhosphatase and tensin homolog
PTPN11Protein phosphatase non-receptor type 11
RB1Retinoblastoma 1
RCCRenal cell carcinoma
SDStable disease
SGCSalivary gland carcinomas
SHHSonic hedgehog
SMARCB1SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1
SMOSmoothened
STK11Serine/threonine kinase 11
TP53Tumor protein 53
TRKNeurotrophin receptor kinases
VEGFRVascular endothelial growth factor
VHLvon Hippel-Lindau
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Funding
This research did not receive any grants or funding.
Funding
Open access funding provided by Medical University of Vienna.
Conflict of interest
H. Taghizadeh, L. Müllauer, R.M. Mader, T. Füreder, and G.W. Prager declare that they have no competing interests. | DOCETAXEL, SUNITINIB | DrugsGivenReaction | CC BY | 33296026 | 19,253,223 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'. | Molecularly guided treatment of metastatic parotid gland carcinoma in adults.
BACKGROUND
Advanced therapy-refractory parotid gland carcinomas have a poor prognosis with limited therapy options. We used molecular profiling to offer molecular guided therapies to patients with advanced metastatic parotid gland malignancies.
METHODS
In this retrospective analysis we describe the molecular profiling of ten patients diagnosed with therapy-refractory metastatic parotid gland malignancies.
RESULTS
We identified seven genetic aberrations in five patients: two mutations in CDKN2A and one mutation in APC, ATM, TP53, SMARCB1 and FGFR1, respectively. No mutations were detected in five patients. The IHC demonstrated frequent expressions of EGFR and p‑mTOR, as well as PTEN in eight patients. For four fifths (n = 8) of the patients, a targeted therapy was suggested. Eventually, three patients received the targeted therapy recommendation and one patient achieved stable disease for 14 months.
CONCLUSIONS
A total of eight therapy recommendations were provided. Based on our observations, molecular-guided therapies may be a feasible treatment approach for this rare disease entity.
Introduction
Salivary gland carcinomas (SGC) comprise rare heterogeneous malignancies that account for only 5% of all head and neck cancers. The SGCs are classified into 24 subtypes according to the World Health Organization (WHO) definition. Likewise, the tumor biology and prognosis of SGCs markedly differ between histological types [1–4]. Among these glands, most malignancies occur in the parotid gland. The parotid gland carcinoma (PGC) is a relatively rare cancer, making up only 0.3% of all cancers combined [5]. The PGC with distant metastases, mainly in the lungs and bones, has a dismal median survival prognosis of 7.3 months despite therapeutic efforts [6].
The mainstay of treatment is complete surgical resection followed by postoperative radiotherapy (depending on the subtype and risk features). In surgical interventions, complete excision of the PGC is carried out with preservation of the functioning facial nerve, provided there is no tumor invasion. Systemic chemotherapy is generally indicated for patients with recurrent and/or metastatic PGC [5, 7–9].
The most common histological subtype in primary PGC is mucoepidermoid carcinoma (MEC) [10, 11]. Given the rarity of this disease, there are, apart from parotidectomy and radiotherapy, few well-established therapy standards for how to treat patients with progressive stage IV PGC [7].
There has recently been an effort to individualize therapy options in cancer diseases. In some instances, tailored therapy attempts with immunotherapeutics or tyrosine kinase inhibitors are used, e.g., trastuzumab in HER2-positive breast cancer or gastric cancer, imatinib in Philadelphia chromosome-positive chronic myeloid leukemia (Ph + CML), BRAF-directed therapy with vemurafenib or dabrafenib/trametinib in melanoma [12–14].
Emerging novel agents, such as the profiling of tumor molecular alterations and mutations as well as the identification of druggable targets and the ground-breaking pilot trial by von Hoff et al. have ushered in a new era of medicine; this approach has received many titles, such as individualized, stratified, tailored, or precision cancer medicine [15]. The main rationale of PCM is to match a therapeutic agent to its corresponding target for precise tailored therapy fitting a specific patient, aiming to achieve a deep durable and sustainable response without damaging healthy cells and tissues. This matches the tailored “therapeutic dress” to the patient [16].
We conducted a retrospective subgroup analysis of our precision molecular register, exclusively focusing on patients with progressive PGC with no available standard treatment options. These patients had been enrolled and whose tumors had been profiled in our special PCM platform. We sought to map the molecular profiles of advanced, relapsed and therapy-refractory PGC to evaluate whether there are any aberrations that can be targeted by a tailored therapy.
Material and methods
Ethics, consent and permission
The study was conducted in accordance with the International Conference on Harmonization E6 requirements for good clinical practice and with the ethical principles outlined in the Declaration of Helsinki. All patients had to provide written informed consent before inclusion in our PCM platform. Furthermore, the institutional ethics committee has also approved this subanalysis (Nr. 1039/2017).
Patients and design of the precision medicine platform
Patients with PGC who had progressed through all standard treatment options were eligible for inclusion in our platform for precision medicine, provided archival tissue samples were available. Patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Our platform for precision medicine is not a clinical trial, but intends to provide the possibility of a targeted therapy to patients where no active anti-tumor treatment is available.
Tissue samples
Formalin-fixed, paraffin-embedded tissue from patients with advanced PGC that were refractory to all available standard treatment lines were sent to or retrieved from the archive of the Department of Pathology.
Cancer gene panel sequencing
DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany) and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA). The panel includes driver mutations, oncogenes, tumor suppressor genes, and gene fusions. See supplementary information for complete list of the gene panel. The Oncomine Comprehensive Assay v3 was optimized for sequencing on an Ion Personal Genome Machine System (Thermo Fisher Scientific). The generated sequencing data were afterwards analyzed with the help of the Ion Reporter Software (Thermo Scientific Fisher). We referred to BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM and 1000 genomes for variant calling and classification. The variants were classified according to a five-tier system comprised of the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. This classification was based on the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.
Immunohistochemistry
The IHC was performed using 2‑μm-thin tissue sections read by a Ventana Benchmark Ultra stainer (Ventana, Tucson, AZ, USA). The following antibodies were applied: anaplastic lymphoma kinase (ALK, clone 1A4; Zytomed, Berlin, Germany), CD20 (clone L26; Dako Omnis from Agilent Technologies, Santa Clara, CA, USA), CD30 (clone BerH2; Agilent Technologies, Vienna, Austria), epidermal growth factor receptor (EGFR, clone 3C6; Ventana), estrogen receptor (clone SP1; Ventana), human epidermal growth factor receptor 2 (HER2, clone 4B5; Ventana), HER3 (clone SP71; Abcam, Cambridge, UK), C‑kit receptor (KIT, clone 9.7; Ventana), MET (clone SP44; Ventana), NTRK (clone EPR17341, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR, clone 49F9; Cell Signaling Technology, Danvers, MA, USA), platelet-derived growth factor alpha (PDGFRA, rabbit polyclonal; Thermo Fisher Scientific), PDGFRB (clone 28E1, Cell Signaling Technology), programmed death-ligand 1 (PD-L1, clone E1L3N; Cell Signaling Technology), progesterone receptor (clone 1E2; Ventana), phosphatase and tensin homolog (PTEN, clone Y184; Abcam) and ROS1 (clone D4D6; Cell Signaling Technology).
To assess the immunostaining intensity for the antigens EGFR, p-mTOR, PDGFRA, PDGFRB and PTEN, a combinative semiquantitative score for immunohistochemistry was used. The immunostaining intensity was graded from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong). To calculate the score, the intensity grade was multiplied by the percentage of corresponding positive cells: (maximum 300) = (% negative × 0) + (% weak × 1) + (% moderate × 2) + (% strong × 3).
The immunohistochemical staining intensity for HER2 was scored from 0 to 3+ (0 = negative, 1+ = negative, 2+ = positive, 3+ = positive) pursuant to the scoring guidelines of the Dako HercepTestR from the company Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA). In the case of HER2 2+, a further test with HER2 in situ hybridization was performed to verify amplification of the HER2 gene.
Estrogen receptor and progesterone receptor staining were graded according to the Allred scoring system [17] from 0 to 8 and MET staining was scored from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong).
For PD-L1, the tumor proportion score was calculated, which is the percentage of viable malignant cells showing membrane staining.
Staining for ALK, CD30, CD20 and ROS1 was classified as positive or negative based on the percentage of reactive tumor cells but without graduation of the staining intensity. In ALK or ROS1 positive cases, the presence of a possible gene translocation was evaluated by fluorescence in situ hybridization (FISH).
All antibodies used in this study were validated and approved at the clinical institute of pathology and are used in routine IHC staining for clinical purposes. The antibodies have been validated, by proper positive and negative tissue controls and by non-IHC methods, such as immunoblotting and flow cytometry, to detect the respective epitope of the antigens. For the control, the use of the antibodies was optimized in terms of intensity, concentration, signal/noise ratio, incubation times and blocking. The negative control was conducted by omitting the primary antibody and by substitution of isotype-specific antibody and serum at the exact same dilution and laboratory conditions as the primary antibody to preclude unspecific binding.
For the positive control, the antibodies were shown not to cross-react with closely related molecules of the target epitope.
Fluorescence in situ hybridization
The FISH was performed with 4‑μm-thick formalin-fixed, paraffin-embedded tissue sections. The following FISH probes were employed: ALK (2p23.1; Abbott, Abbott Park, IL, USA), RET (10q11; Kreatech, Berlin, Germany), PTEN (10q23.31)/centromere 10, and ROS1 (ZytoVision, Bremerhaven, Germany), 200 cell nuclei per tumor were evaluated. The cut-off level for an aberrant ALK, RET, and ROS1 FISH was ≥15% of cells with a split-apart signal. The PTEN FISH was considered positive for PTEN gene loss with ≥30% of cells with only one or no PTEN signals. A chromosome 10 centromere FISH probe served as a control for ploidy of chromosome 10.
Multidisciplinary boards (molecular tumor boards for PCM)
After thorough examination of the molecular profile of each tumor sample by a qualified and competent molecular pathologist, the results and findings were reviewed in multidisciplinary tumor boards (MTB) that were held every other week. Members of the board included molecular pathologists, radiologists, clinical oncologists, biostatisticians, and basic scientists. The MTB recommended the targeted therapy based on the specific molecular profile of each patient. The targeted therapies included tyrosine kinase inhibitors, checkpoint inhibitors (e.g. anti-PD-L1 monoclonal antibodies), and growth factor receptor antibodies with or without endocrine therapy. The treatment recommendations by the MTB were prioritized depending on the level of evidence from high to low according to phase III to phase I trials.
If more than one druggable molecular aberration was identified, the MTB recommended a therapy regimen to target as many molecular aberrations as possible, with special consideration given to the toxicity profile of each antitumor agent and their potential interactions. Since all patients were given all available standard treatment options for their cancer disease prior to their inclusion in our PCM platform, nearly all targeted agents were suggested as off-label use. If the tumor profile and the clinical characteristics of a patient met the requirements of a clinical trial for targeted therapies that was conducted in our cancer center, patients were preferentially asked if they wanted to participate in this trial.
Descriptive statistics
For data description, we used measures of central tendency including the mean and median. We also used the method of frequency distribution to delineate the characteristics of the PGC patients.
Results
All ten patients diagnosed with progressive primary PGC were included in this analysis from our platform for precision medicine that has so far profiled over 600 patients with various advanced solid tumors. All PGC patients were Europeans. Five men and five women were diagnosed with five different histological subtypes of primary PGC. The subtypes were acinic cell carcinoma (n = 1), adenocarcinoma NOS (n = 3), adenoid cystic carcinoma (n = 3), carcinoma ex pleomorphic adenoma (n = 1), and primary squamous cell carcinoma (n = 2). The primary tumor location was the right side in six patients (60%) and the left side in four patients (40%).
At the time of molecular profiling, all patients had an advanced, therapy-refractory and relapsed PGC in stage IV with distant metastases, mainly in the bones and lungs. The whole cohort had undergone parotidectomy and radiation therapy. Four patients had also received prior chemotherapy: two patients were treated with carboplatin and paclitaxel, one patient received cisplatin and cetuximab, and another patient was given a CAP regimen consisting of cyclophosphamid, doxorubicin (trade name Adriamycin) and a platinum-based agent (usually cisplatin).
The median age at the time of initial diagnosis was 59.5 years, ranging from 27 to 82 years, and the median age at the time of molecular profiling was 63 years, ranging from 37 to 83 years (Table 1).Table 1 Patient characteristics (N = 10)
Patient characteristics Number
Median age at first diagnosis (years) 59.5
Median age at molecular profiling (years) 63
Men 5
Women 5
Histological subtypes of parotid gland carcinoma 5
Caucasian 10
Relapsed disease 10
Stage IV 10
Parotid gland carcinoma on the right side 6
Parotid gland carcinoma on the left side 4
Therapy recommendations 8
Of the ten tissue samples, five were from metastatic sites and five from the primary site.
In total, we identified seven molecular aberrations in five patients: two mutations in CDKN2A and one mutation in each of APC, ATM, TP53, SMARCB1, and FGFR1. No mutations were detected in five patients.
Expression of EGFR, p-mTOR and PTEN was detected by IHC in eight patients. The EGFR median score was 120, and 3 patients had a high EGFR score of between 200 and 300. The expression of p-mTOR was lower with a median score of 70, and 2 patients had a high p-mTOR score of between 200 and 300. Expression of MET and PDGFRA was detectable in six and five samples, respectively. MET expression was weak in four patients and moderate in one patient. One sample exhibited a strong MET expression. Less common expressions were observed for KIT and AR which were observed in three and two patients, respectively. The KIT expression was found to be weak in two samples and moderate in one sample, AR was moderately expressed in both patients with adenocarcinoma.
IHC and FISH were not performed in one patient due to insufficient tumor material.
For eight of the ten patients, a targeted therapy was suggested based on their individual molecular profile (Table 1). Androgen deprivation therapy (ADT), crizotinib, and cetuximab each were offered in two cases and imatinib and sunitinib were proposed in one case. We refer here to Tables 2 and 3 for the rationale of the therapy suggestions.Table 2 Rational for therapy recommendations
Therapeutic agent (trade name) Targets Overview of current FDA approval in different entities Overview of current EMA approval in different entities
Cetuximab (Erbitux)
(n = 2)
EGFR CRC, HNSCC CRC, HNSCC
Crizotinib (Xalkori)
(n = 2)
ALK, ROS1
MET overexpression
ALK or ROS1 positive NSCLC ALK or ROS1 positive NSCLC
Imatinib (Gleevec)
(n = 1)
PDGFR, KIT, Bcr/Abl Ph + CML, KIT + GIST, MDS/MPD associated with PDGFR, Ph + ALL Ph + CML, KIT+ GIST, MDS/MPD associated with PDGFR, Ph + ALL
Sunitinib (Sutent)
(n = 1)
PDGFR, KIT, VEGFR, RET, FLT3 RCC, PDAC, GIST RCC, PDAC, GIST
ABL Abelson murine leukemia viral oncogene homolog 1, ALK Anaplastic lymphoma kinase, ALL acute lymphatic leukemia, BCR breakpoint cluster region, CML chronic myleloid leukemia, CRC colorectal cancer, EGFR epidermal growth factor receptor, EMA European Medicines Agency, FDA Food and Drug Administration, FLT3 fms like tyrosine kinase 3, GIST gastrointestinal stromal tumor, HNSCC Head and neck squamous cell carcinoma, MDS/MPD myelodysplastic syndrome/ myeloproliferative disorder, NSCLC Non-small cell lung carcinoma, PDAC pancreatic ductal adenocarcinoma, PDGFR platelet derived growth factor receptor, Ph+ Philadelphia chromosome positive, p‑mTOR phosphorylated mammalian target of rapamycin, RCC renal cell carcinoma, RET rearranged during transfection, TP53 tumor protein 53, VEGFR vascular endothelial growth factor
Table 3 Detailed characteristics of the PGC patients (n = 10)
Patient number, gender and age Histological subtype and Stage and side Site of metastasis Tissue tested Detected mutations by NGS IHC Therapy recommendation
1
Female
61 years
Acinic cell carcinoma IV°
Right
Lung Metastatic No mutation detected Not done (due to insufficient tissue material) No recommendation
2
Male
83 years
Adenocarcinoma IV°
Right
Liver lung Metastatic
(liver)
No mutation detected EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 3+,
AR 2+
Androgen deprivation therapy
3
Female
37 years
Adenocarcinoma IV°
Right
Bone Metastatic No mutation detected EGFR 2+,
KIT 2+,
PTEN 2+,
p‑mTOR 3+
No recommendation
4
Male
71 years
Adenocarcinoma IV°
Left
Bone Primary No mutation detected EGFR 3+,
PTEN 1+,
p‑mTOR 1+,
AR 2+
Androgen deprivation therapy
5
Male
46 years
Adenoid cystic carcinoma IV°
Left
Lung Metastatic No mutation detected KIT 1+,
MET 3+,
PDGFR 1+,
PTEN 1+,
p‑mTOR 2+
Crizotinib
6
Female
56 years
Adenoid cystic carcinoma IV°
Right
Lung Primary ATM: exon 32
c.C9142G (p.Leu3048Val)
EGFR 3+,
MET 1+,
p‑mTOR 1+,
PTEN 1+
Cetuximab
7
Male
47 years
Adenoid cystic carcinoma IV°
Right
Lung Primary APC: exon 16
c.T3920A (p.I1307K)
KIT 1+,
EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 1+
Imatinib
8
Male
65 years
Carcinoma ex pleomorphic adenoma IV°
Right
Lung,
Brain
Metastatic
(lung)
TP53 (exon 7):
c.C742T
(p.R248W)
EGFR 1+,
MET 1+,
PDGFRA 2+,
PTEN 1+,
p‑mTOR 1+
Sunitinib
9
Female
67 years
Primary squamous cell carcinoma IV°
Left
Bone,
lung
Primary CDKN2A (exon 2): c.151_155delGTCT (p.V51 fs);
FGFR1 (exon 5):
c.478_480delGAT (p.Asp.160del);
SMARCB1 (exon 9):
c.G1130A (p.R3677H)
EGFR 3+,
PTEN 2+
Cetuximab
10
Female
72 years
Primary squamous cell carcinoma IV°
Left
Lung Primary CDKN2A (exon 2):
c.C341T,
(p.P114L)
EGFR 1+,
MET 3+,
PDGFRA 1+,
p‑mTOR 1+
Crizotinib
Values in parentheses indicate the immunohistochemical score that was calculated as mentioned in the “Materials and methods” section
APC adenomatous polyposis coli, AR androgen receptor, CDKN2A cyclin-dependent kinase inhibitor 2A, EGFR epidermal growth factor receptor, FiSH fluorescence in situ hybridization, PDGFR platelet derived growth factor receptor, p‑mTOR phosphorylated mammalian target of rapamycin, PTEN phosphatase and tensin homolog, TP53 tumor protein 53, IHC immunohistochemistry, NGS next-generation sequencing
The median turnaround time from the initiation of molecular profiling to therapy initiation was 43 days.
Eventually, three patients received the targeted therapy. One male patient with an adenocarcinoma was administered bicalutamide as ADT but died because of disease progression before restaging was performed. The second patient with a carcinoma ex pleomorphic adenoma received sunitinib 50 mg orally once daily combined with docetaxel every third week but did not respond to this therapy regimen and experienced progressive disease. The third patient had an adenoid cystic carcinoma and was given imatinib 400 mg orally once daily. He achieved a stable disease for 14 months and tolerated the therapy without any treatment-related adverse events.
Discussion
To our knowledge, this is the first study of individual genomic alterations that have been translated into concrete tailored therapy recommendations in a group of patients with exclusively recurrent, progressive, and therapy-refractory PGC in stage IV in a real-world setting. None of these patients had the histological subtype mucoepidermoid carcinoma (MEC); instead, they had rarer subtypes, making this subgroup analysis even more valuable and unique.
In this retrospective single center subgroup analysis, we exclusively present the molecular profile of all ten patients with PGC. Their disease was relapsed, therapy-refractory and advanced. Tumor tissue was obtained from all patients and characterized regarding molecular profiles. Subsequently, the genomic information of the patients was discussed in a multidisciplinary tumor board (MTB) for PCM to evaluate the possibility of a genomic-based therapy concept that is independent of the tumor’s histological classification (tissue-agnostic drugs).
Tumor samples harbored mutations in APC, ATM, CDKN2A, FGFR1, SMARCB1, and TP53. The IHC revealed expressions of EGFR and p-mTOR as well as PTEN in eight patients.
Therapeutic options recommended were ADT, cetuximab, crizotinib, sunitinib, and imatinib. Two patients with AR expression were offered ADT to control the disease. Two patients with strong MET expression were suggested crizotinib as a tailored therapy. For two patients with high EGFR expression cetuximab was recommended. Imatinib was considered in one patient due to expression of KIT and PDGFRA. Sunitinib was proposed to one patient because of PDGFRA overexpression. A treatment recommendation was derived for eight patients from the MTB. The drugs were carefully selected for an individualized treatment with special respect to the patient’s clinical and treatment history and concomitant therapies and comorbidities. Interestingly, all these recommendations were based on the protein expressions obtained by immunohistochemistry. Thus, our analysis underscores the clinical relevance of immunohistochemistry in precision medicine.
Eventually, three patients received the targeted therapy. One patient died before restaging was performed. The second patient received sunitinib and did not respond. Imatinib was applied to the third patient who experienced a stable disease for 14 months. Although this analysis showed that PCM is implementable in daily clinical routine, only one patient had a clinical benefit from this therapy approach. One reason may be the turnaround time: a shorter turnaround time may help to start the targeted therapy earlier and to control the cancer disease. Liquid biopsy may be a viable option to reduce the turnaround time, to monitor the disease and to assess the therapy response. Another reason may be the complexity of PGC. The major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguishes over 20 types of malignant salivary gland tumors [4].
The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance.
The observed genomic aberrations and overexpression of AR, KIT, and EGFR, PTEN, p-mTOR, and PDGFRA in PGC in this analysis are in keeping with previous studies [18–33]. The rationale for the therapy recommendation with ADT was corroborated by a study by Boon et al. They studied the application of ADT in 35 patients with androgen receptor-positive advanced salivary duct carcinoma, which lead to a median overall survival (OS) of 17 months versus 5 months in 43 patients receiving best supportive care [34].
The overexpression of MET was seen in all three patients with adenoid cystic carcinoma and is in line with other studies [35]; however, to our knowledge, this is the first report of an overexpression also in carcinoma ex pleomorphic adenoma and primary squamous cell carcinoma. Crizotinib was offered as a molecularly driven treatment approach. Its clinical efficacy in salivary gland cancers has not yet been described in clinical trials.
Only one study has used molecular profiling to offer an individualized therapy in patients with metastatic salivary gland adenoid cystic carcinoma (ACC). They enrolled a limited 14 patients, of whom 11 actually received the recommended treatment. The investigators reported the clinical benefit of molecularly guided treatment [36]. Imatinib and sunitinib are tyrosine kinase inhibitors that were offered, each in one case, as an alternative therapy in the case of overexpression in PDGFRA/B or KIT. The data pertaining to the use of imatinib in salivary gland cancer are contradictory and unclear. According to two phase II trials that applied imatinib in patients with KIT-positive adenoid cystic cancers of salivary glands, imatinib was not of significant clinical benefit and the best observed response was a stable disease (SD).
As a limitation, however, it should be noted that only ACC was studied, and PDGFR expression of the tumor tissue was not evaluated [37, 38]. In contrast, another phase II trial tested imatinib in 15 patients with ACC of salivary glands and concluded that imatinib was of clinical benefit because it achieved a partial response (PR) in two patients and a SD in five other patients [39]. Likewise, in another study, imatinib achieved significant regression of initially unresectable ACC of salivary glands in two patients, making them eligible for a salvage resection [40].
Similar to imatinib, sunitinib was also tested in a phase II trial in 13 patients with ACC of salivary glands and 11 of these achieved stable disease; however, the investigators did not test the patients’ tumors for KIT or PDGFR expression [41]. Dasatinib is another tyrosine kinase inhibitor that was investigated in a phase II trial for patients with recurrent or metastatic KIT expressing ACC and for nonadenoid cystic malignant salivary tumors. It achieved only one PR in a patient with ACC and the experimental treatment demonstrated no activity in non-ACC salivary gland cancer [42].
In another phase II trial, axitinib was applied in 33 patients with unresectable ACC. Ho et al. reported that axitinib achieved a PR in 3 patients and a SD in 25 patients. The median progression-free survival (PFS) was 5.7 months [43]. Overexpression of EGFR was often observed in salivary gland cancers and provides a solid and sound rationale for the administration of cetuximab [26, 27]. Its clinical efficacy was examined by Locati et al. in 2009 in salivary gland carcinomas, and they reported a clinical benefit rate of 50% [44].
Notably, we identified p-mTOR overexpression in eight patients; however, we did not consider p-mTOR inhibition with everolimus because of the low evidence for clinical efficacy.
Despite great research efforts and the investigated agents in PGC and other salivary gland cancers, progressive recurrent PGC has a dismal prognosis, and because of the rarity of the disease, well-established therapeutic options are scarce.
Great strides in the in-depth analysis of the vast genetic and epigenetic landscape of salivary gland cancers have been made in recent years; however, the PCM approach remains in its infancy when it comes to implementing novel individualized therapeutic strategies and concepts for this malignancy [33, 45, 46].
It is challenging to classify and prioritize the plethora of reported genetic alterations and epigenetic changes to identify actionable targets and to choose adequate tailored therapeutic measures. Thus, the roles of most identified alterations are undefined regarding pathogenesis, therapeutic consequences, and implications [47].
Another major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguished over 20 types of malignant salivary gland tumors [4]. The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance. In light of the complexity of PGC, molecularly driven clinical trials in PCM have to be designed as basket and umbrella trials that take into account the diversity of this malignancy for a better outcome.
Conclusion
This analysis clearly shows that molecular profiling from tumor samples of patients with advanced, heavily pretreated and therapy-refractory PGC in stage IV is feasible and results in meaningful and rational therapy recommendations and strategies; however, the complex tumor biology, heterogeneity, and extreme rarity remain unique challenges for the management of PGC and need to be addressed by further studies seeking a better understanding of this malignancy. In rare diseases such as PGC where randomized trials cannot be performed easily, molecularly driven treatment approaches and strategies may be particularly useful tools and viable options.
Abbreviations
ADTAndrogen deprivation therapy
AKTAlpha serine/threonine-protein kinase
ALKAnaplastic lymphoma kinase
ALLAcute lymphoblastic leukemia
APCAdenomatous polyposis coli
ARAndrogen receptor
ATMAtaxia telangiectasia mutated
BAP1BRCA1 associated protein‑1
BRAFB‑rapidly accelerated fibrosarcoma
BRCABreast cancer 1
CDKN2ACyclin-dependent kinase inhibitor 2A
CMLChronic myeloid leukemia
CRCColorectal cancer
DCRDisease control rate
ECOGEastern Cooperative Oncology Group
EGFREpidermal growth factor receptor
EMAEuropean Medicines Agency
FDAFood and Drug Administration
FGFRFibroblast growth factor receptor
FiSHFluorescence in situ hybridization
FLT3Fms like tyrosine kinase 3
GISTGastrointestinal stromal tumor
HER2Human epidermal growth factor receptor 2
HNSCCHead and neck squamous cell carcinoma
HLHodgkin lymphoma
IDHIsocitrate dehydrogenase 1
IHCImmunohistochemistry
lossPTENLoss of phosphatase and tensin homolog
MDS/MPDMyelodysplastic syndrome/myeloproliferative disorder
MECMucoepidermoid carcinoma
MTBMultidisciplinary tumor board
mTORMammalian target of rapamycin
NF1Neurofibromin 1
NOSNot otherwise specified
NSCLCNon-small cell lung carcinoma
PARPPoly [ADP-ribose] polymerase 1
PCMPrecision cancer medicine
PDACPancreatic ductal adenocarcinoma
PDGFRPlatelet-derived growth factor receptor
PD-L1Programmed death-ligand 1
PGCParotid gland carcinoma
Ph+Philadelphia chromosome positive
PIK3CBPhosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit beta
p-mTORphosphorylated Mammalian target of rapamycin
PRPartial response
PTENPhosphatase and tensin homolog
PTPN11Protein phosphatase non-receptor type 11
RB1Retinoblastoma 1
RCCRenal cell carcinoma
SDStable disease
SGCSalivary gland carcinomas
SHHSonic hedgehog
SMARCB1SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1
SMOSmoothened
STK11Serine/threonine kinase 11
TP53Tumor protein 53
TRKNeurotrophin receptor kinases
VEGFRVascular endothelial growth factor
VHLvon Hippel-Lindau
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Funding
This research did not receive any grants or funding.
Funding
Open access funding provided by Medical University of Vienna.
Conflict of interest
H. Taghizadeh, L. Müllauer, R.M. Mader, T. Füreder, and G.W. Prager declare that they have no competing interests. | DOCETAXEL, SUNITINIB | DrugsGivenReaction | CC BY | 33296026 | 19,253,223 | 2021-01 |
What was the administration route of drug 'SUNITINIB'? | Molecularly guided treatment of metastatic parotid gland carcinoma in adults.
BACKGROUND
Advanced therapy-refractory parotid gland carcinomas have a poor prognosis with limited therapy options. We used molecular profiling to offer molecular guided therapies to patients with advanced metastatic parotid gland malignancies.
METHODS
In this retrospective analysis we describe the molecular profiling of ten patients diagnosed with therapy-refractory metastatic parotid gland malignancies.
RESULTS
We identified seven genetic aberrations in five patients: two mutations in CDKN2A and one mutation in APC, ATM, TP53, SMARCB1 and FGFR1, respectively. No mutations were detected in five patients. The IHC demonstrated frequent expressions of EGFR and p‑mTOR, as well as PTEN in eight patients. For four fifths (n = 8) of the patients, a targeted therapy was suggested. Eventually, three patients received the targeted therapy recommendation and one patient achieved stable disease for 14 months.
CONCLUSIONS
A total of eight therapy recommendations were provided. Based on our observations, molecular-guided therapies may be a feasible treatment approach for this rare disease entity.
Introduction
Salivary gland carcinomas (SGC) comprise rare heterogeneous malignancies that account for only 5% of all head and neck cancers. The SGCs are classified into 24 subtypes according to the World Health Organization (WHO) definition. Likewise, the tumor biology and prognosis of SGCs markedly differ between histological types [1–4]. Among these glands, most malignancies occur in the parotid gland. The parotid gland carcinoma (PGC) is a relatively rare cancer, making up only 0.3% of all cancers combined [5]. The PGC with distant metastases, mainly in the lungs and bones, has a dismal median survival prognosis of 7.3 months despite therapeutic efforts [6].
The mainstay of treatment is complete surgical resection followed by postoperative radiotherapy (depending on the subtype and risk features). In surgical interventions, complete excision of the PGC is carried out with preservation of the functioning facial nerve, provided there is no tumor invasion. Systemic chemotherapy is generally indicated for patients with recurrent and/or metastatic PGC [5, 7–9].
The most common histological subtype in primary PGC is mucoepidermoid carcinoma (MEC) [10, 11]. Given the rarity of this disease, there are, apart from parotidectomy and radiotherapy, few well-established therapy standards for how to treat patients with progressive stage IV PGC [7].
There has recently been an effort to individualize therapy options in cancer diseases. In some instances, tailored therapy attempts with immunotherapeutics or tyrosine kinase inhibitors are used, e.g., trastuzumab in HER2-positive breast cancer or gastric cancer, imatinib in Philadelphia chromosome-positive chronic myeloid leukemia (Ph + CML), BRAF-directed therapy with vemurafenib or dabrafenib/trametinib in melanoma [12–14].
Emerging novel agents, such as the profiling of tumor molecular alterations and mutations as well as the identification of druggable targets and the ground-breaking pilot trial by von Hoff et al. have ushered in a new era of medicine; this approach has received many titles, such as individualized, stratified, tailored, or precision cancer medicine [15]. The main rationale of PCM is to match a therapeutic agent to its corresponding target for precise tailored therapy fitting a specific patient, aiming to achieve a deep durable and sustainable response without damaging healthy cells and tissues. This matches the tailored “therapeutic dress” to the patient [16].
We conducted a retrospective subgroup analysis of our precision molecular register, exclusively focusing on patients with progressive PGC with no available standard treatment options. These patients had been enrolled and whose tumors had been profiled in our special PCM platform. We sought to map the molecular profiles of advanced, relapsed and therapy-refractory PGC to evaluate whether there are any aberrations that can be targeted by a tailored therapy.
Material and methods
Ethics, consent and permission
The study was conducted in accordance with the International Conference on Harmonization E6 requirements for good clinical practice and with the ethical principles outlined in the Declaration of Helsinki. All patients had to provide written informed consent before inclusion in our PCM platform. Furthermore, the institutional ethics committee has also approved this subanalysis (Nr. 1039/2017).
Patients and design of the precision medicine platform
Patients with PGC who had progressed through all standard treatment options were eligible for inclusion in our platform for precision medicine, provided archival tissue samples were available. Patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Our platform for precision medicine is not a clinical trial, but intends to provide the possibility of a targeted therapy to patients where no active anti-tumor treatment is available.
Tissue samples
Formalin-fixed, paraffin-embedded tissue from patients with advanced PGC that were refractory to all available standard treatment lines were sent to or retrieved from the archive of the Department of Pathology.
Cancer gene panel sequencing
DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany) and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA). The panel includes driver mutations, oncogenes, tumor suppressor genes, and gene fusions. See supplementary information for complete list of the gene panel. The Oncomine Comprehensive Assay v3 was optimized for sequencing on an Ion Personal Genome Machine System (Thermo Fisher Scientific). The generated sequencing data were afterwards analyzed with the help of the Ion Reporter Software (Thermo Scientific Fisher). We referred to BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM and 1000 genomes for variant calling and classification. The variants were classified according to a five-tier system comprised of the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. This classification was based on the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.
Immunohistochemistry
The IHC was performed using 2‑μm-thin tissue sections read by a Ventana Benchmark Ultra stainer (Ventana, Tucson, AZ, USA). The following antibodies were applied: anaplastic lymphoma kinase (ALK, clone 1A4; Zytomed, Berlin, Germany), CD20 (clone L26; Dako Omnis from Agilent Technologies, Santa Clara, CA, USA), CD30 (clone BerH2; Agilent Technologies, Vienna, Austria), epidermal growth factor receptor (EGFR, clone 3C6; Ventana), estrogen receptor (clone SP1; Ventana), human epidermal growth factor receptor 2 (HER2, clone 4B5; Ventana), HER3 (clone SP71; Abcam, Cambridge, UK), C‑kit receptor (KIT, clone 9.7; Ventana), MET (clone SP44; Ventana), NTRK (clone EPR17341, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR, clone 49F9; Cell Signaling Technology, Danvers, MA, USA), platelet-derived growth factor alpha (PDGFRA, rabbit polyclonal; Thermo Fisher Scientific), PDGFRB (clone 28E1, Cell Signaling Technology), programmed death-ligand 1 (PD-L1, clone E1L3N; Cell Signaling Technology), progesterone receptor (clone 1E2; Ventana), phosphatase and tensin homolog (PTEN, clone Y184; Abcam) and ROS1 (clone D4D6; Cell Signaling Technology).
To assess the immunostaining intensity for the antigens EGFR, p-mTOR, PDGFRA, PDGFRB and PTEN, a combinative semiquantitative score for immunohistochemistry was used. The immunostaining intensity was graded from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong). To calculate the score, the intensity grade was multiplied by the percentage of corresponding positive cells: (maximum 300) = (% negative × 0) + (% weak × 1) + (% moderate × 2) + (% strong × 3).
The immunohistochemical staining intensity for HER2 was scored from 0 to 3+ (0 = negative, 1+ = negative, 2+ = positive, 3+ = positive) pursuant to the scoring guidelines of the Dako HercepTestR from the company Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA). In the case of HER2 2+, a further test with HER2 in situ hybridization was performed to verify amplification of the HER2 gene.
Estrogen receptor and progesterone receptor staining were graded according to the Allred scoring system [17] from 0 to 8 and MET staining was scored from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong).
For PD-L1, the tumor proportion score was calculated, which is the percentage of viable malignant cells showing membrane staining.
Staining for ALK, CD30, CD20 and ROS1 was classified as positive or negative based on the percentage of reactive tumor cells but without graduation of the staining intensity. In ALK or ROS1 positive cases, the presence of a possible gene translocation was evaluated by fluorescence in situ hybridization (FISH).
All antibodies used in this study were validated and approved at the clinical institute of pathology and are used in routine IHC staining for clinical purposes. The antibodies have been validated, by proper positive and negative tissue controls and by non-IHC methods, such as immunoblotting and flow cytometry, to detect the respective epitope of the antigens. For the control, the use of the antibodies was optimized in terms of intensity, concentration, signal/noise ratio, incubation times and blocking. The negative control was conducted by omitting the primary antibody and by substitution of isotype-specific antibody and serum at the exact same dilution and laboratory conditions as the primary antibody to preclude unspecific binding.
For the positive control, the antibodies were shown not to cross-react with closely related molecules of the target epitope.
Fluorescence in situ hybridization
The FISH was performed with 4‑μm-thick formalin-fixed, paraffin-embedded tissue sections. The following FISH probes were employed: ALK (2p23.1; Abbott, Abbott Park, IL, USA), RET (10q11; Kreatech, Berlin, Germany), PTEN (10q23.31)/centromere 10, and ROS1 (ZytoVision, Bremerhaven, Germany), 200 cell nuclei per tumor were evaluated. The cut-off level for an aberrant ALK, RET, and ROS1 FISH was ≥15% of cells with a split-apart signal. The PTEN FISH was considered positive for PTEN gene loss with ≥30% of cells with only one or no PTEN signals. A chromosome 10 centromere FISH probe served as a control for ploidy of chromosome 10.
Multidisciplinary boards (molecular tumor boards for PCM)
After thorough examination of the molecular profile of each tumor sample by a qualified and competent molecular pathologist, the results and findings were reviewed in multidisciplinary tumor boards (MTB) that were held every other week. Members of the board included molecular pathologists, radiologists, clinical oncologists, biostatisticians, and basic scientists. The MTB recommended the targeted therapy based on the specific molecular profile of each patient. The targeted therapies included tyrosine kinase inhibitors, checkpoint inhibitors (e.g. anti-PD-L1 monoclonal antibodies), and growth factor receptor antibodies with or without endocrine therapy. The treatment recommendations by the MTB were prioritized depending on the level of evidence from high to low according to phase III to phase I trials.
If more than one druggable molecular aberration was identified, the MTB recommended a therapy regimen to target as many molecular aberrations as possible, with special consideration given to the toxicity profile of each antitumor agent and their potential interactions. Since all patients were given all available standard treatment options for their cancer disease prior to their inclusion in our PCM platform, nearly all targeted agents were suggested as off-label use. If the tumor profile and the clinical characteristics of a patient met the requirements of a clinical trial for targeted therapies that was conducted in our cancer center, patients were preferentially asked if they wanted to participate in this trial.
Descriptive statistics
For data description, we used measures of central tendency including the mean and median. We also used the method of frequency distribution to delineate the characteristics of the PGC patients.
Results
All ten patients diagnosed with progressive primary PGC were included in this analysis from our platform for precision medicine that has so far profiled over 600 patients with various advanced solid tumors. All PGC patients were Europeans. Five men and five women were diagnosed with five different histological subtypes of primary PGC. The subtypes were acinic cell carcinoma (n = 1), adenocarcinoma NOS (n = 3), adenoid cystic carcinoma (n = 3), carcinoma ex pleomorphic adenoma (n = 1), and primary squamous cell carcinoma (n = 2). The primary tumor location was the right side in six patients (60%) and the left side in four patients (40%).
At the time of molecular profiling, all patients had an advanced, therapy-refractory and relapsed PGC in stage IV with distant metastases, mainly in the bones and lungs. The whole cohort had undergone parotidectomy and radiation therapy. Four patients had also received prior chemotherapy: two patients were treated with carboplatin and paclitaxel, one patient received cisplatin and cetuximab, and another patient was given a CAP regimen consisting of cyclophosphamid, doxorubicin (trade name Adriamycin) and a platinum-based agent (usually cisplatin).
The median age at the time of initial diagnosis was 59.5 years, ranging from 27 to 82 years, and the median age at the time of molecular profiling was 63 years, ranging from 37 to 83 years (Table 1).Table 1 Patient characteristics (N = 10)
Patient characteristics Number
Median age at first diagnosis (years) 59.5
Median age at molecular profiling (years) 63
Men 5
Women 5
Histological subtypes of parotid gland carcinoma 5
Caucasian 10
Relapsed disease 10
Stage IV 10
Parotid gland carcinoma on the right side 6
Parotid gland carcinoma on the left side 4
Therapy recommendations 8
Of the ten tissue samples, five were from metastatic sites and five from the primary site.
In total, we identified seven molecular aberrations in five patients: two mutations in CDKN2A and one mutation in each of APC, ATM, TP53, SMARCB1, and FGFR1. No mutations were detected in five patients.
Expression of EGFR, p-mTOR and PTEN was detected by IHC in eight patients. The EGFR median score was 120, and 3 patients had a high EGFR score of between 200 and 300. The expression of p-mTOR was lower with a median score of 70, and 2 patients had a high p-mTOR score of between 200 and 300. Expression of MET and PDGFRA was detectable in six and five samples, respectively. MET expression was weak in four patients and moderate in one patient. One sample exhibited a strong MET expression. Less common expressions were observed for KIT and AR which were observed in three and two patients, respectively. The KIT expression was found to be weak in two samples and moderate in one sample, AR was moderately expressed in both patients with adenocarcinoma.
IHC and FISH were not performed in one patient due to insufficient tumor material.
For eight of the ten patients, a targeted therapy was suggested based on their individual molecular profile (Table 1). Androgen deprivation therapy (ADT), crizotinib, and cetuximab each were offered in two cases and imatinib and sunitinib were proposed in one case. We refer here to Tables 2 and 3 for the rationale of the therapy suggestions.Table 2 Rational for therapy recommendations
Therapeutic agent (trade name) Targets Overview of current FDA approval in different entities Overview of current EMA approval in different entities
Cetuximab (Erbitux)
(n = 2)
EGFR CRC, HNSCC CRC, HNSCC
Crizotinib (Xalkori)
(n = 2)
ALK, ROS1
MET overexpression
ALK or ROS1 positive NSCLC ALK or ROS1 positive NSCLC
Imatinib (Gleevec)
(n = 1)
PDGFR, KIT, Bcr/Abl Ph + CML, KIT + GIST, MDS/MPD associated with PDGFR, Ph + ALL Ph + CML, KIT+ GIST, MDS/MPD associated with PDGFR, Ph + ALL
Sunitinib (Sutent)
(n = 1)
PDGFR, KIT, VEGFR, RET, FLT3 RCC, PDAC, GIST RCC, PDAC, GIST
ABL Abelson murine leukemia viral oncogene homolog 1, ALK Anaplastic lymphoma kinase, ALL acute lymphatic leukemia, BCR breakpoint cluster region, CML chronic myleloid leukemia, CRC colorectal cancer, EGFR epidermal growth factor receptor, EMA European Medicines Agency, FDA Food and Drug Administration, FLT3 fms like tyrosine kinase 3, GIST gastrointestinal stromal tumor, HNSCC Head and neck squamous cell carcinoma, MDS/MPD myelodysplastic syndrome/ myeloproliferative disorder, NSCLC Non-small cell lung carcinoma, PDAC pancreatic ductal adenocarcinoma, PDGFR platelet derived growth factor receptor, Ph+ Philadelphia chromosome positive, p‑mTOR phosphorylated mammalian target of rapamycin, RCC renal cell carcinoma, RET rearranged during transfection, TP53 tumor protein 53, VEGFR vascular endothelial growth factor
Table 3 Detailed characteristics of the PGC patients (n = 10)
Patient number, gender and age Histological subtype and Stage and side Site of metastasis Tissue tested Detected mutations by NGS IHC Therapy recommendation
1
Female
61 years
Acinic cell carcinoma IV°
Right
Lung Metastatic No mutation detected Not done (due to insufficient tissue material) No recommendation
2
Male
83 years
Adenocarcinoma IV°
Right
Liver lung Metastatic
(liver)
No mutation detected EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 3+,
AR 2+
Androgen deprivation therapy
3
Female
37 years
Adenocarcinoma IV°
Right
Bone Metastatic No mutation detected EGFR 2+,
KIT 2+,
PTEN 2+,
p‑mTOR 3+
No recommendation
4
Male
71 years
Adenocarcinoma IV°
Left
Bone Primary No mutation detected EGFR 3+,
PTEN 1+,
p‑mTOR 1+,
AR 2+
Androgen deprivation therapy
5
Male
46 years
Adenoid cystic carcinoma IV°
Left
Lung Metastatic No mutation detected KIT 1+,
MET 3+,
PDGFR 1+,
PTEN 1+,
p‑mTOR 2+
Crizotinib
6
Female
56 years
Adenoid cystic carcinoma IV°
Right
Lung Primary ATM: exon 32
c.C9142G (p.Leu3048Val)
EGFR 3+,
MET 1+,
p‑mTOR 1+,
PTEN 1+
Cetuximab
7
Male
47 years
Adenoid cystic carcinoma IV°
Right
Lung Primary APC: exon 16
c.T3920A (p.I1307K)
KIT 1+,
EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 1+
Imatinib
8
Male
65 years
Carcinoma ex pleomorphic adenoma IV°
Right
Lung,
Brain
Metastatic
(lung)
TP53 (exon 7):
c.C742T
(p.R248W)
EGFR 1+,
MET 1+,
PDGFRA 2+,
PTEN 1+,
p‑mTOR 1+
Sunitinib
9
Female
67 years
Primary squamous cell carcinoma IV°
Left
Bone,
lung
Primary CDKN2A (exon 2): c.151_155delGTCT (p.V51 fs);
FGFR1 (exon 5):
c.478_480delGAT (p.Asp.160del);
SMARCB1 (exon 9):
c.G1130A (p.R3677H)
EGFR 3+,
PTEN 2+
Cetuximab
10
Female
72 years
Primary squamous cell carcinoma IV°
Left
Lung Primary CDKN2A (exon 2):
c.C341T,
(p.P114L)
EGFR 1+,
MET 3+,
PDGFRA 1+,
p‑mTOR 1+
Crizotinib
Values in parentheses indicate the immunohistochemical score that was calculated as mentioned in the “Materials and methods” section
APC adenomatous polyposis coli, AR androgen receptor, CDKN2A cyclin-dependent kinase inhibitor 2A, EGFR epidermal growth factor receptor, FiSH fluorescence in situ hybridization, PDGFR platelet derived growth factor receptor, p‑mTOR phosphorylated mammalian target of rapamycin, PTEN phosphatase and tensin homolog, TP53 tumor protein 53, IHC immunohistochemistry, NGS next-generation sequencing
The median turnaround time from the initiation of molecular profiling to therapy initiation was 43 days.
Eventually, three patients received the targeted therapy. One male patient with an adenocarcinoma was administered bicalutamide as ADT but died because of disease progression before restaging was performed. The second patient with a carcinoma ex pleomorphic adenoma received sunitinib 50 mg orally once daily combined with docetaxel every third week but did not respond to this therapy regimen and experienced progressive disease. The third patient had an adenoid cystic carcinoma and was given imatinib 400 mg orally once daily. He achieved a stable disease for 14 months and tolerated the therapy without any treatment-related adverse events.
Discussion
To our knowledge, this is the first study of individual genomic alterations that have been translated into concrete tailored therapy recommendations in a group of patients with exclusively recurrent, progressive, and therapy-refractory PGC in stage IV in a real-world setting. None of these patients had the histological subtype mucoepidermoid carcinoma (MEC); instead, they had rarer subtypes, making this subgroup analysis even more valuable and unique.
In this retrospective single center subgroup analysis, we exclusively present the molecular profile of all ten patients with PGC. Their disease was relapsed, therapy-refractory and advanced. Tumor tissue was obtained from all patients and characterized regarding molecular profiles. Subsequently, the genomic information of the patients was discussed in a multidisciplinary tumor board (MTB) for PCM to evaluate the possibility of a genomic-based therapy concept that is independent of the tumor’s histological classification (tissue-agnostic drugs).
Tumor samples harbored mutations in APC, ATM, CDKN2A, FGFR1, SMARCB1, and TP53. The IHC revealed expressions of EGFR and p-mTOR as well as PTEN in eight patients.
Therapeutic options recommended were ADT, cetuximab, crizotinib, sunitinib, and imatinib. Two patients with AR expression were offered ADT to control the disease. Two patients with strong MET expression were suggested crizotinib as a tailored therapy. For two patients with high EGFR expression cetuximab was recommended. Imatinib was considered in one patient due to expression of KIT and PDGFRA. Sunitinib was proposed to one patient because of PDGFRA overexpression. A treatment recommendation was derived for eight patients from the MTB. The drugs were carefully selected for an individualized treatment with special respect to the patient’s clinical and treatment history and concomitant therapies and comorbidities. Interestingly, all these recommendations were based on the protein expressions obtained by immunohistochemistry. Thus, our analysis underscores the clinical relevance of immunohistochemistry in precision medicine.
Eventually, three patients received the targeted therapy. One patient died before restaging was performed. The second patient received sunitinib and did not respond. Imatinib was applied to the third patient who experienced a stable disease for 14 months. Although this analysis showed that PCM is implementable in daily clinical routine, only one patient had a clinical benefit from this therapy approach. One reason may be the turnaround time: a shorter turnaround time may help to start the targeted therapy earlier and to control the cancer disease. Liquid biopsy may be a viable option to reduce the turnaround time, to monitor the disease and to assess the therapy response. Another reason may be the complexity of PGC. The major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguishes over 20 types of malignant salivary gland tumors [4].
The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance.
The observed genomic aberrations and overexpression of AR, KIT, and EGFR, PTEN, p-mTOR, and PDGFRA in PGC in this analysis are in keeping with previous studies [18–33]. The rationale for the therapy recommendation with ADT was corroborated by a study by Boon et al. They studied the application of ADT in 35 patients with androgen receptor-positive advanced salivary duct carcinoma, which lead to a median overall survival (OS) of 17 months versus 5 months in 43 patients receiving best supportive care [34].
The overexpression of MET was seen in all three patients with adenoid cystic carcinoma and is in line with other studies [35]; however, to our knowledge, this is the first report of an overexpression also in carcinoma ex pleomorphic adenoma and primary squamous cell carcinoma. Crizotinib was offered as a molecularly driven treatment approach. Its clinical efficacy in salivary gland cancers has not yet been described in clinical trials.
Only one study has used molecular profiling to offer an individualized therapy in patients with metastatic salivary gland adenoid cystic carcinoma (ACC). They enrolled a limited 14 patients, of whom 11 actually received the recommended treatment. The investigators reported the clinical benefit of molecularly guided treatment [36]. Imatinib and sunitinib are tyrosine kinase inhibitors that were offered, each in one case, as an alternative therapy in the case of overexpression in PDGFRA/B or KIT. The data pertaining to the use of imatinib in salivary gland cancer are contradictory and unclear. According to two phase II trials that applied imatinib in patients with KIT-positive adenoid cystic cancers of salivary glands, imatinib was not of significant clinical benefit and the best observed response was a stable disease (SD).
As a limitation, however, it should be noted that only ACC was studied, and PDGFR expression of the tumor tissue was not evaluated [37, 38]. In contrast, another phase II trial tested imatinib in 15 patients with ACC of salivary glands and concluded that imatinib was of clinical benefit because it achieved a partial response (PR) in two patients and a SD in five other patients [39]. Likewise, in another study, imatinib achieved significant regression of initially unresectable ACC of salivary glands in two patients, making them eligible for a salvage resection [40].
Similar to imatinib, sunitinib was also tested in a phase II trial in 13 patients with ACC of salivary glands and 11 of these achieved stable disease; however, the investigators did not test the patients’ tumors for KIT or PDGFR expression [41]. Dasatinib is another tyrosine kinase inhibitor that was investigated in a phase II trial for patients with recurrent or metastatic KIT expressing ACC and for nonadenoid cystic malignant salivary tumors. It achieved only one PR in a patient with ACC and the experimental treatment demonstrated no activity in non-ACC salivary gland cancer [42].
In another phase II trial, axitinib was applied in 33 patients with unresectable ACC. Ho et al. reported that axitinib achieved a PR in 3 patients and a SD in 25 patients. The median progression-free survival (PFS) was 5.7 months [43]. Overexpression of EGFR was often observed in salivary gland cancers and provides a solid and sound rationale for the administration of cetuximab [26, 27]. Its clinical efficacy was examined by Locati et al. in 2009 in salivary gland carcinomas, and they reported a clinical benefit rate of 50% [44].
Notably, we identified p-mTOR overexpression in eight patients; however, we did not consider p-mTOR inhibition with everolimus because of the low evidence for clinical efficacy.
Despite great research efforts and the investigated agents in PGC and other salivary gland cancers, progressive recurrent PGC has a dismal prognosis, and because of the rarity of the disease, well-established therapeutic options are scarce.
Great strides in the in-depth analysis of the vast genetic and epigenetic landscape of salivary gland cancers have been made in recent years; however, the PCM approach remains in its infancy when it comes to implementing novel individualized therapeutic strategies and concepts for this malignancy [33, 45, 46].
It is challenging to classify and prioritize the plethora of reported genetic alterations and epigenetic changes to identify actionable targets and to choose adequate tailored therapeutic measures. Thus, the roles of most identified alterations are undefined regarding pathogenesis, therapeutic consequences, and implications [47].
Another major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguished over 20 types of malignant salivary gland tumors [4]. The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance. In light of the complexity of PGC, molecularly driven clinical trials in PCM have to be designed as basket and umbrella trials that take into account the diversity of this malignancy for a better outcome.
Conclusion
This analysis clearly shows that molecular profiling from tumor samples of patients with advanced, heavily pretreated and therapy-refractory PGC in stage IV is feasible and results in meaningful and rational therapy recommendations and strategies; however, the complex tumor biology, heterogeneity, and extreme rarity remain unique challenges for the management of PGC and need to be addressed by further studies seeking a better understanding of this malignancy. In rare diseases such as PGC where randomized trials cannot be performed easily, molecularly driven treatment approaches and strategies may be particularly useful tools and viable options.
Abbreviations
ADTAndrogen deprivation therapy
AKTAlpha serine/threonine-protein kinase
ALKAnaplastic lymphoma kinase
ALLAcute lymphoblastic leukemia
APCAdenomatous polyposis coli
ARAndrogen receptor
ATMAtaxia telangiectasia mutated
BAP1BRCA1 associated protein‑1
BRAFB‑rapidly accelerated fibrosarcoma
BRCABreast cancer 1
CDKN2ACyclin-dependent kinase inhibitor 2A
CMLChronic myeloid leukemia
CRCColorectal cancer
DCRDisease control rate
ECOGEastern Cooperative Oncology Group
EGFREpidermal growth factor receptor
EMAEuropean Medicines Agency
FDAFood and Drug Administration
FGFRFibroblast growth factor receptor
FiSHFluorescence in situ hybridization
FLT3Fms like tyrosine kinase 3
GISTGastrointestinal stromal tumor
HER2Human epidermal growth factor receptor 2
HNSCCHead and neck squamous cell carcinoma
HLHodgkin lymphoma
IDHIsocitrate dehydrogenase 1
IHCImmunohistochemistry
lossPTENLoss of phosphatase and tensin homolog
MDS/MPDMyelodysplastic syndrome/myeloproliferative disorder
MECMucoepidermoid carcinoma
MTBMultidisciplinary tumor board
mTORMammalian target of rapamycin
NF1Neurofibromin 1
NOSNot otherwise specified
NSCLCNon-small cell lung carcinoma
PARPPoly [ADP-ribose] polymerase 1
PCMPrecision cancer medicine
PDACPancreatic ductal adenocarcinoma
PDGFRPlatelet-derived growth factor receptor
PD-L1Programmed death-ligand 1
PGCParotid gland carcinoma
Ph+Philadelphia chromosome positive
PIK3CBPhosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit beta
p-mTORphosphorylated Mammalian target of rapamycin
PRPartial response
PTENPhosphatase and tensin homolog
PTPN11Protein phosphatase non-receptor type 11
RB1Retinoblastoma 1
RCCRenal cell carcinoma
SDStable disease
SGCSalivary gland carcinomas
SHHSonic hedgehog
SMARCB1SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1
SMOSmoothened
STK11Serine/threonine kinase 11
TP53Tumor protein 53
TRKNeurotrophin receptor kinases
VEGFRVascular endothelial growth factor
VHLvon Hippel-Lindau
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Funding
This research did not receive any grants or funding.
Funding
Open access funding provided by Medical University of Vienna.
Conflict of interest
H. Taghizadeh, L. Müllauer, R.M. Mader, T. Füreder, and G.W. Prager declare that they have no competing interests. | Oral | DrugAdministrationRoute | CC BY | 33296026 | 19,253,223 | 2021-01 |
What was the dosage of drug 'SUNITINIB'? | Molecularly guided treatment of metastatic parotid gland carcinoma in adults.
BACKGROUND
Advanced therapy-refractory parotid gland carcinomas have a poor prognosis with limited therapy options. We used molecular profiling to offer molecular guided therapies to patients with advanced metastatic parotid gland malignancies.
METHODS
In this retrospective analysis we describe the molecular profiling of ten patients diagnosed with therapy-refractory metastatic parotid gland malignancies.
RESULTS
We identified seven genetic aberrations in five patients: two mutations in CDKN2A and one mutation in APC, ATM, TP53, SMARCB1 and FGFR1, respectively. No mutations were detected in five patients. The IHC demonstrated frequent expressions of EGFR and p‑mTOR, as well as PTEN in eight patients. For four fifths (n = 8) of the patients, a targeted therapy was suggested. Eventually, three patients received the targeted therapy recommendation and one patient achieved stable disease for 14 months.
CONCLUSIONS
A total of eight therapy recommendations were provided. Based on our observations, molecular-guided therapies may be a feasible treatment approach for this rare disease entity.
Introduction
Salivary gland carcinomas (SGC) comprise rare heterogeneous malignancies that account for only 5% of all head and neck cancers. The SGCs are classified into 24 subtypes according to the World Health Organization (WHO) definition. Likewise, the tumor biology and prognosis of SGCs markedly differ between histological types [1–4]. Among these glands, most malignancies occur in the parotid gland. The parotid gland carcinoma (PGC) is a relatively rare cancer, making up only 0.3% of all cancers combined [5]. The PGC with distant metastases, mainly in the lungs and bones, has a dismal median survival prognosis of 7.3 months despite therapeutic efforts [6].
The mainstay of treatment is complete surgical resection followed by postoperative radiotherapy (depending on the subtype and risk features). In surgical interventions, complete excision of the PGC is carried out with preservation of the functioning facial nerve, provided there is no tumor invasion. Systemic chemotherapy is generally indicated for patients with recurrent and/or metastatic PGC [5, 7–9].
The most common histological subtype in primary PGC is mucoepidermoid carcinoma (MEC) [10, 11]. Given the rarity of this disease, there are, apart from parotidectomy and radiotherapy, few well-established therapy standards for how to treat patients with progressive stage IV PGC [7].
There has recently been an effort to individualize therapy options in cancer diseases. In some instances, tailored therapy attempts with immunotherapeutics or tyrosine kinase inhibitors are used, e.g., trastuzumab in HER2-positive breast cancer or gastric cancer, imatinib in Philadelphia chromosome-positive chronic myeloid leukemia (Ph + CML), BRAF-directed therapy with vemurafenib or dabrafenib/trametinib in melanoma [12–14].
Emerging novel agents, such as the profiling of tumor molecular alterations and mutations as well as the identification of druggable targets and the ground-breaking pilot trial by von Hoff et al. have ushered in a new era of medicine; this approach has received many titles, such as individualized, stratified, tailored, or precision cancer medicine [15]. The main rationale of PCM is to match a therapeutic agent to its corresponding target for precise tailored therapy fitting a specific patient, aiming to achieve a deep durable and sustainable response without damaging healthy cells and tissues. This matches the tailored “therapeutic dress” to the patient [16].
We conducted a retrospective subgroup analysis of our precision molecular register, exclusively focusing on patients with progressive PGC with no available standard treatment options. These patients had been enrolled and whose tumors had been profiled in our special PCM platform. We sought to map the molecular profiles of advanced, relapsed and therapy-refractory PGC to evaluate whether there are any aberrations that can be targeted by a tailored therapy.
Material and methods
Ethics, consent and permission
The study was conducted in accordance with the International Conference on Harmonization E6 requirements for good clinical practice and with the ethical principles outlined in the Declaration of Helsinki. All patients had to provide written informed consent before inclusion in our PCM platform. Furthermore, the institutional ethics committee has also approved this subanalysis (Nr. 1039/2017).
Patients and design of the precision medicine platform
Patients with PGC who had progressed through all standard treatment options were eligible for inclusion in our platform for precision medicine, provided archival tissue samples were available. Patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Our platform for precision medicine is not a clinical trial, but intends to provide the possibility of a targeted therapy to patients where no active anti-tumor treatment is available.
Tissue samples
Formalin-fixed, paraffin-embedded tissue from patients with advanced PGC that were refractory to all available standard treatment lines were sent to or retrieved from the archive of the Department of Pathology.
Cancer gene panel sequencing
DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany) and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA). The panel includes driver mutations, oncogenes, tumor suppressor genes, and gene fusions. See supplementary information for complete list of the gene panel. The Oncomine Comprehensive Assay v3 was optimized for sequencing on an Ion Personal Genome Machine System (Thermo Fisher Scientific). The generated sequencing data were afterwards analyzed with the help of the Ion Reporter Software (Thermo Scientific Fisher). We referred to BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM and 1000 genomes for variant calling and classification. The variants were classified according to a five-tier system comprised of the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. This classification was based on the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.
Immunohistochemistry
The IHC was performed using 2‑μm-thin tissue sections read by a Ventana Benchmark Ultra stainer (Ventana, Tucson, AZ, USA). The following antibodies were applied: anaplastic lymphoma kinase (ALK, clone 1A4; Zytomed, Berlin, Germany), CD20 (clone L26; Dako Omnis from Agilent Technologies, Santa Clara, CA, USA), CD30 (clone BerH2; Agilent Technologies, Vienna, Austria), epidermal growth factor receptor (EGFR, clone 3C6; Ventana), estrogen receptor (clone SP1; Ventana), human epidermal growth factor receptor 2 (HER2, clone 4B5; Ventana), HER3 (clone SP71; Abcam, Cambridge, UK), C‑kit receptor (KIT, clone 9.7; Ventana), MET (clone SP44; Ventana), NTRK (clone EPR17341, Abcam), phosphorylated mammalian target of rapamycin (p-mTOR, clone 49F9; Cell Signaling Technology, Danvers, MA, USA), platelet-derived growth factor alpha (PDGFRA, rabbit polyclonal; Thermo Fisher Scientific), PDGFRB (clone 28E1, Cell Signaling Technology), programmed death-ligand 1 (PD-L1, clone E1L3N; Cell Signaling Technology), progesterone receptor (clone 1E2; Ventana), phosphatase and tensin homolog (PTEN, clone Y184; Abcam) and ROS1 (clone D4D6; Cell Signaling Technology).
To assess the immunostaining intensity for the antigens EGFR, p-mTOR, PDGFRA, PDGFRB and PTEN, a combinative semiquantitative score for immunohistochemistry was used. The immunostaining intensity was graded from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong). To calculate the score, the intensity grade was multiplied by the percentage of corresponding positive cells: (maximum 300) = (% negative × 0) + (% weak × 1) + (% moderate × 2) + (% strong × 3).
The immunohistochemical staining intensity for HER2 was scored from 0 to 3+ (0 = negative, 1+ = negative, 2+ = positive, 3+ = positive) pursuant to the scoring guidelines of the Dako HercepTestR from the company Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA). In the case of HER2 2+, a further test with HER2 in situ hybridization was performed to verify amplification of the HER2 gene.
Estrogen receptor and progesterone receptor staining were graded according to the Allred scoring system [17] from 0 to 8 and MET staining was scored from 0 to 3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong).
For PD-L1, the tumor proportion score was calculated, which is the percentage of viable malignant cells showing membrane staining.
Staining for ALK, CD30, CD20 and ROS1 was classified as positive or negative based on the percentage of reactive tumor cells but without graduation of the staining intensity. In ALK or ROS1 positive cases, the presence of a possible gene translocation was evaluated by fluorescence in situ hybridization (FISH).
All antibodies used in this study were validated and approved at the clinical institute of pathology and are used in routine IHC staining for clinical purposes. The antibodies have been validated, by proper positive and negative tissue controls and by non-IHC methods, such as immunoblotting and flow cytometry, to detect the respective epitope of the antigens. For the control, the use of the antibodies was optimized in terms of intensity, concentration, signal/noise ratio, incubation times and blocking. The negative control was conducted by omitting the primary antibody and by substitution of isotype-specific antibody and serum at the exact same dilution and laboratory conditions as the primary antibody to preclude unspecific binding.
For the positive control, the antibodies were shown not to cross-react with closely related molecules of the target epitope.
Fluorescence in situ hybridization
The FISH was performed with 4‑μm-thick formalin-fixed, paraffin-embedded tissue sections. The following FISH probes were employed: ALK (2p23.1; Abbott, Abbott Park, IL, USA), RET (10q11; Kreatech, Berlin, Germany), PTEN (10q23.31)/centromere 10, and ROS1 (ZytoVision, Bremerhaven, Germany), 200 cell nuclei per tumor were evaluated. The cut-off level for an aberrant ALK, RET, and ROS1 FISH was ≥15% of cells with a split-apart signal. The PTEN FISH was considered positive for PTEN gene loss with ≥30% of cells with only one or no PTEN signals. A chromosome 10 centromere FISH probe served as a control for ploidy of chromosome 10.
Multidisciplinary boards (molecular tumor boards for PCM)
After thorough examination of the molecular profile of each tumor sample by a qualified and competent molecular pathologist, the results and findings were reviewed in multidisciplinary tumor boards (MTB) that were held every other week. Members of the board included molecular pathologists, radiologists, clinical oncologists, biostatisticians, and basic scientists. The MTB recommended the targeted therapy based on the specific molecular profile of each patient. The targeted therapies included tyrosine kinase inhibitors, checkpoint inhibitors (e.g. anti-PD-L1 monoclonal antibodies), and growth factor receptor antibodies with or without endocrine therapy. The treatment recommendations by the MTB were prioritized depending on the level of evidence from high to low according to phase III to phase I trials.
If more than one druggable molecular aberration was identified, the MTB recommended a therapy regimen to target as many molecular aberrations as possible, with special consideration given to the toxicity profile of each antitumor agent and their potential interactions. Since all patients were given all available standard treatment options for their cancer disease prior to their inclusion in our PCM platform, nearly all targeted agents were suggested as off-label use. If the tumor profile and the clinical characteristics of a patient met the requirements of a clinical trial for targeted therapies that was conducted in our cancer center, patients were preferentially asked if they wanted to participate in this trial.
Descriptive statistics
For data description, we used measures of central tendency including the mean and median. We also used the method of frequency distribution to delineate the characteristics of the PGC patients.
Results
All ten patients diagnosed with progressive primary PGC were included in this analysis from our platform for precision medicine that has so far profiled over 600 patients with various advanced solid tumors. All PGC patients were Europeans. Five men and five women were diagnosed with five different histological subtypes of primary PGC. The subtypes were acinic cell carcinoma (n = 1), adenocarcinoma NOS (n = 3), adenoid cystic carcinoma (n = 3), carcinoma ex pleomorphic adenoma (n = 1), and primary squamous cell carcinoma (n = 2). The primary tumor location was the right side in six patients (60%) and the left side in four patients (40%).
At the time of molecular profiling, all patients had an advanced, therapy-refractory and relapsed PGC in stage IV with distant metastases, mainly in the bones and lungs. The whole cohort had undergone parotidectomy and radiation therapy. Four patients had also received prior chemotherapy: two patients were treated with carboplatin and paclitaxel, one patient received cisplatin and cetuximab, and another patient was given a CAP regimen consisting of cyclophosphamid, doxorubicin (trade name Adriamycin) and a platinum-based agent (usually cisplatin).
The median age at the time of initial diagnosis was 59.5 years, ranging from 27 to 82 years, and the median age at the time of molecular profiling was 63 years, ranging from 37 to 83 years (Table 1).Table 1 Patient characteristics (N = 10)
Patient characteristics Number
Median age at first diagnosis (years) 59.5
Median age at molecular profiling (years) 63
Men 5
Women 5
Histological subtypes of parotid gland carcinoma 5
Caucasian 10
Relapsed disease 10
Stage IV 10
Parotid gland carcinoma on the right side 6
Parotid gland carcinoma on the left side 4
Therapy recommendations 8
Of the ten tissue samples, five were from metastatic sites and five from the primary site.
In total, we identified seven molecular aberrations in five patients: two mutations in CDKN2A and one mutation in each of APC, ATM, TP53, SMARCB1, and FGFR1. No mutations were detected in five patients.
Expression of EGFR, p-mTOR and PTEN was detected by IHC in eight patients. The EGFR median score was 120, and 3 patients had a high EGFR score of between 200 and 300. The expression of p-mTOR was lower with a median score of 70, and 2 patients had a high p-mTOR score of between 200 and 300. Expression of MET and PDGFRA was detectable in six and five samples, respectively. MET expression was weak in four patients and moderate in one patient. One sample exhibited a strong MET expression. Less common expressions were observed for KIT and AR which were observed in three and two patients, respectively. The KIT expression was found to be weak in two samples and moderate in one sample, AR was moderately expressed in both patients with adenocarcinoma.
IHC and FISH were not performed in one patient due to insufficient tumor material.
For eight of the ten patients, a targeted therapy was suggested based on their individual molecular profile (Table 1). Androgen deprivation therapy (ADT), crizotinib, and cetuximab each were offered in two cases and imatinib and sunitinib were proposed in one case. We refer here to Tables 2 and 3 for the rationale of the therapy suggestions.Table 2 Rational for therapy recommendations
Therapeutic agent (trade name) Targets Overview of current FDA approval in different entities Overview of current EMA approval in different entities
Cetuximab (Erbitux)
(n = 2)
EGFR CRC, HNSCC CRC, HNSCC
Crizotinib (Xalkori)
(n = 2)
ALK, ROS1
MET overexpression
ALK or ROS1 positive NSCLC ALK or ROS1 positive NSCLC
Imatinib (Gleevec)
(n = 1)
PDGFR, KIT, Bcr/Abl Ph + CML, KIT + GIST, MDS/MPD associated with PDGFR, Ph + ALL Ph + CML, KIT+ GIST, MDS/MPD associated with PDGFR, Ph + ALL
Sunitinib (Sutent)
(n = 1)
PDGFR, KIT, VEGFR, RET, FLT3 RCC, PDAC, GIST RCC, PDAC, GIST
ABL Abelson murine leukemia viral oncogene homolog 1, ALK Anaplastic lymphoma kinase, ALL acute lymphatic leukemia, BCR breakpoint cluster region, CML chronic myleloid leukemia, CRC colorectal cancer, EGFR epidermal growth factor receptor, EMA European Medicines Agency, FDA Food and Drug Administration, FLT3 fms like tyrosine kinase 3, GIST gastrointestinal stromal tumor, HNSCC Head and neck squamous cell carcinoma, MDS/MPD myelodysplastic syndrome/ myeloproliferative disorder, NSCLC Non-small cell lung carcinoma, PDAC pancreatic ductal adenocarcinoma, PDGFR platelet derived growth factor receptor, Ph+ Philadelphia chromosome positive, p‑mTOR phosphorylated mammalian target of rapamycin, RCC renal cell carcinoma, RET rearranged during transfection, TP53 tumor protein 53, VEGFR vascular endothelial growth factor
Table 3 Detailed characteristics of the PGC patients (n = 10)
Patient number, gender and age Histological subtype and Stage and side Site of metastasis Tissue tested Detected mutations by NGS IHC Therapy recommendation
1
Female
61 years
Acinic cell carcinoma IV°
Right
Lung Metastatic No mutation detected Not done (due to insufficient tissue material) No recommendation
2
Male
83 years
Adenocarcinoma IV°
Right
Liver lung Metastatic
(liver)
No mutation detected EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 3+,
AR 2+
Androgen deprivation therapy
3
Female
37 years
Adenocarcinoma IV°
Right
Bone Metastatic No mutation detected EGFR 2+,
KIT 2+,
PTEN 2+,
p‑mTOR 3+
No recommendation
4
Male
71 years
Adenocarcinoma IV°
Left
Bone Primary No mutation detected EGFR 3+,
PTEN 1+,
p‑mTOR 1+,
AR 2+
Androgen deprivation therapy
5
Male
46 years
Adenoid cystic carcinoma IV°
Left
Lung Metastatic No mutation detected KIT 1+,
MET 3+,
PDGFR 1+,
PTEN 1+,
p‑mTOR 2+
Crizotinib
6
Female
56 years
Adenoid cystic carcinoma IV°
Right
Lung Primary ATM: exon 32
c.C9142G (p.Leu3048Val)
EGFR 3+,
MET 1+,
p‑mTOR 1+,
PTEN 1+
Cetuximab
7
Male
47 years
Adenoid cystic carcinoma IV°
Right
Lung Primary APC: exon 16
c.T3920A (p.I1307K)
KIT 1+,
EGFR 2+,
MET 1+,
PDGFRA 1+,
PTEN 1+,
p‑mTOR 1+
Imatinib
8
Male
65 years
Carcinoma ex pleomorphic adenoma IV°
Right
Lung,
Brain
Metastatic
(lung)
TP53 (exon 7):
c.C742T
(p.R248W)
EGFR 1+,
MET 1+,
PDGFRA 2+,
PTEN 1+,
p‑mTOR 1+
Sunitinib
9
Female
67 years
Primary squamous cell carcinoma IV°
Left
Bone,
lung
Primary CDKN2A (exon 2): c.151_155delGTCT (p.V51 fs);
FGFR1 (exon 5):
c.478_480delGAT (p.Asp.160del);
SMARCB1 (exon 9):
c.G1130A (p.R3677H)
EGFR 3+,
PTEN 2+
Cetuximab
10
Female
72 years
Primary squamous cell carcinoma IV°
Left
Lung Primary CDKN2A (exon 2):
c.C341T,
(p.P114L)
EGFR 1+,
MET 3+,
PDGFRA 1+,
p‑mTOR 1+
Crizotinib
Values in parentheses indicate the immunohistochemical score that was calculated as mentioned in the “Materials and methods” section
APC adenomatous polyposis coli, AR androgen receptor, CDKN2A cyclin-dependent kinase inhibitor 2A, EGFR epidermal growth factor receptor, FiSH fluorescence in situ hybridization, PDGFR platelet derived growth factor receptor, p‑mTOR phosphorylated mammalian target of rapamycin, PTEN phosphatase and tensin homolog, TP53 tumor protein 53, IHC immunohistochemistry, NGS next-generation sequencing
The median turnaround time from the initiation of molecular profiling to therapy initiation was 43 days.
Eventually, three patients received the targeted therapy. One male patient with an adenocarcinoma was administered bicalutamide as ADT but died because of disease progression before restaging was performed. The second patient with a carcinoma ex pleomorphic adenoma received sunitinib 50 mg orally once daily combined with docetaxel every third week but did not respond to this therapy regimen and experienced progressive disease. The third patient had an adenoid cystic carcinoma and was given imatinib 400 mg orally once daily. He achieved a stable disease for 14 months and tolerated the therapy without any treatment-related adverse events.
Discussion
To our knowledge, this is the first study of individual genomic alterations that have been translated into concrete tailored therapy recommendations in a group of patients with exclusively recurrent, progressive, and therapy-refractory PGC in stage IV in a real-world setting. None of these patients had the histological subtype mucoepidermoid carcinoma (MEC); instead, they had rarer subtypes, making this subgroup analysis even more valuable and unique.
In this retrospective single center subgroup analysis, we exclusively present the molecular profile of all ten patients with PGC. Their disease was relapsed, therapy-refractory and advanced. Tumor tissue was obtained from all patients and characterized regarding molecular profiles. Subsequently, the genomic information of the patients was discussed in a multidisciplinary tumor board (MTB) for PCM to evaluate the possibility of a genomic-based therapy concept that is independent of the tumor’s histological classification (tissue-agnostic drugs).
Tumor samples harbored mutations in APC, ATM, CDKN2A, FGFR1, SMARCB1, and TP53. The IHC revealed expressions of EGFR and p-mTOR as well as PTEN in eight patients.
Therapeutic options recommended were ADT, cetuximab, crizotinib, sunitinib, and imatinib. Two patients with AR expression were offered ADT to control the disease. Two patients with strong MET expression were suggested crizotinib as a tailored therapy. For two patients with high EGFR expression cetuximab was recommended. Imatinib was considered in one patient due to expression of KIT and PDGFRA. Sunitinib was proposed to one patient because of PDGFRA overexpression. A treatment recommendation was derived for eight patients from the MTB. The drugs were carefully selected for an individualized treatment with special respect to the patient’s clinical and treatment history and concomitant therapies and comorbidities. Interestingly, all these recommendations were based on the protein expressions obtained by immunohistochemistry. Thus, our analysis underscores the clinical relevance of immunohistochemistry in precision medicine.
Eventually, three patients received the targeted therapy. One patient died before restaging was performed. The second patient received sunitinib and did not respond. Imatinib was applied to the third patient who experienced a stable disease for 14 months. Although this analysis showed that PCM is implementable in daily clinical routine, only one patient had a clinical benefit from this therapy approach. One reason may be the turnaround time: a shorter turnaround time may help to start the targeted therapy earlier and to control the cancer disease. Liquid biopsy may be a viable option to reduce the turnaround time, to monitor the disease and to assess the therapy response. Another reason may be the complexity of PGC. The major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguishes over 20 types of malignant salivary gland tumors [4].
The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance.
The observed genomic aberrations and overexpression of AR, KIT, and EGFR, PTEN, p-mTOR, and PDGFRA in PGC in this analysis are in keeping with previous studies [18–33]. The rationale for the therapy recommendation with ADT was corroborated by a study by Boon et al. They studied the application of ADT in 35 patients with androgen receptor-positive advanced salivary duct carcinoma, which lead to a median overall survival (OS) of 17 months versus 5 months in 43 patients receiving best supportive care [34].
The overexpression of MET was seen in all three patients with adenoid cystic carcinoma and is in line with other studies [35]; however, to our knowledge, this is the first report of an overexpression also in carcinoma ex pleomorphic adenoma and primary squamous cell carcinoma. Crizotinib was offered as a molecularly driven treatment approach. Its clinical efficacy in salivary gland cancers has not yet been described in clinical trials.
Only one study has used molecular profiling to offer an individualized therapy in patients with metastatic salivary gland adenoid cystic carcinoma (ACC). They enrolled a limited 14 patients, of whom 11 actually received the recommended treatment. The investigators reported the clinical benefit of molecularly guided treatment [36]. Imatinib and sunitinib are tyrosine kinase inhibitors that were offered, each in one case, as an alternative therapy in the case of overexpression in PDGFRA/B or KIT. The data pertaining to the use of imatinib in salivary gland cancer are contradictory and unclear. According to two phase II trials that applied imatinib in patients with KIT-positive adenoid cystic cancers of salivary glands, imatinib was not of significant clinical benefit and the best observed response was a stable disease (SD).
As a limitation, however, it should be noted that only ACC was studied, and PDGFR expression of the tumor tissue was not evaluated [37, 38]. In contrast, another phase II trial tested imatinib in 15 patients with ACC of salivary glands and concluded that imatinib was of clinical benefit because it achieved a partial response (PR) in two patients and a SD in five other patients [39]. Likewise, in another study, imatinib achieved significant regression of initially unresectable ACC of salivary glands in two patients, making them eligible for a salvage resection [40].
Similar to imatinib, sunitinib was also tested in a phase II trial in 13 patients with ACC of salivary glands and 11 of these achieved stable disease; however, the investigators did not test the patients’ tumors for KIT or PDGFR expression [41]. Dasatinib is another tyrosine kinase inhibitor that was investigated in a phase II trial for patients with recurrent or metastatic KIT expressing ACC and for nonadenoid cystic malignant salivary tumors. It achieved only one PR in a patient with ACC and the experimental treatment demonstrated no activity in non-ACC salivary gland cancer [42].
In another phase II trial, axitinib was applied in 33 patients with unresectable ACC. Ho et al. reported that axitinib achieved a PR in 3 patients and a SD in 25 patients. The median progression-free survival (PFS) was 5.7 months [43]. Overexpression of EGFR was often observed in salivary gland cancers and provides a solid and sound rationale for the administration of cetuximab [26, 27]. Its clinical efficacy was examined by Locati et al. in 2009 in salivary gland carcinomas, and they reported a clinical benefit rate of 50% [44].
Notably, we identified p-mTOR overexpression in eight patients; however, we did not consider p-mTOR inhibition with everolimus because of the low evidence for clinical efficacy.
Despite great research efforts and the investigated agents in PGC and other salivary gland cancers, progressive recurrent PGC has a dismal prognosis, and because of the rarity of the disease, well-established therapeutic options are scarce.
Great strides in the in-depth analysis of the vast genetic and epigenetic landscape of salivary gland cancers have been made in recent years; however, the PCM approach remains in its infancy when it comes to implementing novel individualized therapeutic strategies and concepts for this malignancy [33, 45, 46].
It is challenging to classify and prioritize the plethora of reported genetic alterations and epigenetic changes to identify actionable targets and to choose adequate tailored therapeutic measures. Thus, the roles of most identified alterations are undefined regarding pathogenesis, therapeutic consequences, and implications [47].
Another major challenge is the extreme and complex phenotypical, morphological, histological, clinical, and even intertumor and intratumor heterogeneity within the same tumor tissue [45]. The WHO classification of salivary gland tumors 2017 distinguished over 20 types of malignant salivary gland tumors [4]. The heterogeneity, diversity and the multitude of biological differences between patients may urge the development of novel drugs that are capable of targeting various alterations to increase the efficacy of therapeutic agents and to minimize the risk of drug resistance. In light of the complexity of PGC, molecularly driven clinical trials in PCM have to be designed as basket and umbrella trials that take into account the diversity of this malignancy for a better outcome.
Conclusion
This analysis clearly shows that molecular profiling from tumor samples of patients with advanced, heavily pretreated and therapy-refractory PGC in stage IV is feasible and results in meaningful and rational therapy recommendations and strategies; however, the complex tumor biology, heterogeneity, and extreme rarity remain unique challenges for the management of PGC and need to be addressed by further studies seeking a better understanding of this malignancy. In rare diseases such as PGC where randomized trials cannot be performed easily, molecularly driven treatment approaches and strategies may be particularly useful tools and viable options.
Abbreviations
ADTAndrogen deprivation therapy
AKTAlpha serine/threonine-protein kinase
ALKAnaplastic lymphoma kinase
ALLAcute lymphoblastic leukemia
APCAdenomatous polyposis coli
ARAndrogen receptor
ATMAtaxia telangiectasia mutated
BAP1BRCA1 associated protein‑1
BRAFB‑rapidly accelerated fibrosarcoma
BRCABreast cancer 1
CDKN2ACyclin-dependent kinase inhibitor 2A
CMLChronic myeloid leukemia
CRCColorectal cancer
DCRDisease control rate
ECOGEastern Cooperative Oncology Group
EGFREpidermal growth factor receptor
EMAEuropean Medicines Agency
FDAFood and Drug Administration
FGFRFibroblast growth factor receptor
FiSHFluorescence in situ hybridization
FLT3Fms like tyrosine kinase 3
GISTGastrointestinal stromal tumor
HER2Human epidermal growth factor receptor 2
HNSCCHead and neck squamous cell carcinoma
HLHodgkin lymphoma
IDHIsocitrate dehydrogenase 1
IHCImmunohistochemistry
lossPTENLoss of phosphatase and tensin homolog
MDS/MPDMyelodysplastic syndrome/myeloproliferative disorder
MECMucoepidermoid carcinoma
MTBMultidisciplinary tumor board
mTORMammalian target of rapamycin
NF1Neurofibromin 1
NOSNot otherwise specified
NSCLCNon-small cell lung carcinoma
PARPPoly [ADP-ribose] polymerase 1
PCMPrecision cancer medicine
PDACPancreatic ductal adenocarcinoma
PDGFRPlatelet-derived growth factor receptor
PD-L1Programmed death-ligand 1
PGCParotid gland carcinoma
Ph+Philadelphia chromosome positive
PIK3CBPhosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit beta
p-mTORphosphorylated Mammalian target of rapamycin
PRPartial response
PTENPhosphatase and tensin homolog
PTPN11Protein phosphatase non-receptor type 11
RB1Retinoblastoma 1
RCCRenal cell carcinoma
SDStable disease
SGCSalivary gland carcinomas
SHHSonic hedgehog
SMARCB1SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1
SMOSmoothened
STK11Serine/threonine kinase 11
TP53Tumor protein 53
TRKNeurotrophin receptor kinases
VEGFRVascular endothelial growth factor
VHLvon Hippel-Lindau
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Funding
This research did not receive any grants or funding.
Funding
Open access funding provided by Medical University of Vienna.
Conflict of interest
H. Taghizadeh, L. Müllauer, R.M. Mader, T. Füreder, and G.W. Prager declare that they have no competing interests. | 50 mg (milligrams). | DrugDosage | CC BY | 33296026 | 19,253,223 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Fungal infection'. | Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells. The phase II HORIZON trial evaluated the efficacy of melflufen plus dexamethasone in relapsed and refractory multiple myeloma (RRMM), a population with an important unmet medical need.
Patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody received melflufen 40 mg intravenously on day 1 of each 28-day cycle plus once weekly oral dexamethasone at a dose of 40 mg (20 mg in patients older than 75 years). The primary end point was overall response rate (partial response or better) assessed by the investigator and confirmed by independent review. Secondary end points included duration of response, progression-free survival, overall survival, and safety. The primary analysis is complete with long-term follow-up ongoing.
Of 157 patients (median age 65 years; median five prior lines of therapy) enrolled and treated, 119 patients (76%) had triple-class-refractory disease, 55 (35%) had extramedullary disease, and 92 (59%) were refractory to previous alkylator therapy. The overall response rate was 29% in the all-treated population, with 26% in the triple-class-refractory population. In the all-treated population, median duration of response was 5.5 months, median progression-free survival was 4.2 months, and median overall survival was 11.6 months at a median follow-up of 14 months. Grade ≥ 3 treatment-emergent adverse events occurred in 96% of patients, most commonly neutropenia (79%), thrombocytopenia (76%), and anemia (43%). Pneumonia (10%) was the most common grade 3/4 nonhematologic event. Thrombocytopenia and bleeding (both grade 3/4 but fully reversible) occurred concomitantly in four patients. GI events, reported in 97 patients (62%), were predominantly grade 1/2 (93%); none were grade 4.
Melflufen plus dexamethasone showed clinically meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM, including those with triple-class-refractory and extramedullary disease.
pmcINTRODUCTION
Despite the introduction of novel therapies and regimens that have improved outcomes in multiple myeloma (MM),1,2 almost all patients will relapse.1,3 After relapse, treatment choice is usually determined by the class of and response to previous treatment and patient characteristics.2,3 Although class switching is generally prioritized, this is becoming increasingly difficult, not least because novel agents are commonly administered in combination in earlier treatment lines, resulting in disease resistant to multiple drug classes as early as second-line therapy.2,3
CONTEXT
Key Objective
To evaluate whether melphalan flufenamide (melflufen) plus dexamethasone is effective and safe in patients with heavily pretreated relapsed and refractory multiple myeloma (RRMM), a population with a high unmet medical need.
Knowledge Generated
In this pivotal, phase II study, melflufen plus dexamethasone showed meaningful efficacy in heavily pretreated patients with RRMM, including patients with triple-class–refractory disease and those with extramedullary disease. The safety profile of melflufen plus dexamethasone was consistent with previously reported data and was characterized primarily by clinically manageable hematologic toxicities.
Relevance
As new combinations of antimyeloma drugs are introduced in earlier lines of therapy, patients with RRMM often have disease that is refractory to multiple drugs. Therefore, drugs with novel mechanisms of action are urgently needed. Melflufen, when combined with dexamethasone, has the potential to fill this unmet medical need by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety in patients with RRMM.
Outcomes are particularly poor for patients with high-risk cytogenetics, extramedullary disease, and MM resistant to multiple drug classes, including those with triple-class–refractory disease who represent groups with a high unmet need.1,3,4 Furthermore, patients with relapsed and refractory multiple myeloma (RRMM) may have comorbidities because of age, disease symptoms, and cumulative toxicities stemming from previous therapies.5,6 There is an urgent requirement for agents with novel mechanisms of action that are effective, safe, and tolerable and that maintain quality of life in patients with aggressive and resistant disease.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells.7-12 Melflufen is rapidly and passively taken up by cells because of its high lipophilicity, thereby circumventing the development of transporter-associated resistance.8,11,13 Intracellular aminopeptidases hydrolyze melflufen to release hydrophilic alkylating moieties.11 Melflufen and its metabolites melphalan and desethyl-melflufen have equipotent alkylating potential.11 Unlike previous aminopeptidase-targeting therapies that directly inhibit aminopeptidase activity, melflufen takes a novel approach by leveraging increased aminopeptidase activity to selectively direct potent cytotoxic agents into tumor cells.11,14,15 Melflufen and its metabolites trigger robust and irreversible DNA damage, have antiangiogenic effects, induce apoptosis—resulting in potent antitumor activity in myeloma cells, including those with resistance to melphalan, bortezomib, and dexamethasone—and, importantly, retain activity in myeloma cells with absent or impaired p53 function.8-10,16 Melflufen may also have activity in other hematologic malignancies (including immunoglobulin light chain amyloidosis and leukemia) and solid tumors (including breast cancer and ovarian cancer).11
The phase I/II, multicenter O-12-M1 trial established the dosage of melflufen plus dexamethasone in patients who had RRMM, received a median of four previous lines of therapy (including lenalidomide and bortezomib), and had disease refractory to their last line of therapy.17 In 45 patients treated with infusional melflufen 40 mg administered on day 1 of each 28-day cycle and once weekly dexamethasone dosed at 40 mg, the overall response rate (ORR) was 31%, the median duration of response (DOR) was 8.4 months, the median progression-free survival (PFS) was 5.7 months, and the median overall survival (OS) was encouraging at 20.7 months. The safety profile of melflufen was characterized primarily by hematologic toxicities that were clinically manageable with appropriate dose delays, dose reductions, and supportive care. Based on these results, the efficacy and safety of melflufen plus dexamethasone were therefore evaluated in the current study in a larger population with heavily pretreated, resistant, and poor-risk RRMM, including those with triple-class–refractory disease, for whom few effective treatment options exist.3
PATIENTS AND METHODS
Study Design and Participants
HORIZON (OP-106; ClinicalTrials.gov identifier: NCT02963493) was a pivotal, single-arm, multicenter, phase II study of melflufen plus dexamethasone in patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. Patients were enrolled from December 28, 2016, to October 14, 2019, at 17 sites (see the Data Supplement, online only). Eligible adult patients had an Eastern Cooperative Oncology Group performance status score of 0-2, a previous diagnosis of MM with disease progression, and measurable disease (serum monoclonal protein ≥ 5 g/L, urine monoclonal protein ≥ 200 mg per 24 hours, or serum immunoglobulin-free light chain ≥ 100 mg/L, and abnormal serum immunoglobulin kappa to lambda–free light chain ratio) at study entry. Patients had received at least two prior lines of therapy, including an immunomodulatory agent and proteasome inhibitor, and were refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. RRMM was defined as disease that was nonresponsive (ie, did not achieve a minimal response or better, or developed progressive disease with treatment) while on primary or salvage therapy or progressed within 60 days of last therapy.18 Please see the Data Supplement for full eligibility criteria. Patients received once-monthly melflufen 40 mg as a 30-minute central intravenous infusion on day 1 of each 28-day cycle in combination with oral dexamethasone 40 mg (20 mg for patients age ≥ 75 years) once-weekly administered on days 1, 8, 15, and 22 of each 28-day cycle until disease progression, unacceptable toxicity, or the patient or treating physician determined it was not in the patient's best interest to continue. Melflufen dose reduction for drug-related toxicities was allowed in 10 mg increments each cycle from 40 mg down to 30 mg and from 30 mg down to 20 mg (see the Data Supplement).
This study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation guidelines for Good Clinical Practice. The Protocol was reviewed and approved by national regulatory authorities and an independent ethics committee or institutional review board at each study center. Each patient provided written informed consent.
Outcomes
The primary end point was ORR, defined as the proportion of patients achieving a confirmed response of stringent complete response (sCR), complete response (CR), very good partial response (VGPR), or partial response (PR) as their best response per International Myeloma Working Group (IMWG) uniform response criteria, as assessed by the investigator.18 Response, confirmed response, and confirmed progression were subsequently verified by an independent review committee.18 Secondary end points included DOR, PFS, OS, clinical benefit rate (CBR), best response, time to response, time to progression, time to next treatment, and safety (defined in the Data Supplement). All response categories required confirmation with two consecutive assessments (see the Data Supplement). Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events, version 4.03. AE frequency and relationship to study treatment were summarized.
Statistical Analysis
Planned enrollment was 150 patients. ORR and associated two-sided exact 95% CI19 were estimated for all patients treated (all-treated population). With a sample size of 150 patients and an assumed ORR of 30%, the exact 95% CI was estimated to range between 23% and 38%. CBR and disease stabilization were also summarized. Time-to-event end points were summarized using the Kaplan-Meier method in the all-treated population. Median and estimated 95% CIs were constructed using the methods of Brookmeyer and Crowley20; duration of follow-up was estimated by the reverse Kaplan-Meier methods of Schemper and Smith.21 See the Data Supplement for patient censoring and handling of missing data.
A preplanned subgroup analysis was performed in patients with triple-class–refractory MM (refractory to or intolerant of at least one immunomodulatory drug, at least one proteasome inhibitor, and at least one anti-CD38 monoclonal antibody). With a sample size of 150 patients, 104-120 patients with triple-class–refractory disease were expected; the primary end point was considered met if the lower bound of the 95% CI for the ORR was higher than 15%. Additional subgroup analyses, including extramedullary disease, are described in the Data Supplement. Extramedullary disease was assessed at baseline for patients with known or suspected extramedullary disease and to confirm a response achieved by M-protein or for suspected progression per IMWG uniform response criteria.18
RESULTS
Patients
In total, 157 patients were enrolled in the study, received at least one dose of study medication, and were included in the all-treated population. At the data cutoff date (January 14, 2020), 131 patients (83%) had discontinued treatment—the most common primary reasons for discontinuation were disease progression (n = 88; 56%) and AEs (n = 26; 17%)—and 26 patients (17%) remained on treatment (Fig 1). The median duration of treatment with melflufen plus dexamethasone was 3.8 months (range, 0.9-22.7 months). At baseline, the median age was 65 years, patients had received a median of five prior lines of therapy, 154 patients (98%) had disease that was refractory to the last line of therapy received, 119 (76%) had triple-class–refractory disease, and 92 (59%) had MM that was refractory to prior alkylator therapy (Table 1). Overall, 59 patients (38%) had high-risk cytogenetics, 39 (25%) had International Staging System stage III disease, and 55 (35%) had extramedullary disease.
FIG 1. Trial profile. OS, overall survival; PFS, progression-free survival.
TABLE 1. Baseline Demographics and Clinical Characteristics in the Overall Population
Efficacy
The ORR per investigator assessment was 29% (95% CI, 22% to 37%), with one patient achieving an sCR, 17 a VGPR, and 28 a PR (Table 2). An additional 25 patients achieved a minimal response for a CBR of 45% (95% CI, 37% to 53%). In the triple-class–refractory population, the ORR was 26% (95% CI, 18% to 35%), with 13 patients achieving a VGPR and 18 a PR. The ORR per independent review committee was 30% (95% CI, 23% to 38%) overall and 26% (95% CI, 18% to 35%) in the triple-class–refractory population (Data Supplement). Reduction in M-protein was observed in 118 of the 145 patients (81.4%) (Data Supplement). In the all-treated and triple-class–refractory populations, the median time to PR or better was 1.9 months (range, 1.0-7.4 months) and 1.9 months (range, 1.0-6.1 months), respectively, and the median duration of PR or better was 5.5 months (95% CI, 3.9 to 7.6 months) and 4.4 months (95% CI, 3.4 to 7.6 months), respectively (Fig 2 and Data Supplement).
TABLE 2. Overall Response and Clinical Benefit Rate
FIG 2. Duration of response to melflufen plus dexamethasone. Data on patients in the all-treated population (n = 46), triple-class–refractory population (asterisk; n = 31), and extramedullary subgroup (dagger; n = 13) who achieved a PR or better as the best response. Open circles indicate the latest dose of melflufen received; arrows indicate patients still receiving treatment at the data cutoff date; orange Xs indicate progression-free survival events. CR, complete response; MR, minimal response; PR, partial response; sCR, stringent complete response; SD, stable disease; VGPR, very good partial response.
In the all-treated and triple-class–refractory populations, the median PFS was 4.2 months (95% CI, 3.4 to 4.9 months) and 3.9 months (95% CI, 3.0 to 4.6 months), respectively (Fig 3A). The median OS was 11.6 months (95% CI, 9.3 to 15.4 months) and 11.2 months (95% CI, 7.7 to 13.2 months), with an estimated 1-year event-free rate of 48.8% (95% CI, 39.6% to 57.4%) and 41.9% (95% CI, 31.6% to 51.8%), respectively (Fig 3B), at a median follow-up of 14 months (range, 10.8-18.7 months). Among responders, the median PFS was 8.5 months (95% CI, 5.4 to 13.4 months) and 8.5 months (95% CI, 5.3 to 13.4 months), and the median OS was 17.6 months (95% CI, 13.2 to 28.9 months) and 16.5 months (95% CI, 11.5 to 18.5 months) in the all-treated and triple-class–refractory populations, respectively (Data Supplement). Among patients in the all-treated population and the triple-class–refractory group (n = 70 and n = 52, respectively) who discontinued the study and initiated a new myeloma therapy, the median time to next therapy was 8.2 months (95% CI, 7.2 to 10.8 months) and 7.9 months (95% CI, 6.9 to 10.9 months), respectively. The median time to next therapy or death was 5.8 months (95% CI, 4.8 to 7.1 months) in the all-treated population and 5.3 months (95% CI, 4.5 to 6.3 months) in the triple-class–refractory group.
FIG 3. PFS and OS. Kaplan-Meier analysis of (A) PFS and (B) OS in the all-treated (N = 157) and triple-class–refractory (n = 119) populations. OS, overall survival; PFS, progression-free survival.
In a subgroup analysis, 19 of the 54 patients (35%) age 65-74 years and 8 of the 25 patients (32%) older than 75 years achieved a PR or better. In addition, a PR or better was achieved in 13 of the 55 patients (24%) with extramedullary disease and 12 of the 59 patients (20%) with high-risk cytogenetics (Data Supplement). Among patients with MM refractory to previous alkylator therapy, the ORR was 21% (19 of the 92 patients achieved a PR or better, including one sCR, six VGPRs, and 12 PRs) and the CBR was 34% (Data Supplement). Among patients refractory to an alkylator in one previous line of therapy (n = 60), the ORR was 28% (CBR, 40%). In patients refractory to alkylators in multiple previous lines of therapy (n = 32), the ORR was 6% (CBR, 22%). Median PFS and OS in the subgroups analyzed were consistent with those of the all-treated population (Data Supplement).
Safety
Treatment-emergent AEs (TEAEs) were reported in all 157 patients (100%) in the all-treated population, with 149 (95%) reporting at least one melflufen-related TEAE (Table 3 and Data Supplement). Grade ≥ 3 TEAEs occurred in 150 patients (96%), most commonly neutropenia (124 [79%]), thrombocytopenia (120 [76%]), and anemia (67 [43%]). Any-grade and grade 3/4 bleeding events with concurrent grade 3/4 thrombocytopenia occurred in 25 patients (16%) and four patients (3%), respectively. The most common nonhematologic treatment-emergent grade 3/4 events included pneumonia (16 [10%]; grade 3, 14 [9%]; grade 4, two [1%]) and hypophosphatemia (eight [5%]; grade 3, eight [5%]; grade 4, 0). Grade 3/4 neutropenia with concurrent grade 3/4 infections occurred in 18 patients (11%); of these, 11 (7%) had pneumonia (Data Supplement). GI events occurred in 97 patients overall and were grade 1/2 in 90 of the 97 patients (93%) and grade 3 in seven of the 97 patients (7%). No grade 4 events were reported. The most common any-grade GI events included nausea (50 [32%]), diarrhea (42 [27%]), constipation (23 [15%]), and vomiting (21 [13%]). Mucositis occurred in one patient (1%; grade 1 event), and there were no reports of alopecia or neuropathy.
TABLE 3. TEAEs (Occurring in ≥ 10% of Patients) in the All-Treated Population
Serious TEAEs occurred in 77 patients (49%), most commonly pneumonia (14 [9%]) and febrile neutropenia (eight [5%]; Data Supplement). Second primary malignancies occurred in five patients; of these, four had malignancies with cutaneous manifestations (two patients with basal cell carcinoma, one patient with squamous cell carcinoma, and one patient with basal cell carcinoma, squamous cell carcinoma, and malignant melanoma; see the Data Supplement). One patient developed myelodysplasia after having received 17 cycles of study medication and in the context of multiple prior cycles of alkylator-based therapy, including stem-cell transplant prior to study entry. Moreover, the review of fluorescence in situ hybridization studies from the screening bone marrow confirmed pre-existing abnormalities supporting a subclinical myelodysplastic syndrome that was likely treatment-related and not otherwise apparent. No other cases of myelodysplastic syndromes were seen. Overall, 10 patients (6%) died from TEAEs. Most commonly, general physical health deterioration was associated with progressive disease (n = 3; 2%) and respiratory failure (n = 2; 1%; Data Supplement). None of the deaths were considered related to melflufen.
The average (standard deviation) monthly dose of melflufen received was 37.8 mg (± 4.0). TEAEs leading to melflufen dose reductions occurred in 42 patients (27%), most commonly thrombocytopenia (n = 22; 14%) and neutropenia (n = 5; 3%). While on study, 102 patients (65%) received concomitant RBC or platelet transfusion support, with 68 (43%) receiving platelet transfusion support only and 106 (68%) receiving concomitant growth factor support (Data Supplement). Overall, 34 patients (22%) had at least one TEAE leading to melflufen treatment discontinuation, most commonly thrombocytopenia (n = 16) and neutropenia (n = 5; Data Supplement). Overall, 95 patients (61%) experienced at least one dose delay, and the median number of treatment cycles with a dose delay was one (range, 0-9).
DISCUSSION
In this study, melflufen plus dexamethasone demonstrated meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM. These findings build substantially on previously reported results17 but in a population that is more aligned with current treatment practice in the relapsed and refractory as well as highly resistant disease setting (ie, patients refractory to an anti-CD38 monoclonal antibody and/or pomalidomide, as well as exposed and refractory to prior lenalidomide, dexamethasone, and proteasome inhibitors). Durable responses were seen in this heavily pretreated population with a high proportion of extramedullary disease and high-risk cytogenetic features. Although the median DOR was 5.5 months, the median PFS among responders was encouragingly longer at 8.5 months. Furthermore, the median time to first response was 1.9 months, but many patients achieved their best response beyond 2 months of treatment. Altogether, these data support the notion that the clinical benefit of melflufen plus dexamethasone improves with longer treatment duration.
The ORR of 29% was consistent among high-risk patient subgroups, including those with triple-class–refractory disease (26%), those with extramedullary disease (24%), and patients age 75 years or older (32%), which is encouraging given the reported ORRs (10%-31%) in patients refractory to anti-CD38 monoclonal antibody therapy and/or with extramedullary disease at relapse.3,4,23-25 In fact, this is the largest population with extramedullary disease reported to date in a prospective study.4,26,27 Subgroup analyses showed sufficient efficacy in 60 patients refractory to an alkylator in one previous line of therapy with an ORR of 28%, while the ORR was only 6% in the 32 patients refractory to alkylators in two or more previous lines. Melflufen may have a mechanism of action that is different from that of other alkylators.8,11 For example, melflufen induced cell death more effectively than melphalan in TP53-mutated cell lines and in cells from patients with TP53-mutated RRMM, suggesting that the mechanism of cytotoxicity of melflufen—but not that of other alkylators—is independent of p53 function.8,11,16 Unlike other newer agents that work via immune-based mechanisms (including chimeric antigen receptor T cell therapy, belantamab mafodotin, iberdomide, and isatuximab), melflufen adds a unique mechanism of action to the treatment landscape in relapsed disease as a potent and novel cytotoxic agent targeting myeloma more broadly while providing meaningful clinical efficacy and a manageable safety profile for heavily pretreated RRMM.8,10,28-30
The safety profile of melflufen primarily consisted of hematologic AEs, consistent with previous results.17 Despite cytopenias being common, the incidence of significant bleeding events or infections was low. Hematologic AEs were generally reversible and clinically manageable with dose adjustments, dose delays, growth factor use, platelet transfusions, and appropriate supportive care. Nonhematologic grade 3/4 AEs were infrequent, with infections being the most common. Moreover, the frequency of infections was generally consistent with the expected rates of infections in heavily pretreated patients.23,27,31 Specifically, the 10% rate of grade 3/4 pneumonia reported in HORIZON was similar to 9%-11% reported with pomalidomide plus dexamethasone, bortezomib plus dexamethasone, and selinexor plus dexamethasone in RRMM.23,27,31 GI toxicities, a common reason for treatment discontinuation with other agents,23 were infrequent, primarily grade 1/2, and did not lead to melflufen treatment cessation in HORIZON in any patient. Encouragingly, alopecia and treatment-emergent peripheral neuropathy were not reported. Patients were therefore able to tolerate treatment, with rates of discontinuation from AEs lower than or comparable with other studies (which range from 6% to 33%) in this patient population and with a prolonged median duration of treatment, together with the added convenience of monthly infusions, which is an especially important consideration in the current era of COVID-19.23,27,28
In conclusion, the results from HORIZON suggest that melflufen has the potential to be an important therapeutic option in RRMM by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety when combined with dexamethasone in heavily pretreated patients.32 Based on these results, the efficacy and safety of melflufen plus dexamethasone versus pomalidomide plus dexamethasone are being further evaluated in OCEAN (OP-103), a randomized, global, phase III multicenter study (ClinicalTrials.gov identifier: NCT03151811) for patients in earlier relapse.33 Studies of melflufen plus dexamethasone in combination with bortezomib or daratumumab are also ongoing, with promising results to date.34
PRIOR PRESENTATION
SUPPORT
CLINICAL TRIAL INFORMATION
ACKNOWLEDGMENT
The authors especially thank the patients and their families for participating in this trial and all the study investigators and coordinators for their contributions to this work. The authors also thank Jakob Obermüller and Hanan Zubair (Oncopeptides AB, Stockholm, Sweden) for data management as well as Katherine Mills-Lujan, PhD, CMPP, and Jennifer Leslie, PhD, CMPP, of Team 9 Science for providing medical editorial assistance under the guidance of the authors, which was funded by Oncopeptides AB in accordance with Good Publications Practice (GPP3) guidelines.
DATA SHARING STATEMENT
Oncopeptides commits to share clinical study data with qualified researchers to enable enhancement of public health. As such, Oncopeptides will share anonymized patient-level data on request or if required by law or regulation. Qualified scientific and medical researchers can request patient-level data for studies of Oncopeptides pharmaceutical substances listed on ClinicalTrials.gov and approved by health authorities in the United States and the EU. Patient-level data for studies of newly approved pharmaceutical substances or indications can be requested 9 months after US Food and Drug Administration and European Medicines Agency approval. Such requests are assessed at Oncopeptides' discretion, and the decisions depend on the scientific merit of the proposed request, data availability, and the purpose of the proposal. The applicants should be willing to submit both positive and negative findings to a scientific journal. If Oncopeptides agrees to share clinical data for research purposes, the applicant is required to sign an agreement for data sharing before data release, to ensure that the patient data are de-identified. In case of any risk of re-identification on anonymized data despite measures to protect patient confidentiality, the data will not be shared. The patients' informed consent will always be respected. If the anonymization process will provide futile data, Oncopeptides will have the right to refuse the request. Oncopeptides will provide access to patient-level clinical trial analysis datasets in a secured environment upon execution of the data sharing agreement.
Oncopeptides will also provide the Protocol, statistical analysis plan, and the clinical study report synopsis if needed. For additional information or requests for access to Oncopeptides clinical trial data for research purposes, please contact us at medinfo@oncopeptides.com.
AUTHOR CONTRIBUTIONS
Conception and design: Paul G. Richardson, Catriona Byrne, Johan Harmenberg, María-Victoria Mateos
Provision of study materials or patients: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, María-Victoria Mateos
Collection and assembly of data: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Marcus Thuresson, María-Victoria Mateos
Data analysis and interpretation: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, John W. Hiemenz, Cyrille Touzeau, Adrián Alegre, Agne Paner, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Sara Thuresson, Marcus Thuresson, Catriona Byrne, Johan Harmenberg, Nicolaas A. Bakker, María-Victoria Mateos
Manuscript writing: All authors
Final approval of manuscript: All authors
Accountable for all aspects for the work: All authors
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma
The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I =Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/authors/author-center.
Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).
Paul G. Richardson
Consulting or Advisory Role: Celgene, Janssen, Takeda, Karyopharm Therapeutics, Oncopeptides, Sanofi, Jazz Pharmaceuticals, SecuraBio
Research Funding: Celgene, Takeda, Bristol-Myers Squibb, Oncopeptides
Albert Oriol
Consulting or Advisory Role: Celgene, Janssen, Amgen, Sanofi, GlaxoSmithKline
Speakers' Bureau: Amgen, Celgene
Alessandra Larocca
Honoraria: Amgen, Bristol-Myers Squibb, Celgene, Janssen, GlaxoSmithKline
Consulting or Advisory Role: Bristol-Myers Squibb, Celgene, Janssen, Takeda
Joan Bladé
Honoraria: Janssen, Celgene, Amgen, Takeda, Oncopeptides
Michele Cavo
Honoraria: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Consulting or Advisory Role: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Speakers' Bureau: Janssen, Celgene
Paula Rodriguez-Otero
Honoraria: Janssen, Celgene, Amgen, Oncopeptides, Sanofi, Abbvie, GlaxoSmithKline, Kite Pharma
Consulting or Advisory Role: Janssen, Celgene, Amgen, Takeda, Oncopeptides, Sanofi, AbbVie, GlaxoSmithKline, Kite Pharma
Xavier Leleu
Honoraria: Janssen-Cilag, Celgene, Amgen, Novartis, Bristol-Myers Squibb, Takeda, Sanofi, Abbvie, Merck, Roche, Karyopharm Therapeutics, Carsgen Therapeutics Ltd, Oncopeptides, GlaxoSmithKline
Consulting or Advisory Role: Janssen-Cilag, Celgene, Amgen, Takeda, Bristol-Myers Squibb, Novartis, Merck, Gilead Sciences, Abbvie, Roche, Karyopharm Therapeutics, Oncopeptides, Carsgen Therapeutics Ltd, GlaxoSmithKline
Travel, Accommodations, Expenses: Takeda
Omar Nadeem
Consulting or Advisory Role: Janssen, Celgene, Sanofi, Takeda, Adaptive Biotechnologies
Hani Hassoun
Consulting or Advisory Role: Novartis
Research Funding: Takeda, Janssen
Cyrille Touzeau
Honoraria: Abbvie, Celgene, Amgen, Takeda, Janssen, Sanofi, Novartis, GlaxoSmithKline
Consulting or Advisory Role: Novartis, Amgen, Celgene, Abbvie, Takeda, Janssen, GlaxoSmithKline
Research Funding: Abbvie
Adrián Alegre
Leadership: Amgen, Janssen-Cilag, Celgene-BMS, Takeda, Sanofi, GlaxoSmithKline, ONCOPETIDES
Agne Paner
Honoraria: Amgen, Celgene, Janssen
Consulting or Advisory Role: Takeda, Celgene, Amgen, Karyopharm Therapeutics, Oncopetides
Christopher Maisel
Stock and Other Ownership Interests: Actinium Pharmaceuticals, Karyopharm Therapeutics, Amgen
Honoraria: Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead, Oncopetides, GlaxoSmithKline
Speakers' Bureau: Amgen, Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead
Amitabha Mazumder
Honoraria: Karyopharm Therapeutics
Speakers' Bureau: Karyopharm Therapeutics
Anastasios Raptis
Consulting or Advisory Role: intellisphere, integra
Jan S. Moreb
Consulting or Advisory Role: Oncopeptide
Kenneth C. Anderson
Stock and Other Ownership Interests: C4 Therapeutics, OncoPep
Consulting or Advisory Role: Celgene, Millennium, Gilead Sciences, Bristol-Myers Squibb, Janssen Oncology, Sanofi, Tolero Pharmaceuticals, Precision Biosciences
Patents, Royalties, Other Intellectual Property: C4 Therapeutics, OncoPep
Jacob P. Laubach
Research Funding: Abbvie, Bristol-Myers Squibb, Genentech, Janssen Research & Development, Carsgen, Millennium
Sara Thuresson
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Marcus Thuresson
Employment Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Catriona Byrne
Consulting or Advisory Role: Oncopeptides
Travel, Accommodations, Expenses: Oncopeptides
Johan Harmenberg
Leadership: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides, Ectin AB
Travel, Accommodations, Expenses: Oncopeptides
Nicolaas A. Bakker
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Honoraria: Oncopeptides
María-Victoria Mateos
Honoraria: Janssen-Cilag, Celgene, Amgen, Takeda, GlaxoSmithKline, Abbvie/Genentech, Adaptive Biotechnologies
Consulting or Advisory Role: Takeda, Janssen-Cilag, Celgene, Amgen, Abbvie, GlaxoSmithKline, Pharmamar-zeltia
No other potential conflicts of interest were reported.
APPENDIX
TABLE A1. HORIZON (OP-106) Investigators and Recruitment Sites
Presented in part at the 25th European Hematology Association annual congress, virtual format, June 11-21, 2020; abstract EP945.
This study was sponsored by Oncopeptides AB, which also provided support for manuscript editorial assistance.
NCT02963493
See accompanying article on page 836 | AZACITIDINE | DrugsGivenReaction | CC BY-NC-ND | 33296242 | 19,180,518 | 2021-03-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Neutropenia'. | Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells. The phase II HORIZON trial evaluated the efficacy of melflufen plus dexamethasone in relapsed and refractory multiple myeloma (RRMM), a population with an important unmet medical need.
Patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody received melflufen 40 mg intravenously on day 1 of each 28-day cycle plus once weekly oral dexamethasone at a dose of 40 mg (20 mg in patients older than 75 years). The primary end point was overall response rate (partial response or better) assessed by the investigator and confirmed by independent review. Secondary end points included duration of response, progression-free survival, overall survival, and safety. The primary analysis is complete with long-term follow-up ongoing.
Of 157 patients (median age 65 years; median five prior lines of therapy) enrolled and treated, 119 patients (76%) had triple-class-refractory disease, 55 (35%) had extramedullary disease, and 92 (59%) were refractory to previous alkylator therapy. The overall response rate was 29% in the all-treated population, with 26% in the triple-class-refractory population. In the all-treated population, median duration of response was 5.5 months, median progression-free survival was 4.2 months, and median overall survival was 11.6 months at a median follow-up of 14 months. Grade ≥ 3 treatment-emergent adverse events occurred in 96% of patients, most commonly neutropenia (79%), thrombocytopenia (76%), and anemia (43%). Pneumonia (10%) was the most common grade 3/4 nonhematologic event. Thrombocytopenia and bleeding (both grade 3/4 but fully reversible) occurred concomitantly in four patients. GI events, reported in 97 patients (62%), were predominantly grade 1/2 (93%); none were grade 4.
Melflufen plus dexamethasone showed clinically meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM, including those with triple-class-refractory and extramedullary disease.
pmcINTRODUCTION
Despite the introduction of novel therapies and regimens that have improved outcomes in multiple myeloma (MM),1,2 almost all patients will relapse.1,3 After relapse, treatment choice is usually determined by the class of and response to previous treatment and patient characteristics.2,3 Although class switching is generally prioritized, this is becoming increasingly difficult, not least because novel agents are commonly administered in combination in earlier treatment lines, resulting in disease resistant to multiple drug classes as early as second-line therapy.2,3
CONTEXT
Key Objective
To evaluate whether melphalan flufenamide (melflufen) plus dexamethasone is effective and safe in patients with heavily pretreated relapsed and refractory multiple myeloma (RRMM), a population with a high unmet medical need.
Knowledge Generated
In this pivotal, phase II study, melflufen plus dexamethasone showed meaningful efficacy in heavily pretreated patients with RRMM, including patients with triple-class–refractory disease and those with extramedullary disease. The safety profile of melflufen plus dexamethasone was consistent with previously reported data and was characterized primarily by clinically manageable hematologic toxicities.
Relevance
As new combinations of antimyeloma drugs are introduced in earlier lines of therapy, patients with RRMM often have disease that is refractory to multiple drugs. Therefore, drugs with novel mechanisms of action are urgently needed. Melflufen, when combined with dexamethasone, has the potential to fill this unmet medical need by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety in patients with RRMM.
Outcomes are particularly poor for patients with high-risk cytogenetics, extramedullary disease, and MM resistant to multiple drug classes, including those with triple-class–refractory disease who represent groups with a high unmet need.1,3,4 Furthermore, patients with relapsed and refractory multiple myeloma (RRMM) may have comorbidities because of age, disease symptoms, and cumulative toxicities stemming from previous therapies.5,6 There is an urgent requirement for agents with novel mechanisms of action that are effective, safe, and tolerable and that maintain quality of life in patients with aggressive and resistant disease.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells.7-12 Melflufen is rapidly and passively taken up by cells because of its high lipophilicity, thereby circumventing the development of transporter-associated resistance.8,11,13 Intracellular aminopeptidases hydrolyze melflufen to release hydrophilic alkylating moieties.11 Melflufen and its metabolites melphalan and desethyl-melflufen have equipotent alkylating potential.11 Unlike previous aminopeptidase-targeting therapies that directly inhibit aminopeptidase activity, melflufen takes a novel approach by leveraging increased aminopeptidase activity to selectively direct potent cytotoxic agents into tumor cells.11,14,15 Melflufen and its metabolites trigger robust and irreversible DNA damage, have antiangiogenic effects, induce apoptosis—resulting in potent antitumor activity in myeloma cells, including those with resistance to melphalan, bortezomib, and dexamethasone—and, importantly, retain activity in myeloma cells with absent or impaired p53 function.8-10,16 Melflufen may also have activity in other hematologic malignancies (including immunoglobulin light chain amyloidosis and leukemia) and solid tumors (including breast cancer and ovarian cancer).11
The phase I/II, multicenter O-12-M1 trial established the dosage of melflufen plus dexamethasone in patients who had RRMM, received a median of four previous lines of therapy (including lenalidomide and bortezomib), and had disease refractory to their last line of therapy.17 In 45 patients treated with infusional melflufen 40 mg administered on day 1 of each 28-day cycle and once weekly dexamethasone dosed at 40 mg, the overall response rate (ORR) was 31%, the median duration of response (DOR) was 8.4 months, the median progression-free survival (PFS) was 5.7 months, and the median overall survival (OS) was encouraging at 20.7 months. The safety profile of melflufen was characterized primarily by hematologic toxicities that were clinically manageable with appropriate dose delays, dose reductions, and supportive care. Based on these results, the efficacy and safety of melflufen plus dexamethasone were therefore evaluated in the current study in a larger population with heavily pretreated, resistant, and poor-risk RRMM, including those with triple-class–refractory disease, for whom few effective treatment options exist.3
PATIENTS AND METHODS
Study Design and Participants
HORIZON (OP-106; ClinicalTrials.gov identifier: NCT02963493) was a pivotal, single-arm, multicenter, phase II study of melflufen plus dexamethasone in patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. Patients were enrolled from December 28, 2016, to October 14, 2019, at 17 sites (see the Data Supplement, online only). Eligible adult patients had an Eastern Cooperative Oncology Group performance status score of 0-2, a previous diagnosis of MM with disease progression, and measurable disease (serum monoclonal protein ≥ 5 g/L, urine monoclonal protein ≥ 200 mg per 24 hours, or serum immunoglobulin-free light chain ≥ 100 mg/L, and abnormal serum immunoglobulin kappa to lambda–free light chain ratio) at study entry. Patients had received at least two prior lines of therapy, including an immunomodulatory agent and proteasome inhibitor, and were refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. RRMM was defined as disease that was nonresponsive (ie, did not achieve a minimal response or better, or developed progressive disease with treatment) while on primary or salvage therapy or progressed within 60 days of last therapy.18 Please see the Data Supplement for full eligibility criteria. Patients received once-monthly melflufen 40 mg as a 30-minute central intravenous infusion on day 1 of each 28-day cycle in combination with oral dexamethasone 40 mg (20 mg for patients age ≥ 75 years) once-weekly administered on days 1, 8, 15, and 22 of each 28-day cycle until disease progression, unacceptable toxicity, or the patient or treating physician determined it was not in the patient's best interest to continue. Melflufen dose reduction for drug-related toxicities was allowed in 10 mg increments each cycle from 40 mg down to 30 mg and from 30 mg down to 20 mg (see the Data Supplement).
This study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation guidelines for Good Clinical Practice. The Protocol was reviewed and approved by national regulatory authorities and an independent ethics committee or institutional review board at each study center. Each patient provided written informed consent.
Outcomes
The primary end point was ORR, defined as the proportion of patients achieving a confirmed response of stringent complete response (sCR), complete response (CR), very good partial response (VGPR), or partial response (PR) as their best response per International Myeloma Working Group (IMWG) uniform response criteria, as assessed by the investigator.18 Response, confirmed response, and confirmed progression were subsequently verified by an independent review committee.18 Secondary end points included DOR, PFS, OS, clinical benefit rate (CBR), best response, time to response, time to progression, time to next treatment, and safety (defined in the Data Supplement). All response categories required confirmation with two consecutive assessments (see the Data Supplement). Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events, version 4.03. AE frequency and relationship to study treatment were summarized.
Statistical Analysis
Planned enrollment was 150 patients. ORR and associated two-sided exact 95% CI19 were estimated for all patients treated (all-treated population). With a sample size of 150 patients and an assumed ORR of 30%, the exact 95% CI was estimated to range between 23% and 38%. CBR and disease stabilization were also summarized. Time-to-event end points were summarized using the Kaplan-Meier method in the all-treated population. Median and estimated 95% CIs were constructed using the methods of Brookmeyer and Crowley20; duration of follow-up was estimated by the reverse Kaplan-Meier methods of Schemper and Smith.21 See the Data Supplement for patient censoring and handling of missing data.
A preplanned subgroup analysis was performed in patients with triple-class–refractory MM (refractory to or intolerant of at least one immunomodulatory drug, at least one proteasome inhibitor, and at least one anti-CD38 monoclonal antibody). With a sample size of 150 patients, 104-120 patients with triple-class–refractory disease were expected; the primary end point was considered met if the lower bound of the 95% CI for the ORR was higher than 15%. Additional subgroup analyses, including extramedullary disease, are described in the Data Supplement. Extramedullary disease was assessed at baseline for patients with known or suspected extramedullary disease and to confirm a response achieved by M-protein or for suspected progression per IMWG uniform response criteria.18
RESULTS
Patients
In total, 157 patients were enrolled in the study, received at least one dose of study medication, and were included in the all-treated population. At the data cutoff date (January 14, 2020), 131 patients (83%) had discontinued treatment—the most common primary reasons for discontinuation were disease progression (n = 88; 56%) and AEs (n = 26; 17%)—and 26 patients (17%) remained on treatment (Fig 1). The median duration of treatment with melflufen plus dexamethasone was 3.8 months (range, 0.9-22.7 months). At baseline, the median age was 65 years, patients had received a median of five prior lines of therapy, 154 patients (98%) had disease that was refractory to the last line of therapy received, 119 (76%) had triple-class–refractory disease, and 92 (59%) had MM that was refractory to prior alkylator therapy (Table 1). Overall, 59 patients (38%) had high-risk cytogenetics, 39 (25%) had International Staging System stage III disease, and 55 (35%) had extramedullary disease.
FIG 1. Trial profile. OS, overall survival; PFS, progression-free survival.
TABLE 1. Baseline Demographics and Clinical Characteristics in the Overall Population
Efficacy
The ORR per investigator assessment was 29% (95% CI, 22% to 37%), with one patient achieving an sCR, 17 a VGPR, and 28 a PR (Table 2). An additional 25 patients achieved a minimal response for a CBR of 45% (95% CI, 37% to 53%). In the triple-class–refractory population, the ORR was 26% (95% CI, 18% to 35%), with 13 patients achieving a VGPR and 18 a PR. The ORR per independent review committee was 30% (95% CI, 23% to 38%) overall and 26% (95% CI, 18% to 35%) in the triple-class–refractory population (Data Supplement). Reduction in M-protein was observed in 118 of the 145 patients (81.4%) (Data Supplement). In the all-treated and triple-class–refractory populations, the median time to PR or better was 1.9 months (range, 1.0-7.4 months) and 1.9 months (range, 1.0-6.1 months), respectively, and the median duration of PR or better was 5.5 months (95% CI, 3.9 to 7.6 months) and 4.4 months (95% CI, 3.4 to 7.6 months), respectively (Fig 2 and Data Supplement).
TABLE 2. Overall Response and Clinical Benefit Rate
FIG 2. Duration of response to melflufen plus dexamethasone. Data on patients in the all-treated population (n = 46), triple-class–refractory population (asterisk; n = 31), and extramedullary subgroup (dagger; n = 13) who achieved a PR or better as the best response. Open circles indicate the latest dose of melflufen received; arrows indicate patients still receiving treatment at the data cutoff date; orange Xs indicate progression-free survival events. CR, complete response; MR, minimal response; PR, partial response; sCR, stringent complete response; SD, stable disease; VGPR, very good partial response.
In the all-treated and triple-class–refractory populations, the median PFS was 4.2 months (95% CI, 3.4 to 4.9 months) and 3.9 months (95% CI, 3.0 to 4.6 months), respectively (Fig 3A). The median OS was 11.6 months (95% CI, 9.3 to 15.4 months) and 11.2 months (95% CI, 7.7 to 13.2 months), with an estimated 1-year event-free rate of 48.8% (95% CI, 39.6% to 57.4%) and 41.9% (95% CI, 31.6% to 51.8%), respectively (Fig 3B), at a median follow-up of 14 months (range, 10.8-18.7 months). Among responders, the median PFS was 8.5 months (95% CI, 5.4 to 13.4 months) and 8.5 months (95% CI, 5.3 to 13.4 months), and the median OS was 17.6 months (95% CI, 13.2 to 28.9 months) and 16.5 months (95% CI, 11.5 to 18.5 months) in the all-treated and triple-class–refractory populations, respectively (Data Supplement). Among patients in the all-treated population and the triple-class–refractory group (n = 70 and n = 52, respectively) who discontinued the study and initiated a new myeloma therapy, the median time to next therapy was 8.2 months (95% CI, 7.2 to 10.8 months) and 7.9 months (95% CI, 6.9 to 10.9 months), respectively. The median time to next therapy or death was 5.8 months (95% CI, 4.8 to 7.1 months) in the all-treated population and 5.3 months (95% CI, 4.5 to 6.3 months) in the triple-class–refractory group.
FIG 3. PFS and OS. Kaplan-Meier analysis of (A) PFS and (B) OS in the all-treated (N = 157) and triple-class–refractory (n = 119) populations. OS, overall survival; PFS, progression-free survival.
In a subgroup analysis, 19 of the 54 patients (35%) age 65-74 years and 8 of the 25 patients (32%) older than 75 years achieved a PR or better. In addition, a PR or better was achieved in 13 of the 55 patients (24%) with extramedullary disease and 12 of the 59 patients (20%) with high-risk cytogenetics (Data Supplement). Among patients with MM refractory to previous alkylator therapy, the ORR was 21% (19 of the 92 patients achieved a PR or better, including one sCR, six VGPRs, and 12 PRs) and the CBR was 34% (Data Supplement). Among patients refractory to an alkylator in one previous line of therapy (n = 60), the ORR was 28% (CBR, 40%). In patients refractory to alkylators in multiple previous lines of therapy (n = 32), the ORR was 6% (CBR, 22%). Median PFS and OS in the subgroups analyzed were consistent with those of the all-treated population (Data Supplement).
Safety
Treatment-emergent AEs (TEAEs) were reported in all 157 patients (100%) in the all-treated population, with 149 (95%) reporting at least one melflufen-related TEAE (Table 3 and Data Supplement). Grade ≥ 3 TEAEs occurred in 150 patients (96%), most commonly neutropenia (124 [79%]), thrombocytopenia (120 [76%]), and anemia (67 [43%]). Any-grade and grade 3/4 bleeding events with concurrent grade 3/4 thrombocytopenia occurred in 25 patients (16%) and four patients (3%), respectively. The most common nonhematologic treatment-emergent grade 3/4 events included pneumonia (16 [10%]; grade 3, 14 [9%]; grade 4, two [1%]) and hypophosphatemia (eight [5%]; grade 3, eight [5%]; grade 4, 0). Grade 3/4 neutropenia with concurrent grade 3/4 infections occurred in 18 patients (11%); of these, 11 (7%) had pneumonia (Data Supplement). GI events occurred in 97 patients overall and were grade 1/2 in 90 of the 97 patients (93%) and grade 3 in seven of the 97 patients (7%). No grade 4 events were reported. The most common any-grade GI events included nausea (50 [32%]), diarrhea (42 [27%]), constipation (23 [15%]), and vomiting (21 [13%]). Mucositis occurred in one patient (1%; grade 1 event), and there were no reports of alopecia or neuropathy.
TABLE 3. TEAEs (Occurring in ≥ 10% of Patients) in the All-Treated Population
Serious TEAEs occurred in 77 patients (49%), most commonly pneumonia (14 [9%]) and febrile neutropenia (eight [5%]; Data Supplement). Second primary malignancies occurred in five patients; of these, four had malignancies with cutaneous manifestations (two patients with basal cell carcinoma, one patient with squamous cell carcinoma, and one patient with basal cell carcinoma, squamous cell carcinoma, and malignant melanoma; see the Data Supplement). One patient developed myelodysplasia after having received 17 cycles of study medication and in the context of multiple prior cycles of alkylator-based therapy, including stem-cell transplant prior to study entry. Moreover, the review of fluorescence in situ hybridization studies from the screening bone marrow confirmed pre-existing abnormalities supporting a subclinical myelodysplastic syndrome that was likely treatment-related and not otherwise apparent. No other cases of myelodysplastic syndromes were seen. Overall, 10 patients (6%) died from TEAEs. Most commonly, general physical health deterioration was associated with progressive disease (n = 3; 2%) and respiratory failure (n = 2; 1%; Data Supplement). None of the deaths were considered related to melflufen.
The average (standard deviation) monthly dose of melflufen received was 37.8 mg (± 4.0). TEAEs leading to melflufen dose reductions occurred in 42 patients (27%), most commonly thrombocytopenia (n = 22; 14%) and neutropenia (n = 5; 3%). While on study, 102 patients (65%) received concomitant RBC or platelet transfusion support, with 68 (43%) receiving platelet transfusion support only and 106 (68%) receiving concomitant growth factor support (Data Supplement). Overall, 34 patients (22%) had at least one TEAE leading to melflufen treatment discontinuation, most commonly thrombocytopenia (n = 16) and neutropenia (n = 5; Data Supplement). Overall, 95 patients (61%) experienced at least one dose delay, and the median number of treatment cycles with a dose delay was one (range, 0-9).
DISCUSSION
In this study, melflufen plus dexamethasone demonstrated meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM. These findings build substantially on previously reported results17 but in a population that is more aligned with current treatment practice in the relapsed and refractory as well as highly resistant disease setting (ie, patients refractory to an anti-CD38 monoclonal antibody and/or pomalidomide, as well as exposed and refractory to prior lenalidomide, dexamethasone, and proteasome inhibitors). Durable responses were seen in this heavily pretreated population with a high proportion of extramedullary disease and high-risk cytogenetic features. Although the median DOR was 5.5 months, the median PFS among responders was encouragingly longer at 8.5 months. Furthermore, the median time to first response was 1.9 months, but many patients achieved their best response beyond 2 months of treatment. Altogether, these data support the notion that the clinical benefit of melflufen plus dexamethasone improves with longer treatment duration.
The ORR of 29% was consistent among high-risk patient subgroups, including those with triple-class–refractory disease (26%), those with extramedullary disease (24%), and patients age 75 years or older (32%), which is encouraging given the reported ORRs (10%-31%) in patients refractory to anti-CD38 monoclonal antibody therapy and/or with extramedullary disease at relapse.3,4,23-25 In fact, this is the largest population with extramedullary disease reported to date in a prospective study.4,26,27 Subgroup analyses showed sufficient efficacy in 60 patients refractory to an alkylator in one previous line of therapy with an ORR of 28%, while the ORR was only 6% in the 32 patients refractory to alkylators in two or more previous lines. Melflufen may have a mechanism of action that is different from that of other alkylators.8,11 For example, melflufen induced cell death more effectively than melphalan in TP53-mutated cell lines and in cells from patients with TP53-mutated RRMM, suggesting that the mechanism of cytotoxicity of melflufen—but not that of other alkylators—is independent of p53 function.8,11,16 Unlike other newer agents that work via immune-based mechanisms (including chimeric antigen receptor T cell therapy, belantamab mafodotin, iberdomide, and isatuximab), melflufen adds a unique mechanism of action to the treatment landscape in relapsed disease as a potent and novel cytotoxic agent targeting myeloma more broadly while providing meaningful clinical efficacy and a manageable safety profile for heavily pretreated RRMM.8,10,28-30
The safety profile of melflufen primarily consisted of hematologic AEs, consistent with previous results.17 Despite cytopenias being common, the incidence of significant bleeding events or infections was low. Hematologic AEs were generally reversible and clinically manageable with dose adjustments, dose delays, growth factor use, platelet transfusions, and appropriate supportive care. Nonhematologic grade 3/4 AEs were infrequent, with infections being the most common. Moreover, the frequency of infections was generally consistent with the expected rates of infections in heavily pretreated patients.23,27,31 Specifically, the 10% rate of grade 3/4 pneumonia reported in HORIZON was similar to 9%-11% reported with pomalidomide plus dexamethasone, bortezomib plus dexamethasone, and selinexor plus dexamethasone in RRMM.23,27,31 GI toxicities, a common reason for treatment discontinuation with other agents,23 were infrequent, primarily grade 1/2, and did not lead to melflufen treatment cessation in HORIZON in any patient. Encouragingly, alopecia and treatment-emergent peripheral neuropathy were not reported. Patients were therefore able to tolerate treatment, with rates of discontinuation from AEs lower than or comparable with other studies (which range from 6% to 33%) in this patient population and with a prolonged median duration of treatment, together with the added convenience of monthly infusions, which is an especially important consideration in the current era of COVID-19.23,27,28
In conclusion, the results from HORIZON suggest that melflufen has the potential to be an important therapeutic option in RRMM by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety when combined with dexamethasone in heavily pretreated patients.32 Based on these results, the efficacy and safety of melflufen plus dexamethasone versus pomalidomide plus dexamethasone are being further evaluated in OCEAN (OP-103), a randomized, global, phase III multicenter study (ClinicalTrials.gov identifier: NCT03151811) for patients in earlier relapse.33 Studies of melflufen plus dexamethasone in combination with bortezomib or daratumumab are also ongoing, with promising results to date.34
PRIOR PRESENTATION
SUPPORT
CLINICAL TRIAL INFORMATION
ACKNOWLEDGMENT
The authors especially thank the patients and their families for participating in this trial and all the study investigators and coordinators for their contributions to this work. The authors also thank Jakob Obermüller and Hanan Zubair (Oncopeptides AB, Stockholm, Sweden) for data management as well as Katherine Mills-Lujan, PhD, CMPP, and Jennifer Leslie, PhD, CMPP, of Team 9 Science for providing medical editorial assistance under the guidance of the authors, which was funded by Oncopeptides AB in accordance with Good Publications Practice (GPP3) guidelines.
DATA SHARING STATEMENT
Oncopeptides commits to share clinical study data with qualified researchers to enable enhancement of public health. As such, Oncopeptides will share anonymized patient-level data on request or if required by law or regulation. Qualified scientific and medical researchers can request patient-level data for studies of Oncopeptides pharmaceutical substances listed on ClinicalTrials.gov and approved by health authorities in the United States and the EU. Patient-level data for studies of newly approved pharmaceutical substances or indications can be requested 9 months after US Food and Drug Administration and European Medicines Agency approval. Such requests are assessed at Oncopeptides' discretion, and the decisions depend on the scientific merit of the proposed request, data availability, and the purpose of the proposal. The applicants should be willing to submit both positive and negative findings to a scientific journal. If Oncopeptides agrees to share clinical data for research purposes, the applicant is required to sign an agreement for data sharing before data release, to ensure that the patient data are de-identified. In case of any risk of re-identification on anonymized data despite measures to protect patient confidentiality, the data will not be shared. The patients' informed consent will always be respected. If the anonymization process will provide futile data, Oncopeptides will have the right to refuse the request. Oncopeptides will provide access to patient-level clinical trial analysis datasets in a secured environment upon execution of the data sharing agreement.
Oncopeptides will also provide the Protocol, statistical analysis plan, and the clinical study report synopsis if needed. For additional information or requests for access to Oncopeptides clinical trial data for research purposes, please contact us at medinfo@oncopeptides.com.
AUTHOR CONTRIBUTIONS
Conception and design: Paul G. Richardson, Catriona Byrne, Johan Harmenberg, María-Victoria Mateos
Provision of study materials or patients: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, María-Victoria Mateos
Collection and assembly of data: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Marcus Thuresson, María-Victoria Mateos
Data analysis and interpretation: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, John W. Hiemenz, Cyrille Touzeau, Adrián Alegre, Agne Paner, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Sara Thuresson, Marcus Thuresson, Catriona Byrne, Johan Harmenberg, Nicolaas A. Bakker, María-Victoria Mateos
Manuscript writing: All authors
Final approval of manuscript: All authors
Accountable for all aspects for the work: All authors
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma
The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I =Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/authors/author-center.
Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).
Paul G. Richardson
Consulting or Advisory Role: Celgene, Janssen, Takeda, Karyopharm Therapeutics, Oncopeptides, Sanofi, Jazz Pharmaceuticals, SecuraBio
Research Funding: Celgene, Takeda, Bristol-Myers Squibb, Oncopeptides
Albert Oriol
Consulting or Advisory Role: Celgene, Janssen, Amgen, Sanofi, GlaxoSmithKline
Speakers' Bureau: Amgen, Celgene
Alessandra Larocca
Honoraria: Amgen, Bristol-Myers Squibb, Celgene, Janssen, GlaxoSmithKline
Consulting or Advisory Role: Bristol-Myers Squibb, Celgene, Janssen, Takeda
Joan Bladé
Honoraria: Janssen, Celgene, Amgen, Takeda, Oncopeptides
Michele Cavo
Honoraria: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Consulting or Advisory Role: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Speakers' Bureau: Janssen, Celgene
Paula Rodriguez-Otero
Honoraria: Janssen, Celgene, Amgen, Oncopeptides, Sanofi, Abbvie, GlaxoSmithKline, Kite Pharma
Consulting or Advisory Role: Janssen, Celgene, Amgen, Takeda, Oncopeptides, Sanofi, AbbVie, GlaxoSmithKline, Kite Pharma
Xavier Leleu
Honoraria: Janssen-Cilag, Celgene, Amgen, Novartis, Bristol-Myers Squibb, Takeda, Sanofi, Abbvie, Merck, Roche, Karyopharm Therapeutics, Carsgen Therapeutics Ltd, Oncopeptides, GlaxoSmithKline
Consulting or Advisory Role: Janssen-Cilag, Celgene, Amgen, Takeda, Bristol-Myers Squibb, Novartis, Merck, Gilead Sciences, Abbvie, Roche, Karyopharm Therapeutics, Oncopeptides, Carsgen Therapeutics Ltd, GlaxoSmithKline
Travel, Accommodations, Expenses: Takeda
Omar Nadeem
Consulting or Advisory Role: Janssen, Celgene, Sanofi, Takeda, Adaptive Biotechnologies
Hani Hassoun
Consulting or Advisory Role: Novartis
Research Funding: Takeda, Janssen
Cyrille Touzeau
Honoraria: Abbvie, Celgene, Amgen, Takeda, Janssen, Sanofi, Novartis, GlaxoSmithKline
Consulting or Advisory Role: Novartis, Amgen, Celgene, Abbvie, Takeda, Janssen, GlaxoSmithKline
Research Funding: Abbvie
Adrián Alegre
Leadership: Amgen, Janssen-Cilag, Celgene-BMS, Takeda, Sanofi, GlaxoSmithKline, ONCOPETIDES
Agne Paner
Honoraria: Amgen, Celgene, Janssen
Consulting or Advisory Role: Takeda, Celgene, Amgen, Karyopharm Therapeutics, Oncopetides
Christopher Maisel
Stock and Other Ownership Interests: Actinium Pharmaceuticals, Karyopharm Therapeutics, Amgen
Honoraria: Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead, Oncopetides, GlaxoSmithKline
Speakers' Bureau: Amgen, Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead
Amitabha Mazumder
Honoraria: Karyopharm Therapeutics
Speakers' Bureau: Karyopharm Therapeutics
Anastasios Raptis
Consulting or Advisory Role: intellisphere, integra
Jan S. Moreb
Consulting or Advisory Role: Oncopeptide
Kenneth C. Anderson
Stock and Other Ownership Interests: C4 Therapeutics, OncoPep
Consulting or Advisory Role: Celgene, Millennium, Gilead Sciences, Bristol-Myers Squibb, Janssen Oncology, Sanofi, Tolero Pharmaceuticals, Precision Biosciences
Patents, Royalties, Other Intellectual Property: C4 Therapeutics, OncoPep
Jacob P. Laubach
Research Funding: Abbvie, Bristol-Myers Squibb, Genentech, Janssen Research & Development, Carsgen, Millennium
Sara Thuresson
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Marcus Thuresson
Employment Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Catriona Byrne
Consulting or Advisory Role: Oncopeptides
Travel, Accommodations, Expenses: Oncopeptides
Johan Harmenberg
Leadership: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides, Ectin AB
Travel, Accommodations, Expenses: Oncopeptides
Nicolaas A. Bakker
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Honoraria: Oncopeptides
María-Victoria Mateos
Honoraria: Janssen-Cilag, Celgene, Amgen, Takeda, GlaxoSmithKline, Abbvie/Genentech, Adaptive Biotechnologies
Consulting or Advisory Role: Takeda, Janssen-Cilag, Celgene, Amgen, Abbvie, GlaxoSmithKline, Pharmamar-zeltia
No other potential conflicts of interest were reported.
APPENDIX
TABLE A1. HORIZON (OP-106) Investigators and Recruitment Sites
Presented in part at the 25th European Hematology Association annual congress, virtual format, June 11-21, 2020; abstract EP945.
This study was sponsored by Oncopeptides AB, which also provided support for manuscript editorial assistance.
NCT02963493
See accompanying article on page 836 | AZACITIDINE | DrugsGivenReaction | CC BY-NC-ND | 33296242 | 19,180,518 | 2021-03-01 |
What was the outcome of reaction 'Fungal infection'? | Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells. The phase II HORIZON trial evaluated the efficacy of melflufen plus dexamethasone in relapsed and refractory multiple myeloma (RRMM), a population with an important unmet medical need.
Patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody received melflufen 40 mg intravenously on day 1 of each 28-day cycle plus once weekly oral dexamethasone at a dose of 40 mg (20 mg in patients older than 75 years). The primary end point was overall response rate (partial response or better) assessed by the investigator and confirmed by independent review. Secondary end points included duration of response, progression-free survival, overall survival, and safety. The primary analysis is complete with long-term follow-up ongoing.
Of 157 patients (median age 65 years; median five prior lines of therapy) enrolled and treated, 119 patients (76%) had triple-class-refractory disease, 55 (35%) had extramedullary disease, and 92 (59%) were refractory to previous alkylator therapy. The overall response rate was 29% in the all-treated population, with 26% in the triple-class-refractory population. In the all-treated population, median duration of response was 5.5 months, median progression-free survival was 4.2 months, and median overall survival was 11.6 months at a median follow-up of 14 months. Grade ≥ 3 treatment-emergent adverse events occurred in 96% of patients, most commonly neutropenia (79%), thrombocytopenia (76%), and anemia (43%). Pneumonia (10%) was the most common grade 3/4 nonhematologic event. Thrombocytopenia and bleeding (both grade 3/4 but fully reversible) occurred concomitantly in four patients. GI events, reported in 97 patients (62%), were predominantly grade 1/2 (93%); none were grade 4.
Melflufen plus dexamethasone showed clinically meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM, including those with triple-class-refractory and extramedullary disease.
pmcINTRODUCTION
Despite the introduction of novel therapies and regimens that have improved outcomes in multiple myeloma (MM),1,2 almost all patients will relapse.1,3 After relapse, treatment choice is usually determined by the class of and response to previous treatment and patient characteristics.2,3 Although class switching is generally prioritized, this is becoming increasingly difficult, not least because novel agents are commonly administered in combination in earlier treatment lines, resulting in disease resistant to multiple drug classes as early as second-line therapy.2,3
CONTEXT
Key Objective
To evaluate whether melphalan flufenamide (melflufen) plus dexamethasone is effective and safe in patients with heavily pretreated relapsed and refractory multiple myeloma (RRMM), a population with a high unmet medical need.
Knowledge Generated
In this pivotal, phase II study, melflufen plus dexamethasone showed meaningful efficacy in heavily pretreated patients with RRMM, including patients with triple-class–refractory disease and those with extramedullary disease. The safety profile of melflufen plus dexamethasone was consistent with previously reported data and was characterized primarily by clinically manageable hematologic toxicities.
Relevance
As new combinations of antimyeloma drugs are introduced in earlier lines of therapy, patients with RRMM often have disease that is refractory to multiple drugs. Therefore, drugs with novel mechanisms of action are urgently needed. Melflufen, when combined with dexamethasone, has the potential to fill this unmet medical need by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety in patients with RRMM.
Outcomes are particularly poor for patients with high-risk cytogenetics, extramedullary disease, and MM resistant to multiple drug classes, including those with triple-class–refractory disease who represent groups with a high unmet need.1,3,4 Furthermore, patients with relapsed and refractory multiple myeloma (RRMM) may have comorbidities because of age, disease symptoms, and cumulative toxicities stemming from previous therapies.5,6 There is an urgent requirement for agents with novel mechanisms of action that are effective, safe, and tolerable and that maintain quality of life in patients with aggressive and resistant disease.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells.7-12 Melflufen is rapidly and passively taken up by cells because of its high lipophilicity, thereby circumventing the development of transporter-associated resistance.8,11,13 Intracellular aminopeptidases hydrolyze melflufen to release hydrophilic alkylating moieties.11 Melflufen and its metabolites melphalan and desethyl-melflufen have equipotent alkylating potential.11 Unlike previous aminopeptidase-targeting therapies that directly inhibit aminopeptidase activity, melflufen takes a novel approach by leveraging increased aminopeptidase activity to selectively direct potent cytotoxic agents into tumor cells.11,14,15 Melflufen and its metabolites trigger robust and irreversible DNA damage, have antiangiogenic effects, induce apoptosis—resulting in potent antitumor activity in myeloma cells, including those with resistance to melphalan, bortezomib, and dexamethasone—and, importantly, retain activity in myeloma cells with absent or impaired p53 function.8-10,16 Melflufen may also have activity in other hematologic malignancies (including immunoglobulin light chain amyloidosis and leukemia) and solid tumors (including breast cancer and ovarian cancer).11
The phase I/II, multicenter O-12-M1 trial established the dosage of melflufen plus dexamethasone in patients who had RRMM, received a median of four previous lines of therapy (including lenalidomide and bortezomib), and had disease refractory to their last line of therapy.17 In 45 patients treated with infusional melflufen 40 mg administered on day 1 of each 28-day cycle and once weekly dexamethasone dosed at 40 mg, the overall response rate (ORR) was 31%, the median duration of response (DOR) was 8.4 months, the median progression-free survival (PFS) was 5.7 months, and the median overall survival (OS) was encouraging at 20.7 months. The safety profile of melflufen was characterized primarily by hematologic toxicities that were clinically manageable with appropriate dose delays, dose reductions, and supportive care. Based on these results, the efficacy and safety of melflufen plus dexamethasone were therefore evaluated in the current study in a larger population with heavily pretreated, resistant, and poor-risk RRMM, including those with triple-class–refractory disease, for whom few effective treatment options exist.3
PATIENTS AND METHODS
Study Design and Participants
HORIZON (OP-106; ClinicalTrials.gov identifier: NCT02963493) was a pivotal, single-arm, multicenter, phase II study of melflufen plus dexamethasone in patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. Patients were enrolled from December 28, 2016, to October 14, 2019, at 17 sites (see the Data Supplement, online only). Eligible adult patients had an Eastern Cooperative Oncology Group performance status score of 0-2, a previous diagnosis of MM with disease progression, and measurable disease (serum monoclonal protein ≥ 5 g/L, urine monoclonal protein ≥ 200 mg per 24 hours, or serum immunoglobulin-free light chain ≥ 100 mg/L, and abnormal serum immunoglobulin kappa to lambda–free light chain ratio) at study entry. Patients had received at least two prior lines of therapy, including an immunomodulatory agent and proteasome inhibitor, and were refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. RRMM was defined as disease that was nonresponsive (ie, did not achieve a minimal response or better, or developed progressive disease with treatment) while on primary or salvage therapy or progressed within 60 days of last therapy.18 Please see the Data Supplement for full eligibility criteria. Patients received once-monthly melflufen 40 mg as a 30-minute central intravenous infusion on day 1 of each 28-day cycle in combination with oral dexamethasone 40 mg (20 mg for patients age ≥ 75 years) once-weekly administered on days 1, 8, 15, and 22 of each 28-day cycle until disease progression, unacceptable toxicity, or the patient or treating physician determined it was not in the patient's best interest to continue. Melflufen dose reduction for drug-related toxicities was allowed in 10 mg increments each cycle from 40 mg down to 30 mg and from 30 mg down to 20 mg (see the Data Supplement).
This study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation guidelines for Good Clinical Practice. The Protocol was reviewed and approved by national regulatory authorities and an independent ethics committee or institutional review board at each study center. Each patient provided written informed consent.
Outcomes
The primary end point was ORR, defined as the proportion of patients achieving a confirmed response of stringent complete response (sCR), complete response (CR), very good partial response (VGPR), or partial response (PR) as their best response per International Myeloma Working Group (IMWG) uniform response criteria, as assessed by the investigator.18 Response, confirmed response, and confirmed progression were subsequently verified by an independent review committee.18 Secondary end points included DOR, PFS, OS, clinical benefit rate (CBR), best response, time to response, time to progression, time to next treatment, and safety (defined in the Data Supplement). All response categories required confirmation with two consecutive assessments (see the Data Supplement). Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events, version 4.03. AE frequency and relationship to study treatment were summarized.
Statistical Analysis
Planned enrollment was 150 patients. ORR and associated two-sided exact 95% CI19 were estimated for all patients treated (all-treated population). With a sample size of 150 patients and an assumed ORR of 30%, the exact 95% CI was estimated to range between 23% and 38%. CBR and disease stabilization were also summarized. Time-to-event end points were summarized using the Kaplan-Meier method in the all-treated population. Median and estimated 95% CIs were constructed using the methods of Brookmeyer and Crowley20; duration of follow-up was estimated by the reverse Kaplan-Meier methods of Schemper and Smith.21 See the Data Supplement for patient censoring and handling of missing data.
A preplanned subgroup analysis was performed in patients with triple-class–refractory MM (refractory to or intolerant of at least one immunomodulatory drug, at least one proteasome inhibitor, and at least one anti-CD38 monoclonal antibody). With a sample size of 150 patients, 104-120 patients with triple-class–refractory disease were expected; the primary end point was considered met if the lower bound of the 95% CI for the ORR was higher than 15%. Additional subgroup analyses, including extramedullary disease, are described in the Data Supplement. Extramedullary disease was assessed at baseline for patients with known or suspected extramedullary disease and to confirm a response achieved by M-protein or for suspected progression per IMWG uniform response criteria.18
RESULTS
Patients
In total, 157 patients were enrolled in the study, received at least one dose of study medication, and were included in the all-treated population. At the data cutoff date (January 14, 2020), 131 patients (83%) had discontinued treatment—the most common primary reasons for discontinuation were disease progression (n = 88; 56%) and AEs (n = 26; 17%)—and 26 patients (17%) remained on treatment (Fig 1). The median duration of treatment with melflufen plus dexamethasone was 3.8 months (range, 0.9-22.7 months). At baseline, the median age was 65 years, patients had received a median of five prior lines of therapy, 154 patients (98%) had disease that was refractory to the last line of therapy received, 119 (76%) had triple-class–refractory disease, and 92 (59%) had MM that was refractory to prior alkylator therapy (Table 1). Overall, 59 patients (38%) had high-risk cytogenetics, 39 (25%) had International Staging System stage III disease, and 55 (35%) had extramedullary disease.
FIG 1. Trial profile. OS, overall survival; PFS, progression-free survival.
TABLE 1. Baseline Demographics and Clinical Characteristics in the Overall Population
Efficacy
The ORR per investigator assessment was 29% (95% CI, 22% to 37%), with one patient achieving an sCR, 17 a VGPR, and 28 a PR (Table 2). An additional 25 patients achieved a minimal response for a CBR of 45% (95% CI, 37% to 53%). In the triple-class–refractory population, the ORR was 26% (95% CI, 18% to 35%), with 13 patients achieving a VGPR and 18 a PR. The ORR per independent review committee was 30% (95% CI, 23% to 38%) overall and 26% (95% CI, 18% to 35%) in the triple-class–refractory population (Data Supplement). Reduction in M-protein was observed in 118 of the 145 patients (81.4%) (Data Supplement). In the all-treated and triple-class–refractory populations, the median time to PR or better was 1.9 months (range, 1.0-7.4 months) and 1.9 months (range, 1.0-6.1 months), respectively, and the median duration of PR or better was 5.5 months (95% CI, 3.9 to 7.6 months) and 4.4 months (95% CI, 3.4 to 7.6 months), respectively (Fig 2 and Data Supplement).
TABLE 2. Overall Response and Clinical Benefit Rate
FIG 2. Duration of response to melflufen plus dexamethasone. Data on patients in the all-treated population (n = 46), triple-class–refractory population (asterisk; n = 31), and extramedullary subgroup (dagger; n = 13) who achieved a PR or better as the best response. Open circles indicate the latest dose of melflufen received; arrows indicate patients still receiving treatment at the data cutoff date; orange Xs indicate progression-free survival events. CR, complete response; MR, minimal response; PR, partial response; sCR, stringent complete response; SD, stable disease; VGPR, very good partial response.
In the all-treated and triple-class–refractory populations, the median PFS was 4.2 months (95% CI, 3.4 to 4.9 months) and 3.9 months (95% CI, 3.0 to 4.6 months), respectively (Fig 3A). The median OS was 11.6 months (95% CI, 9.3 to 15.4 months) and 11.2 months (95% CI, 7.7 to 13.2 months), with an estimated 1-year event-free rate of 48.8% (95% CI, 39.6% to 57.4%) and 41.9% (95% CI, 31.6% to 51.8%), respectively (Fig 3B), at a median follow-up of 14 months (range, 10.8-18.7 months). Among responders, the median PFS was 8.5 months (95% CI, 5.4 to 13.4 months) and 8.5 months (95% CI, 5.3 to 13.4 months), and the median OS was 17.6 months (95% CI, 13.2 to 28.9 months) and 16.5 months (95% CI, 11.5 to 18.5 months) in the all-treated and triple-class–refractory populations, respectively (Data Supplement). Among patients in the all-treated population and the triple-class–refractory group (n = 70 and n = 52, respectively) who discontinued the study and initiated a new myeloma therapy, the median time to next therapy was 8.2 months (95% CI, 7.2 to 10.8 months) and 7.9 months (95% CI, 6.9 to 10.9 months), respectively. The median time to next therapy or death was 5.8 months (95% CI, 4.8 to 7.1 months) in the all-treated population and 5.3 months (95% CI, 4.5 to 6.3 months) in the triple-class–refractory group.
FIG 3. PFS and OS. Kaplan-Meier analysis of (A) PFS and (B) OS in the all-treated (N = 157) and triple-class–refractory (n = 119) populations. OS, overall survival; PFS, progression-free survival.
In a subgroup analysis, 19 of the 54 patients (35%) age 65-74 years and 8 of the 25 patients (32%) older than 75 years achieved a PR or better. In addition, a PR or better was achieved in 13 of the 55 patients (24%) with extramedullary disease and 12 of the 59 patients (20%) with high-risk cytogenetics (Data Supplement). Among patients with MM refractory to previous alkylator therapy, the ORR was 21% (19 of the 92 patients achieved a PR or better, including one sCR, six VGPRs, and 12 PRs) and the CBR was 34% (Data Supplement). Among patients refractory to an alkylator in one previous line of therapy (n = 60), the ORR was 28% (CBR, 40%). In patients refractory to alkylators in multiple previous lines of therapy (n = 32), the ORR was 6% (CBR, 22%). Median PFS and OS in the subgroups analyzed were consistent with those of the all-treated population (Data Supplement).
Safety
Treatment-emergent AEs (TEAEs) were reported in all 157 patients (100%) in the all-treated population, with 149 (95%) reporting at least one melflufen-related TEAE (Table 3 and Data Supplement). Grade ≥ 3 TEAEs occurred in 150 patients (96%), most commonly neutropenia (124 [79%]), thrombocytopenia (120 [76%]), and anemia (67 [43%]). Any-grade and grade 3/4 bleeding events with concurrent grade 3/4 thrombocytopenia occurred in 25 patients (16%) and four patients (3%), respectively. The most common nonhematologic treatment-emergent grade 3/4 events included pneumonia (16 [10%]; grade 3, 14 [9%]; grade 4, two [1%]) and hypophosphatemia (eight [5%]; grade 3, eight [5%]; grade 4, 0). Grade 3/4 neutropenia with concurrent grade 3/4 infections occurred in 18 patients (11%); of these, 11 (7%) had pneumonia (Data Supplement). GI events occurred in 97 patients overall and were grade 1/2 in 90 of the 97 patients (93%) and grade 3 in seven of the 97 patients (7%). No grade 4 events were reported. The most common any-grade GI events included nausea (50 [32%]), diarrhea (42 [27%]), constipation (23 [15%]), and vomiting (21 [13%]). Mucositis occurred in one patient (1%; grade 1 event), and there were no reports of alopecia or neuropathy.
TABLE 3. TEAEs (Occurring in ≥ 10% of Patients) in the All-Treated Population
Serious TEAEs occurred in 77 patients (49%), most commonly pneumonia (14 [9%]) and febrile neutropenia (eight [5%]; Data Supplement). Second primary malignancies occurred in five patients; of these, four had malignancies with cutaneous manifestations (two patients with basal cell carcinoma, one patient with squamous cell carcinoma, and one patient with basal cell carcinoma, squamous cell carcinoma, and malignant melanoma; see the Data Supplement). One patient developed myelodysplasia after having received 17 cycles of study medication and in the context of multiple prior cycles of alkylator-based therapy, including stem-cell transplant prior to study entry. Moreover, the review of fluorescence in situ hybridization studies from the screening bone marrow confirmed pre-existing abnormalities supporting a subclinical myelodysplastic syndrome that was likely treatment-related and not otherwise apparent. No other cases of myelodysplastic syndromes were seen. Overall, 10 patients (6%) died from TEAEs. Most commonly, general physical health deterioration was associated with progressive disease (n = 3; 2%) and respiratory failure (n = 2; 1%; Data Supplement). None of the deaths were considered related to melflufen.
The average (standard deviation) monthly dose of melflufen received was 37.8 mg (± 4.0). TEAEs leading to melflufen dose reductions occurred in 42 patients (27%), most commonly thrombocytopenia (n = 22; 14%) and neutropenia (n = 5; 3%). While on study, 102 patients (65%) received concomitant RBC or platelet transfusion support, with 68 (43%) receiving platelet transfusion support only and 106 (68%) receiving concomitant growth factor support (Data Supplement). Overall, 34 patients (22%) had at least one TEAE leading to melflufen treatment discontinuation, most commonly thrombocytopenia (n = 16) and neutropenia (n = 5; Data Supplement). Overall, 95 patients (61%) experienced at least one dose delay, and the median number of treatment cycles with a dose delay was one (range, 0-9).
DISCUSSION
In this study, melflufen plus dexamethasone demonstrated meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM. These findings build substantially on previously reported results17 but in a population that is more aligned with current treatment practice in the relapsed and refractory as well as highly resistant disease setting (ie, patients refractory to an anti-CD38 monoclonal antibody and/or pomalidomide, as well as exposed and refractory to prior lenalidomide, dexamethasone, and proteasome inhibitors). Durable responses were seen in this heavily pretreated population with a high proportion of extramedullary disease and high-risk cytogenetic features. Although the median DOR was 5.5 months, the median PFS among responders was encouragingly longer at 8.5 months. Furthermore, the median time to first response was 1.9 months, but many patients achieved their best response beyond 2 months of treatment. Altogether, these data support the notion that the clinical benefit of melflufen plus dexamethasone improves with longer treatment duration.
The ORR of 29% was consistent among high-risk patient subgroups, including those with triple-class–refractory disease (26%), those with extramedullary disease (24%), and patients age 75 years or older (32%), which is encouraging given the reported ORRs (10%-31%) in patients refractory to anti-CD38 monoclonal antibody therapy and/or with extramedullary disease at relapse.3,4,23-25 In fact, this is the largest population with extramedullary disease reported to date in a prospective study.4,26,27 Subgroup analyses showed sufficient efficacy in 60 patients refractory to an alkylator in one previous line of therapy with an ORR of 28%, while the ORR was only 6% in the 32 patients refractory to alkylators in two or more previous lines. Melflufen may have a mechanism of action that is different from that of other alkylators.8,11 For example, melflufen induced cell death more effectively than melphalan in TP53-mutated cell lines and in cells from patients with TP53-mutated RRMM, suggesting that the mechanism of cytotoxicity of melflufen—but not that of other alkylators—is independent of p53 function.8,11,16 Unlike other newer agents that work via immune-based mechanisms (including chimeric antigen receptor T cell therapy, belantamab mafodotin, iberdomide, and isatuximab), melflufen adds a unique mechanism of action to the treatment landscape in relapsed disease as a potent and novel cytotoxic agent targeting myeloma more broadly while providing meaningful clinical efficacy and a manageable safety profile for heavily pretreated RRMM.8,10,28-30
The safety profile of melflufen primarily consisted of hematologic AEs, consistent with previous results.17 Despite cytopenias being common, the incidence of significant bleeding events or infections was low. Hematologic AEs were generally reversible and clinically manageable with dose adjustments, dose delays, growth factor use, platelet transfusions, and appropriate supportive care. Nonhematologic grade 3/4 AEs were infrequent, with infections being the most common. Moreover, the frequency of infections was generally consistent with the expected rates of infections in heavily pretreated patients.23,27,31 Specifically, the 10% rate of grade 3/4 pneumonia reported in HORIZON was similar to 9%-11% reported with pomalidomide plus dexamethasone, bortezomib plus dexamethasone, and selinexor plus dexamethasone in RRMM.23,27,31 GI toxicities, a common reason for treatment discontinuation with other agents,23 were infrequent, primarily grade 1/2, and did not lead to melflufen treatment cessation in HORIZON in any patient. Encouragingly, alopecia and treatment-emergent peripheral neuropathy were not reported. Patients were therefore able to tolerate treatment, with rates of discontinuation from AEs lower than or comparable with other studies (which range from 6% to 33%) in this patient population and with a prolonged median duration of treatment, together with the added convenience of monthly infusions, which is an especially important consideration in the current era of COVID-19.23,27,28
In conclusion, the results from HORIZON suggest that melflufen has the potential to be an important therapeutic option in RRMM by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety when combined with dexamethasone in heavily pretreated patients.32 Based on these results, the efficacy and safety of melflufen plus dexamethasone versus pomalidomide plus dexamethasone are being further evaluated in OCEAN (OP-103), a randomized, global, phase III multicenter study (ClinicalTrials.gov identifier: NCT03151811) for patients in earlier relapse.33 Studies of melflufen plus dexamethasone in combination with bortezomib or daratumumab are also ongoing, with promising results to date.34
PRIOR PRESENTATION
SUPPORT
CLINICAL TRIAL INFORMATION
ACKNOWLEDGMENT
The authors especially thank the patients and their families for participating in this trial and all the study investigators and coordinators for their contributions to this work. The authors also thank Jakob Obermüller and Hanan Zubair (Oncopeptides AB, Stockholm, Sweden) for data management as well as Katherine Mills-Lujan, PhD, CMPP, and Jennifer Leslie, PhD, CMPP, of Team 9 Science for providing medical editorial assistance under the guidance of the authors, which was funded by Oncopeptides AB in accordance with Good Publications Practice (GPP3) guidelines.
DATA SHARING STATEMENT
Oncopeptides commits to share clinical study data with qualified researchers to enable enhancement of public health. As such, Oncopeptides will share anonymized patient-level data on request or if required by law or regulation. Qualified scientific and medical researchers can request patient-level data for studies of Oncopeptides pharmaceutical substances listed on ClinicalTrials.gov and approved by health authorities in the United States and the EU. Patient-level data for studies of newly approved pharmaceutical substances or indications can be requested 9 months after US Food and Drug Administration and European Medicines Agency approval. Such requests are assessed at Oncopeptides' discretion, and the decisions depend on the scientific merit of the proposed request, data availability, and the purpose of the proposal. The applicants should be willing to submit both positive and negative findings to a scientific journal. If Oncopeptides agrees to share clinical data for research purposes, the applicant is required to sign an agreement for data sharing before data release, to ensure that the patient data are de-identified. In case of any risk of re-identification on anonymized data despite measures to protect patient confidentiality, the data will not be shared. The patients' informed consent will always be respected. If the anonymization process will provide futile data, Oncopeptides will have the right to refuse the request. Oncopeptides will provide access to patient-level clinical trial analysis datasets in a secured environment upon execution of the data sharing agreement.
Oncopeptides will also provide the Protocol, statistical analysis plan, and the clinical study report synopsis if needed. For additional information or requests for access to Oncopeptides clinical trial data for research purposes, please contact us at medinfo@oncopeptides.com.
AUTHOR CONTRIBUTIONS
Conception and design: Paul G. Richardson, Catriona Byrne, Johan Harmenberg, María-Victoria Mateos
Provision of study materials or patients: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, María-Victoria Mateos
Collection and assembly of data: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Marcus Thuresson, María-Victoria Mateos
Data analysis and interpretation: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, John W. Hiemenz, Cyrille Touzeau, Adrián Alegre, Agne Paner, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Sara Thuresson, Marcus Thuresson, Catriona Byrne, Johan Harmenberg, Nicolaas A. Bakker, María-Victoria Mateos
Manuscript writing: All authors
Final approval of manuscript: All authors
Accountable for all aspects for the work: All authors
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma
The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I =Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/authors/author-center.
Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).
Paul G. Richardson
Consulting or Advisory Role: Celgene, Janssen, Takeda, Karyopharm Therapeutics, Oncopeptides, Sanofi, Jazz Pharmaceuticals, SecuraBio
Research Funding: Celgene, Takeda, Bristol-Myers Squibb, Oncopeptides
Albert Oriol
Consulting or Advisory Role: Celgene, Janssen, Amgen, Sanofi, GlaxoSmithKline
Speakers' Bureau: Amgen, Celgene
Alessandra Larocca
Honoraria: Amgen, Bristol-Myers Squibb, Celgene, Janssen, GlaxoSmithKline
Consulting or Advisory Role: Bristol-Myers Squibb, Celgene, Janssen, Takeda
Joan Bladé
Honoraria: Janssen, Celgene, Amgen, Takeda, Oncopeptides
Michele Cavo
Honoraria: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Consulting or Advisory Role: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Speakers' Bureau: Janssen, Celgene
Paula Rodriguez-Otero
Honoraria: Janssen, Celgene, Amgen, Oncopeptides, Sanofi, Abbvie, GlaxoSmithKline, Kite Pharma
Consulting or Advisory Role: Janssen, Celgene, Amgen, Takeda, Oncopeptides, Sanofi, AbbVie, GlaxoSmithKline, Kite Pharma
Xavier Leleu
Honoraria: Janssen-Cilag, Celgene, Amgen, Novartis, Bristol-Myers Squibb, Takeda, Sanofi, Abbvie, Merck, Roche, Karyopharm Therapeutics, Carsgen Therapeutics Ltd, Oncopeptides, GlaxoSmithKline
Consulting or Advisory Role: Janssen-Cilag, Celgene, Amgen, Takeda, Bristol-Myers Squibb, Novartis, Merck, Gilead Sciences, Abbvie, Roche, Karyopharm Therapeutics, Oncopeptides, Carsgen Therapeutics Ltd, GlaxoSmithKline
Travel, Accommodations, Expenses: Takeda
Omar Nadeem
Consulting or Advisory Role: Janssen, Celgene, Sanofi, Takeda, Adaptive Biotechnologies
Hani Hassoun
Consulting or Advisory Role: Novartis
Research Funding: Takeda, Janssen
Cyrille Touzeau
Honoraria: Abbvie, Celgene, Amgen, Takeda, Janssen, Sanofi, Novartis, GlaxoSmithKline
Consulting or Advisory Role: Novartis, Amgen, Celgene, Abbvie, Takeda, Janssen, GlaxoSmithKline
Research Funding: Abbvie
Adrián Alegre
Leadership: Amgen, Janssen-Cilag, Celgene-BMS, Takeda, Sanofi, GlaxoSmithKline, ONCOPETIDES
Agne Paner
Honoraria: Amgen, Celgene, Janssen
Consulting or Advisory Role: Takeda, Celgene, Amgen, Karyopharm Therapeutics, Oncopetides
Christopher Maisel
Stock and Other Ownership Interests: Actinium Pharmaceuticals, Karyopharm Therapeutics, Amgen
Honoraria: Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead, Oncopetides, GlaxoSmithKline
Speakers' Bureau: Amgen, Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead
Amitabha Mazumder
Honoraria: Karyopharm Therapeutics
Speakers' Bureau: Karyopharm Therapeutics
Anastasios Raptis
Consulting or Advisory Role: intellisphere, integra
Jan S. Moreb
Consulting or Advisory Role: Oncopeptide
Kenneth C. Anderson
Stock and Other Ownership Interests: C4 Therapeutics, OncoPep
Consulting or Advisory Role: Celgene, Millennium, Gilead Sciences, Bristol-Myers Squibb, Janssen Oncology, Sanofi, Tolero Pharmaceuticals, Precision Biosciences
Patents, Royalties, Other Intellectual Property: C4 Therapeutics, OncoPep
Jacob P. Laubach
Research Funding: Abbvie, Bristol-Myers Squibb, Genentech, Janssen Research & Development, Carsgen, Millennium
Sara Thuresson
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Marcus Thuresson
Employment Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Catriona Byrne
Consulting or Advisory Role: Oncopeptides
Travel, Accommodations, Expenses: Oncopeptides
Johan Harmenberg
Leadership: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides, Ectin AB
Travel, Accommodations, Expenses: Oncopeptides
Nicolaas A. Bakker
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Honoraria: Oncopeptides
María-Victoria Mateos
Honoraria: Janssen-Cilag, Celgene, Amgen, Takeda, GlaxoSmithKline, Abbvie/Genentech, Adaptive Biotechnologies
Consulting or Advisory Role: Takeda, Janssen-Cilag, Celgene, Amgen, Abbvie, GlaxoSmithKline, Pharmamar-zeltia
No other potential conflicts of interest were reported.
APPENDIX
TABLE A1. HORIZON (OP-106) Investigators and Recruitment Sites
Presented in part at the 25th European Hematology Association annual congress, virtual format, June 11-21, 2020; abstract EP945.
This study was sponsored by Oncopeptides AB, which also provided support for manuscript editorial assistance.
NCT02963493
See accompanying article on page 836 | Fatal | ReactionOutcome | CC BY-NC-ND | 33296242 | 19,180,518 | 2021-03-01 |
What was the outcome of reaction 'Neutropenia'? | Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells. The phase II HORIZON trial evaluated the efficacy of melflufen plus dexamethasone in relapsed and refractory multiple myeloma (RRMM), a population with an important unmet medical need.
Patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody received melflufen 40 mg intravenously on day 1 of each 28-day cycle plus once weekly oral dexamethasone at a dose of 40 mg (20 mg in patients older than 75 years). The primary end point was overall response rate (partial response or better) assessed by the investigator and confirmed by independent review. Secondary end points included duration of response, progression-free survival, overall survival, and safety. The primary analysis is complete with long-term follow-up ongoing.
Of 157 patients (median age 65 years; median five prior lines of therapy) enrolled and treated, 119 patients (76%) had triple-class-refractory disease, 55 (35%) had extramedullary disease, and 92 (59%) were refractory to previous alkylator therapy. The overall response rate was 29% in the all-treated population, with 26% in the triple-class-refractory population. In the all-treated population, median duration of response was 5.5 months, median progression-free survival was 4.2 months, and median overall survival was 11.6 months at a median follow-up of 14 months. Grade ≥ 3 treatment-emergent adverse events occurred in 96% of patients, most commonly neutropenia (79%), thrombocytopenia (76%), and anemia (43%). Pneumonia (10%) was the most common grade 3/4 nonhematologic event. Thrombocytopenia and bleeding (both grade 3/4 but fully reversible) occurred concomitantly in four patients. GI events, reported in 97 patients (62%), were predominantly grade 1/2 (93%); none were grade 4.
Melflufen plus dexamethasone showed clinically meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM, including those with triple-class-refractory and extramedullary disease.
pmcINTRODUCTION
Despite the introduction of novel therapies and regimens that have improved outcomes in multiple myeloma (MM),1,2 almost all patients will relapse.1,3 After relapse, treatment choice is usually determined by the class of and response to previous treatment and patient characteristics.2,3 Although class switching is generally prioritized, this is becoming increasingly difficult, not least because novel agents are commonly administered in combination in earlier treatment lines, resulting in disease resistant to multiple drug classes as early as second-line therapy.2,3
CONTEXT
Key Objective
To evaluate whether melphalan flufenamide (melflufen) plus dexamethasone is effective and safe in patients with heavily pretreated relapsed and refractory multiple myeloma (RRMM), a population with a high unmet medical need.
Knowledge Generated
In this pivotal, phase II study, melflufen plus dexamethasone showed meaningful efficacy in heavily pretreated patients with RRMM, including patients with triple-class–refractory disease and those with extramedullary disease. The safety profile of melflufen plus dexamethasone was consistent with previously reported data and was characterized primarily by clinically manageable hematologic toxicities.
Relevance
As new combinations of antimyeloma drugs are introduced in earlier lines of therapy, patients with RRMM often have disease that is refractory to multiple drugs. Therefore, drugs with novel mechanisms of action are urgently needed. Melflufen, when combined with dexamethasone, has the potential to fill this unmet medical need by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety in patients with RRMM.
Outcomes are particularly poor for patients with high-risk cytogenetics, extramedullary disease, and MM resistant to multiple drug classes, including those with triple-class–refractory disease who represent groups with a high unmet need.1,3,4 Furthermore, patients with relapsed and refractory multiple myeloma (RRMM) may have comorbidities because of age, disease symptoms, and cumulative toxicities stemming from previous therapies.5,6 There is an urgent requirement for agents with novel mechanisms of action that are effective, safe, and tolerable and that maintain quality of life in patients with aggressive and resistant disease.
Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells.7-12 Melflufen is rapidly and passively taken up by cells because of its high lipophilicity, thereby circumventing the development of transporter-associated resistance.8,11,13 Intracellular aminopeptidases hydrolyze melflufen to release hydrophilic alkylating moieties.11 Melflufen and its metabolites melphalan and desethyl-melflufen have equipotent alkylating potential.11 Unlike previous aminopeptidase-targeting therapies that directly inhibit aminopeptidase activity, melflufen takes a novel approach by leveraging increased aminopeptidase activity to selectively direct potent cytotoxic agents into tumor cells.11,14,15 Melflufen and its metabolites trigger robust and irreversible DNA damage, have antiangiogenic effects, induce apoptosis—resulting in potent antitumor activity in myeloma cells, including those with resistance to melphalan, bortezomib, and dexamethasone—and, importantly, retain activity in myeloma cells with absent or impaired p53 function.8-10,16 Melflufen may also have activity in other hematologic malignancies (including immunoglobulin light chain amyloidosis and leukemia) and solid tumors (including breast cancer and ovarian cancer).11
The phase I/II, multicenter O-12-M1 trial established the dosage of melflufen plus dexamethasone in patients who had RRMM, received a median of four previous lines of therapy (including lenalidomide and bortezomib), and had disease refractory to their last line of therapy.17 In 45 patients treated with infusional melflufen 40 mg administered on day 1 of each 28-day cycle and once weekly dexamethasone dosed at 40 mg, the overall response rate (ORR) was 31%, the median duration of response (DOR) was 8.4 months, the median progression-free survival (PFS) was 5.7 months, and the median overall survival (OS) was encouraging at 20.7 months. The safety profile of melflufen was characterized primarily by hematologic toxicities that were clinically manageable with appropriate dose delays, dose reductions, and supportive care. Based on these results, the efficacy and safety of melflufen plus dexamethasone were therefore evaluated in the current study in a larger population with heavily pretreated, resistant, and poor-risk RRMM, including those with triple-class–refractory disease, for whom few effective treatment options exist.3
PATIENTS AND METHODS
Study Design and Participants
HORIZON (OP-106; ClinicalTrials.gov identifier: NCT02963493) was a pivotal, single-arm, multicenter, phase II study of melflufen plus dexamethasone in patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. Patients were enrolled from December 28, 2016, to October 14, 2019, at 17 sites (see the Data Supplement, online only). Eligible adult patients had an Eastern Cooperative Oncology Group performance status score of 0-2, a previous diagnosis of MM with disease progression, and measurable disease (serum monoclonal protein ≥ 5 g/L, urine monoclonal protein ≥ 200 mg per 24 hours, or serum immunoglobulin-free light chain ≥ 100 mg/L, and abnormal serum immunoglobulin kappa to lambda–free light chain ratio) at study entry. Patients had received at least two prior lines of therapy, including an immunomodulatory agent and proteasome inhibitor, and were refractory to pomalidomide and/or an anti-CD38 monoclonal antibody. RRMM was defined as disease that was nonresponsive (ie, did not achieve a minimal response or better, or developed progressive disease with treatment) while on primary or salvage therapy or progressed within 60 days of last therapy.18 Please see the Data Supplement for full eligibility criteria. Patients received once-monthly melflufen 40 mg as a 30-minute central intravenous infusion on day 1 of each 28-day cycle in combination with oral dexamethasone 40 mg (20 mg for patients age ≥ 75 years) once-weekly administered on days 1, 8, 15, and 22 of each 28-day cycle until disease progression, unacceptable toxicity, or the patient or treating physician determined it was not in the patient's best interest to continue. Melflufen dose reduction for drug-related toxicities was allowed in 10 mg increments each cycle from 40 mg down to 30 mg and from 30 mg down to 20 mg (see the Data Supplement).
This study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation guidelines for Good Clinical Practice. The Protocol was reviewed and approved by national regulatory authorities and an independent ethics committee or institutional review board at each study center. Each patient provided written informed consent.
Outcomes
The primary end point was ORR, defined as the proportion of patients achieving a confirmed response of stringent complete response (sCR), complete response (CR), very good partial response (VGPR), or partial response (PR) as their best response per International Myeloma Working Group (IMWG) uniform response criteria, as assessed by the investigator.18 Response, confirmed response, and confirmed progression were subsequently verified by an independent review committee.18 Secondary end points included DOR, PFS, OS, clinical benefit rate (CBR), best response, time to response, time to progression, time to next treatment, and safety (defined in the Data Supplement). All response categories required confirmation with two consecutive assessments (see the Data Supplement). Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events, version 4.03. AE frequency and relationship to study treatment were summarized.
Statistical Analysis
Planned enrollment was 150 patients. ORR and associated two-sided exact 95% CI19 were estimated for all patients treated (all-treated population). With a sample size of 150 patients and an assumed ORR of 30%, the exact 95% CI was estimated to range between 23% and 38%. CBR and disease stabilization were also summarized. Time-to-event end points were summarized using the Kaplan-Meier method in the all-treated population. Median and estimated 95% CIs were constructed using the methods of Brookmeyer and Crowley20; duration of follow-up was estimated by the reverse Kaplan-Meier methods of Schemper and Smith.21 See the Data Supplement for patient censoring and handling of missing data.
A preplanned subgroup analysis was performed in patients with triple-class–refractory MM (refractory to or intolerant of at least one immunomodulatory drug, at least one proteasome inhibitor, and at least one anti-CD38 monoclonal antibody). With a sample size of 150 patients, 104-120 patients with triple-class–refractory disease were expected; the primary end point was considered met if the lower bound of the 95% CI for the ORR was higher than 15%. Additional subgroup analyses, including extramedullary disease, are described in the Data Supplement. Extramedullary disease was assessed at baseline for patients with known or suspected extramedullary disease and to confirm a response achieved by M-protein or for suspected progression per IMWG uniform response criteria.18
RESULTS
Patients
In total, 157 patients were enrolled in the study, received at least one dose of study medication, and were included in the all-treated population. At the data cutoff date (January 14, 2020), 131 patients (83%) had discontinued treatment—the most common primary reasons for discontinuation were disease progression (n = 88; 56%) and AEs (n = 26; 17%)—and 26 patients (17%) remained on treatment (Fig 1). The median duration of treatment with melflufen plus dexamethasone was 3.8 months (range, 0.9-22.7 months). At baseline, the median age was 65 years, patients had received a median of five prior lines of therapy, 154 patients (98%) had disease that was refractory to the last line of therapy received, 119 (76%) had triple-class–refractory disease, and 92 (59%) had MM that was refractory to prior alkylator therapy (Table 1). Overall, 59 patients (38%) had high-risk cytogenetics, 39 (25%) had International Staging System stage III disease, and 55 (35%) had extramedullary disease.
FIG 1. Trial profile. OS, overall survival; PFS, progression-free survival.
TABLE 1. Baseline Demographics and Clinical Characteristics in the Overall Population
Efficacy
The ORR per investigator assessment was 29% (95% CI, 22% to 37%), with one patient achieving an sCR, 17 a VGPR, and 28 a PR (Table 2). An additional 25 patients achieved a minimal response for a CBR of 45% (95% CI, 37% to 53%). In the triple-class–refractory population, the ORR was 26% (95% CI, 18% to 35%), with 13 patients achieving a VGPR and 18 a PR. The ORR per independent review committee was 30% (95% CI, 23% to 38%) overall and 26% (95% CI, 18% to 35%) in the triple-class–refractory population (Data Supplement). Reduction in M-protein was observed in 118 of the 145 patients (81.4%) (Data Supplement). In the all-treated and triple-class–refractory populations, the median time to PR or better was 1.9 months (range, 1.0-7.4 months) and 1.9 months (range, 1.0-6.1 months), respectively, and the median duration of PR or better was 5.5 months (95% CI, 3.9 to 7.6 months) and 4.4 months (95% CI, 3.4 to 7.6 months), respectively (Fig 2 and Data Supplement).
TABLE 2. Overall Response and Clinical Benefit Rate
FIG 2. Duration of response to melflufen plus dexamethasone. Data on patients in the all-treated population (n = 46), triple-class–refractory population (asterisk; n = 31), and extramedullary subgroup (dagger; n = 13) who achieved a PR or better as the best response. Open circles indicate the latest dose of melflufen received; arrows indicate patients still receiving treatment at the data cutoff date; orange Xs indicate progression-free survival events. CR, complete response; MR, minimal response; PR, partial response; sCR, stringent complete response; SD, stable disease; VGPR, very good partial response.
In the all-treated and triple-class–refractory populations, the median PFS was 4.2 months (95% CI, 3.4 to 4.9 months) and 3.9 months (95% CI, 3.0 to 4.6 months), respectively (Fig 3A). The median OS was 11.6 months (95% CI, 9.3 to 15.4 months) and 11.2 months (95% CI, 7.7 to 13.2 months), with an estimated 1-year event-free rate of 48.8% (95% CI, 39.6% to 57.4%) and 41.9% (95% CI, 31.6% to 51.8%), respectively (Fig 3B), at a median follow-up of 14 months (range, 10.8-18.7 months). Among responders, the median PFS was 8.5 months (95% CI, 5.4 to 13.4 months) and 8.5 months (95% CI, 5.3 to 13.4 months), and the median OS was 17.6 months (95% CI, 13.2 to 28.9 months) and 16.5 months (95% CI, 11.5 to 18.5 months) in the all-treated and triple-class–refractory populations, respectively (Data Supplement). Among patients in the all-treated population and the triple-class–refractory group (n = 70 and n = 52, respectively) who discontinued the study and initiated a new myeloma therapy, the median time to next therapy was 8.2 months (95% CI, 7.2 to 10.8 months) and 7.9 months (95% CI, 6.9 to 10.9 months), respectively. The median time to next therapy or death was 5.8 months (95% CI, 4.8 to 7.1 months) in the all-treated population and 5.3 months (95% CI, 4.5 to 6.3 months) in the triple-class–refractory group.
FIG 3. PFS and OS. Kaplan-Meier analysis of (A) PFS and (B) OS in the all-treated (N = 157) and triple-class–refractory (n = 119) populations. OS, overall survival; PFS, progression-free survival.
In a subgroup analysis, 19 of the 54 patients (35%) age 65-74 years and 8 of the 25 patients (32%) older than 75 years achieved a PR or better. In addition, a PR or better was achieved in 13 of the 55 patients (24%) with extramedullary disease and 12 of the 59 patients (20%) with high-risk cytogenetics (Data Supplement). Among patients with MM refractory to previous alkylator therapy, the ORR was 21% (19 of the 92 patients achieved a PR or better, including one sCR, six VGPRs, and 12 PRs) and the CBR was 34% (Data Supplement). Among patients refractory to an alkylator in one previous line of therapy (n = 60), the ORR was 28% (CBR, 40%). In patients refractory to alkylators in multiple previous lines of therapy (n = 32), the ORR was 6% (CBR, 22%). Median PFS and OS in the subgroups analyzed were consistent with those of the all-treated population (Data Supplement).
Safety
Treatment-emergent AEs (TEAEs) were reported in all 157 patients (100%) in the all-treated population, with 149 (95%) reporting at least one melflufen-related TEAE (Table 3 and Data Supplement). Grade ≥ 3 TEAEs occurred in 150 patients (96%), most commonly neutropenia (124 [79%]), thrombocytopenia (120 [76%]), and anemia (67 [43%]). Any-grade and grade 3/4 bleeding events with concurrent grade 3/4 thrombocytopenia occurred in 25 patients (16%) and four patients (3%), respectively. The most common nonhematologic treatment-emergent grade 3/4 events included pneumonia (16 [10%]; grade 3, 14 [9%]; grade 4, two [1%]) and hypophosphatemia (eight [5%]; grade 3, eight [5%]; grade 4, 0). Grade 3/4 neutropenia with concurrent grade 3/4 infections occurred in 18 patients (11%); of these, 11 (7%) had pneumonia (Data Supplement). GI events occurred in 97 patients overall and were grade 1/2 in 90 of the 97 patients (93%) and grade 3 in seven of the 97 patients (7%). No grade 4 events were reported. The most common any-grade GI events included nausea (50 [32%]), diarrhea (42 [27%]), constipation (23 [15%]), and vomiting (21 [13%]). Mucositis occurred in one patient (1%; grade 1 event), and there were no reports of alopecia or neuropathy.
TABLE 3. TEAEs (Occurring in ≥ 10% of Patients) in the All-Treated Population
Serious TEAEs occurred in 77 patients (49%), most commonly pneumonia (14 [9%]) and febrile neutropenia (eight [5%]; Data Supplement). Second primary malignancies occurred in five patients; of these, four had malignancies with cutaneous manifestations (two patients with basal cell carcinoma, one patient with squamous cell carcinoma, and one patient with basal cell carcinoma, squamous cell carcinoma, and malignant melanoma; see the Data Supplement). One patient developed myelodysplasia after having received 17 cycles of study medication and in the context of multiple prior cycles of alkylator-based therapy, including stem-cell transplant prior to study entry. Moreover, the review of fluorescence in situ hybridization studies from the screening bone marrow confirmed pre-existing abnormalities supporting a subclinical myelodysplastic syndrome that was likely treatment-related and not otherwise apparent. No other cases of myelodysplastic syndromes were seen. Overall, 10 patients (6%) died from TEAEs. Most commonly, general physical health deterioration was associated with progressive disease (n = 3; 2%) and respiratory failure (n = 2; 1%; Data Supplement). None of the deaths were considered related to melflufen.
The average (standard deviation) monthly dose of melflufen received was 37.8 mg (± 4.0). TEAEs leading to melflufen dose reductions occurred in 42 patients (27%), most commonly thrombocytopenia (n = 22; 14%) and neutropenia (n = 5; 3%). While on study, 102 patients (65%) received concomitant RBC or platelet transfusion support, with 68 (43%) receiving platelet transfusion support only and 106 (68%) receiving concomitant growth factor support (Data Supplement). Overall, 34 patients (22%) had at least one TEAE leading to melflufen treatment discontinuation, most commonly thrombocytopenia (n = 16) and neutropenia (n = 5; Data Supplement). Overall, 95 patients (61%) experienced at least one dose delay, and the median number of treatment cycles with a dose delay was one (range, 0-9).
DISCUSSION
In this study, melflufen plus dexamethasone demonstrated meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM. These findings build substantially on previously reported results17 but in a population that is more aligned with current treatment practice in the relapsed and refractory as well as highly resistant disease setting (ie, patients refractory to an anti-CD38 monoclonal antibody and/or pomalidomide, as well as exposed and refractory to prior lenalidomide, dexamethasone, and proteasome inhibitors). Durable responses were seen in this heavily pretreated population with a high proportion of extramedullary disease and high-risk cytogenetic features. Although the median DOR was 5.5 months, the median PFS among responders was encouragingly longer at 8.5 months. Furthermore, the median time to first response was 1.9 months, but many patients achieved their best response beyond 2 months of treatment. Altogether, these data support the notion that the clinical benefit of melflufen plus dexamethasone improves with longer treatment duration.
The ORR of 29% was consistent among high-risk patient subgroups, including those with triple-class–refractory disease (26%), those with extramedullary disease (24%), and patients age 75 years or older (32%), which is encouraging given the reported ORRs (10%-31%) in patients refractory to anti-CD38 monoclonal antibody therapy and/or with extramedullary disease at relapse.3,4,23-25 In fact, this is the largest population with extramedullary disease reported to date in a prospective study.4,26,27 Subgroup analyses showed sufficient efficacy in 60 patients refractory to an alkylator in one previous line of therapy with an ORR of 28%, while the ORR was only 6% in the 32 patients refractory to alkylators in two or more previous lines. Melflufen may have a mechanism of action that is different from that of other alkylators.8,11 For example, melflufen induced cell death more effectively than melphalan in TP53-mutated cell lines and in cells from patients with TP53-mutated RRMM, suggesting that the mechanism of cytotoxicity of melflufen—but not that of other alkylators—is independent of p53 function.8,11,16 Unlike other newer agents that work via immune-based mechanisms (including chimeric antigen receptor T cell therapy, belantamab mafodotin, iberdomide, and isatuximab), melflufen adds a unique mechanism of action to the treatment landscape in relapsed disease as a potent and novel cytotoxic agent targeting myeloma more broadly while providing meaningful clinical efficacy and a manageable safety profile for heavily pretreated RRMM.8,10,28-30
The safety profile of melflufen primarily consisted of hematologic AEs, consistent with previous results.17 Despite cytopenias being common, the incidence of significant bleeding events or infections was low. Hematologic AEs were generally reversible and clinically manageable with dose adjustments, dose delays, growth factor use, platelet transfusions, and appropriate supportive care. Nonhematologic grade 3/4 AEs were infrequent, with infections being the most common. Moreover, the frequency of infections was generally consistent with the expected rates of infections in heavily pretreated patients.23,27,31 Specifically, the 10% rate of grade 3/4 pneumonia reported in HORIZON was similar to 9%-11% reported with pomalidomide plus dexamethasone, bortezomib plus dexamethasone, and selinexor plus dexamethasone in RRMM.23,27,31 GI toxicities, a common reason for treatment discontinuation with other agents,23 were infrequent, primarily grade 1/2, and did not lead to melflufen treatment cessation in HORIZON in any patient. Encouragingly, alopecia and treatment-emergent peripheral neuropathy were not reported. Patients were therefore able to tolerate treatment, with rates of discontinuation from AEs lower than or comparable with other studies (which range from 6% to 33%) in this patient population and with a prolonged median duration of treatment, together with the added convenience of monthly infusions, which is an especially important consideration in the current era of COVID-19.23,27,28
In conclusion, the results from HORIZON suggest that melflufen has the potential to be an important therapeutic option in RRMM by providing a novel mechanism of action, clinically meaningful efficacy, and manageable safety when combined with dexamethasone in heavily pretreated patients.32 Based on these results, the efficacy and safety of melflufen plus dexamethasone versus pomalidomide plus dexamethasone are being further evaluated in OCEAN (OP-103), a randomized, global, phase III multicenter study (ClinicalTrials.gov identifier: NCT03151811) for patients in earlier relapse.33 Studies of melflufen plus dexamethasone in combination with bortezomib or daratumumab are also ongoing, with promising results to date.34
PRIOR PRESENTATION
SUPPORT
CLINICAL TRIAL INFORMATION
ACKNOWLEDGMENT
The authors especially thank the patients and their families for participating in this trial and all the study investigators and coordinators for their contributions to this work. The authors also thank Jakob Obermüller and Hanan Zubair (Oncopeptides AB, Stockholm, Sweden) for data management as well as Katherine Mills-Lujan, PhD, CMPP, and Jennifer Leslie, PhD, CMPP, of Team 9 Science for providing medical editorial assistance under the guidance of the authors, which was funded by Oncopeptides AB in accordance with Good Publications Practice (GPP3) guidelines.
DATA SHARING STATEMENT
Oncopeptides commits to share clinical study data with qualified researchers to enable enhancement of public health. As such, Oncopeptides will share anonymized patient-level data on request or if required by law or regulation. Qualified scientific and medical researchers can request patient-level data for studies of Oncopeptides pharmaceutical substances listed on ClinicalTrials.gov and approved by health authorities in the United States and the EU. Patient-level data for studies of newly approved pharmaceutical substances or indications can be requested 9 months after US Food and Drug Administration and European Medicines Agency approval. Such requests are assessed at Oncopeptides' discretion, and the decisions depend on the scientific merit of the proposed request, data availability, and the purpose of the proposal. The applicants should be willing to submit both positive and negative findings to a scientific journal. If Oncopeptides agrees to share clinical data for research purposes, the applicant is required to sign an agreement for data sharing before data release, to ensure that the patient data are de-identified. In case of any risk of re-identification on anonymized data despite measures to protect patient confidentiality, the data will not be shared. The patients' informed consent will always be respected. If the anonymization process will provide futile data, Oncopeptides will have the right to refuse the request. Oncopeptides will provide access to patient-level clinical trial analysis datasets in a secured environment upon execution of the data sharing agreement.
Oncopeptides will also provide the Protocol, statistical analysis plan, and the clinical study report synopsis if needed. For additional information or requests for access to Oncopeptides clinical trial data for research purposes, please contact us at medinfo@oncopeptides.com.
AUTHOR CONTRIBUTIONS
Conception and design: Paul G. Richardson, Catriona Byrne, Johan Harmenberg, María-Victoria Mateos
Provision of study materials or patients: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, María-Victoria Mateos
Collection and assembly of data: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, Omar Nadeem, John W. Hiemenz, Hani Hassoun, Cyrille Touzeau, Adrián Alegre, Agner Paner, Christopher Maisel, Amitabha Mazumder, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Marcus Thuresson, María-Victoria Mateos
Data analysis and interpretation: Paul G. Richardson, Albert Oriol, Alessandra Larocca, Joan Bladé, Michele Cavo, Paula Rodriguez-Otero, Xavier Leleu, John W. Hiemenz, Cyrille Touzeau, Adrián Alegre, Agne Paner, Anastasios Raptis, Jan S. Moreb, Kenneth C. Anderson, Jacob P. Laubach, Sara Thuresson, Marcus Thuresson, Catriona Byrne, Johan Harmenberg, Nicolaas A. Bakker, María-Victoria Mateos
Manuscript writing: All authors
Final approval of manuscript: All authors
Accountable for all aspects for the work: All authors
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma
The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I =Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/authors/author-center.
Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).
Paul G. Richardson
Consulting or Advisory Role: Celgene, Janssen, Takeda, Karyopharm Therapeutics, Oncopeptides, Sanofi, Jazz Pharmaceuticals, SecuraBio
Research Funding: Celgene, Takeda, Bristol-Myers Squibb, Oncopeptides
Albert Oriol
Consulting or Advisory Role: Celgene, Janssen, Amgen, Sanofi, GlaxoSmithKline
Speakers' Bureau: Amgen, Celgene
Alessandra Larocca
Honoraria: Amgen, Bristol-Myers Squibb, Celgene, Janssen, GlaxoSmithKline
Consulting or Advisory Role: Bristol-Myers Squibb, Celgene, Janssen, Takeda
Joan Bladé
Honoraria: Janssen, Celgene, Amgen, Takeda, Oncopeptides
Michele Cavo
Honoraria: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Consulting or Advisory Role: Janssen, Bristol-Myers Squibb, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, Abbvie, Karyopharm Therapeutics, Adaptive Biotechnologies
Speakers' Bureau: Janssen, Celgene
Paula Rodriguez-Otero
Honoraria: Janssen, Celgene, Amgen, Oncopeptides, Sanofi, Abbvie, GlaxoSmithKline, Kite Pharma
Consulting or Advisory Role: Janssen, Celgene, Amgen, Takeda, Oncopeptides, Sanofi, AbbVie, GlaxoSmithKline, Kite Pharma
Xavier Leleu
Honoraria: Janssen-Cilag, Celgene, Amgen, Novartis, Bristol-Myers Squibb, Takeda, Sanofi, Abbvie, Merck, Roche, Karyopharm Therapeutics, Carsgen Therapeutics Ltd, Oncopeptides, GlaxoSmithKline
Consulting or Advisory Role: Janssen-Cilag, Celgene, Amgen, Takeda, Bristol-Myers Squibb, Novartis, Merck, Gilead Sciences, Abbvie, Roche, Karyopharm Therapeutics, Oncopeptides, Carsgen Therapeutics Ltd, GlaxoSmithKline
Travel, Accommodations, Expenses: Takeda
Omar Nadeem
Consulting or Advisory Role: Janssen, Celgene, Sanofi, Takeda, Adaptive Biotechnologies
Hani Hassoun
Consulting or Advisory Role: Novartis
Research Funding: Takeda, Janssen
Cyrille Touzeau
Honoraria: Abbvie, Celgene, Amgen, Takeda, Janssen, Sanofi, Novartis, GlaxoSmithKline
Consulting or Advisory Role: Novartis, Amgen, Celgene, Abbvie, Takeda, Janssen, GlaxoSmithKline
Research Funding: Abbvie
Adrián Alegre
Leadership: Amgen, Janssen-Cilag, Celgene-BMS, Takeda, Sanofi, GlaxoSmithKline, ONCOPETIDES
Agne Paner
Honoraria: Amgen, Celgene, Janssen
Consulting or Advisory Role: Takeda, Celgene, Amgen, Karyopharm Therapeutics, Oncopetides
Christopher Maisel
Stock and Other Ownership Interests: Actinium Pharmaceuticals, Karyopharm Therapeutics, Amgen
Honoraria: Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead, Oncopetides, GlaxoSmithKline
Speakers' Bureau: Amgen, Bristol-Myers Squibb, Karyopharm Therapeutics, Takeda, Janssen Oncology, Kite/Gilead
Amitabha Mazumder
Honoraria: Karyopharm Therapeutics
Speakers' Bureau: Karyopharm Therapeutics
Anastasios Raptis
Consulting or Advisory Role: intellisphere, integra
Jan S. Moreb
Consulting or Advisory Role: Oncopeptide
Kenneth C. Anderson
Stock and Other Ownership Interests: C4 Therapeutics, OncoPep
Consulting or Advisory Role: Celgene, Millennium, Gilead Sciences, Bristol-Myers Squibb, Janssen Oncology, Sanofi, Tolero Pharmaceuticals, Precision Biosciences
Patents, Royalties, Other Intellectual Property: C4 Therapeutics, OncoPep
Jacob P. Laubach
Research Funding: Abbvie, Bristol-Myers Squibb, Genentech, Janssen Research & Development, Carsgen, Millennium
Sara Thuresson
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Marcus Thuresson
Employment Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides
Catriona Byrne
Consulting or Advisory Role: Oncopeptides
Travel, Accommodations, Expenses: Oncopeptides
Johan Harmenberg
Leadership: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Consulting or Advisory Role: Oncopeptides, Ectin AB
Travel, Accommodations, Expenses: Oncopeptides
Nicolaas A. Bakker
Employment: Oncopeptides
Stock and Other Ownership Interests: Oncopeptides
Honoraria: Oncopeptides
María-Victoria Mateos
Honoraria: Janssen-Cilag, Celgene, Amgen, Takeda, GlaxoSmithKline, Abbvie/Genentech, Adaptive Biotechnologies
Consulting or Advisory Role: Takeda, Janssen-Cilag, Celgene, Amgen, Abbvie, GlaxoSmithKline, Pharmamar-zeltia
No other potential conflicts of interest were reported.
APPENDIX
TABLE A1. HORIZON (OP-106) Investigators and Recruitment Sites
Presented in part at the 25th European Hematology Association annual congress, virtual format, June 11-21, 2020; abstract EP945.
This study was sponsored by Oncopeptides AB, which also provided support for manuscript editorial assistance.
NCT02963493
See accompanying article on page 836 | Fatal | ReactionOutcome | CC BY-NC-ND | 33296242 | 19,180,518 | 2021-03-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Bacterial sepsis'. | Experience of treating Candida auris cases at a general hospital in the state of Qatar.
So far there have been no studies on Candida auris in Qatar. This study aimed to describe the clinical spectrum and outcome of C. auris infection in patients admitted to a general hospital in Qatar.
We conducted this descriptive observational study in a general hospital in Qatar. We have involved all patients with C. auris infection and colonization admitted to a general hospital from December 2018 to August 2019.
We identified 13 patients with confirmed C.auris infection/colonization, of which five cases represented an actual C. auris infection, while the remaining eight cases were considered as colonization. The mean age of the patients with infection was 76.6 ± 8.4 years, while the mean age of the patients with colonization was 66.4 ± 24.7 years. Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All strains of C. auris were susceptible to echinocandins, flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Of the patients with C. auris infection who received systemic antifungal therapy, three (60%) died during antifungal therapy.
Our study showed that C. auris can cause a wide variety of invasive infections, including bloodstream infection, urinary tract infection, skin infection, and lower respiratory tract infections, especially in critically ill patients. In addition, our isolates showed resistance to the most common antifungal agents such as fluconazole and amphotericin B.
Introduction
Candida auris is a novel multidrug-resistant yeast with high overall mortality that was first isolated from the external auditory canal of a patient in Japan in 2009 [1]. Since then, this fungal infection has been reported from various countries across the world [[2], [3], [4]], and over time it has become a serious global health concern as one of the most serious emerging pathogens that critical care physicians should be aware of [5].
C. auris being resistant to major antifungal classes used to treat Candida including azole antifungal agents, poses a challenge to routine microbiology laboratories, as C. auris can be misidentified with standard laboratory techniques, and have a tendency to cause outbreaks in healthcare settings especially critical care areas despite adequate infection prevention and control measures [4,5].
In Qatar, there is no published data on Candida aurisso far. In this series, we reported the first outbreak of C. auris infection in Qatar, to describe the clinical spectrum and outcome of this infection in the affected patients.
Methods and patients
We conducted this descriptive observational study in a general hospital in Qatar. We involved all patients with Candida Auris infection and colonization in the intensive care units and other wards from December 2018 to August 2019. This study was given ethical approval by the medical research committee at Hamad Medical Corporation, under number: MRC-01-19-503..
Definitions
Colonization is defined as isolation of C. auris from endotracheal aspiration fluid, throat swabs, sputum, urine, and samples from central venous catheters or other parts of the body in absence of clinical signs or symptomes of infection. C.auris infection is defined as the isolation of Cauris from clinical specimens with compatible clinical signs and symptoms of infection [5].
Candida auris identification
All clinical specimens, from different sites, were cultured by quantitative technique on Sabouraud Dextrose Agar (OXOID, UK) and incubated at 35−37 °C for 48 h. Preliminary fungal strain identification was based on colony morphology on Chromogenic Candida Agar (OXOID, UK), while the identification to the species level was confirmed by Vitek 2 XL automated system (bioMerieux). Susceptibility of strains to Amphotericin B, Fluconazole, 5-fluorocytosine, and voriconazole was determined by using Sensititre™ YeastOne™ plate and by interpreting results according to closely related Candida species and on expert opinion. As per the Centers for Disease Control and Prevention (CDC), there are currently no established C. auris-specific susceptibility breakpoints [6].
Pulsed-field gel electrophoresis (PFGE) typing, which consisted of electrophoretic karyotyping (EK), was performed to compare the isolates from different sites. Following the results of the PFGE, an outbreak of C. auris infection in critical care unit and medical unit was confirmed by identifying five5 cases and patient screening revealed colonization of eight additional patients. Intensive efforts were done to find out the cause of cross-transmission and environmental and surface swabbing was done in affected areas, but all results were negative.
Data analysis
The results of analyses of continuous variables are expressed as means and standard deviations (SD) unless otherwise specified.
Results
During the study period, we identified 13 patients with confirmed C. auris infection/colonization, of which five cases represented an actual Candida infection, while the remaining eight cases were considered colonization. The mean age of the patients with infection was 76.6 ± 8.4 years (range: 65–90 years), while the mean age of the patients with colonization was 66.4 ± 24.7 years (range: 23–91 years). Table 1 describes the demographic characteristics of the patients involved in this study.Table 1 Candida auris infection/colonization patients details.
Table 1Case/No Age Sex Site of infection/ or site of Candida isolation Type of infection Pre or co-infection Co-morbidity Treatment provided Outcome
1 78 years Male Tracheal aspirate and urine Lower respiratory tract infection Corona virus 229 E PCR positive from nasal swab Interstitial lung disease Anidulafungin Died of hypoxic respiratory failure
2 79 years Male Nose and decubitus ulcer Skin soft tissue infection Pseudomonas MDR and Morganella morganii from decubitus ulcer Diabetes mellitus, sacral bed sores Flucytosine Died of bacterial/fungal sepsis
3 71 years Male Nose, throat, tracheal aspirate, and decubitus ulcer Candidemia Pseudomonas aeruginosa MDR and ESBL Klebsiella pneumoniae from sputum Diabetes mellitus, sacral bed sores Anidulafungin and posaconazole Cured
4 90 years Male urine, throat and nose Urinary tract infection Klebsiella pneumoniae and carbapenem resistant Pseudomonas aeruginosa from sputum, Pseudomonas aeruginosa MDR from a bedsore Cerebrovascular accident, dementia Anidulafungin Cured
5 65 years Male Throat, sputum, groin and urine Urinary tract infection Pseudomonas aeruginosa multidrug resistant Motor neuron disease, hospital-acquired pneumonia Anidulafungin Died of bacterial pneumonia
6 29 years Male Groin Colonization ESBL Klebsiella Acute liver failure secondary to hepatitis C, acute kidney injury, critical care polyneuropathy Terbinafine spray Discharged home
7 86 years Male Axilla, urine Colonization Pseudomonas aeruginosa COPD, vascular dementia, bedbound on tracheostomy to Terbinafine spray, nystatin application Died due to aspiration pneumonia and hypoxic respiratory failure
8 80 years Female Nose, tracheostomy site Colonization ESBL Klebsiella Chronic kidney disease, coronary artery disease, on tracheostomy Terbinafine spray, nystatin application Transfer to geriatric ward
9 62 years Female Axilla Colonization Pseudomonas multi drug-resistant Chronic kidney disease, necrotizing fasciitis Terbinafine spray, nystatin application Died due to bacterial sepsis
10 91 years Female Groin area Colonization None COPD, hypertension Terbinafine spray, nystatin application Discharged home
11 23 years Male Nose, axilla Colonization Escherichia coli Hypoxic brain injury, recurrent urinary tract infection Terbinafine spray, nystatin application Transfer to long-term unit
12 75 years Male Nose, groin Colonization Pseudomonas aeruginosa Diabetes mellitus, chronic kidney disease, recurrent pneumonia Terbinafine spray, nystatin application Discharged home
13 85 years Male urine Colonization Pseudomonas aeruginosa Parkinson’s disease, cerebrovascular accident Terbinafine spray, nystatin application Transfer to geriatric unit
PCR: polymerase chain reaction, MRD: multi-drug resiatant, ESBL: extended spectrum beta lactamase, COPD: chronic obstructive pulmonary disease.
Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All patients had bacterial or viral infections prior to or concomitantly with C. auris infection/colonization, as shown in Table 1.
For the typing of C. auris isolates, the molecular technique PFGE, which consisted of electrophoretic karyotyping (EK), was utilized to compare the isolates from different sites. The PFGE karyotype of the outbreak isolates of C. auris in our series is shown in Fig. 1. Antifungal susceptibility tests were performed on isolates from infected subjects. All strains of C. auris shared the same susceptibility profile, being susceptible to echinocandins (especially anidulafungin), flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Table 2 shows the susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates. All patients with C. auris infection received systemic antifungal drugs, while the eight patients who were colonized were appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC (Table 1).Fig. 1 Electrophoretic karyotypes of C. auris isolates. Karyotypes of representative outbreak isolates from five patients in the intensive care unit. Lane 1, 2 and 8 are control specimens which served as comparison for different genotypes. Lane 3 to 7 strains (specimens from the five C. auris cases) show no single band variation and are likely representing the same strain.
Fig. 1Table 2 Susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates from subjects with infection.
Table 2Antifungal drugs Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Amphotericin 4-R 4-R 2-R 4-R 2-R
Caspofungin 0.25 8-R 8 0.5 8
Fluconazole 64 128-R 128-R 128-R 128-R
Flucytocin 0.125 0.5-S 0.12 0.12 0.12
Itraconazole 0.125-R 16-R 0.12 0.12 16
Posaconazole 0.012 8-R 0.06 0.06-S 8
Voriconazole 0.25 8-R 0.25 0.5 8
Anidulafungin 0.125-S 0.5-I 0.25 0.12-S 0.5-I
Micafungin 0.25 0.12 0.25
R: resistant, S: sensitive, I: intermediate.
Among the patients with C. auris infection who received systemic antifungal therapy, three (60 %) died during antifungal therapy. The other two patients were successfully treated and appropriately decolonized of C. auris (Table 1).
Discussion
Recent reports showed that C. auris is an emerging yeast that has been identified worldwide as a cause of severe invasive healthcare infections, which mostly affect critically ill patients and cause substantial morbidity and mortality [7,8]. To our knowledge, our series is the first designed to study this infection in Qatar.
Many C. auris outbreaks have been reported worldwide. In India, the first C. auris outbreak was reported in 2013 by Chowdhary et al. [9] who identified 12 patients with positive microbiological clinical specimens collected between 2009 and 2012. While Calvo et al. reported the first outbreak of C. auris infection in Venezuela between March 2012 and July 2013 [10]. All the isolates were initially identified as C. haemulonii. However, the isolation of C. auris was later confirmed by genome sequencing [9,10]. Similarly, we have reported the first outbreak of C. auris infection in Qatar, identifying 13 patients. The emergence of C. auris in our hospital raises concerns that this fungus may spread to other healthcare settings, particularly critical care facilities in Qatar, requiring intensified measures to control the spread of this infection. Therefore, knowing the source of infection and detection of possible routes of transmission can help in preventing the clonal spread of this infection and hospital outbreaks among various health facilities in Qatar [5,7,8]. Similarly, intensive efforts have been made in our hospital to find the cause of the cross-transmission. Environmental and surface swabs were carried out in the affected areas, but all results were negative.
Diagnosing C. auris infection is difficult because the clinical presentation is non-specific or may not be recognizable since patients infected with C. auris often have another serious illness or condition. Moreover, C. auris can be misidentified with standard laboratory techniques as C. haemulonii [11,12]. As a result, a high index of suspicion is required to diagnose this infection. In addition, accurate identification of C. auris through specialized laboratory methods is required to avoid misidentification and inappropriate treatment that may make it difficult to control the spread of C. auris in the healthcare settings [10]. In this study, the diagnosis of C. auris infection was suspected because of the resistance of the isolates to fluconazole and amphotericin B. The diagnosis was confirmed by molecular methods.
The spectrum of C. auris infection ranges from superficial infections that affect the skin to widespread infections that affect the brain, heart, lungs, liver, spleen, and kidneys [5]. Antifungal therapy should be administered to eradicate and control C. auris infection. On the other hand, C. auris can be isolated from the skin, rectum, wounds or mouth of some patients who do not show symptoms of infection. This condition is referred to as asymptomatic colonization and treatment with antifungal drugs does not eradicate C. auris colonization. However, the identification of C. auris colonization is significant because it carries the risk of transmission, which requires the immediate implementation of adequate infection control measures [13]. Likewise, our patients showed different clinical presentations, and cases with colonization were identified and appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC.
In agreement with other reports [[3], [4], [5],7,13], our isolates showed resistance to the most important antifungal agents such as fluconazole and amphotericin B. The all cause mortality among our patients was 60 % which is in line with the mortality rate seen in other studies ranging from 30 to 60% [3].
One of the limitations of this study is the retrospective nature of the research. In addition, the small sample size is another factor that limits the generalizability of these findings.
Conclusion
C. auris can cause a wide variety of invasive infections, including bloodstream infections, urinary tract infection, skin infection, and lower respiratory tract infection, especially in critically ill patients. In addition, all isolates showed resistance to fluconazole and amphotericin B and were sensitive to echinocandins especially anidulafungin.
Authors contribution (Authorship)
Adila Shaukat: Desgning, interpretation of data, revising and approving the final draft.
Nasir Al Ansari: conception of the study, revising and approving the final draft.
Walid Al Wali: interpretation of data, revising and approving the final draft.
Edin Karic: interpretation of data, revising and approving the final draft.
Ihab El Madhoun: acquisition of data, revising and approving the final draft.
Hassan Mitwally: interpretation of data, revising and approving the final draft.
Manal Hamed: acquisition of data, revising and approving the final draft.
Feah Alutra- Visan: interpretation of data, drafting the article and approving the final draft.
Conflict of interest
All authors report no conflict of interest.
Acknowledgement
Open Access funding provided by the Qatar National Library.
We are alsothankful for Dr Jameela Al Ajmi,from Corporate Infection prevention and control dept and Ms Tahani M. Al Saadi from Laboratory department for their corporation and support. | NYSTATIN, TERBINAFINE | DrugsGivenReaction | CC BY | 33299794 | 18,688,888 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pneumonia aspiration'. | Experience of treating Candida auris cases at a general hospital in the state of Qatar.
So far there have been no studies on Candida auris in Qatar. This study aimed to describe the clinical spectrum and outcome of C. auris infection in patients admitted to a general hospital in Qatar.
We conducted this descriptive observational study in a general hospital in Qatar. We have involved all patients with C. auris infection and colonization admitted to a general hospital from December 2018 to August 2019.
We identified 13 patients with confirmed C.auris infection/colonization, of which five cases represented an actual C. auris infection, while the remaining eight cases were considered as colonization. The mean age of the patients with infection was 76.6 ± 8.4 years, while the mean age of the patients with colonization was 66.4 ± 24.7 years. Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All strains of C. auris were susceptible to echinocandins, flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Of the patients with C. auris infection who received systemic antifungal therapy, three (60%) died during antifungal therapy.
Our study showed that C. auris can cause a wide variety of invasive infections, including bloodstream infection, urinary tract infection, skin infection, and lower respiratory tract infections, especially in critically ill patients. In addition, our isolates showed resistance to the most common antifungal agents such as fluconazole and amphotericin B.
Introduction
Candida auris is a novel multidrug-resistant yeast with high overall mortality that was first isolated from the external auditory canal of a patient in Japan in 2009 [1]. Since then, this fungal infection has been reported from various countries across the world [[2], [3], [4]], and over time it has become a serious global health concern as one of the most serious emerging pathogens that critical care physicians should be aware of [5].
C. auris being resistant to major antifungal classes used to treat Candida including azole antifungal agents, poses a challenge to routine microbiology laboratories, as C. auris can be misidentified with standard laboratory techniques, and have a tendency to cause outbreaks in healthcare settings especially critical care areas despite adequate infection prevention and control measures [4,5].
In Qatar, there is no published data on Candida aurisso far. In this series, we reported the first outbreak of C. auris infection in Qatar, to describe the clinical spectrum and outcome of this infection in the affected patients.
Methods and patients
We conducted this descriptive observational study in a general hospital in Qatar. We involved all patients with Candida Auris infection and colonization in the intensive care units and other wards from December 2018 to August 2019. This study was given ethical approval by the medical research committee at Hamad Medical Corporation, under number: MRC-01-19-503..
Definitions
Colonization is defined as isolation of C. auris from endotracheal aspiration fluid, throat swabs, sputum, urine, and samples from central venous catheters or other parts of the body in absence of clinical signs or symptomes of infection. C.auris infection is defined as the isolation of Cauris from clinical specimens with compatible clinical signs and symptoms of infection [5].
Candida auris identification
All clinical specimens, from different sites, were cultured by quantitative technique on Sabouraud Dextrose Agar (OXOID, UK) and incubated at 35−37 °C for 48 h. Preliminary fungal strain identification was based on colony morphology on Chromogenic Candida Agar (OXOID, UK), while the identification to the species level was confirmed by Vitek 2 XL automated system (bioMerieux). Susceptibility of strains to Amphotericin B, Fluconazole, 5-fluorocytosine, and voriconazole was determined by using Sensititre™ YeastOne™ plate and by interpreting results according to closely related Candida species and on expert opinion. As per the Centers for Disease Control and Prevention (CDC), there are currently no established C. auris-specific susceptibility breakpoints [6].
Pulsed-field gel electrophoresis (PFGE) typing, which consisted of electrophoretic karyotyping (EK), was performed to compare the isolates from different sites. Following the results of the PFGE, an outbreak of C. auris infection in critical care unit and medical unit was confirmed by identifying five5 cases and patient screening revealed colonization of eight additional patients. Intensive efforts were done to find out the cause of cross-transmission and environmental and surface swabbing was done in affected areas, but all results were negative.
Data analysis
The results of analyses of continuous variables are expressed as means and standard deviations (SD) unless otherwise specified.
Results
During the study period, we identified 13 patients with confirmed C. auris infection/colonization, of which five cases represented an actual Candida infection, while the remaining eight cases were considered colonization. The mean age of the patients with infection was 76.6 ± 8.4 years (range: 65–90 years), while the mean age of the patients with colonization was 66.4 ± 24.7 years (range: 23–91 years). Table 1 describes the demographic characteristics of the patients involved in this study.Table 1 Candida auris infection/colonization patients details.
Table 1Case/No Age Sex Site of infection/ or site of Candida isolation Type of infection Pre or co-infection Co-morbidity Treatment provided Outcome
1 78 years Male Tracheal aspirate and urine Lower respiratory tract infection Corona virus 229 E PCR positive from nasal swab Interstitial lung disease Anidulafungin Died of hypoxic respiratory failure
2 79 years Male Nose and decubitus ulcer Skin soft tissue infection Pseudomonas MDR and Morganella morganii from decubitus ulcer Diabetes mellitus, sacral bed sores Flucytosine Died of bacterial/fungal sepsis
3 71 years Male Nose, throat, tracheal aspirate, and decubitus ulcer Candidemia Pseudomonas aeruginosa MDR and ESBL Klebsiella pneumoniae from sputum Diabetes mellitus, sacral bed sores Anidulafungin and posaconazole Cured
4 90 years Male urine, throat and nose Urinary tract infection Klebsiella pneumoniae and carbapenem resistant Pseudomonas aeruginosa from sputum, Pseudomonas aeruginosa MDR from a bedsore Cerebrovascular accident, dementia Anidulafungin Cured
5 65 years Male Throat, sputum, groin and urine Urinary tract infection Pseudomonas aeruginosa multidrug resistant Motor neuron disease, hospital-acquired pneumonia Anidulafungin Died of bacterial pneumonia
6 29 years Male Groin Colonization ESBL Klebsiella Acute liver failure secondary to hepatitis C, acute kidney injury, critical care polyneuropathy Terbinafine spray Discharged home
7 86 years Male Axilla, urine Colonization Pseudomonas aeruginosa COPD, vascular dementia, bedbound on tracheostomy to Terbinafine spray, nystatin application Died due to aspiration pneumonia and hypoxic respiratory failure
8 80 years Female Nose, tracheostomy site Colonization ESBL Klebsiella Chronic kidney disease, coronary artery disease, on tracheostomy Terbinafine spray, nystatin application Transfer to geriatric ward
9 62 years Female Axilla Colonization Pseudomonas multi drug-resistant Chronic kidney disease, necrotizing fasciitis Terbinafine spray, nystatin application Died due to bacterial sepsis
10 91 years Female Groin area Colonization None COPD, hypertension Terbinafine spray, nystatin application Discharged home
11 23 years Male Nose, axilla Colonization Escherichia coli Hypoxic brain injury, recurrent urinary tract infection Terbinafine spray, nystatin application Transfer to long-term unit
12 75 years Male Nose, groin Colonization Pseudomonas aeruginosa Diabetes mellitus, chronic kidney disease, recurrent pneumonia Terbinafine spray, nystatin application Discharged home
13 85 years Male urine Colonization Pseudomonas aeruginosa Parkinson’s disease, cerebrovascular accident Terbinafine spray, nystatin application Transfer to geriatric unit
PCR: polymerase chain reaction, MRD: multi-drug resiatant, ESBL: extended spectrum beta lactamase, COPD: chronic obstructive pulmonary disease.
Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All patients had bacterial or viral infections prior to or concomitantly with C. auris infection/colonization, as shown in Table 1.
For the typing of C. auris isolates, the molecular technique PFGE, which consisted of electrophoretic karyotyping (EK), was utilized to compare the isolates from different sites. The PFGE karyotype of the outbreak isolates of C. auris in our series is shown in Fig. 1. Antifungal susceptibility tests were performed on isolates from infected subjects. All strains of C. auris shared the same susceptibility profile, being susceptible to echinocandins (especially anidulafungin), flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Table 2 shows the susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates. All patients with C. auris infection received systemic antifungal drugs, while the eight patients who were colonized were appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC (Table 1).Fig. 1 Electrophoretic karyotypes of C. auris isolates. Karyotypes of representative outbreak isolates from five patients in the intensive care unit. Lane 1, 2 and 8 are control specimens which served as comparison for different genotypes. Lane 3 to 7 strains (specimens from the five C. auris cases) show no single band variation and are likely representing the same strain.
Fig. 1Table 2 Susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates from subjects with infection.
Table 2Antifungal drugs Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Amphotericin 4-R 4-R 2-R 4-R 2-R
Caspofungin 0.25 8-R 8 0.5 8
Fluconazole 64 128-R 128-R 128-R 128-R
Flucytocin 0.125 0.5-S 0.12 0.12 0.12
Itraconazole 0.125-R 16-R 0.12 0.12 16
Posaconazole 0.012 8-R 0.06 0.06-S 8
Voriconazole 0.25 8-R 0.25 0.5 8
Anidulafungin 0.125-S 0.5-I 0.25 0.12-S 0.5-I
Micafungin 0.25 0.12 0.25
R: resistant, S: sensitive, I: intermediate.
Among the patients with C. auris infection who received systemic antifungal therapy, three (60 %) died during antifungal therapy. The other two patients were successfully treated and appropriately decolonized of C. auris (Table 1).
Discussion
Recent reports showed that C. auris is an emerging yeast that has been identified worldwide as a cause of severe invasive healthcare infections, which mostly affect critically ill patients and cause substantial morbidity and mortality [7,8]. To our knowledge, our series is the first designed to study this infection in Qatar.
Many C. auris outbreaks have been reported worldwide. In India, the first C. auris outbreak was reported in 2013 by Chowdhary et al. [9] who identified 12 patients with positive microbiological clinical specimens collected between 2009 and 2012. While Calvo et al. reported the first outbreak of C. auris infection in Venezuela between March 2012 and July 2013 [10]. All the isolates were initially identified as C. haemulonii. However, the isolation of C. auris was later confirmed by genome sequencing [9,10]. Similarly, we have reported the first outbreak of C. auris infection in Qatar, identifying 13 patients. The emergence of C. auris in our hospital raises concerns that this fungus may spread to other healthcare settings, particularly critical care facilities in Qatar, requiring intensified measures to control the spread of this infection. Therefore, knowing the source of infection and detection of possible routes of transmission can help in preventing the clonal spread of this infection and hospital outbreaks among various health facilities in Qatar [5,7,8]. Similarly, intensive efforts have been made in our hospital to find the cause of the cross-transmission. Environmental and surface swabs were carried out in the affected areas, but all results were negative.
Diagnosing C. auris infection is difficult because the clinical presentation is non-specific or may not be recognizable since patients infected with C. auris often have another serious illness or condition. Moreover, C. auris can be misidentified with standard laboratory techniques as C. haemulonii [11,12]. As a result, a high index of suspicion is required to diagnose this infection. In addition, accurate identification of C. auris through specialized laboratory methods is required to avoid misidentification and inappropriate treatment that may make it difficult to control the spread of C. auris in the healthcare settings [10]. In this study, the diagnosis of C. auris infection was suspected because of the resistance of the isolates to fluconazole and amphotericin B. The diagnosis was confirmed by molecular methods.
The spectrum of C. auris infection ranges from superficial infections that affect the skin to widespread infections that affect the brain, heart, lungs, liver, spleen, and kidneys [5]. Antifungal therapy should be administered to eradicate and control C. auris infection. On the other hand, C. auris can be isolated from the skin, rectum, wounds or mouth of some patients who do not show symptoms of infection. This condition is referred to as asymptomatic colonization and treatment with antifungal drugs does not eradicate C. auris colonization. However, the identification of C. auris colonization is significant because it carries the risk of transmission, which requires the immediate implementation of adequate infection control measures [13]. Likewise, our patients showed different clinical presentations, and cases with colonization were identified and appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC.
In agreement with other reports [[3], [4], [5],7,13], our isolates showed resistance to the most important antifungal agents such as fluconazole and amphotericin B. The all cause mortality among our patients was 60 % which is in line with the mortality rate seen in other studies ranging from 30 to 60% [3].
One of the limitations of this study is the retrospective nature of the research. In addition, the small sample size is another factor that limits the generalizability of these findings.
Conclusion
C. auris can cause a wide variety of invasive infections, including bloodstream infections, urinary tract infection, skin infection, and lower respiratory tract infection, especially in critically ill patients. In addition, all isolates showed resistance to fluconazole and amphotericin B and were sensitive to echinocandins especially anidulafungin.
Authors contribution (Authorship)
Adila Shaukat: Desgning, interpretation of data, revising and approving the final draft.
Nasir Al Ansari: conception of the study, revising and approving the final draft.
Walid Al Wali: interpretation of data, revising and approving the final draft.
Edin Karic: interpretation of data, revising and approving the final draft.
Ihab El Madhoun: acquisition of data, revising and approving the final draft.
Hassan Mitwally: interpretation of data, revising and approving the final draft.
Manal Hamed: acquisition of data, revising and approving the final draft.
Feah Alutra- Visan: interpretation of data, drafting the article and approving the final draft.
Conflict of interest
All authors report no conflict of interest.
Acknowledgement
Open Access funding provided by the Qatar National Library.
We are alsothankful for Dr Jameela Al Ajmi,from Corporate Infection prevention and control dept and Ms Tahani M. Al Saadi from Laboratory department for their corporation and support. | NYSTATIN, TERBINAFINE | DrugsGivenReaction | CC BY | 33299794 | 18,688,885 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Respiratory failure'. | Experience of treating Candida auris cases at a general hospital in the state of Qatar.
So far there have been no studies on Candida auris in Qatar. This study aimed to describe the clinical spectrum and outcome of C. auris infection in patients admitted to a general hospital in Qatar.
We conducted this descriptive observational study in a general hospital in Qatar. We have involved all patients with C. auris infection and colonization admitted to a general hospital from December 2018 to August 2019.
We identified 13 patients with confirmed C.auris infection/colonization, of which five cases represented an actual C. auris infection, while the remaining eight cases were considered as colonization. The mean age of the patients with infection was 76.6 ± 8.4 years, while the mean age of the patients with colonization was 66.4 ± 24.7 years. Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All strains of C. auris were susceptible to echinocandins, flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Of the patients with C. auris infection who received systemic antifungal therapy, three (60%) died during antifungal therapy.
Our study showed that C. auris can cause a wide variety of invasive infections, including bloodstream infection, urinary tract infection, skin infection, and lower respiratory tract infections, especially in critically ill patients. In addition, our isolates showed resistance to the most common antifungal agents such as fluconazole and amphotericin B.
Introduction
Candida auris is a novel multidrug-resistant yeast with high overall mortality that was first isolated from the external auditory canal of a patient in Japan in 2009 [1]. Since then, this fungal infection has been reported from various countries across the world [[2], [3], [4]], and over time it has become a serious global health concern as one of the most serious emerging pathogens that critical care physicians should be aware of [5].
C. auris being resistant to major antifungal classes used to treat Candida including azole antifungal agents, poses a challenge to routine microbiology laboratories, as C. auris can be misidentified with standard laboratory techniques, and have a tendency to cause outbreaks in healthcare settings especially critical care areas despite adequate infection prevention and control measures [4,5].
In Qatar, there is no published data on Candida aurisso far. In this series, we reported the first outbreak of C. auris infection in Qatar, to describe the clinical spectrum and outcome of this infection in the affected patients.
Methods and patients
We conducted this descriptive observational study in a general hospital in Qatar. We involved all patients with Candida Auris infection and colonization in the intensive care units and other wards from December 2018 to August 2019. This study was given ethical approval by the medical research committee at Hamad Medical Corporation, under number: MRC-01-19-503..
Definitions
Colonization is defined as isolation of C. auris from endotracheal aspiration fluid, throat swabs, sputum, urine, and samples from central venous catheters or other parts of the body in absence of clinical signs or symptomes of infection. C.auris infection is defined as the isolation of Cauris from clinical specimens with compatible clinical signs and symptoms of infection [5].
Candida auris identification
All clinical specimens, from different sites, were cultured by quantitative technique on Sabouraud Dextrose Agar (OXOID, UK) and incubated at 35−37 °C for 48 h. Preliminary fungal strain identification was based on colony morphology on Chromogenic Candida Agar (OXOID, UK), while the identification to the species level was confirmed by Vitek 2 XL automated system (bioMerieux). Susceptibility of strains to Amphotericin B, Fluconazole, 5-fluorocytosine, and voriconazole was determined by using Sensititre™ YeastOne™ plate and by interpreting results according to closely related Candida species and on expert opinion. As per the Centers for Disease Control and Prevention (CDC), there are currently no established C. auris-specific susceptibility breakpoints [6].
Pulsed-field gel electrophoresis (PFGE) typing, which consisted of electrophoretic karyotyping (EK), was performed to compare the isolates from different sites. Following the results of the PFGE, an outbreak of C. auris infection in critical care unit and medical unit was confirmed by identifying five5 cases and patient screening revealed colonization of eight additional patients. Intensive efforts were done to find out the cause of cross-transmission and environmental and surface swabbing was done in affected areas, but all results were negative.
Data analysis
The results of analyses of continuous variables are expressed as means and standard deviations (SD) unless otherwise specified.
Results
During the study period, we identified 13 patients with confirmed C. auris infection/colonization, of which five cases represented an actual Candida infection, while the remaining eight cases were considered colonization. The mean age of the patients with infection was 76.6 ± 8.4 years (range: 65–90 years), while the mean age of the patients with colonization was 66.4 ± 24.7 years (range: 23–91 years). Table 1 describes the demographic characteristics of the patients involved in this study.Table 1 Candida auris infection/colonization patients details.
Table 1Case/No Age Sex Site of infection/ or site of Candida isolation Type of infection Pre or co-infection Co-morbidity Treatment provided Outcome
1 78 years Male Tracheal aspirate and urine Lower respiratory tract infection Corona virus 229 E PCR positive from nasal swab Interstitial lung disease Anidulafungin Died of hypoxic respiratory failure
2 79 years Male Nose and decubitus ulcer Skin soft tissue infection Pseudomonas MDR and Morganella morganii from decubitus ulcer Diabetes mellitus, sacral bed sores Flucytosine Died of bacterial/fungal sepsis
3 71 years Male Nose, throat, tracheal aspirate, and decubitus ulcer Candidemia Pseudomonas aeruginosa MDR and ESBL Klebsiella pneumoniae from sputum Diabetes mellitus, sacral bed sores Anidulafungin and posaconazole Cured
4 90 years Male urine, throat and nose Urinary tract infection Klebsiella pneumoniae and carbapenem resistant Pseudomonas aeruginosa from sputum, Pseudomonas aeruginosa MDR from a bedsore Cerebrovascular accident, dementia Anidulafungin Cured
5 65 years Male Throat, sputum, groin and urine Urinary tract infection Pseudomonas aeruginosa multidrug resistant Motor neuron disease, hospital-acquired pneumonia Anidulafungin Died of bacterial pneumonia
6 29 years Male Groin Colonization ESBL Klebsiella Acute liver failure secondary to hepatitis C, acute kidney injury, critical care polyneuropathy Terbinafine spray Discharged home
7 86 years Male Axilla, urine Colonization Pseudomonas aeruginosa COPD, vascular dementia, bedbound on tracheostomy to Terbinafine spray, nystatin application Died due to aspiration pneumonia and hypoxic respiratory failure
8 80 years Female Nose, tracheostomy site Colonization ESBL Klebsiella Chronic kidney disease, coronary artery disease, on tracheostomy Terbinafine spray, nystatin application Transfer to geriatric ward
9 62 years Female Axilla Colonization Pseudomonas multi drug-resistant Chronic kidney disease, necrotizing fasciitis Terbinafine spray, nystatin application Died due to bacterial sepsis
10 91 years Female Groin area Colonization None COPD, hypertension Terbinafine spray, nystatin application Discharged home
11 23 years Male Nose, axilla Colonization Escherichia coli Hypoxic brain injury, recurrent urinary tract infection Terbinafine spray, nystatin application Transfer to long-term unit
12 75 years Male Nose, groin Colonization Pseudomonas aeruginosa Diabetes mellitus, chronic kidney disease, recurrent pneumonia Terbinafine spray, nystatin application Discharged home
13 85 years Male urine Colonization Pseudomonas aeruginosa Parkinson’s disease, cerebrovascular accident Terbinafine spray, nystatin application Transfer to geriatric unit
PCR: polymerase chain reaction, MRD: multi-drug resiatant, ESBL: extended spectrum beta lactamase, COPD: chronic obstructive pulmonary disease.
Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All patients had bacterial or viral infections prior to or concomitantly with C. auris infection/colonization, as shown in Table 1.
For the typing of C. auris isolates, the molecular technique PFGE, which consisted of electrophoretic karyotyping (EK), was utilized to compare the isolates from different sites. The PFGE karyotype of the outbreak isolates of C. auris in our series is shown in Fig. 1. Antifungal susceptibility tests were performed on isolates from infected subjects. All strains of C. auris shared the same susceptibility profile, being susceptible to echinocandins (especially anidulafungin), flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Table 2 shows the susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates. All patients with C. auris infection received systemic antifungal drugs, while the eight patients who were colonized were appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC (Table 1).Fig. 1 Electrophoretic karyotypes of C. auris isolates. Karyotypes of representative outbreak isolates from five patients in the intensive care unit. Lane 1, 2 and 8 are control specimens which served as comparison for different genotypes. Lane 3 to 7 strains (specimens from the five C. auris cases) show no single band variation and are likely representing the same strain.
Fig. 1Table 2 Susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates from subjects with infection.
Table 2Antifungal drugs Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Amphotericin 4-R 4-R 2-R 4-R 2-R
Caspofungin 0.25 8-R 8 0.5 8
Fluconazole 64 128-R 128-R 128-R 128-R
Flucytocin 0.125 0.5-S 0.12 0.12 0.12
Itraconazole 0.125-R 16-R 0.12 0.12 16
Posaconazole 0.012 8-R 0.06 0.06-S 8
Voriconazole 0.25 8-R 0.25 0.5 8
Anidulafungin 0.125-S 0.5-I 0.25 0.12-S 0.5-I
Micafungin 0.25 0.12 0.25
R: resistant, S: sensitive, I: intermediate.
Among the patients with C. auris infection who received systemic antifungal therapy, three (60 %) died during antifungal therapy. The other two patients were successfully treated and appropriately decolonized of C. auris (Table 1).
Discussion
Recent reports showed that C. auris is an emerging yeast that has been identified worldwide as a cause of severe invasive healthcare infections, which mostly affect critically ill patients and cause substantial morbidity and mortality [7,8]. To our knowledge, our series is the first designed to study this infection in Qatar.
Many C. auris outbreaks have been reported worldwide. In India, the first C. auris outbreak was reported in 2013 by Chowdhary et al. [9] who identified 12 patients with positive microbiological clinical specimens collected between 2009 and 2012. While Calvo et al. reported the first outbreak of C. auris infection in Venezuela between March 2012 and July 2013 [10]. All the isolates were initially identified as C. haemulonii. However, the isolation of C. auris was later confirmed by genome sequencing [9,10]. Similarly, we have reported the first outbreak of C. auris infection in Qatar, identifying 13 patients. The emergence of C. auris in our hospital raises concerns that this fungus may spread to other healthcare settings, particularly critical care facilities in Qatar, requiring intensified measures to control the spread of this infection. Therefore, knowing the source of infection and detection of possible routes of transmission can help in preventing the clonal spread of this infection and hospital outbreaks among various health facilities in Qatar [5,7,8]. Similarly, intensive efforts have been made in our hospital to find the cause of the cross-transmission. Environmental and surface swabs were carried out in the affected areas, but all results were negative.
Diagnosing C. auris infection is difficult because the clinical presentation is non-specific or may not be recognizable since patients infected with C. auris often have another serious illness or condition. Moreover, C. auris can be misidentified with standard laboratory techniques as C. haemulonii [11,12]. As a result, a high index of suspicion is required to diagnose this infection. In addition, accurate identification of C. auris through specialized laboratory methods is required to avoid misidentification and inappropriate treatment that may make it difficult to control the spread of C. auris in the healthcare settings [10]. In this study, the diagnosis of C. auris infection was suspected because of the resistance of the isolates to fluconazole and amphotericin B. The diagnosis was confirmed by molecular methods.
The spectrum of C. auris infection ranges from superficial infections that affect the skin to widespread infections that affect the brain, heart, lungs, liver, spleen, and kidneys [5]. Antifungal therapy should be administered to eradicate and control C. auris infection. On the other hand, C. auris can be isolated from the skin, rectum, wounds or mouth of some patients who do not show symptoms of infection. This condition is referred to as asymptomatic colonization and treatment with antifungal drugs does not eradicate C. auris colonization. However, the identification of C. auris colonization is significant because it carries the risk of transmission, which requires the immediate implementation of adequate infection control measures [13]. Likewise, our patients showed different clinical presentations, and cases with colonization were identified and appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC.
In agreement with other reports [[3], [4], [5],7,13], our isolates showed resistance to the most important antifungal agents such as fluconazole and amphotericin B. The all cause mortality among our patients was 60 % which is in line with the mortality rate seen in other studies ranging from 30 to 60% [3].
One of the limitations of this study is the retrospective nature of the research. In addition, the small sample size is another factor that limits the generalizability of these findings.
Conclusion
C. auris can cause a wide variety of invasive infections, including bloodstream infections, urinary tract infection, skin infection, and lower respiratory tract infection, especially in critically ill patients. In addition, all isolates showed resistance to fluconazole and amphotericin B and were sensitive to echinocandins especially anidulafungin.
Authors contribution (Authorship)
Adila Shaukat: Desgning, interpretation of data, revising and approving the final draft.
Nasir Al Ansari: conception of the study, revising and approving the final draft.
Walid Al Wali: interpretation of data, revising and approving the final draft.
Edin Karic: interpretation of data, revising and approving the final draft.
Ihab El Madhoun: acquisition of data, revising and approving the final draft.
Hassan Mitwally: interpretation of data, revising and approving the final draft.
Manal Hamed: acquisition of data, revising and approving the final draft.
Feah Alutra- Visan: interpretation of data, drafting the article and approving the final draft.
Conflict of interest
All authors report no conflict of interest.
Acknowledgement
Open Access funding provided by the Qatar National Library.
We are alsothankful for Dr Jameela Al Ajmi,from Corporate Infection prevention and control dept and Ms Tahani M. Al Saadi from Laboratory department for their corporation and support. | NYSTATIN, TERBINAFINE | DrugsGivenReaction | CC BY | 33299794 | 18,688,885 | 2021 |
What was the outcome of reaction 'Bacterial sepsis'? | Experience of treating Candida auris cases at a general hospital in the state of Qatar.
So far there have been no studies on Candida auris in Qatar. This study aimed to describe the clinical spectrum and outcome of C. auris infection in patients admitted to a general hospital in Qatar.
We conducted this descriptive observational study in a general hospital in Qatar. We have involved all patients with C. auris infection and colonization admitted to a general hospital from December 2018 to August 2019.
We identified 13 patients with confirmed C.auris infection/colonization, of which five cases represented an actual C. auris infection, while the remaining eight cases were considered as colonization. The mean age of the patients with infection was 76.6 ± 8.4 years, while the mean age of the patients with colonization was 66.4 ± 24.7 years. Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All strains of C. auris were susceptible to echinocandins, flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Of the patients with C. auris infection who received systemic antifungal therapy, three (60%) died during antifungal therapy.
Our study showed that C. auris can cause a wide variety of invasive infections, including bloodstream infection, urinary tract infection, skin infection, and lower respiratory tract infections, especially in critically ill patients. In addition, our isolates showed resistance to the most common antifungal agents such as fluconazole and amphotericin B.
Introduction
Candida auris is a novel multidrug-resistant yeast with high overall mortality that was first isolated from the external auditory canal of a patient in Japan in 2009 [1]. Since then, this fungal infection has been reported from various countries across the world [[2], [3], [4]], and over time it has become a serious global health concern as one of the most serious emerging pathogens that critical care physicians should be aware of [5].
C. auris being resistant to major antifungal classes used to treat Candida including azole antifungal agents, poses a challenge to routine microbiology laboratories, as C. auris can be misidentified with standard laboratory techniques, and have a tendency to cause outbreaks in healthcare settings especially critical care areas despite adequate infection prevention and control measures [4,5].
In Qatar, there is no published data on Candida aurisso far. In this series, we reported the first outbreak of C. auris infection in Qatar, to describe the clinical spectrum and outcome of this infection in the affected patients.
Methods and patients
We conducted this descriptive observational study in a general hospital in Qatar. We involved all patients with Candida Auris infection and colonization in the intensive care units and other wards from December 2018 to August 2019. This study was given ethical approval by the medical research committee at Hamad Medical Corporation, under number: MRC-01-19-503..
Definitions
Colonization is defined as isolation of C. auris from endotracheal aspiration fluid, throat swabs, sputum, urine, and samples from central venous catheters or other parts of the body in absence of clinical signs or symptomes of infection. C.auris infection is defined as the isolation of Cauris from clinical specimens with compatible clinical signs and symptoms of infection [5].
Candida auris identification
All clinical specimens, from different sites, were cultured by quantitative technique on Sabouraud Dextrose Agar (OXOID, UK) and incubated at 35−37 °C for 48 h. Preliminary fungal strain identification was based on colony morphology on Chromogenic Candida Agar (OXOID, UK), while the identification to the species level was confirmed by Vitek 2 XL automated system (bioMerieux). Susceptibility of strains to Amphotericin B, Fluconazole, 5-fluorocytosine, and voriconazole was determined by using Sensititre™ YeastOne™ plate and by interpreting results according to closely related Candida species and on expert opinion. As per the Centers for Disease Control and Prevention (CDC), there are currently no established C. auris-specific susceptibility breakpoints [6].
Pulsed-field gel electrophoresis (PFGE) typing, which consisted of electrophoretic karyotyping (EK), was performed to compare the isolates from different sites. Following the results of the PFGE, an outbreak of C. auris infection in critical care unit and medical unit was confirmed by identifying five5 cases and patient screening revealed colonization of eight additional patients. Intensive efforts were done to find out the cause of cross-transmission and environmental and surface swabbing was done in affected areas, but all results were negative.
Data analysis
The results of analyses of continuous variables are expressed as means and standard deviations (SD) unless otherwise specified.
Results
During the study period, we identified 13 patients with confirmed C. auris infection/colonization, of which five cases represented an actual Candida infection, while the remaining eight cases were considered colonization. The mean age of the patients with infection was 76.6 ± 8.4 years (range: 65–90 years), while the mean age of the patients with colonization was 66.4 ± 24.7 years (range: 23–91 years). Table 1 describes the demographic characteristics of the patients involved in this study.Table 1 Candida auris infection/colonization patients details.
Table 1Case/No Age Sex Site of infection/ or site of Candida isolation Type of infection Pre or co-infection Co-morbidity Treatment provided Outcome
1 78 years Male Tracheal aspirate and urine Lower respiratory tract infection Corona virus 229 E PCR positive from nasal swab Interstitial lung disease Anidulafungin Died of hypoxic respiratory failure
2 79 years Male Nose and decubitus ulcer Skin soft tissue infection Pseudomonas MDR and Morganella morganii from decubitus ulcer Diabetes mellitus, sacral bed sores Flucytosine Died of bacterial/fungal sepsis
3 71 years Male Nose, throat, tracheal aspirate, and decubitus ulcer Candidemia Pseudomonas aeruginosa MDR and ESBL Klebsiella pneumoniae from sputum Diabetes mellitus, sacral bed sores Anidulafungin and posaconazole Cured
4 90 years Male urine, throat and nose Urinary tract infection Klebsiella pneumoniae and carbapenem resistant Pseudomonas aeruginosa from sputum, Pseudomonas aeruginosa MDR from a bedsore Cerebrovascular accident, dementia Anidulafungin Cured
5 65 years Male Throat, sputum, groin and urine Urinary tract infection Pseudomonas aeruginosa multidrug resistant Motor neuron disease, hospital-acquired pneumonia Anidulafungin Died of bacterial pneumonia
6 29 years Male Groin Colonization ESBL Klebsiella Acute liver failure secondary to hepatitis C, acute kidney injury, critical care polyneuropathy Terbinafine spray Discharged home
7 86 years Male Axilla, urine Colonization Pseudomonas aeruginosa COPD, vascular dementia, bedbound on tracheostomy to Terbinafine spray, nystatin application Died due to aspiration pneumonia and hypoxic respiratory failure
8 80 years Female Nose, tracheostomy site Colonization ESBL Klebsiella Chronic kidney disease, coronary artery disease, on tracheostomy Terbinafine spray, nystatin application Transfer to geriatric ward
9 62 years Female Axilla Colonization Pseudomonas multi drug-resistant Chronic kidney disease, necrotizing fasciitis Terbinafine spray, nystatin application Died due to bacterial sepsis
10 91 years Female Groin area Colonization None COPD, hypertension Terbinafine spray, nystatin application Discharged home
11 23 years Male Nose, axilla Colonization Escherichia coli Hypoxic brain injury, recurrent urinary tract infection Terbinafine spray, nystatin application Transfer to long-term unit
12 75 years Male Nose, groin Colonization Pseudomonas aeruginosa Diabetes mellitus, chronic kidney disease, recurrent pneumonia Terbinafine spray, nystatin application Discharged home
13 85 years Male urine Colonization Pseudomonas aeruginosa Parkinson’s disease, cerebrovascular accident Terbinafine spray, nystatin application Transfer to geriatric unit
PCR: polymerase chain reaction, MRD: multi-drug resiatant, ESBL: extended spectrum beta lactamase, COPD: chronic obstructive pulmonary disease.
Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All patients had bacterial or viral infections prior to or concomitantly with C. auris infection/colonization, as shown in Table 1.
For the typing of C. auris isolates, the molecular technique PFGE, which consisted of electrophoretic karyotyping (EK), was utilized to compare the isolates from different sites. The PFGE karyotype of the outbreak isolates of C. auris in our series is shown in Fig. 1. Antifungal susceptibility tests were performed on isolates from infected subjects. All strains of C. auris shared the same susceptibility profile, being susceptible to echinocandins (especially anidulafungin), flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Table 2 shows the susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates. All patients with C. auris infection received systemic antifungal drugs, while the eight patients who were colonized were appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC (Table 1).Fig. 1 Electrophoretic karyotypes of C. auris isolates. Karyotypes of representative outbreak isolates from five patients in the intensive care unit. Lane 1, 2 and 8 are control specimens which served as comparison for different genotypes. Lane 3 to 7 strains (specimens from the five C. auris cases) show no single band variation and are likely representing the same strain.
Fig. 1Table 2 Susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates from subjects with infection.
Table 2Antifungal drugs Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Amphotericin 4-R 4-R 2-R 4-R 2-R
Caspofungin 0.25 8-R 8 0.5 8
Fluconazole 64 128-R 128-R 128-R 128-R
Flucytocin 0.125 0.5-S 0.12 0.12 0.12
Itraconazole 0.125-R 16-R 0.12 0.12 16
Posaconazole 0.012 8-R 0.06 0.06-S 8
Voriconazole 0.25 8-R 0.25 0.5 8
Anidulafungin 0.125-S 0.5-I 0.25 0.12-S 0.5-I
Micafungin 0.25 0.12 0.25
R: resistant, S: sensitive, I: intermediate.
Among the patients with C. auris infection who received systemic antifungal therapy, three (60 %) died during antifungal therapy. The other two patients were successfully treated and appropriately decolonized of C. auris (Table 1).
Discussion
Recent reports showed that C. auris is an emerging yeast that has been identified worldwide as a cause of severe invasive healthcare infections, which mostly affect critically ill patients and cause substantial morbidity and mortality [7,8]. To our knowledge, our series is the first designed to study this infection in Qatar.
Many C. auris outbreaks have been reported worldwide. In India, the first C. auris outbreak was reported in 2013 by Chowdhary et al. [9] who identified 12 patients with positive microbiological clinical specimens collected between 2009 and 2012. While Calvo et al. reported the first outbreak of C. auris infection in Venezuela between March 2012 and July 2013 [10]. All the isolates were initially identified as C. haemulonii. However, the isolation of C. auris was later confirmed by genome sequencing [9,10]. Similarly, we have reported the first outbreak of C. auris infection in Qatar, identifying 13 patients. The emergence of C. auris in our hospital raises concerns that this fungus may spread to other healthcare settings, particularly critical care facilities in Qatar, requiring intensified measures to control the spread of this infection. Therefore, knowing the source of infection and detection of possible routes of transmission can help in preventing the clonal spread of this infection and hospital outbreaks among various health facilities in Qatar [5,7,8]. Similarly, intensive efforts have been made in our hospital to find the cause of the cross-transmission. Environmental and surface swabs were carried out in the affected areas, but all results were negative.
Diagnosing C. auris infection is difficult because the clinical presentation is non-specific or may not be recognizable since patients infected with C. auris often have another serious illness or condition. Moreover, C. auris can be misidentified with standard laboratory techniques as C. haemulonii [11,12]. As a result, a high index of suspicion is required to diagnose this infection. In addition, accurate identification of C. auris through specialized laboratory methods is required to avoid misidentification and inappropriate treatment that may make it difficult to control the spread of C. auris in the healthcare settings [10]. In this study, the diagnosis of C. auris infection was suspected because of the resistance of the isolates to fluconazole and amphotericin B. The diagnosis was confirmed by molecular methods.
The spectrum of C. auris infection ranges from superficial infections that affect the skin to widespread infections that affect the brain, heart, lungs, liver, spleen, and kidneys [5]. Antifungal therapy should be administered to eradicate and control C. auris infection. On the other hand, C. auris can be isolated from the skin, rectum, wounds or mouth of some patients who do not show symptoms of infection. This condition is referred to as asymptomatic colonization and treatment with antifungal drugs does not eradicate C. auris colonization. However, the identification of C. auris colonization is significant because it carries the risk of transmission, which requires the immediate implementation of adequate infection control measures [13]. Likewise, our patients showed different clinical presentations, and cases with colonization were identified and appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC.
In agreement with other reports [[3], [4], [5],7,13], our isolates showed resistance to the most important antifungal agents such as fluconazole and amphotericin B. The all cause mortality among our patients was 60 % which is in line with the mortality rate seen in other studies ranging from 30 to 60% [3].
One of the limitations of this study is the retrospective nature of the research. In addition, the small sample size is another factor that limits the generalizability of these findings.
Conclusion
C. auris can cause a wide variety of invasive infections, including bloodstream infections, urinary tract infection, skin infection, and lower respiratory tract infection, especially in critically ill patients. In addition, all isolates showed resistance to fluconazole and amphotericin B and were sensitive to echinocandins especially anidulafungin.
Authors contribution (Authorship)
Adila Shaukat: Desgning, interpretation of data, revising and approving the final draft.
Nasir Al Ansari: conception of the study, revising and approving the final draft.
Walid Al Wali: interpretation of data, revising and approving the final draft.
Edin Karic: interpretation of data, revising and approving the final draft.
Ihab El Madhoun: acquisition of data, revising and approving the final draft.
Hassan Mitwally: interpretation of data, revising and approving the final draft.
Manal Hamed: acquisition of data, revising and approving the final draft.
Feah Alutra- Visan: interpretation of data, drafting the article and approving the final draft.
Conflict of interest
All authors report no conflict of interest.
Acknowledgement
Open Access funding provided by the Qatar National Library.
We are alsothankful for Dr Jameela Al Ajmi,from Corporate Infection prevention and control dept and Ms Tahani M. Al Saadi from Laboratory department for their corporation and support. | Fatal | ReactionOutcome | CC BY | 33299794 | 18,688,888 | 2021 |
What was the outcome of reaction 'Pneumonia aspiration'? | Experience of treating Candida auris cases at a general hospital in the state of Qatar.
So far there have been no studies on Candida auris in Qatar. This study aimed to describe the clinical spectrum and outcome of C. auris infection in patients admitted to a general hospital in Qatar.
We conducted this descriptive observational study in a general hospital in Qatar. We have involved all patients with C. auris infection and colonization admitted to a general hospital from December 2018 to August 2019.
We identified 13 patients with confirmed C.auris infection/colonization, of which five cases represented an actual C. auris infection, while the remaining eight cases were considered as colonization. The mean age of the patients with infection was 76.6 ± 8.4 years, while the mean age of the patients with colonization was 66.4 ± 24.7 years. Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All strains of C. auris were susceptible to echinocandins, flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Of the patients with C. auris infection who received systemic antifungal therapy, three (60%) died during antifungal therapy.
Our study showed that C. auris can cause a wide variety of invasive infections, including bloodstream infection, urinary tract infection, skin infection, and lower respiratory tract infections, especially in critically ill patients. In addition, our isolates showed resistance to the most common antifungal agents such as fluconazole and amphotericin B.
Introduction
Candida auris is a novel multidrug-resistant yeast with high overall mortality that was first isolated from the external auditory canal of a patient in Japan in 2009 [1]. Since then, this fungal infection has been reported from various countries across the world [[2], [3], [4]], and over time it has become a serious global health concern as one of the most serious emerging pathogens that critical care physicians should be aware of [5].
C. auris being resistant to major antifungal classes used to treat Candida including azole antifungal agents, poses a challenge to routine microbiology laboratories, as C. auris can be misidentified with standard laboratory techniques, and have a tendency to cause outbreaks in healthcare settings especially critical care areas despite adequate infection prevention and control measures [4,5].
In Qatar, there is no published data on Candida aurisso far. In this series, we reported the first outbreak of C. auris infection in Qatar, to describe the clinical spectrum and outcome of this infection in the affected patients.
Methods and patients
We conducted this descriptive observational study in a general hospital in Qatar. We involved all patients with Candida Auris infection and colonization in the intensive care units and other wards from December 2018 to August 2019. This study was given ethical approval by the medical research committee at Hamad Medical Corporation, under number: MRC-01-19-503..
Definitions
Colonization is defined as isolation of C. auris from endotracheal aspiration fluid, throat swabs, sputum, urine, and samples from central venous catheters or other parts of the body in absence of clinical signs or symptomes of infection. C.auris infection is defined as the isolation of Cauris from clinical specimens with compatible clinical signs and symptoms of infection [5].
Candida auris identification
All clinical specimens, from different sites, were cultured by quantitative technique on Sabouraud Dextrose Agar (OXOID, UK) and incubated at 35−37 °C for 48 h. Preliminary fungal strain identification was based on colony morphology on Chromogenic Candida Agar (OXOID, UK), while the identification to the species level was confirmed by Vitek 2 XL automated system (bioMerieux). Susceptibility of strains to Amphotericin B, Fluconazole, 5-fluorocytosine, and voriconazole was determined by using Sensititre™ YeastOne™ plate and by interpreting results according to closely related Candida species and on expert opinion. As per the Centers for Disease Control and Prevention (CDC), there are currently no established C. auris-specific susceptibility breakpoints [6].
Pulsed-field gel electrophoresis (PFGE) typing, which consisted of electrophoretic karyotyping (EK), was performed to compare the isolates from different sites. Following the results of the PFGE, an outbreak of C. auris infection in critical care unit and medical unit was confirmed by identifying five5 cases and patient screening revealed colonization of eight additional patients. Intensive efforts were done to find out the cause of cross-transmission and environmental and surface swabbing was done in affected areas, but all results were negative.
Data analysis
The results of analyses of continuous variables are expressed as means and standard deviations (SD) unless otherwise specified.
Results
During the study period, we identified 13 patients with confirmed C. auris infection/colonization, of which five cases represented an actual Candida infection, while the remaining eight cases were considered colonization. The mean age of the patients with infection was 76.6 ± 8.4 years (range: 65–90 years), while the mean age of the patients with colonization was 66.4 ± 24.7 years (range: 23–91 years). Table 1 describes the demographic characteristics of the patients involved in this study.Table 1 Candida auris infection/colonization patients details.
Table 1Case/No Age Sex Site of infection/ or site of Candida isolation Type of infection Pre or co-infection Co-morbidity Treatment provided Outcome
1 78 years Male Tracheal aspirate and urine Lower respiratory tract infection Corona virus 229 E PCR positive from nasal swab Interstitial lung disease Anidulafungin Died of hypoxic respiratory failure
2 79 years Male Nose and decubitus ulcer Skin soft tissue infection Pseudomonas MDR and Morganella morganii from decubitus ulcer Diabetes mellitus, sacral bed sores Flucytosine Died of bacterial/fungal sepsis
3 71 years Male Nose, throat, tracheal aspirate, and decubitus ulcer Candidemia Pseudomonas aeruginosa MDR and ESBL Klebsiella pneumoniae from sputum Diabetes mellitus, sacral bed sores Anidulafungin and posaconazole Cured
4 90 years Male urine, throat and nose Urinary tract infection Klebsiella pneumoniae and carbapenem resistant Pseudomonas aeruginosa from sputum, Pseudomonas aeruginosa MDR from a bedsore Cerebrovascular accident, dementia Anidulafungin Cured
5 65 years Male Throat, sputum, groin and urine Urinary tract infection Pseudomonas aeruginosa multidrug resistant Motor neuron disease, hospital-acquired pneumonia Anidulafungin Died of bacterial pneumonia
6 29 years Male Groin Colonization ESBL Klebsiella Acute liver failure secondary to hepatitis C, acute kidney injury, critical care polyneuropathy Terbinafine spray Discharged home
7 86 years Male Axilla, urine Colonization Pseudomonas aeruginosa COPD, vascular dementia, bedbound on tracheostomy to Terbinafine spray, nystatin application Died due to aspiration pneumonia and hypoxic respiratory failure
8 80 years Female Nose, tracheostomy site Colonization ESBL Klebsiella Chronic kidney disease, coronary artery disease, on tracheostomy Terbinafine spray, nystatin application Transfer to geriatric ward
9 62 years Female Axilla Colonization Pseudomonas multi drug-resistant Chronic kidney disease, necrotizing fasciitis Terbinafine spray, nystatin application Died due to bacterial sepsis
10 91 years Female Groin area Colonization None COPD, hypertension Terbinafine spray, nystatin application Discharged home
11 23 years Male Nose, axilla Colonization Escherichia coli Hypoxic brain injury, recurrent urinary tract infection Terbinafine spray, nystatin application Transfer to long-term unit
12 75 years Male Nose, groin Colonization Pseudomonas aeruginosa Diabetes mellitus, chronic kidney disease, recurrent pneumonia Terbinafine spray, nystatin application Discharged home
13 85 years Male urine Colonization Pseudomonas aeruginosa Parkinson’s disease, cerebrovascular accident Terbinafine spray, nystatin application Transfer to geriatric unit
PCR: polymerase chain reaction, MRD: multi-drug resiatant, ESBL: extended spectrum beta lactamase, COPD: chronic obstructive pulmonary disease.
Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All patients had bacterial or viral infections prior to or concomitantly with C. auris infection/colonization, as shown in Table 1.
For the typing of C. auris isolates, the molecular technique PFGE, which consisted of electrophoretic karyotyping (EK), was utilized to compare the isolates from different sites. The PFGE karyotype of the outbreak isolates of C. auris in our series is shown in Fig. 1. Antifungal susceptibility tests were performed on isolates from infected subjects. All strains of C. auris shared the same susceptibility profile, being susceptible to echinocandins (especially anidulafungin), flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Table 2 shows the susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates. All patients with C. auris infection received systemic antifungal drugs, while the eight patients who were colonized were appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC (Table 1).Fig. 1 Electrophoretic karyotypes of C. auris isolates. Karyotypes of representative outbreak isolates from five patients in the intensive care unit. Lane 1, 2 and 8 are control specimens which served as comparison for different genotypes. Lane 3 to 7 strains (specimens from the five C. auris cases) show no single band variation and are likely representing the same strain.
Fig. 1Table 2 Susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates from subjects with infection.
Table 2Antifungal drugs Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Amphotericin 4-R 4-R 2-R 4-R 2-R
Caspofungin 0.25 8-R 8 0.5 8
Fluconazole 64 128-R 128-R 128-R 128-R
Flucytocin 0.125 0.5-S 0.12 0.12 0.12
Itraconazole 0.125-R 16-R 0.12 0.12 16
Posaconazole 0.012 8-R 0.06 0.06-S 8
Voriconazole 0.25 8-R 0.25 0.5 8
Anidulafungin 0.125-S 0.5-I 0.25 0.12-S 0.5-I
Micafungin 0.25 0.12 0.25
R: resistant, S: sensitive, I: intermediate.
Among the patients with C. auris infection who received systemic antifungal therapy, three (60 %) died during antifungal therapy. The other two patients were successfully treated and appropriately decolonized of C. auris (Table 1).
Discussion
Recent reports showed that C. auris is an emerging yeast that has been identified worldwide as a cause of severe invasive healthcare infections, which mostly affect critically ill patients and cause substantial morbidity and mortality [7,8]. To our knowledge, our series is the first designed to study this infection in Qatar.
Many C. auris outbreaks have been reported worldwide. In India, the first C. auris outbreak was reported in 2013 by Chowdhary et al. [9] who identified 12 patients with positive microbiological clinical specimens collected between 2009 and 2012. While Calvo et al. reported the first outbreak of C. auris infection in Venezuela between March 2012 and July 2013 [10]. All the isolates were initially identified as C. haemulonii. However, the isolation of C. auris was later confirmed by genome sequencing [9,10]. Similarly, we have reported the first outbreak of C. auris infection in Qatar, identifying 13 patients. The emergence of C. auris in our hospital raises concerns that this fungus may spread to other healthcare settings, particularly critical care facilities in Qatar, requiring intensified measures to control the spread of this infection. Therefore, knowing the source of infection and detection of possible routes of transmission can help in preventing the clonal spread of this infection and hospital outbreaks among various health facilities in Qatar [5,7,8]. Similarly, intensive efforts have been made in our hospital to find the cause of the cross-transmission. Environmental and surface swabs were carried out in the affected areas, but all results were negative.
Diagnosing C. auris infection is difficult because the clinical presentation is non-specific or may not be recognizable since patients infected with C. auris often have another serious illness or condition. Moreover, C. auris can be misidentified with standard laboratory techniques as C. haemulonii [11,12]. As a result, a high index of suspicion is required to diagnose this infection. In addition, accurate identification of C. auris through specialized laboratory methods is required to avoid misidentification and inappropriate treatment that may make it difficult to control the spread of C. auris in the healthcare settings [10]. In this study, the diagnosis of C. auris infection was suspected because of the resistance of the isolates to fluconazole and amphotericin B. The diagnosis was confirmed by molecular methods.
The spectrum of C. auris infection ranges from superficial infections that affect the skin to widespread infections that affect the brain, heart, lungs, liver, spleen, and kidneys [5]. Antifungal therapy should be administered to eradicate and control C. auris infection. On the other hand, C. auris can be isolated from the skin, rectum, wounds or mouth of some patients who do not show symptoms of infection. This condition is referred to as asymptomatic colonization and treatment with antifungal drugs does not eradicate C. auris colonization. However, the identification of C. auris colonization is significant because it carries the risk of transmission, which requires the immediate implementation of adequate infection control measures [13]. Likewise, our patients showed different clinical presentations, and cases with colonization were identified and appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC.
In agreement with other reports [[3], [4], [5],7,13], our isolates showed resistance to the most important antifungal agents such as fluconazole and amphotericin B. The all cause mortality among our patients was 60 % which is in line with the mortality rate seen in other studies ranging from 30 to 60% [3].
One of the limitations of this study is the retrospective nature of the research. In addition, the small sample size is another factor that limits the generalizability of these findings.
Conclusion
C. auris can cause a wide variety of invasive infections, including bloodstream infections, urinary tract infection, skin infection, and lower respiratory tract infection, especially in critically ill patients. In addition, all isolates showed resistance to fluconazole and amphotericin B and were sensitive to echinocandins especially anidulafungin.
Authors contribution (Authorship)
Adila Shaukat: Desgning, interpretation of data, revising and approving the final draft.
Nasir Al Ansari: conception of the study, revising and approving the final draft.
Walid Al Wali: interpretation of data, revising and approving the final draft.
Edin Karic: interpretation of data, revising and approving the final draft.
Ihab El Madhoun: acquisition of data, revising and approving the final draft.
Hassan Mitwally: interpretation of data, revising and approving the final draft.
Manal Hamed: acquisition of data, revising and approving the final draft.
Feah Alutra- Visan: interpretation of data, drafting the article and approving the final draft.
Conflict of interest
All authors report no conflict of interest.
Acknowledgement
Open Access funding provided by the Qatar National Library.
We are alsothankful for Dr Jameela Al Ajmi,from Corporate Infection prevention and control dept and Ms Tahani M. Al Saadi from Laboratory department for their corporation and support. | Fatal | ReactionOutcome | CC BY | 33299794 | 18,688,885 | 2021 |
What was the outcome of reaction 'Respiratory failure'? | Experience of treating Candida auris cases at a general hospital in the state of Qatar.
So far there have been no studies on Candida auris in Qatar. This study aimed to describe the clinical spectrum and outcome of C. auris infection in patients admitted to a general hospital in Qatar.
We conducted this descriptive observational study in a general hospital in Qatar. We have involved all patients with C. auris infection and colonization admitted to a general hospital from December 2018 to August 2019.
We identified 13 patients with confirmed C.auris infection/colonization, of which five cases represented an actual C. auris infection, while the remaining eight cases were considered as colonization. The mean age of the patients with infection was 76.6 ± 8.4 years, while the mean age of the patients with colonization was 66.4 ± 24.7 years. Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All strains of C. auris were susceptible to echinocandins, flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Of the patients with C. auris infection who received systemic antifungal therapy, three (60%) died during antifungal therapy.
Our study showed that C. auris can cause a wide variety of invasive infections, including bloodstream infection, urinary tract infection, skin infection, and lower respiratory tract infections, especially in critically ill patients. In addition, our isolates showed resistance to the most common antifungal agents such as fluconazole and amphotericin B.
Introduction
Candida auris is a novel multidrug-resistant yeast with high overall mortality that was first isolated from the external auditory canal of a patient in Japan in 2009 [1]. Since then, this fungal infection has been reported from various countries across the world [[2], [3], [4]], and over time it has become a serious global health concern as one of the most serious emerging pathogens that critical care physicians should be aware of [5].
C. auris being resistant to major antifungal classes used to treat Candida including azole antifungal agents, poses a challenge to routine microbiology laboratories, as C. auris can be misidentified with standard laboratory techniques, and have a tendency to cause outbreaks in healthcare settings especially critical care areas despite adequate infection prevention and control measures [4,5].
In Qatar, there is no published data on Candida aurisso far. In this series, we reported the first outbreak of C. auris infection in Qatar, to describe the clinical spectrum and outcome of this infection in the affected patients.
Methods and patients
We conducted this descriptive observational study in a general hospital in Qatar. We involved all patients with Candida Auris infection and colonization in the intensive care units and other wards from December 2018 to August 2019. This study was given ethical approval by the medical research committee at Hamad Medical Corporation, under number: MRC-01-19-503..
Definitions
Colonization is defined as isolation of C. auris from endotracheal aspiration fluid, throat swabs, sputum, urine, and samples from central venous catheters or other parts of the body in absence of clinical signs or symptomes of infection. C.auris infection is defined as the isolation of Cauris from clinical specimens with compatible clinical signs and symptoms of infection [5].
Candida auris identification
All clinical specimens, from different sites, were cultured by quantitative technique on Sabouraud Dextrose Agar (OXOID, UK) and incubated at 35−37 °C for 48 h. Preliminary fungal strain identification was based on colony morphology on Chromogenic Candida Agar (OXOID, UK), while the identification to the species level was confirmed by Vitek 2 XL automated system (bioMerieux). Susceptibility of strains to Amphotericin B, Fluconazole, 5-fluorocytosine, and voriconazole was determined by using Sensititre™ YeastOne™ plate and by interpreting results according to closely related Candida species and on expert opinion. As per the Centers for Disease Control and Prevention (CDC), there are currently no established C. auris-specific susceptibility breakpoints [6].
Pulsed-field gel electrophoresis (PFGE) typing, which consisted of electrophoretic karyotyping (EK), was performed to compare the isolates from different sites. Following the results of the PFGE, an outbreak of C. auris infection in critical care unit and medical unit was confirmed by identifying five5 cases and patient screening revealed colonization of eight additional patients. Intensive efforts were done to find out the cause of cross-transmission and environmental and surface swabbing was done in affected areas, but all results were negative.
Data analysis
The results of analyses of continuous variables are expressed as means and standard deviations (SD) unless otherwise specified.
Results
During the study period, we identified 13 patients with confirmed C. auris infection/colonization, of which five cases represented an actual Candida infection, while the remaining eight cases were considered colonization. The mean age of the patients with infection was 76.6 ± 8.4 years (range: 65–90 years), while the mean age of the patients with colonization was 66.4 ± 24.7 years (range: 23–91 years). Table 1 describes the demographic characteristics of the patients involved in this study.Table 1 Candida auris infection/colonization patients details.
Table 1Case/No Age Sex Site of infection/ or site of Candida isolation Type of infection Pre or co-infection Co-morbidity Treatment provided Outcome
1 78 years Male Tracheal aspirate and urine Lower respiratory tract infection Corona virus 229 E PCR positive from nasal swab Interstitial lung disease Anidulafungin Died of hypoxic respiratory failure
2 79 years Male Nose and decubitus ulcer Skin soft tissue infection Pseudomonas MDR and Morganella morganii from decubitus ulcer Diabetes mellitus, sacral bed sores Flucytosine Died of bacterial/fungal sepsis
3 71 years Male Nose, throat, tracheal aspirate, and decubitus ulcer Candidemia Pseudomonas aeruginosa MDR and ESBL Klebsiella pneumoniae from sputum Diabetes mellitus, sacral bed sores Anidulafungin and posaconazole Cured
4 90 years Male urine, throat and nose Urinary tract infection Klebsiella pneumoniae and carbapenem resistant Pseudomonas aeruginosa from sputum, Pseudomonas aeruginosa MDR from a bedsore Cerebrovascular accident, dementia Anidulafungin Cured
5 65 years Male Throat, sputum, groin and urine Urinary tract infection Pseudomonas aeruginosa multidrug resistant Motor neuron disease, hospital-acquired pneumonia Anidulafungin Died of bacterial pneumonia
6 29 years Male Groin Colonization ESBL Klebsiella Acute liver failure secondary to hepatitis C, acute kidney injury, critical care polyneuropathy Terbinafine spray Discharged home
7 86 years Male Axilla, urine Colonization Pseudomonas aeruginosa COPD, vascular dementia, bedbound on tracheostomy to Terbinafine spray, nystatin application Died due to aspiration pneumonia and hypoxic respiratory failure
8 80 years Female Nose, tracheostomy site Colonization ESBL Klebsiella Chronic kidney disease, coronary artery disease, on tracheostomy Terbinafine spray, nystatin application Transfer to geriatric ward
9 62 years Female Axilla Colonization Pseudomonas multi drug-resistant Chronic kidney disease, necrotizing fasciitis Terbinafine spray, nystatin application Died due to bacterial sepsis
10 91 years Female Groin area Colonization None COPD, hypertension Terbinafine spray, nystatin application Discharged home
11 23 years Male Nose, axilla Colonization Escherichia coli Hypoxic brain injury, recurrent urinary tract infection Terbinafine spray, nystatin application Transfer to long-term unit
12 75 years Male Nose, groin Colonization Pseudomonas aeruginosa Diabetes mellitus, chronic kidney disease, recurrent pneumonia Terbinafine spray, nystatin application Discharged home
13 85 years Male urine Colonization Pseudomonas aeruginosa Parkinson’s disease, cerebrovascular accident Terbinafine spray, nystatin application Transfer to geriatric unit
PCR: polymerase chain reaction, MRD: multi-drug resiatant, ESBL: extended spectrum beta lactamase, COPD: chronic obstructive pulmonary disease.
Among the individuals clinically infected with C. auris, two had urinary tract infections, one had candidemia, one acquired soft tissue infection, and one had a lower respiratory tract infection. All patients had bacterial or viral infections prior to or concomitantly with C. auris infection/colonization, as shown in Table 1.
For the typing of C. auris isolates, the molecular technique PFGE, which consisted of electrophoretic karyotyping (EK), was utilized to compare the isolates from different sites. The PFGE karyotype of the outbreak isolates of C. auris in our series is shown in Fig. 1. Antifungal susceptibility tests were performed on isolates from infected subjects. All strains of C. auris shared the same susceptibility profile, being susceptible to echinocandins (especially anidulafungin), flucytosine, and posaconazole while resistance to fluconazole and amphotericin B. Table 2 shows the susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates. All patients with C. auris infection received systemic antifungal drugs, while the eight patients who were colonized were appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC (Table 1).Fig. 1 Electrophoretic karyotypes of C. auris isolates. Karyotypes of representative outbreak isolates from five patients in the intensive care unit. Lane 1, 2 and 8 are control specimens which served as comparison for different genotypes. Lane 3 to 7 strains (specimens from the five C. auris cases) show no single band variation and are likely representing the same strain.
Fig. 1Table 2 Susceptibility pattern in the form of minimal inhibitory concentrations (MIC) of antifungal agents for the C. auris isolates from subjects with infection.
Table 2Antifungal drugs Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Amphotericin 4-R 4-R 2-R 4-R 2-R
Caspofungin 0.25 8-R 8 0.5 8
Fluconazole 64 128-R 128-R 128-R 128-R
Flucytocin 0.125 0.5-S 0.12 0.12 0.12
Itraconazole 0.125-R 16-R 0.12 0.12 16
Posaconazole 0.012 8-R 0.06 0.06-S 8
Voriconazole 0.25 8-R 0.25 0.5 8
Anidulafungin 0.125-S 0.5-I 0.25 0.12-S 0.5-I
Micafungin 0.25 0.12 0.25
R: resistant, S: sensitive, I: intermediate.
Among the patients with C. auris infection who received systemic antifungal therapy, three (60 %) died during antifungal therapy. The other two patients were successfully treated and appropriately decolonized of C. auris (Table 1).
Discussion
Recent reports showed that C. auris is an emerging yeast that has been identified worldwide as a cause of severe invasive healthcare infections, which mostly affect critically ill patients and cause substantial morbidity and mortality [7,8]. To our knowledge, our series is the first designed to study this infection in Qatar.
Many C. auris outbreaks have been reported worldwide. In India, the first C. auris outbreak was reported in 2013 by Chowdhary et al. [9] who identified 12 patients with positive microbiological clinical specimens collected between 2009 and 2012. While Calvo et al. reported the first outbreak of C. auris infection in Venezuela between March 2012 and July 2013 [10]. All the isolates were initially identified as C. haemulonii. However, the isolation of C. auris was later confirmed by genome sequencing [9,10]. Similarly, we have reported the first outbreak of C. auris infection in Qatar, identifying 13 patients. The emergence of C. auris in our hospital raises concerns that this fungus may spread to other healthcare settings, particularly critical care facilities in Qatar, requiring intensified measures to control the spread of this infection. Therefore, knowing the source of infection and detection of possible routes of transmission can help in preventing the clonal spread of this infection and hospital outbreaks among various health facilities in Qatar [5,7,8]. Similarly, intensive efforts have been made in our hospital to find the cause of the cross-transmission. Environmental and surface swabs were carried out in the affected areas, but all results were negative.
Diagnosing C. auris infection is difficult because the clinical presentation is non-specific or may not be recognizable since patients infected with C. auris often have another serious illness or condition. Moreover, C. auris can be misidentified with standard laboratory techniques as C. haemulonii [11,12]. As a result, a high index of suspicion is required to diagnose this infection. In addition, accurate identification of C. auris through specialized laboratory methods is required to avoid misidentification and inappropriate treatment that may make it difficult to control the spread of C. auris in the healthcare settings [10]. In this study, the diagnosis of C. auris infection was suspected because of the resistance of the isolates to fluconazole and amphotericin B. The diagnosis was confirmed by molecular methods.
The spectrum of C. auris infection ranges from superficial infections that affect the skin to widespread infections that affect the brain, heart, lungs, liver, spleen, and kidneys [5]. Antifungal therapy should be administered to eradicate and control C. auris infection. On the other hand, C. auris can be isolated from the skin, rectum, wounds or mouth of some patients who do not show symptoms of infection. This condition is referred to as asymptomatic colonization and treatment with antifungal drugs does not eradicate C. auris colonization. However, the identification of C. auris colonization is significant because it carries the risk of transmission, which requires the immediate implementation of adequate infection control measures [13]. Likewise, our patients showed different clinical presentations, and cases with colonization were identified and appropriately decolonized with topical nystatin and terbinafine as recommended by the CDC.
In agreement with other reports [[3], [4], [5],7,13], our isolates showed resistance to the most important antifungal agents such as fluconazole and amphotericin B. The all cause mortality among our patients was 60 % which is in line with the mortality rate seen in other studies ranging from 30 to 60% [3].
One of the limitations of this study is the retrospective nature of the research. In addition, the small sample size is another factor that limits the generalizability of these findings.
Conclusion
C. auris can cause a wide variety of invasive infections, including bloodstream infections, urinary tract infection, skin infection, and lower respiratory tract infection, especially in critically ill patients. In addition, all isolates showed resistance to fluconazole and amphotericin B and were sensitive to echinocandins especially anidulafungin.
Authors contribution (Authorship)
Adila Shaukat: Desgning, interpretation of data, revising and approving the final draft.
Nasir Al Ansari: conception of the study, revising and approving the final draft.
Walid Al Wali: interpretation of data, revising and approving the final draft.
Edin Karic: interpretation of data, revising and approving the final draft.
Ihab El Madhoun: acquisition of data, revising and approving the final draft.
Hassan Mitwally: interpretation of data, revising and approving the final draft.
Manal Hamed: acquisition of data, revising and approving the final draft.
Feah Alutra- Visan: interpretation of data, drafting the article and approving the final draft.
Conflict of interest
All authors report no conflict of interest.
Acknowledgement
Open Access funding provided by the Qatar National Library.
We are alsothankful for Dr Jameela Al Ajmi,from Corporate Infection prevention and control dept and Ms Tahani M. Al Saadi from Laboratory department for their corporation and support. | Fatal | ReactionOutcome | CC BY | 33299794 | 18,688,885 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Autoimmune hepatitis'. | Onset of azoospermia in man treated with ipilimumab/nivolumab for BRAF negative metastatic melanoma.
Azoospermia is classified as the complete absence of sperm in ejaculate and accounts for 10-15% of male infertility. Many anticancer drugs are known to cause defects in spermatogenesis, but the effects of immune checkpoint inhibitor cancer therapy on spermatogenesis remains largely unknown. Presented here is a normozoospermic man (60 million sperm/cc of ejaculate) who received a trial combination treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma. Two years after the treatment, the patient presented as completely azoospermic. The patient subsequently underwent microdissection testicular sperm extraction, during which no sperm was retrieved, and sertoli-only pathology was elucidated.
Introduction
Infertility is defined as the inability to conceive after twelve months of unprotected intercourse or 6 months of unprotected sex in the setting of advanced maternal age.1 Infertility affects 8–12% of all couples worldwide, of which male factor accounts for 40–50% of cases.1 Azoospermia occurs due to an inadequate production of spermatozoa, such that spermatozoa are totally absent from the ejaculate, and can appear through pharmacological origins.2
Several anti-neoplastic agents have been identified that impair spermatogenesis and decrease sperm count including dabrafenib, a BRAF inhibitor used to treat metastatic melanomas containing a mutated Raf gene.2 For metastatic melanomas lacking the BRAF mutation, Nivolumab, a PD-1 monoclonal antibody, and Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody, were FDA approved as a combination therapy in 2015.3 These drugs, known as immune checkpoint inhibitors (ICI), mediate tumor destruction through potentiation of the T-cell anti-tumor response via the removal of coinhibitory signaling. However, the effect of Ipilimumab/Nivolumab treatment on spermatogenesis remains largely unknown. Here we present a case in which a previously normozoospermic man became azoospermic after receiving Ipilimumab/Nivolumab therapy for metastatic melanoma.
Case presentation
Here we present a 30 year-old-male who was diagnosed with mediastinal adenopathy on physical examination. He subsequently underwent a PET-CT scan which delineated multiple enlarged hypermetabolic lymph nodes (the largest being 2.4 × 3.0 cm) localized in the aortopulmonic mediastinum and left hilum, along with pleural findings. A lymph node biopsy was performed via a left anterior thoracostomy which revealed a node positive for melanoma, confirming the diagnosis of stage IV metastatic melanoma. Biopsy of the patient's melanoma revealed an absence of a BRAF mutation.
The patient subsequently began a clinical trial that involved Ipilimumab/Nivolumab combination therapy, which started one month after his diagnosis was confirmed. In the four months that followed, the patient underwent therapy with mycophenolate (2mg everyday) and prednisone for autoimmune hepatitis, during which he developed decreased libido that resolved once the treatment had concluded. The patient denied symptoms of orchalgia during therapy and did not visualize any signs of epididymo-orchititis during his ICI therapy. The patient responded to the combination and entered remission, which he has remained in for five years.
Nearly two years after initiation of the patient's ICI treatment, he presented to a urologist for a male factor infertility evaluation. The now 32-year-old patient and his 30-year-old wife, neither of whom had been pregnant prior, had been having unprotected intercourse for two years trying to achieve pregnancy. The patient had previously demonstrated normozoospermia eleven years prior as part of an evaluation for testicular pain and small varicocele veins. The semen analysis (SA) yielded 60 million sperm/cc of ejaculate. On re-presentation after ICI therapy, the SA at this time demonstrated normal volume azoospermia despite centrifugation. A genitourinary exam revealed bilaterally descended testes, 18 cc bilaterally. The vas deferens and epididymis were palpable bilaterally. Bilateral varicoceles were also palpated by the urologist; the left varicocele was documented as grade 2. Hormone analysis showed total testosterone of 556.74 ng/dL, FSH of 20.95 mIU/mL, LH of 5.68 mIU/mL, and E2 of 31 pg/mL.
As a result of these exam findings, the urologist recommended a bilateral varicocelectomy. At the time of varicocelectomy a testicular biopsy was performed demonstrating a sertoli-only pathology. Stains were performed to assess for lymphocytes and neutrophils; however, no inflammatory cells were visualized in the peritubular areas of the testis. Six months following the varicocele repair, the SA was repeated, and the patient remained azoospermic.
The patient subsequently underwent a microscopic testicular sperm extraction (mTESE), a procedure performed on azoospermic men with successful sperm retrieval rates of approximately 52% over all populations.4 Despite an uncomplicated surgery, no viable sperm were obtained. Testicular biopsy from the procedure once again displayed sertoli-only pathology (Fig. 1).Fig. 1 Sertoli-Only Biopsy (2020) Histology from testicular biopsy during early 2020 mTESE procedure illustrating seminiferous tubules with Sertoli-only pattern and interstitium with Leydig cell hyperplasia (H&E, 10X).
Fig. 1
The patient denied any prior exposure to radiation or chemotherapy. He had no history of epididymitis, orchitis, STIs, or trauma to testicles, and denied any history of testicular torsion, postpubertal mumps, or cryptorchidism. The man had no family history of infertility. The patient's previous medications included alprazolam, crisaborole, desonide, fexofenadine, and meclizine. At the time of follow up, the patient was taking alprazolam, fexofenadine HCL, and omeprazole. These medications have not been linked to male infertility or spermatogenesis.2,3
Discussion
It is well understood that chemotherapy may cause deleterious effects on sperm production and fertility. To this end, sperm cryopreservation is an effective fertility preservation technique recommended to postpubertal males receiving certain types of cancer treatment.5 However, the ever-increasing breadth of anticancer targets has harried the identification of interactions potentially harmful to spermatogenesis. The pathways involved in ICI therapies, such as Ipilimumab/Nivolumab, have been understudied in the scope of male fertility. Here we present a case supporting possible correlation between 10.13039/100016531ICI use and male infertility (Fig. 2).Fig. 2 Case OverviewTimeline of patient's semen analyses (SA), cancer diagnosis and immune checkpoint inhibitor (ICI) treatment (2015), bilateral varicocele repair and testicular biopsy (2017), and microdissection testicular sperm extraction (mTESE) (2020).
Fig. 2
Although this combination treatment was FDA approved in 2015, a 2016 review found no available evidence in preclinical animal or human data investigating the effect of Nivolumab on infertility.3 Moreover, the study associated Ipilimumab with positive evidence of fertility risk, citing studies of male monkeys receiving Ipilimumab that yielded evidence of decreased testicular weight without changes to sperm histopathology. Further, in human studies of Ipilimumab, 11% of male and female patients treated exhibited persistent anterior hypophysitis, the portion of the pituitary responsible for gonadotropin production.3 Nonetheless, the effect of Ipilimumab/Nivolumab on spermatogenesis remains largely unknown.
Conclusion
In the circumstances of the patient presented in this case report, a normozoospermic man (60 million sperm/cc) received a trial treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma and presented as completely azoospermic two years later. The mTESE preformed five years after the patient's ICI therapy also failed to retrieve any viable sperm. This case report highlights another significant adverse event in spermatogenesis seemingly correlated with ICI use.
Consent
Written consent was obtained from the patient referenced in this case report for the use of health information and chart review.
Declarations of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Author statement
Matthew J. Rabinowitz: Writing - original draft, Investigation, Visualization Taylor P. Kohn: Writing - review & editing, Supervision Vanessa N. Peña: Writing - review & editing Iryna V. Samarska: Resources Andres Matoso: Resources Amin S. Herati: Conceptualization, Project Administration, Validation.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Acknowledgments
None. | IPILIMUMAB, MYCOPHENOLIC ACID, NIVOLUMAB, PREDNISONE | DrugsGivenReaction | CC BY-NC-ND | 33299797 | 18,683,511 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Azoospermia'. | Onset of azoospermia in man treated with ipilimumab/nivolumab for BRAF negative metastatic melanoma.
Azoospermia is classified as the complete absence of sperm in ejaculate and accounts for 10-15% of male infertility. Many anticancer drugs are known to cause defects in spermatogenesis, but the effects of immune checkpoint inhibitor cancer therapy on spermatogenesis remains largely unknown. Presented here is a normozoospermic man (60 million sperm/cc of ejaculate) who received a trial combination treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma. Two years after the treatment, the patient presented as completely azoospermic. The patient subsequently underwent microdissection testicular sperm extraction, during which no sperm was retrieved, and sertoli-only pathology was elucidated.
Introduction
Infertility is defined as the inability to conceive after twelve months of unprotected intercourse or 6 months of unprotected sex in the setting of advanced maternal age.1 Infertility affects 8–12% of all couples worldwide, of which male factor accounts for 40–50% of cases.1 Azoospermia occurs due to an inadequate production of spermatozoa, such that spermatozoa are totally absent from the ejaculate, and can appear through pharmacological origins.2
Several anti-neoplastic agents have been identified that impair spermatogenesis and decrease sperm count including dabrafenib, a BRAF inhibitor used to treat metastatic melanomas containing a mutated Raf gene.2 For metastatic melanomas lacking the BRAF mutation, Nivolumab, a PD-1 monoclonal antibody, and Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody, were FDA approved as a combination therapy in 2015.3 These drugs, known as immune checkpoint inhibitors (ICI), mediate tumor destruction through potentiation of the T-cell anti-tumor response via the removal of coinhibitory signaling. However, the effect of Ipilimumab/Nivolumab treatment on spermatogenesis remains largely unknown. Here we present a case in which a previously normozoospermic man became azoospermic after receiving Ipilimumab/Nivolumab therapy for metastatic melanoma.
Case presentation
Here we present a 30 year-old-male who was diagnosed with mediastinal adenopathy on physical examination. He subsequently underwent a PET-CT scan which delineated multiple enlarged hypermetabolic lymph nodes (the largest being 2.4 × 3.0 cm) localized in the aortopulmonic mediastinum and left hilum, along with pleural findings. A lymph node biopsy was performed via a left anterior thoracostomy which revealed a node positive for melanoma, confirming the diagnosis of stage IV metastatic melanoma. Biopsy of the patient's melanoma revealed an absence of a BRAF mutation.
The patient subsequently began a clinical trial that involved Ipilimumab/Nivolumab combination therapy, which started one month after his diagnosis was confirmed. In the four months that followed, the patient underwent therapy with mycophenolate (2mg everyday) and prednisone for autoimmune hepatitis, during which he developed decreased libido that resolved once the treatment had concluded. The patient denied symptoms of orchalgia during therapy and did not visualize any signs of epididymo-orchititis during his ICI therapy. The patient responded to the combination and entered remission, which he has remained in for five years.
Nearly two years after initiation of the patient's ICI treatment, he presented to a urologist for a male factor infertility evaluation. The now 32-year-old patient and his 30-year-old wife, neither of whom had been pregnant prior, had been having unprotected intercourse for two years trying to achieve pregnancy. The patient had previously demonstrated normozoospermia eleven years prior as part of an evaluation for testicular pain and small varicocele veins. The semen analysis (SA) yielded 60 million sperm/cc of ejaculate. On re-presentation after ICI therapy, the SA at this time demonstrated normal volume azoospermia despite centrifugation. A genitourinary exam revealed bilaterally descended testes, 18 cc bilaterally. The vas deferens and epididymis were palpable bilaterally. Bilateral varicoceles were also palpated by the urologist; the left varicocele was documented as grade 2. Hormone analysis showed total testosterone of 556.74 ng/dL, FSH of 20.95 mIU/mL, LH of 5.68 mIU/mL, and E2 of 31 pg/mL.
As a result of these exam findings, the urologist recommended a bilateral varicocelectomy. At the time of varicocelectomy a testicular biopsy was performed demonstrating a sertoli-only pathology. Stains were performed to assess for lymphocytes and neutrophils; however, no inflammatory cells were visualized in the peritubular areas of the testis. Six months following the varicocele repair, the SA was repeated, and the patient remained azoospermic.
The patient subsequently underwent a microscopic testicular sperm extraction (mTESE), a procedure performed on azoospermic men with successful sperm retrieval rates of approximately 52% over all populations.4 Despite an uncomplicated surgery, no viable sperm were obtained. Testicular biopsy from the procedure once again displayed sertoli-only pathology (Fig. 1).Fig. 1 Sertoli-Only Biopsy (2020) Histology from testicular biopsy during early 2020 mTESE procedure illustrating seminiferous tubules with Sertoli-only pattern and interstitium with Leydig cell hyperplasia (H&E, 10X).
Fig. 1
The patient denied any prior exposure to radiation or chemotherapy. He had no history of epididymitis, orchitis, STIs, or trauma to testicles, and denied any history of testicular torsion, postpubertal mumps, or cryptorchidism. The man had no family history of infertility. The patient's previous medications included alprazolam, crisaborole, desonide, fexofenadine, and meclizine. At the time of follow up, the patient was taking alprazolam, fexofenadine HCL, and omeprazole. These medications have not been linked to male infertility or spermatogenesis.2,3
Discussion
It is well understood that chemotherapy may cause deleterious effects on sperm production and fertility. To this end, sperm cryopreservation is an effective fertility preservation technique recommended to postpubertal males receiving certain types of cancer treatment.5 However, the ever-increasing breadth of anticancer targets has harried the identification of interactions potentially harmful to spermatogenesis. The pathways involved in ICI therapies, such as Ipilimumab/Nivolumab, have been understudied in the scope of male fertility. Here we present a case supporting possible correlation between 10.13039/100016531ICI use and male infertility (Fig. 2).Fig. 2 Case OverviewTimeline of patient's semen analyses (SA), cancer diagnosis and immune checkpoint inhibitor (ICI) treatment (2015), bilateral varicocele repair and testicular biopsy (2017), and microdissection testicular sperm extraction (mTESE) (2020).
Fig. 2
Although this combination treatment was FDA approved in 2015, a 2016 review found no available evidence in preclinical animal or human data investigating the effect of Nivolumab on infertility.3 Moreover, the study associated Ipilimumab with positive evidence of fertility risk, citing studies of male monkeys receiving Ipilimumab that yielded evidence of decreased testicular weight without changes to sperm histopathology. Further, in human studies of Ipilimumab, 11% of male and female patients treated exhibited persistent anterior hypophysitis, the portion of the pituitary responsible for gonadotropin production.3 Nonetheless, the effect of Ipilimumab/Nivolumab on spermatogenesis remains largely unknown.
Conclusion
In the circumstances of the patient presented in this case report, a normozoospermic man (60 million sperm/cc) received a trial treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma and presented as completely azoospermic two years later. The mTESE preformed five years after the patient's ICI therapy also failed to retrieve any viable sperm. This case report highlights another significant adverse event in spermatogenesis seemingly correlated with ICI use.
Consent
Written consent was obtained from the patient referenced in this case report for the use of health information and chart review.
Declarations of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Author statement
Matthew J. Rabinowitz: Writing - original draft, Investigation, Visualization Taylor P. Kohn: Writing - review & editing, Supervision Vanessa N. Peña: Writing - review & editing Iryna V. Samarska: Resources Andres Matoso: Resources Amin S. Herati: Conceptualization, Project Administration, Validation.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Acknowledgments
None. | IPILIMUMAB, MYCOPHENOLIC ACID, NIVOLUMAB, PREDNISONE | DrugsGivenReaction | CC BY-NC-ND | 33299797 | 18,683,511 | 2021-01 |
What was the dosage of drug 'MYCOPHENOLIC ACID'? | Onset of azoospermia in man treated with ipilimumab/nivolumab for BRAF negative metastatic melanoma.
Azoospermia is classified as the complete absence of sperm in ejaculate and accounts for 10-15% of male infertility. Many anticancer drugs are known to cause defects in spermatogenesis, but the effects of immune checkpoint inhibitor cancer therapy on spermatogenesis remains largely unknown. Presented here is a normozoospermic man (60 million sperm/cc of ejaculate) who received a trial combination treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma. Two years after the treatment, the patient presented as completely azoospermic. The patient subsequently underwent microdissection testicular sperm extraction, during which no sperm was retrieved, and sertoli-only pathology was elucidated.
Introduction
Infertility is defined as the inability to conceive after twelve months of unprotected intercourse or 6 months of unprotected sex in the setting of advanced maternal age.1 Infertility affects 8–12% of all couples worldwide, of which male factor accounts for 40–50% of cases.1 Azoospermia occurs due to an inadequate production of spermatozoa, such that spermatozoa are totally absent from the ejaculate, and can appear through pharmacological origins.2
Several anti-neoplastic agents have been identified that impair spermatogenesis and decrease sperm count including dabrafenib, a BRAF inhibitor used to treat metastatic melanomas containing a mutated Raf gene.2 For metastatic melanomas lacking the BRAF mutation, Nivolumab, a PD-1 monoclonal antibody, and Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody, were FDA approved as a combination therapy in 2015.3 These drugs, known as immune checkpoint inhibitors (ICI), mediate tumor destruction through potentiation of the T-cell anti-tumor response via the removal of coinhibitory signaling. However, the effect of Ipilimumab/Nivolumab treatment on spermatogenesis remains largely unknown. Here we present a case in which a previously normozoospermic man became azoospermic after receiving Ipilimumab/Nivolumab therapy for metastatic melanoma.
Case presentation
Here we present a 30 year-old-male who was diagnosed with mediastinal adenopathy on physical examination. He subsequently underwent a PET-CT scan which delineated multiple enlarged hypermetabolic lymph nodes (the largest being 2.4 × 3.0 cm) localized in the aortopulmonic mediastinum and left hilum, along with pleural findings. A lymph node biopsy was performed via a left anterior thoracostomy which revealed a node positive for melanoma, confirming the diagnosis of stage IV metastatic melanoma. Biopsy of the patient's melanoma revealed an absence of a BRAF mutation.
The patient subsequently began a clinical trial that involved Ipilimumab/Nivolumab combination therapy, which started one month after his diagnosis was confirmed. In the four months that followed, the patient underwent therapy with mycophenolate (2mg everyday) and prednisone for autoimmune hepatitis, during which he developed decreased libido that resolved once the treatment had concluded. The patient denied symptoms of orchalgia during therapy and did not visualize any signs of epididymo-orchititis during his ICI therapy. The patient responded to the combination and entered remission, which he has remained in for five years.
Nearly two years after initiation of the patient's ICI treatment, he presented to a urologist for a male factor infertility evaluation. The now 32-year-old patient and his 30-year-old wife, neither of whom had been pregnant prior, had been having unprotected intercourse for two years trying to achieve pregnancy. The patient had previously demonstrated normozoospermia eleven years prior as part of an evaluation for testicular pain and small varicocele veins. The semen analysis (SA) yielded 60 million sperm/cc of ejaculate. On re-presentation after ICI therapy, the SA at this time demonstrated normal volume azoospermia despite centrifugation. A genitourinary exam revealed bilaterally descended testes, 18 cc bilaterally. The vas deferens and epididymis were palpable bilaterally. Bilateral varicoceles were also palpated by the urologist; the left varicocele was documented as grade 2. Hormone analysis showed total testosterone of 556.74 ng/dL, FSH of 20.95 mIU/mL, LH of 5.68 mIU/mL, and E2 of 31 pg/mL.
As a result of these exam findings, the urologist recommended a bilateral varicocelectomy. At the time of varicocelectomy a testicular biopsy was performed demonstrating a sertoli-only pathology. Stains were performed to assess for lymphocytes and neutrophils; however, no inflammatory cells were visualized in the peritubular areas of the testis. Six months following the varicocele repair, the SA was repeated, and the patient remained azoospermic.
The patient subsequently underwent a microscopic testicular sperm extraction (mTESE), a procedure performed on azoospermic men with successful sperm retrieval rates of approximately 52% over all populations.4 Despite an uncomplicated surgery, no viable sperm were obtained. Testicular biopsy from the procedure once again displayed sertoli-only pathology (Fig. 1).Fig. 1 Sertoli-Only Biopsy (2020) Histology from testicular biopsy during early 2020 mTESE procedure illustrating seminiferous tubules with Sertoli-only pattern and interstitium with Leydig cell hyperplasia (H&E, 10X).
Fig. 1
The patient denied any prior exposure to radiation or chemotherapy. He had no history of epididymitis, orchitis, STIs, or trauma to testicles, and denied any history of testicular torsion, postpubertal mumps, or cryptorchidism. The man had no family history of infertility. The patient's previous medications included alprazolam, crisaborole, desonide, fexofenadine, and meclizine. At the time of follow up, the patient was taking alprazolam, fexofenadine HCL, and omeprazole. These medications have not been linked to male infertility or spermatogenesis.2,3
Discussion
It is well understood that chemotherapy may cause deleterious effects on sperm production and fertility. To this end, sperm cryopreservation is an effective fertility preservation technique recommended to postpubertal males receiving certain types of cancer treatment.5 However, the ever-increasing breadth of anticancer targets has harried the identification of interactions potentially harmful to spermatogenesis. The pathways involved in ICI therapies, such as Ipilimumab/Nivolumab, have been understudied in the scope of male fertility. Here we present a case supporting possible correlation between 10.13039/100016531ICI use and male infertility (Fig. 2).Fig. 2 Case OverviewTimeline of patient's semen analyses (SA), cancer diagnosis and immune checkpoint inhibitor (ICI) treatment (2015), bilateral varicocele repair and testicular biopsy (2017), and microdissection testicular sperm extraction (mTESE) (2020).
Fig. 2
Although this combination treatment was FDA approved in 2015, a 2016 review found no available evidence in preclinical animal or human data investigating the effect of Nivolumab on infertility.3 Moreover, the study associated Ipilimumab with positive evidence of fertility risk, citing studies of male monkeys receiving Ipilimumab that yielded evidence of decreased testicular weight without changes to sperm histopathology. Further, in human studies of Ipilimumab, 11% of male and female patients treated exhibited persistent anterior hypophysitis, the portion of the pituitary responsible for gonadotropin production.3 Nonetheless, the effect of Ipilimumab/Nivolumab on spermatogenesis remains largely unknown.
Conclusion
In the circumstances of the patient presented in this case report, a normozoospermic man (60 million sperm/cc) received a trial treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma and presented as completely azoospermic two years later. The mTESE preformed five years after the patient's ICI therapy also failed to retrieve any viable sperm. This case report highlights another significant adverse event in spermatogenesis seemingly correlated with ICI use.
Consent
Written consent was obtained from the patient referenced in this case report for the use of health information and chart review.
Declarations of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Author statement
Matthew J. Rabinowitz: Writing - original draft, Investigation, Visualization Taylor P. Kohn: Writing - review & editing, Supervision Vanessa N. Peña: Writing - review & editing Iryna V. Samarska: Resources Andres Matoso: Resources Amin S. Herati: Conceptualization, Project Administration, Validation.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Acknowledgments
None. | 2MG EVERYDAY | DrugDosageText | CC BY-NC-ND | 33299797 | 18,683,511 | 2021-01 |
What was the outcome of reaction 'Libido decreased'? | Onset of azoospermia in man treated with ipilimumab/nivolumab for BRAF negative metastatic melanoma.
Azoospermia is classified as the complete absence of sperm in ejaculate and accounts for 10-15% of male infertility. Many anticancer drugs are known to cause defects in spermatogenesis, but the effects of immune checkpoint inhibitor cancer therapy on spermatogenesis remains largely unknown. Presented here is a normozoospermic man (60 million sperm/cc of ejaculate) who received a trial combination treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma. Two years after the treatment, the patient presented as completely azoospermic. The patient subsequently underwent microdissection testicular sperm extraction, during which no sperm was retrieved, and sertoli-only pathology was elucidated.
Introduction
Infertility is defined as the inability to conceive after twelve months of unprotected intercourse or 6 months of unprotected sex in the setting of advanced maternal age.1 Infertility affects 8–12% of all couples worldwide, of which male factor accounts for 40–50% of cases.1 Azoospermia occurs due to an inadequate production of spermatozoa, such that spermatozoa are totally absent from the ejaculate, and can appear through pharmacological origins.2
Several anti-neoplastic agents have been identified that impair spermatogenesis and decrease sperm count including dabrafenib, a BRAF inhibitor used to treat metastatic melanomas containing a mutated Raf gene.2 For metastatic melanomas lacking the BRAF mutation, Nivolumab, a PD-1 monoclonal antibody, and Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody, were FDA approved as a combination therapy in 2015.3 These drugs, known as immune checkpoint inhibitors (ICI), mediate tumor destruction through potentiation of the T-cell anti-tumor response via the removal of coinhibitory signaling. However, the effect of Ipilimumab/Nivolumab treatment on spermatogenesis remains largely unknown. Here we present a case in which a previously normozoospermic man became azoospermic after receiving Ipilimumab/Nivolumab therapy for metastatic melanoma.
Case presentation
Here we present a 30 year-old-male who was diagnosed with mediastinal adenopathy on physical examination. He subsequently underwent a PET-CT scan which delineated multiple enlarged hypermetabolic lymph nodes (the largest being 2.4 × 3.0 cm) localized in the aortopulmonic mediastinum and left hilum, along with pleural findings. A lymph node biopsy was performed via a left anterior thoracostomy which revealed a node positive for melanoma, confirming the diagnosis of stage IV metastatic melanoma. Biopsy of the patient's melanoma revealed an absence of a BRAF mutation.
The patient subsequently began a clinical trial that involved Ipilimumab/Nivolumab combination therapy, which started one month after his diagnosis was confirmed. In the four months that followed, the patient underwent therapy with mycophenolate (2mg everyday) and prednisone for autoimmune hepatitis, during which he developed decreased libido that resolved once the treatment had concluded. The patient denied symptoms of orchalgia during therapy and did not visualize any signs of epididymo-orchititis during his ICI therapy. The patient responded to the combination and entered remission, which he has remained in for five years.
Nearly two years after initiation of the patient's ICI treatment, he presented to a urologist for a male factor infertility evaluation. The now 32-year-old patient and his 30-year-old wife, neither of whom had been pregnant prior, had been having unprotected intercourse for two years trying to achieve pregnancy. The patient had previously demonstrated normozoospermia eleven years prior as part of an evaluation for testicular pain and small varicocele veins. The semen analysis (SA) yielded 60 million sperm/cc of ejaculate. On re-presentation after ICI therapy, the SA at this time demonstrated normal volume azoospermia despite centrifugation. A genitourinary exam revealed bilaterally descended testes, 18 cc bilaterally. The vas deferens and epididymis were palpable bilaterally. Bilateral varicoceles were also palpated by the urologist; the left varicocele was documented as grade 2. Hormone analysis showed total testosterone of 556.74 ng/dL, FSH of 20.95 mIU/mL, LH of 5.68 mIU/mL, and E2 of 31 pg/mL.
As a result of these exam findings, the urologist recommended a bilateral varicocelectomy. At the time of varicocelectomy a testicular biopsy was performed demonstrating a sertoli-only pathology. Stains were performed to assess for lymphocytes and neutrophils; however, no inflammatory cells were visualized in the peritubular areas of the testis. Six months following the varicocele repair, the SA was repeated, and the patient remained azoospermic.
The patient subsequently underwent a microscopic testicular sperm extraction (mTESE), a procedure performed on azoospermic men with successful sperm retrieval rates of approximately 52% over all populations.4 Despite an uncomplicated surgery, no viable sperm were obtained. Testicular biopsy from the procedure once again displayed sertoli-only pathology (Fig. 1).Fig. 1 Sertoli-Only Biopsy (2020) Histology from testicular biopsy during early 2020 mTESE procedure illustrating seminiferous tubules with Sertoli-only pattern and interstitium with Leydig cell hyperplasia (H&E, 10X).
Fig. 1
The patient denied any prior exposure to radiation or chemotherapy. He had no history of epididymitis, orchitis, STIs, or trauma to testicles, and denied any history of testicular torsion, postpubertal mumps, or cryptorchidism. The man had no family history of infertility. The patient's previous medications included alprazolam, crisaborole, desonide, fexofenadine, and meclizine. At the time of follow up, the patient was taking alprazolam, fexofenadine HCL, and omeprazole. These medications have not been linked to male infertility or spermatogenesis.2,3
Discussion
It is well understood that chemotherapy may cause deleterious effects on sperm production and fertility. To this end, sperm cryopreservation is an effective fertility preservation technique recommended to postpubertal males receiving certain types of cancer treatment.5 However, the ever-increasing breadth of anticancer targets has harried the identification of interactions potentially harmful to spermatogenesis. The pathways involved in ICI therapies, such as Ipilimumab/Nivolumab, have been understudied in the scope of male fertility. Here we present a case supporting possible correlation between 10.13039/100016531ICI use and male infertility (Fig. 2).Fig. 2 Case OverviewTimeline of patient's semen analyses (SA), cancer diagnosis and immune checkpoint inhibitor (ICI) treatment (2015), bilateral varicocele repair and testicular biopsy (2017), and microdissection testicular sperm extraction (mTESE) (2020).
Fig. 2
Although this combination treatment was FDA approved in 2015, a 2016 review found no available evidence in preclinical animal or human data investigating the effect of Nivolumab on infertility.3 Moreover, the study associated Ipilimumab with positive evidence of fertility risk, citing studies of male monkeys receiving Ipilimumab that yielded evidence of decreased testicular weight without changes to sperm histopathology. Further, in human studies of Ipilimumab, 11% of male and female patients treated exhibited persistent anterior hypophysitis, the portion of the pituitary responsible for gonadotropin production.3 Nonetheless, the effect of Ipilimumab/Nivolumab on spermatogenesis remains largely unknown.
Conclusion
In the circumstances of the patient presented in this case report, a normozoospermic man (60 million sperm/cc) received a trial treatment of Ipilimumab/Nivolumab to treat BRAF negative, stage IV metastatic melanoma and presented as completely azoospermic two years later. The mTESE preformed five years after the patient's ICI therapy also failed to retrieve any viable sperm. This case report highlights another significant adverse event in spermatogenesis seemingly correlated with ICI use.
Consent
Written consent was obtained from the patient referenced in this case report for the use of health information and chart review.
Declarations of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Author statement
Matthew J. Rabinowitz: Writing - original draft, Investigation, Visualization Taylor P. Kohn: Writing - review & editing, Supervision Vanessa N. Peña: Writing - review & editing Iryna V. Samarska: Resources Andres Matoso: Resources Amin S. Herati: Conceptualization, Project Administration, Validation.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Acknowledgments
None. | Recovered | ReactionOutcome | CC BY-NC-ND | 33299797 | 20,229,188 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug resistance'. | Heterogeneous tumor-immune microenvironments between primary and metastatic carcinoid tumors differentially respond to anti-PD-L1 antibody therapy.
A pulmonary carcinoid tumor is a rare tumor that lacks a validated therapeutic approach for unresectable disease. Understanding the intersite tumor-immune heterogeneity is essential to harness the immune system for cancer therapy. However, little is known about the tumor-immune microenvironment (TIME). Here, we describe a patient who had heterogeneous TIME between primary and metastatic carcinoid tumors which differentially responded to chemoimmunotherapy. A 72-year-old man was diagnosed with an advanced pulmonary carcinoid tumor. CT-guided biopsies of lung and scapular tumors confirmed typical carcinoid (PD-L1, 1%-24%) and atypical carcinoid tumors (PD-L1, negative), respectively. Although the primary lung carcinoid tumor showed a partial response, the scapular tumor was significantly enlarged after two cycles of anti-PD-L1 antibody therapy in combination with carboplatin plus etoposide. We performed quantitative pathology imaging analysis with fluorescent multiplex immunohistochemistry. CD8+ T cell infiltration was detected in the PD-L1-positive primary lung tumor nest; however, it was mostly restrained in the stroma in a PD-L1-negative metastatic scapular tumor. Treg infiltrations into both tumor nests and stroma were detected in the lung tumor, which were not detected in the metastatic scapular tumor. This study provides the first evidence of coexistence of heterogeneous TIME within a single individual with a pulmonary carcinoid tumor. This study may provide new insights into the mechanism of primary resistance to chemoimmunotherapy in pulmonary carcinoid tumors.
Introduction
Typical and atypical carcinoids are classified as highly differentiated malignant neuroendocrine tumors (NET).
1
Due to their rarity, pulmonary carcinoids lack a validated therapeutic approach for unresectable disease.
2
,
3
Therefore, development of new therapeutic options is urgently needed.
Although immune checkpoint inhibitors (ICIs) have revolutionized the treatment of non‐small cell lung cancer (NSCLC), their impact on small cell lung cancer, which is in the same spectrum with typical and atypical carcinoids, is limited.
4
,
5
Drug development of immunotherapy targeting pulmonary NET including carcinoids is ongoing.
1
,
3
,
6
However, there is only very limited evidence for immunotherapy in patients with pulmonary carcinoids, with early trials suggesting a low‐level activity in patients treated with single agent therapy,
3
,
6
highlighting the importance of investigating the heterogeneity of tumor‐immune microenvironments (TIME) in carcinoids.
TIME have an influence on tumor initiation and response to therapy.
7
,
8
,
9
Thus, understanding the intersite tumor‐immune heterogeneity is essential to harness the immune system for cancer therapy. However, little is known about TIME in carcinoid tumors.
10
Here, we describe a patient who had heterogeneous TIME between primary and metastatic carcinoid tumors which differentially responded to chemoimmunotherapy. This study may provide new insights into the mechanism of primary and acquired resistance to immunotherapy in carcinoid tumors.
Case report
A 72‐year‐old man was diagnosed with advanced pulmonary carcinoid with multiple bone metastases (Fig 1). CT‐guided biopsies of lung and scapular tumors confirmed the diagnoses of typical carcinoid (PD‐L1, 1%–24%; Oncomine Dx target test, negative; microsatellite instability [MSI], negative) and atypical carcinoid (PD‐L1, negative; Oncomine Dx, negative; MSI, negative), respectively.
Figure 1 Key imaging and clinicopathological results. Chest computed tomography (CT) scan show masses in the left lower lung and right scapular (yellow arrows). Histopathological findings obtained from the left lower primary lung tumor show small to medium‐size round nuclei and lightly acidic cytoplasm distributed in a sheet‐like form. The mitotic count was less than 1 in 10/high power field (HPF). Immunohistochemical staining was positive for thyroid transcription factor‐1 (TTF‐1), synaptophysin and insulinoma‐associated 1 (INSM‐1). The Ki‐67 labeling index was 4%–5%. Based on these findings, the lung tumor was diagnosed as a typical pulmonary carcinoid tumor. The programmed death‐ligand 1 (PD‐L1) tumor proportion score (TPS) of the lung tumor was 1%–24% (clone 22C3, pharmDx kit). Next‐generation sequencing (NGS) (Oncomine Dx Target Test multi‐CDx system) and microsatellite instability (MSI) tests were negative. After two cycles of atezolizumab in combination with carboplatin plus etoposide, CT scan of the chest showed a partial response in the lung lesion according to Response Evaluation Criteria in Solid Tumor (RECIST) version 1.1. In the biopsy sample from the scapular tumor, the mitotic count of the scapular lesion was 2–3/HPF and diagnosed as an atypical carcinoid. The PD‐L1 TPS was negative (<1%). Oncomine Dx Target Test multi‐CDx system and MSI tests were negative. A post‐therapy CT scan showed progressive disease in the scapular lesion.
The patient received atezolizumab in combination with carboplatin plus etoposide as first‐line therapy. Although a CT scan showed a partial response in the primary lung lesion, the scapular tumor was significantly enlarged after two cycles of chemoimmunotherapy. There was no antitumor effect on other bone metastases similar to the scapula tumor. Thus, a progressive disease was evaluated according to RECIST version 1.1.
Understanding the intersite tumor‐immune heterogeneity is essential to harness the immune system for cancer therapy. However, little is known about TIME in pulmonary carcinoid tumors. To further explore this case, we investigated the immune contexture of pretreatment primary and metastatic tumors by fluorescent multiplex immunohistochemistry (Supplementary Text S1), which can capture multidimensional data related to tissue architecture, spatial distribution of multiple cell phenotypes, and co‐expression of signaling.
11
,
12
Synaptophysin and PD‐L1 were simultaneously stained and the tissue localizations of tumor‐infiltrating lymphocytes (TIL) were evaluated. CD8+ T cells and immunosuppressive CD3+FOXP3+ regulatory T cells (Tregs) in the tumor nest and surrounding stroma were profiled and quantified by quantitative pathology imaging system. CD8+ T cell infiltrations were detected in the tumor nest of PD‐L1‐positive primary lung carcinoid; however, they were mostly restrained in the stroma of the PD‐L1‐negative metastatic scapular tumor. Treg infiltrations into both tumor nests and stroma were detected in the primary lung carcinoid tumor, however, which were not detected in the metastatic scapular tumor (Fig 2).
Figure 2 Multiplex fluorescent immunohistochemistry results. (a) Representative images of scapular and lung tissues are shown. Formalin‐fixed paraffin‐embedded sections of scapular and lung tumors were stained by one of two sequences of primary antibodies, PD‐L1 (purple), synaptophysin (light blue) and CD8 (green), or synaptophysin, FOXP3 (red) and CD3 (yellow) respectively. Nuclei were counterstained with DAPI (blue). In the scapular tumor, CD8+ T cells were distributed mainly in the tumor stroma, and CD3+FOXP3+ Tregs were scarcely observed. In lung tumor, CD8+ T cell infiltrations into PD‐L1‐positive tumor cell nest were observed, and Treg also infiltrated into the synaptophysin‐positive tumor cell nest. The insert panel in the right upper panel shows a Treg at high magnification. Scale bars, 50 μm are shown in each panel. (b) Quantification results of CD8+ T cells and CD3+FOXP3+ Tregs are shown. High‐speed scanning of whole slide images was performed on stained tissue sections. Images of full sections were acquired and analyzed with automated quantitative pathology imaging system. All images were analyzed and statistics of the number of CD8+ T cells and CD3+FOXP3+ Tregs were generated automatically. Cells were classified as tumor nest (red) or tumor stroma (blue) according to the relationship with synaptophysin‐positive tumor cells. Most CD8+ T cells in the scapular tumor were classified as tumor stroma, and few Tregs were detected in the scapular tumor. In the lung tumor, CD8+ T cells were distributed almost equally in the tumor nest and stroma. Tregs in lung tumor were counted more than in the scapular tumor. The detailed methods are described in Supplementary Text S1. () Tumor nest, () Tumor stroma
We evaluated a systemic immune response in the peripheral blood. Multiparameter flow cytometric analysis was performed on the peripheral blood mononuclear cells (PBMCs) as previously described (Supplementary Text S1).
13
,
14
,
15
PD‐1 and Ki67 in CD8+ T cells increased after one‐cycle of chemoimmunotherapy, suggesting systemic immune activation was induced by blocking PD‐1/PD‐L1 pathway (Fig 3). However, CTLA‐4 and Ki67 expressions in Tregs simultaneously increased, indicating Treg activation was induced by blocking the PD‐1/PD‐L1 pathway.
Figure 3 Peripheral blood immune subset analyses. Immune checkpoint receptors and Ki67 expressions of peripheral CD8+ T cells and Tregs in response to anti‐PD‐L1 antibody therapy in combination with chemotherapy are shown. Peripheral blood mononuclear cells (PBMCs) were obtained before therapy on cycle one day one (C1D1pre), and prior to treatment on C2D1(C2D1pre). Multiparameter flow cytometric analysis was performed on PMBCs. The following immunophenotypic markers were used to define immune subsets: CD8+ T cells were CD4−CD8+, Tregs were CD8−CD4+CD25highFOXP3+. Cells were incubated with Fc receptor blocking agent and dead cells were excluded from all analyses by the means of LIVE/DEAD stain. The 34 495 CD8+ T cells (C1D1pre) and 23 948 CD8+ T cells (C2D1pre) were acquired and mean fluorescence intensity (MFI) for Ki67 and PD‐1 expressions were calculated. The 987 Treg cells (C1D1pre) and 564 Treg cells (C2D1pre) were acquired and MFI for Ki67 and CTLA‐4 expressions were calculated. (a) Change of Ki67 and PD‐1 expressions in CD8+ T cells after one cycle of atezolizumab plus chemotherapy in a patient with advanced carcinoid. PD‐1 and Ki67 in CD8+ T cells increased after one cycle of therapy. (b) Change of Ki67 and CTLA‐4 expressions in Tregs after one cycle of atezolizumab plus chemotherapy in a patient with advanced carcinoid. Ki67 and CTLA‐4 in Tregs increased after one cycle of therapy. Data were analyzed using FlowJo software (FlowJo LLC, OR, USA). Detailed methods are described in the Supplementary Text S1. () Ki67, () PD‐1, () Ki67, () CTLA‐4
Discussion
Jiménez‐Sánchez et al. reported that multiple distinct TIME co‐exist within a single individual and heterogeneous TIME was associated with the heterogeneous fates of metastatic lesions,
8
which were consistent with our findings. In the current study, a significant CD8+ T cell infiltration was seen in the tumor nest of the PD‐L1‐positive lung carcinoid, which was associated with a significant response to chemoimmunotherapy, despite Treg infiltration in both the tumor nest and stroma. However, CD8+ T cell infiltration was restrained in the surrounding stroma in the PD‐L1‐negative metastatic scapular tumor and which was associated with primary resistance to chemoimmunotherapy, suggesting that the infiltrating CD8+ T cells in the tumor nest may play a key role in response to ICIs in advanced carcinoid tumors.
Tregs have immunosuppressive activity and have been reported to play a critical role in negatively regulating antitumor immune responses.
16
Activation of systemic Tregs with an increase of CTLA‐4 expression was confirmed after treatment with ICI followed by disease progression, suggesting that the CTLA‐4 signaling pathway could be one of the key mechanisms of acquired resistance to PD‐1/PD‐L1 blockade therapy in carcinoid.
Lung carcinoid tumors are categorized as typical (<2 mitoses per 2 mm2 and no necrosis) and atypical (2–10 mitoses per 2 mm2 and/or necrosis), corresponding to low‐grade (grade 1) and intermediate‐grade (grade 2), respectively.
17
These categories were developed for resected primary tumors. A biopsied specimen represents only part of a whole tumor. Tumor heterogeneity within neuroendocrine tumors has been previously reported.
17
Thus, the tumor heterogeneity of tissue specimens obtained by CT‐guided biopsies may have affected the difference of diagnosis in the current study.
Everolimus is an oral mammalian target of rapamycin inhibitor approved for NETs based on a clinical trial (RADIANT‐4).
18
In this study, efficacy of everolimus compared to placebo in previously‐treated patients was reported. The current patient showed rapid progression of tumors at diagnosis. Therefore, we chose atezolizumab in combination with chemotherapy, which is currently used for poorly differentiated NET, small cell lung cancer as the first‐line therapy.
In our study, the PD‐L1‐positive primary tumor responded to therapy; however, the predictive value of PD‐L1 has not yet been validated in NET immunotherapy. Therefore, our findings should be interpreted with caution. Only one patient was involved in this study, thus further studies are needed to determine whether the principles discovered here apply to other patients with carcinoid tumors. Despite such limitations, the present study provides the first evidence of the coexistence of heterogeneous TIME within a single individual with a pulmonary carcinoid tumor. The present study may therefore contribute to our understanding of the TIME of carcinoids and provide new insights into the complex TIME of NETs.
Disclosure
No potential conflicts of interest were disclosed.
Supporting information
Appendix
S1. Supporting Information
Click here for additional data file.
Acknowledgments
We thank Misako Takahashi (Department of Respiratory Medicine, Kumamoto University) for technical assistance in vitro analysis. We are very grateful to our patient for participation in this study. This work was supported by JSPS KAKENHI Grant Number JP18K15928 and Takeda Science Foundation. | ATEZOLIZUMAB, CARBOPLATIN, ETOPOSIDE | DrugsGivenReaction | CC BY | 33300302 | 19,744,836 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'. | Intranasal lidocaine atomization as novel and noninvasive treatment option for postdural puncture headache : Two case reports from obstetric anesthesiology.
Postdural puncture headache (PDPH) occurs in up to 11% of patients after spinal anesthesia and in more than 80% after dural perforation upon epidural anesthesia. It represents a severe anesthesiological complication in obstetric patients. If conservative medication measures do not result in a timely relief of symptoms, the current guidelines recommend the early implementation of an epidural blood patch; however, although performing an epidural blood patch is effective to treat PDPH, potential side effects include neurological complications, spinal hematoma and infections. Assumed to reduce cerebral vasodilatation as a potential pathophysiological driver of PDPH, the transnasal block of the sphenopalatine ganglion with local anesthetics is discussed as an alternative approach.
In this case study a modification of this technique is reported using a mucosal atomization device (MAD) for off-label nasal administration of lidocaine in two obstetric patients suffering from PDPH. Up to now there is no experience with this modified technique in obstetric anesthesiology.
The first patient (25-year-old secundigravida, body mass index [BMI] 54.7 kg/m2) displayed a pronounced PDPH with nausea and vomiting during the first day after a cesarean section under spinal anesthesia (3 attempts). The second patient (32-year-old tertiagravida, BMI 27.3 kg/m2) was readmitted to hospital due to PDPH 4 days after a natural birth under epidural anesthesia. Whereas conservative measures and therapeutic attempts with nonopioid analgesics and caffeine did not result in a sufficient treatment success, intranasal lidocaine administration via a MAD led to an immediate and persisting symptom relief. Both patients could be discharged from hospital after 24 h of surveillance and did not report any relevant side effects of the lidocaine administration.
The described noninvasive and simple procedure represents a valuable addition to previously known treatment options for PDPH and a potential alternative to an epidural blood patch in obstetric patients with PDPH. Prospective studies are needed to validate the findings.
Hintergrund
Bei ca. 22 % aller schwangeren Patientinnen in Deutschland werden zur Milderung des Geburtsschmerzes rückenmarknahe Anästhesieverfahren eingesetzt [1]. Zu den typischen Komplikationen dieser Verfahren gehört der Postpunktionskopfschmerz („postdural puncture headache“ [PDPH]) nach einer Spinalanästhesie (SpA) oder infolge einer akzidentiellen Punktion der Dura mater im Rahmen einer Periduralanästhesie (PDA). Mit einer Häufigkeit zwischen 1,5 und 11,2 % nach SpA [2] resp. >80 % nach ungewollter Duraverletzung während einer PDA [3] stellt der PDPH eine nichtunerhebliche Belastung der betroffenen Patienten dar. Hierbei weisen Frauen ein gegenüber Männern deutlich erhöhtes PDPH-Risiko auf [4].
In der überwiegenden Mehrheit der Fälle tritt ein PDPH innerhalb von 3 Tagen nach stattgehabter Punktion auf [5], wobei potenziell auch ein deutlich verspäteter Beginn der Symptomatik möglich ist [6]. Entgegen der ursprünglichen Annahme, dass es sich bei einem PDPH um ein selbstlimitierendes Phänomen handelt, kommt es nicht selten zu einer Chronifizierung des Schmerzes [7]. Bei mehr als einem Drittel aller Betroffenen führen die Beschwerden zu einer deutlichen Einschränkung der Leistungsfähigkeit [8]. Im Bereich der Geburtshilfe nimmt dies nicht nur direkten Einfluss auf die mütterliche Versorgung des Neugeborenen, sondern kann bei Folgeschwangerschaften zu einer ablehnenden Haltung der Frauen gegenüber rückenmarknahen Anästhesieverfahren beitragen [9].
Wenngleich die zugrunde liegende Pathophysiologie noch nicht abschließend geklärt ist, werden dem Verlust von Liquor und der kompensatorischen intrakraniellen Vasodilatation eine zentrale Bedeutung beigemessen. Zudem kann es durch den Liquorverlust zu einem Zug an Nerven, Gefäßen und einer Reizung der Hirnhaut selbst kommen, weshalb die ausgelösten Symptome typischerweise durch eine aufrechte Körperposition verstärkt werden [5].
Die klinischen Charakteristika und diagnostischen Kriterien des PDPH sind in Tab. 1 zusammengefasst. Bei Zweifel an der klinischen Diagnose/atypischer Klinik sowie bei persistierenden Beschwerden sollte laut der aktuellen S1-Leitlinie Die geburtshilfliche Analgesie und Anästhesie der Deutschen Gesellschaft für Anästhesiologie und Intensivmedizin in Zusammenarbeit mit der Deutschen Gesellschaft für Gynäkologie und Geburtshilfe eine differenzialdiagnostische Abklärung (frühzeitig bildgebende Verfahren sowie die Durchführung eines neurologischen Konsils; Tab. 1) erfolgen [1].Klinische Charakteristika Auftreten bzw. Verschlechterung innerhalb von 15 min nach dem Aufrichten sowie mindestens eines der folgenden Symptome:
Nackensteifigkeit
Tinnitus
Veränderung des Hörens
Fotophobie
Nausea
Vorausgegangene Liquorpunktion
Auftreten innerhalb von 5 Tagen nach Liquorpunktion
Remission spontan oder innerhalb von 48 h nach effektiver Therapie
Bei anhaltender oder atypischer Klinik Bildgebung
Computertomographie
Magnetresonanztomographie
Neurologisches Konsil
Mögliche Differenzialdiagnosen Perfusionsstörung
Intrazerebrale Blutung/subdurales Hämatom
Hirnvenenthrombose
Apoplex
Hypophysenischämie
Intrakranieller Tumor
Sonstiges
Virale, chemische oder bakterielle Meningitis
Migräne
Koffeinentzugskopfschmerz
Drogenentzug
Präeklampsie
Spontanes Liquorunterdrucksyndrom
Hypovolämie
Pneumozephalus
Laktationskopfschmerz
Zur Behandlung des PDPH kommen zunächst konservative Maßnahmen oder medikamentöse Therapieversuche beispielsweise mit Nichtopioidanalgetika oder Koffein zur Anwendung ([1]; Tab. 2). Führen diese nicht innerhalb kurzer Zeit zu einer Linderung der Beschwerden, wird die frühzeitige Beratung der Patientin hinsichtlich der Durchführung eines epiduralen Blut-Patch (EBP) empfohlen [1]. Dies ist auch insofern von Relevanz, als dass Frauen mit PDPH eine erhöhte Inzidenz einer postpartalen Sinusvenenthrombose oder eines Subduralhämatoms aufweisen [11]. Bezüglich dieser Komplikation ist eine prophylaktische Wirkung durch Sistieren des Liquorverlustes nach Anlage eines EBP potenziell denkbar.Substanz Dosierung
Koffein 3- bis 4‑mal 200–300 mg/Tag p.o.
Theophyllin 3‑mal 280–350 mg/Tag p.o.
Gabapentin Ein- bis 4‑mal 300 mg/Tag p.o.
Hydrocortison Ein- bis 3‑mal 10 mg/Tag p.o.
Entgegen weit höherer Erfolgsraten nach Durchführung eines EBP in älteren Untersuchungen zeigen neuere Studien eine komplette und andauernde Beschwerdelinderung in ca. einem Drittel der untersuchten Fälle, während eine zumindest teilweise Besserung in 50–80 % erzielt wird [10]. Damit wird der EBP weiterhin als effektive Therapie des PDPH angesehen, allerdings geht dieses invasive Verfahren per se ebenfalls mit dem Risiko teilweise schwerwiegender Komplikationen wie der Ausbildung von subduralen Hämatomen, Nervenschädigungen, spinalen Infektionen oder einer weiteren Verschlimmerung der bestehenden Symptomatik einher [11, 12].
Als potenzielles Alternativverfahren zum EBP wird die transnasale Blockade des Ganglion sphenopalatinum diskutiert [13, 14], welche bereits bei verschiedenen Formen des Kopfschmerzes eingesetzt wird [15]. Hierbei wird den Patienten ein mit Lokalanästhetikum getränkter Watteträger an der Oberkante der Concha nasalis media entlang in Richtung Nasopharynxhinterwand eingeführt und für einige Minuten im Bereich des Ganglions auf Höhe der Fossa pterygopalatina platziert [16]. Die exakten Mechanismen, über die eine topische Blockade des Ganglion sphenopalatinum zu einer Beschwerdelinderung führt, sind bislang nicht bekannt. Eine mögliche Theorie beinhaltet jedoch die Blockade parasympathischer Ganglionanteile, welche einer intrakraniellen Vasodilatation als Treiber der PDPH-Symptomatik entgegenwirkt [17]. Potenzielle Nebenwirkungen umfassen temporäre nasopharyngeale Missempfindungen, Geschmacksstörungen, Irritationen der Schleimhaut sowie Nasenbluten.
In diesen Fallberichten wird erstmals die Anwendung einer Modifikation dieser Technik zur Therapie des PDPH bei geburtshilflichen Patientinnen beschrieben. Beide Patientinnen wurden zwischen Januar und Juni 2020 am Universitätsklinikum Heidelberg sowie am Klinikum Hanau behandelt. Nach Aufklärung über die Off-label-Nutzung wurde Lidocain 2 % mittels „mucosal atomization device“ (MAD) intranasal vernebelt. Über dieses Verfahren existieren bislang keine Erfahrungen aus der geburtshilflichen Anästhesiologie.
Fallbeschreibung 1
Anamnese
Bei der ersten Patientin handelte es sich um eine 25-jährige Zweitgravida (Tab. 3), welche zur geplanten Re-Sectio caesarea in SpA bei rascher Schwangerschaftsfolge in der 37 + 1 SSW stationär aufgenommen wurde. Die Durchführung der SpA wurde als erschwert dokumentiert; nach insgesamt 2 Punktionsversuchen auf 2 Höhen mit einer 25-G-Sprotte-Nadel gelang schließlich mit einer 12 cm langen 22-G-Sprotte-Nadel die Punktion des Liquorraums. Nach komplikationslosem Eingriff konnte die Patientin in den Kreißsaal und nach Abklingen der SpA auf die Normalstation verlegt werden. Patientin 1 Patientin 2
Alter (Jahre) 25 32
Körpergewicht (kg) 140 70
BMI (kg/m2) 54,7 27,3
Schwangerschaftswoche 37 + 1 38 + 5
Geburtshilfliche Anästhesie Spinalanästhesie Periduralanästhesie
Nadeltyp Sprotte Tuohy
Nadelgröße Initial 25 G, dann 22 G 18 G
PDPH-Symptome
Lageabhängigkeit Ja Ja
Nackensteifigkeit Nein Ja
Tinnitus Nein Ja
Dysakusis Nein Ja
Photophobie Nein Nein
Nausea Ja Nein
Therapie
Initial 4‑mal 600 mg Ibuprofen
4‑mal 1 g Paracetamol
3‑mal 200 mg Koffein
4‑mal 600 mg Ibuprofen
4‑mal 1 g Paracetamol
3‑mal 200 mg Koffein
Lidocain, intranasal Einmal via getränktem Tupfer (ca. 50 mg/Seite)
Einmal 50 mg/Seite (MAD)
100 mg/Seite (MAD)
Zeit Lidocaingabe via MAD bis Symptomlinderung (min) 5 2
NRS vor Lidocaingabe 9/10 7/10
NRS nach Lidocaingabe 0/10–1/10 0/10
BMI body mass index, PDPH postdural puncture headache, MAD mucosal atomization device, NRS numeric rating scale
Befund und Diagnose
Am Morgen des ersten postoperativen Tages klagte die Patientin über lageabhängige stärkste Kopf- und Nackenschmerzen mit Ausbreitung über den Rücken bis ins Gesäß, begleitet von Schwindelgefühl sowie ausgeprägter Nausea und Emesis. Bereits geringe Oberkörperhochlagerung führte zu einer deutlichen Verstärkung der Symptomatik; ein aufrechter Gang war nicht möglich. Es bestanden keine neurologischen Defizite; die Einstichstellen zeigten sich unauffällig. In Zusammenschau der Befunde wurde die Diagnose eines PDPH nach einer erschwerten SpA gestellt.
Therapie und Verlauf
Die symptomatische Therapie wurde um Ibuprofen, Koffein und Dimenhydrinat ergänzt (Tab. 3). Bei ausbleibender Beschwerdelinderung wurden mit der Patientin weitere Therapiemöglichkeiten, inklusive EBP, eruiert; die Patientin entschied sich nach Aufklärung über die Off-label-Gabe zunächst für die beidseitige nasale Einlage lidocaingetränkter Watteträger (ca. 50 mg/Seite). Auch hierunter besserten sich die Beschwerden nur unzureichend. Mit der Patientin wurde daraufhin die Möglichkeit einer weiteren Therapieeskalation mittels EBP besprochen. Bei weiterbestehenden Beschwerden wurde als alternativer Therapieversuch die Lidocainvernebelung mittels MAD angeboten, welcher die Patientin zustimmte. Vier Stunden nach Einlage der lidocaingetränkten Wattetupfer erfolgte die einmalige Vernebelung mittels MAD von jeweils 50 mg Lidocain/Seite. Hierunter zeigte sich die Patientin nach ca. 5 min nahezu symptomfrei; lediglich in aufrechter Position persistierten noch minimale Schmerzen (NRS 1/10). Diese bildeten sich im weiteren Verlauf ebenfalls vollständig zurück, sodass die Patientin am Folgetag schmerz- und beschwerdefrei nach Hause entlassen werden konnte.
Fallbeschreibung 2
Anamnese
Die zweite Patientin (32-jährige Drittgravida) wurde bei vorzeitigem Blasensprung aufgenommen. Unter als problemlos dokumentierter Periduralanästhesie (Anlage mit einer 18-G-Tuohy-Nadel, ein Punktionsversuch) kam es zunächst zu einer komplikationslosen Spontangeburt. Die vollständige Entfernung des Katheters erfolgte bei geplanter ambulanter Behandlung ca. 2 h post partum.
Befund und Diagnose
Zum Zeitpunkt der Katheterentfernung bestand eine leichte Verspannung im Nackenbereich. Weitere 2 h später beklagte die Patientin eine ausgeprägte Nackensteifigkeit, Nackenschmerzen, Bewegungseinschränkungen beider Arme und Schultern sowie im Verlauf starke Kopfschmerzen, wobei eine Lageabhängigkeit der genannten Symptome bestand (Tab. 3). Mit Beginn der Beschwerden wurde neben der symptomatischen Therapie ein neurologisches Konsil initiiert. Hierbei wurde der Verdacht auf Vorliegen eines PDPH, differenzialdiagnostisch einer muskulären Verspannung oder einer meningealen Reizung nach PDK-Anlage gestellt.
Therapie und Verlauf
Trotz leichter Symptomverbesserung unter angepasster Schmerzmedikation (Tab. 3) erfolgten die Aufklärung über eine potenzielle Therapieeskalation (inklusive EBP) sowie am Folgetag eine cMRT-Diagnostik, welche keine pathologischen Befunde ergab. Bei unauffälliger Bildgebung und zunächst weiterer Beschwerdebesserung wurde die Patientin am ersten postpartalen Tag auf eigenen Wunsch nach Hause entlassen.
Im weiteren Verlauf kamen zu den beschriebenen Symptomen ein Tinnitus sowie eine deutliche Hörminderung beidseits hinzu, sodass sich die Patientin am vierten postpartalen Tag erneut in der Klinik vorstellte, um das postpunktionelle Syndrom via Blut-Patch therapieren zu lassen. Hierüber wurde sie nun auch schriftlich aufgeklärt. Aufgrund der über mehrere Tage fortgeführten regelmäßigen Ibuprofeneinnahme wurde ein EBP jedoch nicht am selben Tag durchgeführt. Daher erfolgte bei hohem Leidensdruck nach ausführlicher Aufklärung über die Off-label-Gabe die fraktionierte nasale Applikation von jeweils 100 mg Lidocain/Seite via MAD. Die Option der Anlage eines Blut-Patch am Folgetag bei ausbleibender oder ungenügender Wirkung wurde durch Pausieren der Ibuprofeneinnahme sichergestellt. Nach intranasaler Lidocainvernebelung kam es innerhalb von ca. 2 min zu einem kompletten Sistieren der Kopf- und Nackenschmerzen sowie des Tinnitus; lediglich die Hörminderung blieb bestehen. Am Folgetag war die Patientin schmerzfrei; Tinnitus und Hörvermögen zeigten sich deutlich gebessert, mit lediglich noch vorhandener linksseitiger Einschränkung. Die Patientin konnte schließlich am ersten Tag nach Wiederaufnahme (fünfter postpartaler Tag) in deutlich gebessertem Allgemeinzustand nach Hause entlassen werden. Als einzige Nebenwirkung wurde ein vorübergehendes pharyngeales Taubheitsgefühl berichtet. Die Patientin wurde im weiteren Verlauf noch mehrfach kontaktiert; die noch geringfügig zum Zeitpunkt der Entlassung vorhandenen linksbetonten Einschränkungen des Hörvermögens sowie der Tinnitus verbesserten sich zunehmend und waren ca. 5 Wochen nach Punktion komplett verschwunden.
Diskussion
In diesem Fallbericht wird erstmals der erfolgreiche Einsatz einer intranasalen Lidocainvernebelung mittels MAD zur Therapie eines PDPH nach geburtshilflicher rückenmarknaher Anästhesie geschildert. Bei beiden Patientinnen kam es nach erfolglosen Therapieversuchen mit Nichtopioidanalgetika sowie Koffein durch intranasale Lidocainverabreichung zu einer rasch eintretenden und anhaltenden Symptomlinderung.
Die hier beschriebene Methode weist unseres Erachtens einige Vorteile gegenüber bislang beschriebenen Verfahren zur Blockade des Ganglion sphenopalatinum auf. So kann das Einbringen von Watteträgern aufgrund der anatomischen Lokalisation insbesondere bei Patienten mit anatomischen Besonderheiten wie Polypen oder Septumdeviation erschwert sein, was den ausgebliebenen Therapieeffekt in unserem erstgenannten Fall erklären könnte. Interessanterweise benötigte unsere Patientin zur Einlage der Watteträger keine vorherige Lokalanästhesie, wohingegen in der Literatur teilweise ein Eintröpfeln oder -sprühen von Lokalanästhetika vor Durchführung der eigentlichen Prozedur beschrieben wird [18, 19]. Da das Ganglion sphenopalatinum von einer nur wenige Millimeter messenden Schleimhautschicht überdeckt wird [20], ist nicht auszuschließen, dass durch eine solche Vorabverabreichung von Lokalanästhetika bereits eine (Teil)Wirkung erzielt werden konnte.
Einen aus unserer Sicht wichtigen Aspekt im Vergleich zwischen konventionellen nasalen Blockadeformen und der hier beschriebenen Lokalanästhetikavernebelung stellt der Komfort für die Patientinnen während der Anwendung dar. Während das Einbringen von Watteträgern und deren Verbleib im Nasopharynx über den Zeitraum der Prozedur als sehr unangenehm empfunden werden können, sodass für die Durchführung in Einzelfällen eine Sedierung nötig ist [19], wird das Vernebeln des Lokalanästhetikums als vergleichbar zur Anwendung eines Nasensprays beschrieben. Als mögliche Nebenwirkung kann es durch Schlucken der Lokalanästhetikalösung jedoch zu temporären pharyngealen Missempfindungen kommen. In den hier berichteten Fällen wurde fraktioniert Lidocain 2 % vernebelt; zur Vermeidung der genannten Nebenwirkung wäre daher der zukünftige Einsatz geringerer Mengen höher konzentrierter Lösungen denkbar. So beschreiben Kanai et al. die erfolgreiche Anwendung 8 %igen Lidocainsprays zur Therapie einer Trigeminusneuralgie [21]. Gegenüber vorgefertigten Spraysystemen ermöglichen MAD-Systeme eine individuell angepasste Dosierung. Dies ist insofern relevant, da systemische Nebenwirkungen durch Resorption des intranasal applizierten Lidocains bei unsachgemäßer Handhabung und Überschreitung toxischer Grenzwerte prinzipiell möglich sind. Die von uns eingesetzte Dosis lag jeweils unterhalb der maximalen Einzeldosierung von 300 mg; bei beiden Patientinnen kam es zu keinen systemischen Nebenwirkungen.
Zusammenfassend handelt es sich bei der vorgestellten Maßnahme um eine aus unserer Sicht wertvolle Ergänzung bisheriger Therapieoptionen und eine potenzielle minimal-invasive Alternative zum epiduralen Blutpatch. Die Vernebelung von Lokalanästhetika via MAD ist eine bettseitig durchführbare, kostengünstige und mit einem deutlichen Zugewinn an Patientenkomfort verbundene Methode, deren Anwendung nicht nur bei PDPH in der Geburtshilfe sinnvoll sein kann. Es gilt zu beachten, dass es sich bei der intranasalen Verneblung von Lidocain um eine Off-label-Anwendung handelt, über die entsprechend aufgeklärt und dies auch dokumentiert werden muss. Prospektive Studien sind nötig, um die hier berichteten Erkenntnisse zu validieren und Aussagen zu optimaler Dosierung und Wirksamkeit auch anderer Lokalanästhetika im Rahmen der transnasalen Applikation bei PDPH treffen zu können [22].
Fazit für die Praxis
Es wird erstmals der Einsatz einer nasalen Lidocainvernebelung mittels Mucosal atomization device (MAD) zur Therapie eines Postpunktionskopfschmerzes (PDPH) nach geburtshilflicher rückenmarknaher Anästhesie beschrieben.
Nach vorangegangen frustranen konventionell-medikamentösen Therapieversuchen führte die nasale Lidocaingabe zu einer unmittelbaren und persistierenden Besserung der Symptome; die Anlage eines epiduralen Blutpatch (EBP) konnte in beiden Fällen vermieden werden.
Die geschilderte Methode ist einfach und bettseitig durchführbar, mit einem deutlichen Zugewinn an Patientenkomfort verbunden und sollte als neue potenzielle Therapieoption des PDPH und nichtinvasive Alternative zum EBP prospektiv validiert werden.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Einhaltung ethischer Richtlinien
Interessenkonflikt
B.H. Siegler, M. Gruß, B. Oehler, J. Keßler, H. Fluhr, C. Weis, F. Schulz und M.A. Weigand geben an, dass kein Interessenkonflikt besteht.
Für diesen Beitrag wurden von den Autoren keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien. Für Bildmaterial oder anderweitige Angaben innerhalb des Manuskripts, über die Patienten zu identifizieren sind, liegt von ihnen und/oder ihren gesetzlichen Vertretern eine schriftliche Einwilligung vor.
Die Autoren B.H. Siegler und M. Gruß teilen sich die Erstautorenschaft. | ACETAMINOPHEN, CAFFEINE, DIMENHYDRINATE, IBUPROFEN | DrugsGivenReaction | CC BY | 33301057 | 20,099,098 | 2021-05 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Diabetes mellitus'. | Metastatic renal cell carcinoma to the prostate and seminal vesicle.
Renal cell carcinoma (RCC) a common malignancy with potential to metastasize to visceral organs. However, it uncommonly spreads to the lower genitourinary tract. We present a man with a history of RCC status post radical nephrectomy in April 2012. He presented 8 years later with obstructive lower urinary tract symptoms and an elevated prostate specific antigen (PSA). Further imaging showed a large enhancing mass with internal blood vessels posterior to the left prostate and seminal vesicle. A prostate biopsy was performed and consistent with metastatic RCC. He was ultimately treated with immunotherapy and focal stereotactic radioablation.
Introduction
Renal cell carcinoma (RCC) is the sixth and eighth most frequently diagnosed cancer in men and women, respectively. In 2020, there are an estimated 74,000 new cases and 15,000 deaths from RCC in the United States.1 Clear cell is the most common subtype of RCC comprising up to 85% of renal tumors. Up to 17% of patients have distant metastatic disease upon diagnosis, with the most common sites being lung, bone, liver, brain, and adrenal gland.2
Secondary tumors of the prostate from distant primary neoplasms are very rare. In particular, metastatic RCC to the prostate is an extraordinarily uncommon phenomenon with few cases reported in the literature. We present a case of oligometastatic clear cell RCC discovered at the left prostate and seminal vesicle after a radical nephrectomy of the same pathology 8 years prior.
Case presentation
The patient is a 51-year-old healthy male who initially presented with an incidentally found 7.5 cm right renal mass in April 2012 after workup for suspected cholelithiasis. He underwent a laparoscopic right radical nephrectomy at an outside hospital. The pathology returned as Fuhrman grade II renal cell carcinoma, clear cell type. His stage was pT3NxMx with extension into the perirenal fat. He underwent surveillance after surgery with no symptoms or evidence of recurrence. He last presented to his two-year surveillance visit and was subsequently lost to follow-up.
In early 2018, the patient developed dysuria and obstructive voiding symptoms. He was found to have an elevated prostate specific antigen (PSA) to 5.07 ng/mL. He was treated for presumed prostatitis. Urine cytology at this time was negative. A prostate biopsy was performed in December 2018 which was negative for carcinoma. Of note, a nodule was not noted during the biopsy. His symptoms worsened throughout the year. A repeat PSA was drawn 5 months later and was further elevated to 12.49 ng/mL. A digital rectal exam was performed in September 2019, which demonstrated a suspicious nodule on the left lateral prostate. As a result, an MRI of the prostate was performed, which showed a 3 cm enhancing mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle. The diagnosis was thought to be a paraganglioma based on imaging (Fig. 1). He underwent a repeat transrectal ultrasound guided prostate biopsy with targeted areas within the nodule. His prostate measured 33 cc. Immunohistochemical stains were positive for PAX8 and negative for PSA, consistent with metastatic clear cell RCC (Fig. 2). Further distant metastatic work-up was negative.Fig. 1 An axial (A) and sagittal view (B) of the patient's T2-weighted MRI demonstrating a 2.4 × 3.0 × 2.9 cm mass abutting the left posterior prostate and seminal vesicle. Initial MRI in October 2019 demonstrated a 3 cm avidly enhancing solid mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle.
Fig. 1Fig. 2 The pathology obtained from a transrectal prostate biopsy is shown above. (A) demonstrates the H&E stained tissue that was positive for clear cell metastatic renal cell carcinoma (RCC). This was confirmed with further immunohistochemical stains. (B) demonstrates the tissue that is negative for PSA staining. (C) demonstrates the tissue was positive for PAX8 staining confirming the diagnosis of RCC and the metastatic nature of the tumor.
Fig. 2
The case was presented at a multi-disciplinary tumor board conference. The patient was categorized as favorable risk by MSKCC and IMDC criteria. After discussion and counseling, he was started a combination of ipilimumab and nivolumab immunotherapy. Unfortunately, he developed new onset diabetes mellitus after 1 cycle of immunotherapy resulting in diabetic ketoacidosis. Systemic therapy was discontinued. Given close proximity to the rectum and inflammatory characteristics of the lesion, surgical resection was deferred. Thus, he underwent focal stereotactic radioablation to the peri-prostatic lesion. His most recent follow-up scans showed stable disease.
Discussion
Clear cell lesions of the urinary tract, including the prostate, often present a diagnostic challenge. RCC has the capacity to metastasize to almost anywhere in the body. In fact, some studies suggest that up to 48% of patients present with metastatic disease.3 However, metastasis to the prostate from RCC is very rare. In a large autopsy series, the rate of RCC metastasizing to the prostate was 0.9%.3 Secondary tumors of the prostate are regularly considered a late manifestation of malignant disease and often herald a poor prognosis when discovered in the clinical setting.
The likely etiology for the mass in our patient is “drop metastasis” to the rectovesical pouch that eventually seeded the prostate and seminal vesicle.4 However, because of the limited number of cases, the optimal treatment options are not clear. The treatment of metastatic renal cell cancers traditionally relies on systemic immunotherapy, radiotherapy, or surgical resection. The benefits of metastasectomy for solitary metastases have been firmly established with wide excision being the most common surgical technique for a solitary metastasis. Reports have shown an approximately 35% 5-year survival rate for patients who underwent nephrectomy and surgical resection of a solitary metastasis.5 Unfortunately in our patient, metastasectomy was not feasible due to nearby tissue inflammation and proximity to the rectum.
Conclusion
Metastatic RCC has the capability to spread throughout the body in a delayed fashion. However, involvement of the lower genitourinary tract is a rare phenomenon. Despite the rarity, new symptoms including those of urinary etiology should not be ignored in the setting of a positive oncologic history. | IPILIMUMAB, NIVOLUMAB | DrugsGivenReaction | CC BY-NC-ND | 33304825 | 18,650,969 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Diabetic ketoacidosis'. | Metastatic renal cell carcinoma to the prostate and seminal vesicle.
Renal cell carcinoma (RCC) a common malignancy with potential to metastasize to visceral organs. However, it uncommonly spreads to the lower genitourinary tract. We present a man with a history of RCC status post radical nephrectomy in April 2012. He presented 8 years later with obstructive lower urinary tract symptoms and an elevated prostate specific antigen (PSA). Further imaging showed a large enhancing mass with internal blood vessels posterior to the left prostate and seminal vesicle. A prostate biopsy was performed and consistent with metastatic RCC. He was ultimately treated with immunotherapy and focal stereotactic radioablation.
Introduction
Renal cell carcinoma (RCC) is the sixth and eighth most frequently diagnosed cancer in men and women, respectively. In 2020, there are an estimated 74,000 new cases and 15,000 deaths from RCC in the United States.1 Clear cell is the most common subtype of RCC comprising up to 85% of renal tumors. Up to 17% of patients have distant metastatic disease upon diagnosis, with the most common sites being lung, bone, liver, brain, and adrenal gland.2
Secondary tumors of the prostate from distant primary neoplasms are very rare. In particular, metastatic RCC to the prostate is an extraordinarily uncommon phenomenon with few cases reported in the literature. We present a case of oligometastatic clear cell RCC discovered at the left prostate and seminal vesicle after a radical nephrectomy of the same pathology 8 years prior.
Case presentation
The patient is a 51-year-old healthy male who initially presented with an incidentally found 7.5 cm right renal mass in April 2012 after workup for suspected cholelithiasis. He underwent a laparoscopic right radical nephrectomy at an outside hospital. The pathology returned as Fuhrman grade II renal cell carcinoma, clear cell type. His stage was pT3NxMx with extension into the perirenal fat. He underwent surveillance after surgery with no symptoms or evidence of recurrence. He last presented to his two-year surveillance visit and was subsequently lost to follow-up.
In early 2018, the patient developed dysuria and obstructive voiding symptoms. He was found to have an elevated prostate specific antigen (PSA) to 5.07 ng/mL. He was treated for presumed prostatitis. Urine cytology at this time was negative. A prostate biopsy was performed in December 2018 which was negative for carcinoma. Of note, a nodule was not noted during the biopsy. His symptoms worsened throughout the year. A repeat PSA was drawn 5 months later and was further elevated to 12.49 ng/mL. A digital rectal exam was performed in September 2019, which demonstrated a suspicious nodule on the left lateral prostate. As a result, an MRI of the prostate was performed, which showed a 3 cm enhancing mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle. The diagnosis was thought to be a paraganglioma based on imaging (Fig. 1). He underwent a repeat transrectal ultrasound guided prostate biopsy with targeted areas within the nodule. His prostate measured 33 cc. Immunohistochemical stains were positive for PAX8 and negative for PSA, consistent with metastatic clear cell RCC (Fig. 2). Further distant metastatic work-up was negative.Fig. 1 An axial (A) and sagittal view (B) of the patient's T2-weighted MRI demonstrating a 2.4 × 3.0 × 2.9 cm mass abutting the left posterior prostate and seminal vesicle. Initial MRI in October 2019 demonstrated a 3 cm avidly enhancing solid mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle.
Fig. 1Fig. 2 The pathology obtained from a transrectal prostate biopsy is shown above. (A) demonstrates the H&E stained tissue that was positive for clear cell metastatic renal cell carcinoma (RCC). This was confirmed with further immunohistochemical stains. (B) demonstrates the tissue that is negative for PSA staining. (C) demonstrates the tissue was positive for PAX8 staining confirming the diagnosis of RCC and the metastatic nature of the tumor.
Fig. 2
The case was presented at a multi-disciplinary tumor board conference. The patient was categorized as favorable risk by MSKCC and IMDC criteria. After discussion and counseling, he was started a combination of ipilimumab and nivolumab immunotherapy. Unfortunately, he developed new onset diabetes mellitus after 1 cycle of immunotherapy resulting in diabetic ketoacidosis. Systemic therapy was discontinued. Given close proximity to the rectum and inflammatory characteristics of the lesion, surgical resection was deferred. Thus, he underwent focal stereotactic radioablation to the peri-prostatic lesion. His most recent follow-up scans showed stable disease.
Discussion
Clear cell lesions of the urinary tract, including the prostate, often present a diagnostic challenge. RCC has the capacity to metastasize to almost anywhere in the body. In fact, some studies suggest that up to 48% of patients present with metastatic disease.3 However, metastasis to the prostate from RCC is very rare. In a large autopsy series, the rate of RCC metastasizing to the prostate was 0.9%.3 Secondary tumors of the prostate are regularly considered a late manifestation of malignant disease and often herald a poor prognosis when discovered in the clinical setting.
The likely etiology for the mass in our patient is “drop metastasis” to the rectovesical pouch that eventually seeded the prostate and seminal vesicle.4 However, because of the limited number of cases, the optimal treatment options are not clear. The treatment of metastatic renal cell cancers traditionally relies on systemic immunotherapy, radiotherapy, or surgical resection. The benefits of metastasectomy for solitary metastases have been firmly established with wide excision being the most common surgical technique for a solitary metastasis. Reports have shown an approximately 35% 5-year survival rate for patients who underwent nephrectomy and surgical resection of a solitary metastasis.5 Unfortunately in our patient, metastasectomy was not feasible due to nearby tissue inflammation and proximity to the rectum.
Conclusion
Metastatic RCC has the capability to spread throughout the body in a delayed fashion. However, involvement of the lower genitourinary tract is a rare phenomenon. Despite the rarity, new symptoms including those of urinary etiology should not be ignored in the setting of a positive oncologic history. | IPILIMUMAB, NIVOLUMAB | DrugsGivenReaction | CC BY-NC-ND | 33304825 | 18,650,969 | 2021-01 |
What was the outcome of reaction 'Diabetes mellitus'? | Metastatic renal cell carcinoma to the prostate and seminal vesicle.
Renal cell carcinoma (RCC) a common malignancy with potential to metastasize to visceral organs. However, it uncommonly spreads to the lower genitourinary tract. We present a man with a history of RCC status post radical nephrectomy in April 2012. He presented 8 years later with obstructive lower urinary tract symptoms and an elevated prostate specific antigen (PSA). Further imaging showed a large enhancing mass with internal blood vessels posterior to the left prostate and seminal vesicle. A prostate biopsy was performed and consistent with metastatic RCC. He was ultimately treated with immunotherapy and focal stereotactic radioablation.
Introduction
Renal cell carcinoma (RCC) is the sixth and eighth most frequently diagnosed cancer in men and women, respectively. In 2020, there are an estimated 74,000 new cases and 15,000 deaths from RCC in the United States.1 Clear cell is the most common subtype of RCC comprising up to 85% of renal tumors. Up to 17% of patients have distant metastatic disease upon diagnosis, with the most common sites being lung, bone, liver, brain, and adrenal gland.2
Secondary tumors of the prostate from distant primary neoplasms are very rare. In particular, metastatic RCC to the prostate is an extraordinarily uncommon phenomenon with few cases reported in the literature. We present a case of oligometastatic clear cell RCC discovered at the left prostate and seminal vesicle after a radical nephrectomy of the same pathology 8 years prior.
Case presentation
The patient is a 51-year-old healthy male who initially presented with an incidentally found 7.5 cm right renal mass in April 2012 after workup for suspected cholelithiasis. He underwent a laparoscopic right radical nephrectomy at an outside hospital. The pathology returned as Fuhrman grade II renal cell carcinoma, clear cell type. His stage was pT3NxMx with extension into the perirenal fat. He underwent surveillance after surgery with no symptoms or evidence of recurrence. He last presented to his two-year surveillance visit and was subsequently lost to follow-up.
In early 2018, the patient developed dysuria and obstructive voiding symptoms. He was found to have an elevated prostate specific antigen (PSA) to 5.07 ng/mL. He was treated for presumed prostatitis. Urine cytology at this time was negative. A prostate biopsy was performed in December 2018 which was negative for carcinoma. Of note, a nodule was not noted during the biopsy. His symptoms worsened throughout the year. A repeat PSA was drawn 5 months later and was further elevated to 12.49 ng/mL. A digital rectal exam was performed in September 2019, which demonstrated a suspicious nodule on the left lateral prostate. As a result, an MRI of the prostate was performed, which showed a 3 cm enhancing mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle. The diagnosis was thought to be a paraganglioma based on imaging (Fig. 1). He underwent a repeat transrectal ultrasound guided prostate biopsy with targeted areas within the nodule. His prostate measured 33 cc. Immunohistochemical stains were positive for PAX8 and negative for PSA, consistent with metastatic clear cell RCC (Fig. 2). Further distant metastatic work-up was negative.Fig. 1 An axial (A) and sagittal view (B) of the patient's T2-weighted MRI demonstrating a 2.4 × 3.0 × 2.9 cm mass abutting the left posterior prostate and seminal vesicle. Initial MRI in October 2019 demonstrated a 3 cm avidly enhancing solid mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle.
Fig. 1Fig. 2 The pathology obtained from a transrectal prostate biopsy is shown above. (A) demonstrates the H&E stained tissue that was positive for clear cell metastatic renal cell carcinoma (RCC). This was confirmed with further immunohistochemical stains. (B) demonstrates the tissue that is negative for PSA staining. (C) demonstrates the tissue was positive for PAX8 staining confirming the diagnosis of RCC and the metastatic nature of the tumor.
Fig. 2
The case was presented at a multi-disciplinary tumor board conference. The patient was categorized as favorable risk by MSKCC and IMDC criteria. After discussion and counseling, he was started a combination of ipilimumab and nivolumab immunotherapy. Unfortunately, he developed new onset diabetes mellitus after 1 cycle of immunotherapy resulting in diabetic ketoacidosis. Systemic therapy was discontinued. Given close proximity to the rectum and inflammatory characteristics of the lesion, surgical resection was deferred. Thus, he underwent focal stereotactic radioablation to the peri-prostatic lesion. His most recent follow-up scans showed stable disease.
Discussion
Clear cell lesions of the urinary tract, including the prostate, often present a diagnostic challenge. RCC has the capacity to metastasize to almost anywhere in the body. In fact, some studies suggest that up to 48% of patients present with metastatic disease.3 However, metastasis to the prostate from RCC is very rare. In a large autopsy series, the rate of RCC metastasizing to the prostate was 0.9%.3 Secondary tumors of the prostate are regularly considered a late manifestation of malignant disease and often herald a poor prognosis when discovered in the clinical setting.
The likely etiology for the mass in our patient is “drop metastasis” to the rectovesical pouch that eventually seeded the prostate and seminal vesicle.4 However, because of the limited number of cases, the optimal treatment options are not clear. The treatment of metastatic renal cell cancers traditionally relies on systemic immunotherapy, radiotherapy, or surgical resection. The benefits of metastasectomy for solitary metastases have been firmly established with wide excision being the most common surgical technique for a solitary metastasis. Reports have shown an approximately 35% 5-year survival rate for patients who underwent nephrectomy and surgical resection of a solitary metastasis.5 Unfortunately in our patient, metastasectomy was not feasible due to nearby tissue inflammation and proximity to the rectum.
Conclusion
Metastatic RCC has the capability to spread throughout the body in a delayed fashion. However, involvement of the lower genitourinary tract is a rare phenomenon. Despite the rarity, new symptoms including those of urinary etiology should not be ignored in the setting of a positive oncologic history. | Recovering | ReactionOutcome | CC BY-NC-ND | 33304825 | 18,650,969 | 2021-01 |
What was the outcome of reaction 'Diabetic ketoacidosis'? | Metastatic renal cell carcinoma to the prostate and seminal vesicle.
Renal cell carcinoma (RCC) a common malignancy with potential to metastasize to visceral organs. However, it uncommonly spreads to the lower genitourinary tract. We present a man with a history of RCC status post radical nephrectomy in April 2012. He presented 8 years later with obstructive lower urinary tract symptoms and an elevated prostate specific antigen (PSA). Further imaging showed a large enhancing mass with internal blood vessels posterior to the left prostate and seminal vesicle. A prostate biopsy was performed and consistent with metastatic RCC. He was ultimately treated with immunotherapy and focal stereotactic radioablation.
Introduction
Renal cell carcinoma (RCC) is the sixth and eighth most frequently diagnosed cancer in men and women, respectively. In 2020, there are an estimated 74,000 new cases and 15,000 deaths from RCC in the United States.1 Clear cell is the most common subtype of RCC comprising up to 85% of renal tumors. Up to 17% of patients have distant metastatic disease upon diagnosis, with the most common sites being lung, bone, liver, brain, and adrenal gland.2
Secondary tumors of the prostate from distant primary neoplasms are very rare. In particular, metastatic RCC to the prostate is an extraordinarily uncommon phenomenon with few cases reported in the literature. We present a case of oligometastatic clear cell RCC discovered at the left prostate and seminal vesicle after a radical nephrectomy of the same pathology 8 years prior.
Case presentation
The patient is a 51-year-old healthy male who initially presented with an incidentally found 7.5 cm right renal mass in April 2012 after workup for suspected cholelithiasis. He underwent a laparoscopic right radical nephrectomy at an outside hospital. The pathology returned as Fuhrman grade II renal cell carcinoma, clear cell type. His stage was pT3NxMx with extension into the perirenal fat. He underwent surveillance after surgery with no symptoms or evidence of recurrence. He last presented to his two-year surveillance visit and was subsequently lost to follow-up.
In early 2018, the patient developed dysuria and obstructive voiding symptoms. He was found to have an elevated prostate specific antigen (PSA) to 5.07 ng/mL. He was treated for presumed prostatitis. Urine cytology at this time was negative. A prostate biopsy was performed in December 2018 which was negative for carcinoma. Of note, a nodule was not noted during the biopsy. His symptoms worsened throughout the year. A repeat PSA was drawn 5 months later and was further elevated to 12.49 ng/mL. A digital rectal exam was performed in September 2019, which demonstrated a suspicious nodule on the left lateral prostate. As a result, an MRI of the prostate was performed, which showed a 3 cm enhancing mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle. The diagnosis was thought to be a paraganglioma based on imaging (Fig. 1). He underwent a repeat transrectal ultrasound guided prostate biopsy with targeted areas within the nodule. His prostate measured 33 cc. Immunohistochemical stains were positive for PAX8 and negative for PSA, consistent with metastatic clear cell RCC (Fig. 2). Further distant metastatic work-up was negative.Fig. 1 An axial (A) and sagittal view (B) of the patient's T2-weighted MRI demonstrating a 2.4 × 3.0 × 2.9 cm mass abutting the left posterior prostate and seminal vesicle. Initial MRI in October 2019 demonstrated a 3 cm avidly enhancing solid mass with internal blood vessels posterior to the left peripheral zone and seminal vesicle.
Fig. 1Fig. 2 The pathology obtained from a transrectal prostate biopsy is shown above. (A) demonstrates the H&E stained tissue that was positive for clear cell metastatic renal cell carcinoma (RCC). This was confirmed with further immunohistochemical stains. (B) demonstrates the tissue that is negative for PSA staining. (C) demonstrates the tissue was positive for PAX8 staining confirming the diagnosis of RCC and the metastatic nature of the tumor.
Fig. 2
The case was presented at a multi-disciplinary tumor board conference. The patient was categorized as favorable risk by MSKCC and IMDC criteria. After discussion and counseling, he was started a combination of ipilimumab and nivolumab immunotherapy. Unfortunately, he developed new onset diabetes mellitus after 1 cycle of immunotherapy resulting in diabetic ketoacidosis. Systemic therapy was discontinued. Given close proximity to the rectum and inflammatory characteristics of the lesion, surgical resection was deferred. Thus, he underwent focal stereotactic radioablation to the peri-prostatic lesion. His most recent follow-up scans showed stable disease.
Discussion
Clear cell lesions of the urinary tract, including the prostate, often present a diagnostic challenge. RCC has the capacity to metastasize to almost anywhere in the body. In fact, some studies suggest that up to 48% of patients present with metastatic disease.3 However, metastasis to the prostate from RCC is very rare. In a large autopsy series, the rate of RCC metastasizing to the prostate was 0.9%.3 Secondary tumors of the prostate are regularly considered a late manifestation of malignant disease and often herald a poor prognosis when discovered in the clinical setting.
The likely etiology for the mass in our patient is “drop metastasis” to the rectovesical pouch that eventually seeded the prostate and seminal vesicle.4 However, because of the limited number of cases, the optimal treatment options are not clear. The treatment of metastatic renal cell cancers traditionally relies on systemic immunotherapy, radiotherapy, or surgical resection. The benefits of metastasectomy for solitary metastases have been firmly established with wide excision being the most common surgical technique for a solitary metastasis. Reports have shown an approximately 35% 5-year survival rate for patients who underwent nephrectomy and surgical resection of a solitary metastasis.5 Unfortunately in our patient, metastasectomy was not feasible due to nearby tissue inflammation and proximity to the rectum.
Conclusion
Metastatic RCC has the capability to spread throughout the body in a delayed fashion. However, involvement of the lower genitourinary tract is a rare phenomenon. Despite the rarity, new symptoms including those of urinary etiology should not be ignored in the setting of a positive oncologic history. | Recovering | ReactionOutcome | CC BY-NC-ND | 33304825 | 18,650,969 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Glucocorticoid deficiency'. | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | ASPIRIN, METYRAPONE, NIFEDIPINE | DrugsGivenReaction | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Maternal exposure during pregnancy'. | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | ASPIRIN, METYRAPONE, NIFEDIPINE | DrugsGivenReaction | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
What was the administration route of drug 'ASPIRIN'? | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
What was the administration route of drug 'METYRAPONE'? | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
What was the administration route of drug 'NIFEDIPINE'? | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
What was the dosage of drug 'NIFEDIPINE'? | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | 60 mg (milligrams). | DrugDosage | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
What was the outcome of reaction 'Glucocorticoid deficiency'? | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Recovered | ReactionOutcome | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
What was the outcome of reaction 'Maternal exposure during pregnancy'? | Adrenal Cushing Syndrome Diagnosed During Pregnancy: Successful Medical Management With Metyrapone.
Adrenal Cushing syndrome during pregnancy is rare, and there is limited information on the effect and safety of metyrapone treatment both for mother and fetus. We present a 24-year-old woman diagnosed with adrenal Cushing syndrome at the end of the second trimester. We elected treatment with metyrapone titrated to 250 mg 3 times daily, resulting in good clinical response and maternal serum and saliva cortisol levels in the upper half of the normal pregnancy range. A healthy male infant was born at 35 weeks' gestation, with no clinical signs of adrenal insufficiency, this despite a low cortisol of 5 nmol/L on the first day of life. We measured metyrapone in maternal and umbilical cord blood samples, demonstrating fetal venous metyrapone levels similar to maternal venous concentration, and a fetal arterial cord concentration at about 60% of the fetal venous cord concentration. This case demonstrates that salivary cortisol levels may be used to monitor the effect of metyrapone on adrenal Cushing syndrome during pregnancy. We show, for the first time in humans, that metyrapone does cross the placenta and may suppress fetal cortisol production without necessarily causing clinical signs of adrenal insufficiency.
Cushing syndrome (CS) during pregnancy is rare, with only about 200 cases reported in the literature [1]. Its rarity in pregnancy is multifactorial because of low rates of fertility consequent to CS, and because of the challenging diagnosis [2] caused by the overlapping clinical presentation of CS vs the normal physiological state of hypercortisolism seen in pregnancy. In pregnant women, CS is most commonly adrenal in origin (50%-60% of cases), whereas in nonpregnant women pituitary Cushing disease is found in 70% of cases [2]. Left undiagnosed and untreated, CS can be detrimental both to maternal and fetal health [2].
Literature available to guide therapy is sparse, and no consensus has been established for optimal management of CS during pregnancy. Medical and surgical treatments were both found to be protective in avoiding fetal loss. Surgery tends to be preferred because of reduced complications at time of delivery. However, timing limits its safety to the second and early third trimester [2, 3].
Metyrapone, a steroidogenesis inhibitor, has been safely used in pregnancy for the medical treatment of CS [4, 5]. Metyrapone passes through the placental barrier and may potentially affect adrenal steroid production by the fetus [6]. There are no documented fetal complications with its use; however, transplacental studies have not been reported in humans. Animal studies have demonstrated cross-placental transfer of metyrapone without detrimental effect [7]. Risk of worsening preexisting hypertension leading to preeclampsia has been reported because of increase in the 11-deoxycortisol precursor. This case report explores the challenges of medical management of adrenal CS in pregnancy when surgical intervention is not possible. Metyrapone was used to reduce serum and saliva cortisol levels to the upper limits of expected pregnancy ranges. Unique to this case, additional analyses were performed to measure increase in 11-deoxycortisol as well as analysis of cord blood samples to assess cross-placental transfer of metyrapone.
1. Clinical Case
A 24-year-old pregnant women was referred at 25 weeks’ gestation for concerns about excess cortisol secretion. Her symptoms included rapid weight gain, hirsutism, chronic hypertension, generalized weakness, fatigue, and widespread violaceous striations. Her medications included prenatal vitamins with folic acid, daily low-dose aspirin, and nifedipine XL 60 mg daily. Six years prior to presentation, her first pregnancy was complicated by gestational diabetes, 32-kg weight gain, hypertension, and an eclamptic seizure necessitating an emergency cesarean delivery at 33 weeks’ gestation in a community hospital. A baby boy weighing 3 pounds and 9 ounces was delivered. Post partum, her hypertension persisted and widespread violaceous striations did not substantially improve. Over the next years her obesity remained and she continued antihypertensive treatment with long-acting nifedipine. A glucose tolerance test was normal, and she was followed by her family health team.
At presentation during the second pregnancy, she appeared cushingoid, with a round and flushed face, hirsutism, supraclavicular and dorsal fat pads, widespread violaceous striae, and mild proximal myopathy. Her weight was 123.1 kg with a body mass index of 44 kg/m2, blood pressure of 145/108 mm Hg, and heart rate of 122 per minute. Laboratory investigations showed an increased 24-hour urine cortisol excretion of 1141 nmol/day (nonpregnancy reference range < 275), increased salivary cortisol of 44.5 nmol/L at 8 am and 61.1 nmol/L at 8 pm (< 24.1 nmol/L for 6-8 am and < 9.7 for 4-8 pm) showing loss of diurnal variation, suppressed morning adrenocorticotropin (ACTH) (< 0.3 pmol/L) and low DHEAS (dehydroepiandrosterone sulfate) of 1.2 µmol/L (range, 2.7-9.2 µmol/L). A 2-hour 75-g glucose tolerance test confirmed diabetes mellitus. Testing of 24-hour urinary excretion of catecholamines and metanephrines was normal. Magnetic resonance imaging of the abdomen showed a 3.7-cm left adrenal adenoma.
A diagnosis of adrenal ACTH-independent CS during pregnancy was made. A multidisciplinary case conference, with representation of departments of adult endocrinology, general surgery, maternal fetal medicine, neonatology, anesthesia, and clinical pharmacology, was organized. Although the literature would favor early surgical adrenalectomy during pregnancy, this patient was considered a high-risk surgical candidate because of her high body mass index (possibly necessitating conversion from laparoscopic to open surgery) and the concern for inferior vena cava compression by the gravid uterus when placed in the right lateral decubitus position.
Therefore, we elected to treat this patient medically but decided against ketoconazole because of its reported embryotoxicity [8]. We started the patient on metyrapone, an inhibitor of 11β-hydroxylase in the steroidogenesis pathway of the adrenal cortex, resulting in a reduction in serum cortisol. Because the patient lived in a remote area, this was administered during hospital admission in our center. We performed serial measurements of plasma metyrapone, cortisol and 11-deoxycortisol, and salivary cortisol. We also measured metyrapone, cortisol, and 11-deoxycortisol in fetal (umbilical artery [UA] and vein) and maternal plasma during delivery.
We measured plasma concentrations of metyrapone, cortisol, and 11-deoxycortisol by liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (50 μL) were combined with 150 μL of acetonitrile containing internal standards (10 ng/mL alprazolam and 350 ng/mL D4-cortisol). Alprazolam served as the internal standard for metyrapone, while D4-cortisol was the internal standard used for the analysis of cortisol and 11-deoxycortisol. After centrifugation, the resulting supernatants were dried in a SpeedVac and reconstituted with 150 μL of acetonitrile/water (5%/95%) containing 0.1% formic acid for injection into the liquid chromatograph (Agilent 1100). Analytes were separated with gradient elution using mobile phases A (water with 1% formic acid) and B (acetonitrile with 1% formic acid) on a Hypersil Gold C18 column (50 × 5 mm, 5 μm, Thermo Scientific). Retention times for metyrapone, alprazolam, cortisol, and 11- deoxycortisol were 2.9 minutes, 4.3 minutes, 4.3 minutes, and 4.3 minutes, respectively. Solutes were analyzed by tandem mass spectrometry (TSQ Vantage, Thermo Scientific) by electrospray ionization and detection in positive mode. For metyrapone, alprazolam, cortisol, D4-cortisol and 11-deoxycortisol, the mass transitions used were 227.2 → 121.1 m/z, 309.0 → 280.9 m/z, 363.2 → 121.1 m/z, 367.2 → 121.1 m/z and 347.0 → 97.0 m/z, respectively. Standard curve samples were prepared in charcoal-stripped plasma (BioIVT) and processed in a similar fashion as patient samples. For metyrapone, the limit of detection (LOD) was 0.05 ng/mL, the limit of quantitation (LOQ) was 0.2 ng/mL, precision (coefficient of variation %) was 9.3%, and bias was 11.4%. For cortisol, LOD was 2 ng/mL, LOQ was 5 ng/mL, precision was 2.0%, and bias was 2.3%. Last, for 11-deoxycortisol, LOD was 0.2 ng/mL, LOQ was 0.5 ng/mL, precision was 7.4%, and bias was 9.4%.
As shown in Table 1, a single dose of metyrapone 250 mg resulted in rapid metyrapone absorption within 1 hour, with levels returning to barely detectable 24 hours after ingestion. Maternal and fetal vital signs remained stable during this period. Similar analysis after a cumulative dose of 750 mg demonstrated mean peak plasma concentrations of 4.9 ng/mL.
Table 1. Metyrapone—effect of an initial single 250-mg dose and cumulative 1000-mg dose
Time Metyrapone, ng/mL Cortisol, nmol/L Cortisol saliva, nmol/L 11-Deoxycortisol, ng/mL
Day 1
Baseline 12:30 0 1036 48.4 1.7
1 h post 13:30 35.9 820 28.6 12.4
8 h post 20:30 2.4 1015 40.4 2.8
24 h post 12:30 0.1 950 43.1 0.9
Day 3
Pre-fifth dose 00:30 1.7 1096 3.1
1 h post 01:35 4.9 1141 2.8
The patient received 250 mg metyrapone at 12:30 on day 1, at 8:30 and 16:30 on day 2, and at 00:30 on day 3. All measurements were taken in plasma.
The metyrapone dose was gradually increased to 250 mg TID. This dose resulted in a steady decline both of plasma and salivary cortisol concentrations (Fig. 1), approaching serum cortisol levels around 800 nmol/L and saliva cortisol levels around 30 pmol/L, both in the upper half of the normal pregnancy range [9]. Hypertension remained controlled on nifedipine.
Figure 1. Steady decline both in A, plasma, and B, morning salivary cortisol readings were observed during successful control of hypercortisolism with metyrapone therapy. Note that serum cortisol levels reflect total cortisol (including protein-bound cortisol), whereas salivary cortisol levels reflect free cortisol concentrations.
A. Maternal Outcomes
The overall condition of the patient, including control of gestational diabetes and hypertension, improved during metyrapone therapy. An elective repeat cesarean delivery was performed at 35 weeks’ gestation, after balancing the risks of further maternal medical decompensation with those of late preterm delivery. A healthy male infant was delivered without complication. Serum cortisol level at time of delivery was 2398 nmol/L, remaining around the upper limit of normal pregnancy levels. Postpartum transition to more affordable ketoconazole therapy was well tolerated. Laparoscopic left adrenalectomy was performed without complications at 10 weeks post delivery. Since then, there has been notable improvement of the patient’s initial Cushing features. She was treated with a tapering dose of hydrocortisone for 6 months, at which time she had a normal functioning pituitary–right adrenal axis. Her cushingoid symptoms gradually improved and her weight decreased to 103 kg, her blood pressure normalized to 115/83 mm Hg off medications, and further biochemical evaluation showed normal cortisol secretion.
B. Fetal Outcomes
Fetal growth, well-being, and placental function were monitored during metyrapone treatment using serial ultrasounds. Estimated fetal weights were measured every 3 weeks, with growth parameters remaining within the normal range (70th percentile at 26 weeks, 50th percentile at 35 weeks). Biophysical profile scores and UA Dopplers were performed weekly and consistently within normal limits (UA pulsatility index < 95th percentile).
A male infant was born at 35 weeks’ gestation with a weight of 2.41 kg (33rd percentile) and an Apgar score 9 both at 1 and 5 minutes. There were no signs suggestive of CS or hyperandrogenism, and no neonatal complications. On the first day of life, a random evening venous cortisol concentration was low at 5 nmol/L (normal range, 68-327 nmol/L). On the third day of life, the baseline ACTH was 9.12 pmol/L (range, 1.98-2.47 pmol/L), and the cortisol level improved to 41 nmol/L. A peak cortisol value of 214 nmol/L was observed after ACTH stimulation. The child maintained a normal electrolytes profile and was clinically stable. Therefore, these results were interpreted as appropriate for the child’s gestational age. Defining normal cortisol values is difficult in the neonatal period as studies have shown a large range of reference values [10]. There was no evidence of hypoglycemia, hyponatremia, hyperkalemia, lethargy, or vomiting. He was discharged home on the fourth day of life. He was seen by a pediatric endocrinologist at age 4 weeks and was noted to have had appropriate weight gain, regular feeding patterns, and a normal morning cortisol level (154 nmol/L). There was no clinical evidence of hyperandrogenism. He was discharged from endocrinology follow-up to primary care.
The results of umbilical cord sampling are shown in Table 2 and demonstrate that metyrapone does cross the placenta, with the fetal venous cord concentration being only slightly lower than the maternal venous concentration, and the fetal arterial cord concentration being about 60% of the fetal venous cord concentration.
Table 2. Maternal and fetal effects of metyrapone therapy at delivery
Time Metyrapone, ng/mL Cortisol, nmol/L 11-Deoxycortisol, ng/mL
Sample
Mother 15:00 19.8 2398 7.1
Venous cord 15:00 16.1 843 4.8
Arterial cord 15:00 9.4 924 3.8
All measurements were taken in plasma.
2. Discussion
We describe a patient with adrenal CS diagnosed at the end of the second trimester who elected treatment with metyrapone. Unique features of this case include the monitoring of salivary cortisol in addition to serum cortisol, as well as monitoring of maternal and fetal levels of cortisol and metyrapone at the time of delivery.
Systematic review of published cases until April 2015 have found only 220 patients (263 pregnancies) with active CS in pregnancy [2, 6]. The diagnosis of CS is also confounded by the physiological state of hypercortisolism normally seen in pregnancy. Serum cortisol levels have been noted to increase 2 to 3 times the upper limit of normal, and urine cortisol can rise 180% in pregnancy. Lack of suppression with dexamethasone is often seen because of persistent unsuppressed placental ACTH secretion. The salivary diurnal variation, however, seems to be preserved during pregnancy [9]. Early recognition of CS in pregnancy is imperative. Caimari et al [2] demonstrated a higher risk of gestational diabetes (36.9 vs 2.3% P = .003), gestational hypertension (40.5 vs 2.3% P < .001), and preeclampsia (26.3 vs 2.3% P = .001) in active compared to cured pregnant patients. Fetal outcomes were also negatively affected, with higher rates of fetal loss (23.6 vs 8.5% P = .021) and global fetal morbidities (33.3 vs 4.9% P < .001). The most common fetal morbidities include premature birth, intrauterine growth restriction, stillbirths and, rarely, adrenal insufficiency. No fetal hypercortisolism was reported [2]. Other maternal complications noted in the literature included poor wound healing, osteoporosis, pathological fractures, cardiac failure and, rarely, maternal mortality.
Although the primary treatment of adrenal CS in pregnancy is adrenalectomy, we elected to treat the patient with metyrapone because of the gestational age at diagnosis and the concerns about surgical complications due to maternal body habitus. Serum cortisol levels were monitored but, because serum cortisol measures total cortisol including protein-bound cortisol, interpretation remains challenging. Ambroziak and colleagues [9] described salivary cortisol levels specified by trimester, and reported a morning saliva cortisol of 21.9 nmol/L (range, 8.9-39.7 nmol/L [mean and 2.5 and 97.5th percentile]). During the patient’s metyrapone treatment, we were able to achieve morning saliva cortisol levels in the upper half of this third trimester–specific range. Worsening hypertension and risk of preeclampsia are known risks of metyrapone use. It has been suggested that the increased levels of precursor 11-deoxycortisol during the inhibition of 11β-hydroxylase by metyrapone cause sodium retention, leading to hypertension. In this patient, with the use of liquid chromatography-tandem mass spectrometry, 11-deoxycortisol levels were measured and a slight increase was observed. However, her blood pressure improved once she was on the metyrapone and remained stable throughout the pregnancy. 11-Deoxycortisol may have some glucocorticoid activity (about 15% of that of cortisol) [11] but does not have mineralocorticoid activity. However, the hypertension and hypokalemia may primarily be caused by 11-deoxycorticosterone, which has mineralocorticoid but no glucocorticoid activity [12], and which would be increased due to inhibition of 11β-hydroxylase activity [13]. In the present study we did not measure 11-deoxycorticosterone levels.
In addition, immunoassays for cortisol may have cross-reactivity for 11-deoxycortisol. This cross-reactivity is reported to be low at 4.6%, but indicated to be clinically relevant in patients with 11β-hydroxylase deficiency or following metyrapone challenge [14]. Therefore, the use of liquid chromatography-mass spectrometry to measure 11-deoxycortisol was also important to reduce the risk of overtreatment and potential adrenal insufficiency both for mother and fetus.
We are not aware of any previous human transplacental data on metyrapone. Animal models demonstrate a 50% placental transfer of metyrapone with no pronounced effects on adrenal function [7]. Our data demonstrate that metyrapone does cross the placenta in humans. The metyrapone concentration in venous cord blood was 16.1 ng/mL and 9.4 ng/mL in arterial cord blood. This implies that the theoretical risk of fetal steroid synthesis inhibition is real, with a potential risk of fetal adrenal insufficiency. Indeed, on day 3 of life, neonatal cortisol levels were suboptimal both at baseline and after stimulation, demonstrating a likely transient effect of metyrapone on fetal cortisol production. However, there were no clinical or other biochemical signs of adrenal insufficiency, and this child has never required glucocorticoid administration. Although in this particular case there were no signs of neonatal adrenal insufficiency, we were able to confirm that fetal exposure to metyrapone does occur and careful monitoring of the neonatal hypothalamic-pituitary-adrenal axis post delivery remains important.
The metyrapone plasma concentrations described in the product monograph were based on data published in abstract format in 1967 [15]. Concentrations were measured after a single dose of 750 mg, but the analytic methods were not described, making any direct comparison with our results difficult. The plasma concentrations in our patient are similar to what has been reported for metyrapone in breast milk [16].
During her first pregnancy, our patient developed symptoms and complications that could potentially be explained by CS. Hána and colleagues [17] described a patient with ACTH-independent CS that developed during pregnancy and went into remission within 3 weeks of delivery, with the same pattern developing during her next 2 pregnancies. Searching for an underlying mechanism, Andreescu and colleagues [18] described the presence of abnormal cortisol responses in 3 pregnant patients with CS due to an adrenal adenoma. These patients had an abnormal cortisol response to luteinizing hormone–releasing hormone and human chorionic gonadotropin, and suppressed ACTH levels, and developed CS during pregnancy. Other rare causes of pregnancy-induced CS included placental corticotropin-releasing hormone synthesis or estrogen-dependent nodular adrenal hyperplasia [19]. It is possible that similar mechanisms may explain, at least in part, the presentation in our patient. However, because her symptoms did not completely resolve between pregnancies, this is unlikely to completely explain our patient’s pathophysiology.
In conclusion, this case report illustrates the additional complexity of CS management when detected in late pregnancy. Our case report clearly illustrates that a multidisciplinary approach is critical because it provides the information and expertise required to carefully balance maternal and fetal risks. In a high-risk surgical patient, the literature on medical therapy is sparse. Therefore, multiple key endocrine points required exploration, including the safety of medical treatment in pregnancy, the availability and effectiveness of metyrapone in pregnancy, as well as unknown fetal risk. We have demonstrated the use of saliva cortisol for monitoring the effect of metyrapone, and demonstrated, for the first time, that metyrapone crosses the placenta in humans.
Abbreviations
ACTH adrenocorticotropin
CS Cushing syndrome
LOD limit of detection
LOQ limit of quantitation
UA umbilical artery
Additional Information
Disclosure Summary: The authors have nothing to disclose.
Data Availability
Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study. | Recovered | ReactionOutcome | CC BY-NC-ND | 33305159 | 18,699,300 | 2021-01-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Haemorrhage'. | Undifferentiated carcinoma of the liver in a 3-year-old girl treated by neoadjuvant chemotherapy and complete resection.
BACKGROUND
Undifferentiated carcinoma (UC) of the liver has only been reported in three adults in the English language literature and is so rare it has never been reported in a child. Our management is presented to improve knowledge of its treatment.
METHODS
A 3-year-old previously well Japanese girl was referred for further assessment/management of an abdominal mass. On examination an obvious right hypocostal mass was visible extending across the midline. Diagnostic imaging identified a 12.5 cm mass on the ventral surface of the liver containing multiple cystic lesions extending along Glisson's capsule with invasion to the portal vein. Open biopsy eventually led to a diagnosis of poorly differentiated or UC of the liver with embryonal features. Resection of hepatic segments 4b and 5 after a remarkable initial response to cisplatin/doxorubicin that shrank the tumor substantially, separating it from Glisson's capsule enabled total excision. Surgery was successful and tolerated well with unremarkable postoperative recovery. Unfortunately, ascites due to peritoneal carcinomatosis developed 4 months postoperatively and she died 5 months later.
CONCLUSIONS
The initial impressive response to neoadjuvant chemotherapy and successful surgery was unexpectedly fortuitous but inadequate for controlling such an aggressive malignancy. Our case demonstrates the value of neoadjuvant chemotherapy.
1 Introduction
Undifferentiated carcinoma (UC) of the liver is an extremely rare, aggressively malignant condition reported in only 3 adults in the English literature [[1], [2], [3]]. Nakasuka et al. [1] reported a case with neuroendocrine features characterised by high serum neuron-specific enolase (NSE) that responded well to etoposide + cisplatin (CDDP) with NSE decreasing to normal. Maeda et al. [2] reported a case of fatal acute intra-abdominal haemorrhage from a ruptured liver, that was found to be caused by UC of the liver with diffuse proliferation of anaplastic cells positive for NSE suggesting neuroendocrine differentiation at autopsy; and Hiraki et al. [3] reported a case who had a mass with cystic and partially solid components located in segments 6 and 7 of the liver removed successfully by radical surgery alone, without neoadjuvant or adjuvant chemotherapy, who is well, with no evidence of recurrence some three years postoperatively. Due to its rarity, the natural history (typical mode of presentation, routine investigations, and histopathologic/biologic characteristics) of UC of the liver and management strategies have not been established. To the best of our knowledge this is the first report of UC of the liver in a child.
2 Presentation of case
A previously well 3-year-old Japanese girl of average height and weight was referred to our hospital for further management of an abdominal mass. She had no drug history and no relevant family history. No patient perspective was possible given the age of our case.
On examination at presentation, there was an obvious right hypocostal mass that extended across the midline. She was admitted and blood biochemistry and diagnostic radiology investigations were ordered to obtain basic information about the mass. Serum lactate dehyrogenase was elevated (LDH; 1,339 IU/L) and while specific serum tumor markers such as NSE (224 ng/mL), soluble interleukin-2 receptor (sIL-2R; 982 IU/mL), CA125 (315 IU/mL), CA19-9 (51 IU/mL), and PIVKA-II (84mAU/mL) were elevated, alpha-foetoprotein (AFP; 4 ng/mL) and CEA (1.5 ng/dL) were normal. Computed tomography (CT) confirmed a mass, 12.5 cm in maximum diameter, located on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein (Fig. 1A).Fig. 1 Change in tumor size after neoadjuvant chemotherapy.
A. Computed tomography (CT) scan on admission. The tumor is on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein.
B. CT scan 4 weeks after admission; pre-chemotherapy.
C. CT scan 8 weeks after admission; after 2 cycles of CDDP + DOX.
D. CT scan 16 weeks after admission; after 5 cycles of CDDP + DOX. The tumor has shrunk remarkably separating from Glisson’s capsule.
Fig. 1
Open biopsy of the mass was performed two days after admission to avoid complications of needle biopsy such as bleeding. Microscopic sections of biopsy specimens showed diffuse infiltration of tumor cells with nuclear pleomorphism and cohesiveness (Fig. 2A and B) with no specific structure or differentiation. Immunohistochemistry was positive for epithelial markers (pan cytokeratin (CK) antibodies AE1/AE3, CK19, CK7), vimentin, SALL4, Glypican3, CD10 and p53, while negative for CK20, EMA, CD30, AFP, HCG-β, HepPar1, D2-40, CDX2, β-catenin and p40. A definitive diagnosis could not be made, but a germ cell tumor or carcinoma with germ cell differentiation was suspected because SALL4 and Glypican3 were both positive. Biopsy specimens were sent for Central Pathology Review by paediatric tumor pathology specialists. Additional immunostaining was performed with SMARCB1, SMARCA2, SMARCA4, ARID1A, and LIN28 being positive and Oct3/4, CD117, DLK1, and vimentin being negative, a reflection of how poorly differentiated the tumor was. Their diagnosis, which took almost a month to be finalised, was poorly differentiated or UC of the liver with embryonal features.Fig. 2 Histopathology of open liver (tumor) biopsies.
Micrographs of open liver (tumor) biopsy (A: HE x12.5; B: HE x400) showing diffuse infiltration of tumor cells with nuclear pleomorphism.
Fig. 2
During this time, the tumor had grown to 14.5 cm in maximum diameter (Fig. 1B) with enlargement of multiple mediastinal lymph nodes. Gallium scintigraphy was positive for the liver tumor and the mediastinum. Preoperative liver CT volumetry [4] indicated that the standard liver volume would be less than 20% following surgical resection, a level consistent with high postoperative morbidity and liver failure [5]. Thus, her tumor was group III [6] using the pretreatment extent of disease (PRETEXT) system for classifying primary hepatic malignancies in children, which meant that the tumor was inoperable and that she was also not a viable candidate for primary liver transplantation (LTx).
In the absence of guidelines for the treatment of UC of the liver, we referred to treatment options for hepatoblastoma (HB) and commenced neoadjuvant chemotherapy with cisplatin/doxorubicin (CDDP + DOX) [7,8] starting with one dose of CDDP (4.0 mg/kg/dose) and two doses of DOX (2.0 mg/kg/dose). The response was immediate and impressive. A further four cycles of one dose of CDDP (5.2 mg/kg/dose) and two doses of DOX (2.6 mg/kg/dose) followed by one dose of CDDP (5.2 mg/kg/dose) without DOX to prevent DOX-induced cardiomyopathy shrank the tumor so remarkably (Fig. 1C and D) that it separated from Glisson’s capsule to become a post-treatment extent of disease (POSTEXT) II [6] tumor that was excised completely by open resection of segments 4b and 5 performed by a team comprised of two board-certified paediatric surgeons, one with 34 years’ experience (AY) and one with 11 years’ experience (TO) and one board certified adult hepatobiliary pancreatic surgeon with 26 years’ experience for supervisory support (AS). During surgery, ultrasonography was used as shown in Fig. 3A to confirm the relationship of the tumor to vascular structures and guide dissection for resection. The Pringle maneuver was used during transection (Fig. 3B) enabling the tumor to be excised completely (Fig. 3C). Neither mediastinal dissemination nor ascites were noted. No additional procedures were considered. The abdominal cavity was irrigated and there were no signs of bleeding; a drainage tube was not placed. Total operative time was 5 h and 20 min. Blood loss was 60 mL. The patient tolerated surgery well with no perioperative complications and uneventful postoperative recovery. Adjuvant chemotherapy was not indicated.Fig. 3 Operative findings.
Intraoperative ultrasound was used to confirm the relationship of the tumor to vascular structures, and guide dissection for resection (A). The Pringle maneuver was used during transection (B), and tumor was excised completely by resecting segments 4b and 5 (C).
Fig. 3
Histopathology confirmed all surgical margins were negative for malignancy (Fig. 4A). Microscopic sections showed numerous diffuse cystic lesions lined with tumor cells with focal papillary projections into the cystic spaces (Fig. 4B). Scattered necrosis/haemorrhage secondary to neoadjuvant chemotherapy was observed. Immunohistochemical staining was positive for AE1/AE3, SALL4, Glypican3, CD10 and EMA, and negative for AFP, CD30, HCG-β, HepPar1, CDX2 and podoplanin.Fig. 4 Histopathology of resected specimens.
Macroscopic appearance (A) and micrographs (B: HE x200) of the resected specimen showing cysts lined by tumor cells with focal papillary projections.
Fig. 4
Some four months after surgery, she developed ascites secondary to peritoneal carcinomatosis. Unfortunately, her creatinine clearance deteriorated after only two doses of CDDP (5.8 mg/kg/dose). She was discharged with no active treatment plan about one month before she eventually passed away, nine months after surgery.
The work has been reported in line with the Scare 2018 criteria [9].
3 Discussion
Two of the three cases in the literature had neuroendocrine features and were diffuse and one was more solitary and amenable to surgery. Our case had multiple cystic lesions with partial solid components on CT which would categorise her as being of the solitary type, but her tumor was considered inoperable because of extensive spread within the liver and insufficient volume of future liver remnant.
While some studies recommend LTx for HB patients with PRETEXT/POSTEXT groups III and IV, claiming five-year survival rates of 77%–100% [[10], [11], [12]], others recommend extended hepatic resection, also claiming excellent overall survival rates of 80%–88% [13,14]. We do not know if LTx could have prevented metastases or if immunosuppressants would have been problematic, but in hindsight, complete surgical resection may have been too optimistic for such an aggressive tumor because even though neoadjuvant chemotherapy would have weakened the tumor and all resected edges were negative for abnormal histopathology, she succumbed to overwhelming metastases.
4 Conclusion
UC should be included in the provisional diagnosis of a child with a right upper quadrant mass with or without ascites. Its rarity has prevented appropriate chemotherapeutic and surgical strategies from being established, however, our case demonstrates the importance of gross inspection as a clinical skill to identify masses as early as possible and the value of neoadjuvant chemotherapy for facilitating surgical intervention.
Declaration of Competing Interest
The authors report no declarations of interest.
Funding
None.
Ethical approval
This case report was approved by the Ethics Committee of Juntendo University School of Medicine (approval number: JHS20-010).
Consent
Written informed consent was obtained from the patient’s parents for preparation and publication of this case report and accompanying images.
There is no identifying characteristics in the manuscript.
Author contribution
Akio Saiura, Atsuyuki Yamataka, and Takanori Ochi performed surgery on our patient. Takanori Ochi wrote the manuscript with help from Junya Fujimura and Atsushi Arakawa. Geoffrey J. Lane, Atsuyuki Yamataka, and Akio Saiura edited and revised the manuscript and prepared it for submission.
Registration of research studies
Not applicable.
Guarantor
The Guarantor of this case report is Takanori Ochi (Associate Professor of the Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine).
Acknowledgements
We wish to express our appreciation to Eri Abe, Yuichiro Miyake, and Osamu Tomita for their contributions and commitment to the care of our patient and Shiho Yoshida, Hiroyuki Koga, and Toshiaki Shimizu for their support and encouragement in preparing this report. | CISPLATIN, DOXORUBICIN HYDROCHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33310474 | 19,165,864 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Necrosis'. | Undifferentiated carcinoma of the liver in a 3-year-old girl treated by neoadjuvant chemotherapy and complete resection.
BACKGROUND
Undifferentiated carcinoma (UC) of the liver has only been reported in three adults in the English language literature and is so rare it has never been reported in a child. Our management is presented to improve knowledge of its treatment.
METHODS
A 3-year-old previously well Japanese girl was referred for further assessment/management of an abdominal mass. On examination an obvious right hypocostal mass was visible extending across the midline. Diagnostic imaging identified a 12.5 cm mass on the ventral surface of the liver containing multiple cystic lesions extending along Glisson's capsule with invasion to the portal vein. Open biopsy eventually led to a diagnosis of poorly differentiated or UC of the liver with embryonal features. Resection of hepatic segments 4b and 5 after a remarkable initial response to cisplatin/doxorubicin that shrank the tumor substantially, separating it from Glisson's capsule enabled total excision. Surgery was successful and tolerated well with unremarkable postoperative recovery. Unfortunately, ascites due to peritoneal carcinomatosis developed 4 months postoperatively and she died 5 months later.
CONCLUSIONS
The initial impressive response to neoadjuvant chemotherapy and successful surgery was unexpectedly fortuitous but inadequate for controlling such an aggressive malignancy. Our case demonstrates the value of neoadjuvant chemotherapy.
1 Introduction
Undifferentiated carcinoma (UC) of the liver is an extremely rare, aggressively malignant condition reported in only 3 adults in the English literature [[1], [2], [3]]. Nakasuka et al. [1] reported a case with neuroendocrine features characterised by high serum neuron-specific enolase (NSE) that responded well to etoposide + cisplatin (CDDP) with NSE decreasing to normal. Maeda et al. [2] reported a case of fatal acute intra-abdominal haemorrhage from a ruptured liver, that was found to be caused by UC of the liver with diffuse proliferation of anaplastic cells positive for NSE suggesting neuroendocrine differentiation at autopsy; and Hiraki et al. [3] reported a case who had a mass with cystic and partially solid components located in segments 6 and 7 of the liver removed successfully by radical surgery alone, without neoadjuvant or adjuvant chemotherapy, who is well, with no evidence of recurrence some three years postoperatively. Due to its rarity, the natural history (typical mode of presentation, routine investigations, and histopathologic/biologic characteristics) of UC of the liver and management strategies have not been established. To the best of our knowledge this is the first report of UC of the liver in a child.
2 Presentation of case
A previously well 3-year-old Japanese girl of average height and weight was referred to our hospital for further management of an abdominal mass. She had no drug history and no relevant family history. No patient perspective was possible given the age of our case.
On examination at presentation, there was an obvious right hypocostal mass that extended across the midline. She was admitted and blood biochemistry and diagnostic radiology investigations were ordered to obtain basic information about the mass. Serum lactate dehyrogenase was elevated (LDH; 1,339 IU/L) and while specific serum tumor markers such as NSE (224 ng/mL), soluble interleukin-2 receptor (sIL-2R; 982 IU/mL), CA125 (315 IU/mL), CA19-9 (51 IU/mL), and PIVKA-II (84mAU/mL) were elevated, alpha-foetoprotein (AFP; 4 ng/mL) and CEA (1.5 ng/dL) were normal. Computed tomography (CT) confirmed a mass, 12.5 cm in maximum diameter, located on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein (Fig. 1A).Fig. 1 Change in tumor size after neoadjuvant chemotherapy.
A. Computed tomography (CT) scan on admission. The tumor is on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein.
B. CT scan 4 weeks after admission; pre-chemotherapy.
C. CT scan 8 weeks after admission; after 2 cycles of CDDP + DOX.
D. CT scan 16 weeks after admission; after 5 cycles of CDDP + DOX. The tumor has shrunk remarkably separating from Glisson’s capsule.
Fig. 1
Open biopsy of the mass was performed two days after admission to avoid complications of needle biopsy such as bleeding. Microscopic sections of biopsy specimens showed diffuse infiltration of tumor cells with nuclear pleomorphism and cohesiveness (Fig. 2A and B) with no specific structure or differentiation. Immunohistochemistry was positive for epithelial markers (pan cytokeratin (CK) antibodies AE1/AE3, CK19, CK7), vimentin, SALL4, Glypican3, CD10 and p53, while negative for CK20, EMA, CD30, AFP, HCG-β, HepPar1, D2-40, CDX2, β-catenin and p40. A definitive diagnosis could not be made, but a germ cell tumor or carcinoma with germ cell differentiation was suspected because SALL4 and Glypican3 were both positive. Biopsy specimens were sent for Central Pathology Review by paediatric tumor pathology specialists. Additional immunostaining was performed with SMARCB1, SMARCA2, SMARCA4, ARID1A, and LIN28 being positive and Oct3/4, CD117, DLK1, and vimentin being negative, a reflection of how poorly differentiated the tumor was. Their diagnosis, which took almost a month to be finalised, was poorly differentiated or UC of the liver with embryonal features.Fig. 2 Histopathology of open liver (tumor) biopsies.
Micrographs of open liver (tumor) biopsy (A: HE x12.5; B: HE x400) showing diffuse infiltration of tumor cells with nuclear pleomorphism.
Fig. 2
During this time, the tumor had grown to 14.5 cm in maximum diameter (Fig. 1B) with enlargement of multiple mediastinal lymph nodes. Gallium scintigraphy was positive for the liver tumor and the mediastinum. Preoperative liver CT volumetry [4] indicated that the standard liver volume would be less than 20% following surgical resection, a level consistent with high postoperative morbidity and liver failure [5]. Thus, her tumor was group III [6] using the pretreatment extent of disease (PRETEXT) system for classifying primary hepatic malignancies in children, which meant that the tumor was inoperable and that she was also not a viable candidate for primary liver transplantation (LTx).
In the absence of guidelines for the treatment of UC of the liver, we referred to treatment options for hepatoblastoma (HB) and commenced neoadjuvant chemotherapy with cisplatin/doxorubicin (CDDP + DOX) [7,8] starting with one dose of CDDP (4.0 mg/kg/dose) and two doses of DOX (2.0 mg/kg/dose). The response was immediate and impressive. A further four cycles of one dose of CDDP (5.2 mg/kg/dose) and two doses of DOX (2.6 mg/kg/dose) followed by one dose of CDDP (5.2 mg/kg/dose) without DOX to prevent DOX-induced cardiomyopathy shrank the tumor so remarkably (Fig. 1C and D) that it separated from Glisson’s capsule to become a post-treatment extent of disease (POSTEXT) II [6] tumor that was excised completely by open resection of segments 4b and 5 performed by a team comprised of two board-certified paediatric surgeons, one with 34 years’ experience (AY) and one with 11 years’ experience (TO) and one board certified adult hepatobiliary pancreatic surgeon with 26 years’ experience for supervisory support (AS). During surgery, ultrasonography was used as shown in Fig. 3A to confirm the relationship of the tumor to vascular structures and guide dissection for resection. The Pringle maneuver was used during transection (Fig. 3B) enabling the tumor to be excised completely (Fig. 3C). Neither mediastinal dissemination nor ascites were noted. No additional procedures were considered. The abdominal cavity was irrigated and there were no signs of bleeding; a drainage tube was not placed. Total operative time was 5 h and 20 min. Blood loss was 60 mL. The patient tolerated surgery well with no perioperative complications and uneventful postoperative recovery. Adjuvant chemotherapy was not indicated.Fig. 3 Operative findings.
Intraoperative ultrasound was used to confirm the relationship of the tumor to vascular structures, and guide dissection for resection (A). The Pringle maneuver was used during transection (B), and tumor was excised completely by resecting segments 4b and 5 (C).
Fig. 3
Histopathology confirmed all surgical margins were negative for malignancy (Fig. 4A). Microscopic sections showed numerous diffuse cystic lesions lined with tumor cells with focal papillary projections into the cystic spaces (Fig. 4B). Scattered necrosis/haemorrhage secondary to neoadjuvant chemotherapy was observed. Immunohistochemical staining was positive for AE1/AE3, SALL4, Glypican3, CD10 and EMA, and negative for AFP, CD30, HCG-β, HepPar1, CDX2 and podoplanin.Fig. 4 Histopathology of resected specimens.
Macroscopic appearance (A) and micrographs (B: HE x200) of the resected specimen showing cysts lined by tumor cells with focal papillary projections.
Fig. 4
Some four months after surgery, she developed ascites secondary to peritoneal carcinomatosis. Unfortunately, her creatinine clearance deteriorated after only two doses of CDDP (5.8 mg/kg/dose). She was discharged with no active treatment plan about one month before she eventually passed away, nine months after surgery.
The work has been reported in line with the Scare 2018 criteria [9].
3 Discussion
Two of the three cases in the literature had neuroendocrine features and were diffuse and one was more solitary and amenable to surgery. Our case had multiple cystic lesions with partial solid components on CT which would categorise her as being of the solitary type, but her tumor was considered inoperable because of extensive spread within the liver and insufficient volume of future liver remnant.
While some studies recommend LTx for HB patients with PRETEXT/POSTEXT groups III and IV, claiming five-year survival rates of 77%–100% [[10], [11], [12]], others recommend extended hepatic resection, also claiming excellent overall survival rates of 80%–88% [13,14]. We do not know if LTx could have prevented metastases or if immunosuppressants would have been problematic, but in hindsight, complete surgical resection may have been too optimistic for such an aggressive tumor because even though neoadjuvant chemotherapy would have weakened the tumor and all resected edges were negative for abnormal histopathology, she succumbed to overwhelming metastases.
4 Conclusion
UC should be included in the provisional diagnosis of a child with a right upper quadrant mass with or without ascites. Its rarity has prevented appropriate chemotherapeutic and surgical strategies from being established, however, our case demonstrates the importance of gross inspection as a clinical skill to identify masses as early as possible and the value of neoadjuvant chemotherapy for facilitating surgical intervention.
Declaration of Competing Interest
The authors report no declarations of interest.
Funding
None.
Ethical approval
This case report was approved by the Ethics Committee of Juntendo University School of Medicine (approval number: JHS20-010).
Consent
Written informed consent was obtained from the patient’s parents for preparation and publication of this case report and accompanying images.
There is no identifying characteristics in the manuscript.
Author contribution
Akio Saiura, Atsuyuki Yamataka, and Takanori Ochi performed surgery on our patient. Takanori Ochi wrote the manuscript with help from Junya Fujimura and Atsushi Arakawa. Geoffrey J. Lane, Atsuyuki Yamataka, and Akio Saiura edited and revised the manuscript and prepared it for submission.
Registration of research studies
Not applicable.
Guarantor
The Guarantor of this case report is Takanori Ochi (Associate Professor of the Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine).
Acknowledgements
We wish to express our appreciation to Eri Abe, Yuichiro Miyake, and Osamu Tomita for their contributions and commitment to the care of our patient and Shiho Yoshida, Hiroyuki Koga, and Toshiaki Shimizu for their support and encouragement in preparing this report. | CISPLATIN, DOXORUBICIN HYDROCHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33310474 | 19,165,864 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Neoplasm progression'. | Undifferentiated carcinoma of the liver in a 3-year-old girl treated by neoadjuvant chemotherapy and complete resection.
BACKGROUND
Undifferentiated carcinoma (UC) of the liver has only been reported in three adults in the English language literature and is so rare it has never been reported in a child. Our management is presented to improve knowledge of its treatment.
METHODS
A 3-year-old previously well Japanese girl was referred for further assessment/management of an abdominal mass. On examination an obvious right hypocostal mass was visible extending across the midline. Diagnostic imaging identified a 12.5 cm mass on the ventral surface of the liver containing multiple cystic lesions extending along Glisson's capsule with invasion to the portal vein. Open biopsy eventually led to a diagnosis of poorly differentiated or UC of the liver with embryonal features. Resection of hepatic segments 4b and 5 after a remarkable initial response to cisplatin/doxorubicin that shrank the tumor substantially, separating it from Glisson's capsule enabled total excision. Surgery was successful and tolerated well with unremarkable postoperative recovery. Unfortunately, ascites due to peritoneal carcinomatosis developed 4 months postoperatively and she died 5 months later.
CONCLUSIONS
The initial impressive response to neoadjuvant chemotherapy and successful surgery was unexpectedly fortuitous but inadequate for controlling such an aggressive malignancy. Our case demonstrates the value of neoadjuvant chemotherapy.
1 Introduction
Undifferentiated carcinoma (UC) of the liver is an extremely rare, aggressively malignant condition reported in only 3 adults in the English literature [[1], [2], [3]]. Nakasuka et al. [1] reported a case with neuroendocrine features characterised by high serum neuron-specific enolase (NSE) that responded well to etoposide + cisplatin (CDDP) with NSE decreasing to normal. Maeda et al. [2] reported a case of fatal acute intra-abdominal haemorrhage from a ruptured liver, that was found to be caused by UC of the liver with diffuse proliferation of anaplastic cells positive for NSE suggesting neuroendocrine differentiation at autopsy; and Hiraki et al. [3] reported a case who had a mass with cystic and partially solid components located in segments 6 and 7 of the liver removed successfully by radical surgery alone, without neoadjuvant or adjuvant chemotherapy, who is well, with no evidence of recurrence some three years postoperatively. Due to its rarity, the natural history (typical mode of presentation, routine investigations, and histopathologic/biologic characteristics) of UC of the liver and management strategies have not been established. To the best of our knowledge this is the first report of UC of the liver in a child.
2 Presentation of case
A previously well 3-year-old Japanese girl of average height and weight was referred to our hospital for further management of an abdominal mass. She had no drug history and no relevant family history. No patient perspective was possible given the age of our case.
On examination at presentation, there was an obvious right hypocostal mass that extended across the midline. She was admitted and blood biochemistry and diagnostic radiology investigations were ordered to obtain basic information about the mass. Serum lactate dehyrogenase was elevated (LDH; 1,339 IU/L) and while specific serum tumor markers such as NSE (224 ng/mL), soluble interleukin-2 receptor (sIL-2R; 982 IU/mL), CA125 (315 IU/mL), CA19-9 (51 IU/mL), and PIVKA-II (84mAU/mL) were elevated, alpha-foetoprotein (AFP; 4 ng/mL) and CEA (1.5 ng/dL) were normal. Computed tomography (CT) confirmed a mass, 12.5 cm in maximum diameter, located on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein (Fig. 1A).Fig. 1 Change in tumor size after neoadjuvant chemotherapy.
A. Computed tomography (CT) scan on admission. The tumor is on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein.
B. CT scan 4 weeks after admission; pre-chemotherapy.
C. CT scan 8 weeks after admission; after 2 cycles of CDDP + DOX.
D. CT scan 16 weeks after admission; after 5 cycles of CDDP + DOX. The tumor has shrunk remarkably separating from Glisson’s capsule.
Fig. 1
Open biopsy of the mass was performed two days after admission to avoid complications of needle biopsy such as bleeding. Microscopic sections of biopsy specimens showed diffuse infiltration of tumor cells with nuclear pleomorphism and cohesiveness (Fig. 2A and B) with no specific structure or differentiation. Immunohistochemistry was positive for epithelial markers (pan cytokeratin (CK) antibodies AE1/AE3, CK19, CK7), vimentin, SALL4, Glypican3, CD10 and p53, while negative for CK20, EMA, CD30, AFP, HCG-β, HepPar1, D2-40, CDX2, β-catenin and p40. A definitive diagnosis could not be made, but a germ cell tumor or carcinoma with germ cell differentiation was suspected because SALL4 and Glypican3 were both positive. Biopsy specimens were sent for Central Pathology Review by paediatric tumor pathology specialists. Additional immunostaining was performed with SMARCB1, SMARCA2, SMARCA4, ARID1A, and LIN28 being positive and Oct3/4, CD117, DLK1, and vimentin being negative, a reflection of how poorly differentiated the tumor was. Their diagnosis, which took almost a month to be finalised, was poorly differentiated or UC of the liver with embryonal features.Fig. 2 Histopathology of open liver (tumor) biopsies.
Micrographs of open liver (tumor) biopsy (A: HE x12.5; B: HE x400) showing diffuse infiltration of tumor cells with nuclear pleomorphism.
Fig. 2
During this time, the tumor had grown to 14.5 cm in maximum diameter (Fig. 1B) with enlargement of multiple mediastinal lymph nodes. Gallium scintigraphy was positive for the liver tumor and the mediastinum. Preoperative liver CT volumetry [4] indicated that the standard liver volume would be less than 20% following surgical resection, a level consistent with high postoperative morbidity and liver failure [5]. Thus, her tumor was group III [6] using the pretreatment extent of disease (PRETEXT) system for classifying primary hepatic malignancies in children, which meant that the tumor was inoperable and that she was also not a viable candidate for primary liver transplantation (LTx).
In the absence of guidelines for the treatment of UC of the liver, we referred to treatment options for hepatoblastoma (HB) and commenced neoadjuvant chemotherapy with cisplatin/doxorubicin (CDDP + DOX) [7,8] starting with one dose of CDDP (4.0 mg/kg/dose) and two doses of DOX (2.0 mg/kg/dose). The response was immediate and impressive. A further four cycles of one dose of CDDP (5.2 mg/kg/dose) and two doses of DOX (2.6 mg/kg/dose) followed by one dose of CDDP (5.2 mg/kg/dose) without DOX to prevent DOX-induced cardiomyopathy shrank the tumor so remarkably (Fig. 1C and D) that it separated from Glisson’s capsule to become a post-treatment extent of disease (POSTEXT) II [6] tumor that was excised completely by open resection of segments 4b and 5 performed by a team comprised of two board-certified paediatric surgeons, one with 34 years’ experience (AY) and one with 11 years’ experience (TO) and one board certified adult hepatobiliary pancreatic surgeon with 26 years’ experience for supervisory support (AS). During surgery, ultrasonography was used as shown in Fig. 3A to confirm the relationship of the tumor to vascular structures and guide dissection for resection. The Pringle maneuver was used during transection (Fig. 3B) enabling the tumor to be excised completely (Fig. 3C). Neither mediastinal dissemination nor ascites were noted. No additional procedures were considered. The abdominal cavity was irrigated and there were no signs of bleeding; a drainage tube was not placed. Total operative time was 5 h and 20 min. Blood loss was 60 mL. The patient tolerated surgery well with no perioperative complications and uneventful postoperative recovery. Adjuvant chemotherapy was not indicated.Fig. 3 Operative findings.
Intraoperative ultrasound was used to confirm the relationship of the tumor to vascular structures, and guide dissection for resection (A). The Pringle maneuver was used during transection (B), and tumor was excised completely by resecting segments 4b and 5 (C).
Fig. 3
Histopathology confirmed all surgical margins were negative for malignancy (Fig. 4A). Microscopic sections showed numerous diffuse cystic lesions lined with tumor cells with focal papillary projections into the cystic spaces (Fig. 4B). Scattered necrosis/haemorrhage secondary to neoadjuvant chemotherapy was observed. Immunohistochemical staining was positive for AE1/AE3, SALL4, Glypican3, CD10 and EMA, and negative for AFP, CD30, HCG-β, HepPar1, CDX2 and podoplanin.Fig. 4 Histopathology of resected specimens.
Macroscopic appearance (A) and micrographs (B: HE x200) of the resected specimen showing cysts lined by tumor cells with focal papillary projections.
Fig. 4
Some four months after surgery, she developed ascites secondary to peritoneal carcinomatosis. Unfortunately, her creatinine clearance deteriorated after only two doses of CDDP (5.8 mg/kg/dose). She was discharged with no active treatment plan about one month before she eventually passed away, nine months after surgery.
The work has been reported in line with the Scare 2018 criteria [9].
3 Discussion
Two of the three cases in the literature had neuroendocrine features and were diffuse and one was more solitary and amenable to surgery. Our case had multiple cystic lesions with partial solid components on CT which would categorise her as being of the solitary type, but her tumor was considered inoperable because of extensive spread within the liver and insufficient volume of future liver remnant.
While some studies recommend LTx for HB patients with PRETEXT/POSTEXT groups III and IV, claiming five-year survival rates of 77%–100% [[10], [11], [12]], others recommend extended hepatic resection, also claiming excellent overall survival rates of 80%–88% [13,14]. We do not know if LTx could have prevented metastases or if immunosuppressants would have been problematic, but in hindsight, complete surgical resection may have been too optimistic for such an aggressive tumor because even though neoadjuvant chemotherapy would have weakened the tumor and all resected edges were negative for abnormal histopathology, she succumbed to overwhelming metastases.
4 Conclusion
UC should be included in the provisional diagnosis of a child with a right upper quadrant mass with or without ascites. Its rarity has prevented appropriate chemotherapeutic and surgical strategies from being established, however, our case demonstrates the importance of gross inspection as a clinical skill to identify masses as early as possible and the value of neoadjuvant chemotherapy for facilitating surgical intervention.
Declaration of Competing Interest
The authors report no declarations of interest.
Funding
None.
Ethical approval
This case report was approved by the Ethics Committee of Juntendo University School of Medicine (approval number: JHS20-010).
Consent
Written informed consent was obtained from the patient’s parents for preparation and publication of this case report and accompanying images.
There is no identifying characteristics in the manuscript.
Author contribution
Akio Saiura, Atsuyuki Yamataka, and Takanori Ochi performed surgery on our patient. Takanori Ochi wrote the manuscript with help from Junya Fujimura and Atsushi Arakawa. Geoffrey J. Lane, Atsuyuki Yamataka, and Akio Saiura edited and revised the manuscript and prepared it for submission.
Registration of research studies
Not applicable.
Guarantor
The Guarantor of this case report is Takanori Ochi (Associate Professor of the Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine).
Acknowledgements
We wish to express our appreciation to Eri Abe, Yuichiro Miyake, and Osamu Tomita for their contributions and commitment to the care of our patient and Shiho Yoshida, Hiroyuki Koga, and Toshiaki Shimizu for their support and encouragement in preparing this report. | CISPLATIN, DOXORUBICIN HYDROCHLORIDE | DrugsGivenReaction | CC BY-NC-ND | 33310474 | 19,165,864 | 2021-01 |
What was the outcome of reaction 'Neoplasm progression'? | Undifferentiated carcinoma of the liver in a 3-year-old girl treated by neoadjuvant chemotherapy and complete resection.
BACKGROUND
Undifferentiated carcinoma (UC) of the liver has only been reported in three adults in the English language literature and is so rare it has never been reported in a child. Our management is presented to improve knowledge of its treatment.
METHODS
A 3-year-old previously well Japanese girl was referred for further assessment/management of an abdominal mass. On examination an obvious right hypocostal mass was visible extending across the midline. Diagnostic imaging identified a 12.5 cm mass on the ventral surface of the liver containing multiple cystic lesions extending along Glisson's capsule with invasion to the portal vein. Open biopsy eventually led to a diagnosis of poorly differentiated or UC of the liver with embryonal features. Resection of hepatic segments 4b and 5 after a remarkable initial response to cisplatin/doxorubicin that shrank the tumor substantially, separating it from Glisson's capsule enabled total excision. Surgery was successful and tolerated well with unremarkable postoperative recovery. Unfortunately, ascites due to peritoneal carcinomatosis developed 4 months postoperatively and she died 5 months later.
CONCLUSIONS
The initial impressive response to neoadjuvant chemotherapy and successful surgery was unexpectedly fortuitous but inadequate for controlling such an aggressive malignancy. Our case demonstrates the value of neoadjuvant chemotherapy.
1 Introduction
Undifferentiated carcinoma (UC) of the liver is an extremely rare, aggressively malignant condition reported in only 3 adults in the English literature [[1], [2], [3]]. Nakasuka et al. [1] reported a case with neuroendocrine features characterised by high serum neuron-specific enolase (NSE) that responded well to etoposide + cisplatin (CDDP) with NSE decreasing to normal. Maeda et al. [2] reported a case of fatal acute intra-abdominal haemorrhage from a ruptured liver, that was found to be caused by UC of the liver with diffuse proliferation of anaplastic cells positive for NSE suggesting neuroendocrine differentiation at autopsy; and Hiraki et al. [3] reported a case who had a mass with cystic and partially solid components located in segments 6 and 7 of the liver removed successfully by radical surgery alone, without neoadjuvant or adjuvant chemotherapy, who is well, with no evidence of recurrence some three years postoperatively. Due to its rarity, the natural history (typical mode of presentation, routine investigations, and histopathologic/biologic characteristics) of UC of the liver and management strategies have not been established. To the best of our knowledge this is the first report of UC of the liver in a child.
2 Presentation of case
A previously well 3-year-old Japanese girl of average height and weight was referred to our hospital for further management of an abdominal mass. She had no drug history and no relevant family history. No patient perspective was possible given the age of our case.
On examination at presentation, there was an obvious right hypocostal mass that extended across the midline. She was admitted and blood biochemistry and diagnostic radiology investigations were ordered to obtain basic information about the mass. Serum lactate dehyrogenase was elevated (LDH; 1,339 IU/L) and while specific serum tumor markers such as NSE (224 ng/mL), soluble interleukin-2 receptor (sIL-2R; 982 IU/mL), CA125 (315 IU/mL), CA19-9 (51 IU/mL), and PIVKA-II (84mAU/mL) were elevated, alpha-foetoprotein (AFP; 4 ng/mL) and CEA (1.5 ng/dL) were normal. Computed tomography (CT) confirmed a mass, 12.5 cm in maximum diameter, located on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein (Fig. 1A).Fig. 1 Change in tumor size after neoadjuvant chemotherapy.
A. Computed tomography (CT) scan on admission. The tumor is on the ventral surface of the liver containing multiple cystic lesions with ring enhancement, extending along Glisson’s capsule with invasion to the portal vein.
B. CT scan 4 weeks after admission; pre-chemotherapy.
C. CT scan 8 weeks after admission; after 2 cycles of CDDP + DOX.
D. CT scan 16 weeks after admission; after 5 cycles of CDDP + DOX. The tumor has shrunk remarkably separating from Glisson’s capsule.
Fig. 1
Open biopsy of the mass was performed two days after admission to avoid complications of needle biopsy such as bleeding. Microscopic sections of biopsy specimens showed diffuse infiltration of tumor cells with nuclear pleomorphism and cohesiveness (Fig. 2A and B) with no specific structure or differentiation. Immunohistochemistry was positive for epithelial markers (pan cytokeratin (CK) antibodies AE1/AE3, CK19, CK7), vimentin, SALL4, Glypican3, CD10 and p53, while negative for CK20, EMA, CD30, AFP, HCG-β, HepPar1, D2-40, CDX2, β-catenin and p40. A definitive diagnosis could not be made, but a germ cell tumor or carcinoma with germ cell differentiation was suspected because SALL4 and Glypican3 were both positive. Biopsy specimens were sent for Central Pathology Review by paediatric tumor pathology specialists. Additional immunostaining was performed with SMARCB1, SMARCA2, SMARCA4, ARID1A, and LIN28 being positive and Oct3/4, CD117, DLK1, and vimentin being negative, a reflection of how poorly differentiated the tumor was. Their diagnosis, which took almost a month to be finalised, was poorly differentiated or UC of the liver with embryonal features.Fig. 2 Histopathology of open liver (tumor) biopsies.
Micrographs of open liver (tumor) biopsy (A: HE x12.5; B: HE x400) showing diffuse infiltration of tumor cells with nuclear pleomorphism.
Fig. 2
During this time, the tumor had grown to 14.5 cm in maximum diameter (Fig. 1B) with enlargement of multiple mediastinal lymph nodes. Gallium scintigraphy was positive for the liver tumor and the mediastinum. Preoperative liver CT volumetry [4] indicated that the standard liver volume would be less than 20% following surgical resection, a level consistent with high postoperative morbidity and liver failure [5]. Thus, her tumor was group III [6] using the pretreatment extent of disease (PRETEXT) system for classifying primary hepatic malignancies in children, which meant that the tumor was inoperable and that she was also not a viable candidate for primary liver transplantation (LTx).
In the absence of guidelines for the treatment of UC of the liver, we referred to treatment options for hepatoblastoma (HB) and commenced neoadjuvant chemotherapy with cisplatin/doxorubicin (CDDP + DOX) [7,8] starting with one dose of CDDP (4.0 mg/kg/dose) and two doses of DOX (2.0 mg/kg/dose). The response was immediate and impressive. A further four cycles of one dose of CDDP (5.2 mg/kg/dose) and two doses of DOX (2.6 mg/kg/dose) followed by one dose of CDDP (5.2 mg/kg/dose) without DOX to prevent DOX-induced cardiomyopathy shrank the tumor so remarkably (Fig. 1C and D) that it separated from Glisson’s capsule to become a post-treatment extent of disease (POSTEXT) II [6] tumor that was excised completely by open resection of segments 4b and 5 performed by a team comprised of two board-certified paediatric surgeons, one with 34 years’ experience (AY) and one with 11 years’ experience (TO) and one board certified adult hepatobiliary pancreatic surgeon with 26 years’ experience for supervisory support (AS). During surgery, ultrasonography was used as shown in Fig. 3A to confirm the relationship of the tumor to vascular structures and guide dissection for resection. The Pringle maneuver was used during transection (Fig. 3B) enabling the tumor to be excised completely (Fig. 3C). Neither mediastinal dissemination nor ascites were noted. No additional procedures were considered. The abdominal cavity was irrigated and there were no signs of bleeding; a drainage tube was not placed. Total operative time was 5 h and 20 min. Blood loss was 60 mL. The patient tolerated surgery well with no perioperative complications and uneventful postoperative recovery. Adjuvant chemotherapy was not indicated.Fig. 3 Operative findings.
Intraoperative ultrasound was used to confirm the relationship of the tumor to vascular structures, and guide dissection for resection (A). The Pringle maneuver was used during transection (B), and tumor was excised completely by resecting segments 4b and 5 (C).
Fig. 3
Histopathology confirmed all surgical margins were negative for malignancy (Fig. 4A). Microscopic sections showed numerous diffuse cystic lesions lined with tumor cells with focal papillary projections into the cystic spaces (Fig. 4B). Scattered necrosis/haemorrhage secondary to neoadjuvant chemotherapy was observed. Immunohistochemical staining was positive for AE1/AE3, SALL4, Glypican3, CD10 and EMA, and negative for AFP, CD30, HCG-β, HepPar1, CDX2 and podoplanin.Fig. 4 Histopathology of resected specimens.
Macroscopic appearance (A) and micrographs (B: HE x200) of the resected specimen showing cysts lined by tumor cells with focal papillary projections.
Fig. 4
Some four months after surgery, she developed ascites secondary to peritoneal carcinomatosis. Unfortunately, her creatinine clearance deteriorated after only two doses of CDDP (5.8 mg/kg/dose). She was discharged with no active treatment plan about one month before she eventually passed away, nine months after surgery.
The work has been reported in line with the Scare 2018 criteria [9].
3 Discussion
Two of the three cases in the literature had neuroendocrine features and were diffuse and one was more solitary and amenable to surgery. Our case had multiple cystic lesions with partial solid components on CT which would categorise her as being of the solitary type, but her tumor was considered inoperable because of extensive spread within the liver and insufficient volume of future liver remnant.
While some studies recommend LTx for HB patients with PRETEXT/POSTEXT groups III and IV, claiming five-year survival rates of 77%–100% [[10], [11], [12]], others recommend extended hepatic resection, also claiming excellent overall survival rates of 80%–88% [13,14]. We do not know if LTx could have prevented metastases or if immunosuppressants would have been problematic, but in hindsight, complete surgical resection may have been too optimistic for such an aggressive tumor because even though neoadjuvant chemotherapy would have weakened the tumor and all resected edges were negative for abnormal histopathology, she succumbed to overwhelming metastases.
4 Conclusion
UC should be included in the provisional diagnosis of a child with a right upper quadrant mass with or without ascites. Its rarity has prevented appropriate chemotherapeutic and surgical strategies from being established, however, our case demonstrates the importance of gross inspection as a clinical skill to identify masses as early as possible and the value of neoadjuvant chemotherapy for facilitating surgical intervention.
Declaration of Competing Interest
The authors report no declarations of interest.
Funding
None.
Ethical approval
This case report was approved by the Ethics Committee of Juntendo University School of Medicine (approval number: JHS20-010).
Consent
Written informed consent was obtained from the patient’s parents for preparation and publication of this case report and accompanying images.
There is no identifying characteristics in the manuscript.
Author contribution
Akio Saiura, Atsuyuki Yamataka, and Takanori Ochi performed surgery on our patient. Takanori Ochi wrote the manuscript with help from Junya Fujimura and Atsushi Arakawa. Geoffrey J. Lane, Atsuyuki Yamataka, and Akio Saiura edited and revised the manuscript and prepared it for submission.
Registration of research studies
Not applicable.
Guarantor
The Guarantor of this case report is Takanori Ochi (Associate Professor of the Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine).
Acknowledgements
We wish to express our appreciation to Eri Abe, Yuichiro Miyake, and Osamu Tomita for their contributions and commitment to the care of our patient and Shiho Yoshida, Hiroyuki Koga, and Toshiaki Shimizu for their support and encouragement in preparing this report. | Fatal | ReactionOutcome | CC BY-NC-ND | 33310474 | 19,165,864 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Low birth weight baby'. | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | ASPIRIN | DrugsGivenReaction | CC BY | 33313931 | 20,187,437 | 2021-07-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Small for dates baby'. | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | ASPIRIN | DrugsGivenReaction | CC BY | 33313931 | 20,187,437 | 2021-07-01 |
What is the weight of the patient? | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | .511 kg. | Weight | CC BY | 33313931 | 20,187,437 | 2021-07-01 |
What was the administration route of drug 'ASPIRIN'? | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | Transplacental | DrugAdministrationRoute | CC BY | 33313931 | 20,187,437 | 2021-07-01 |
What was the administration route of drug 'HYDROXYCHLOROQUINE SULFATE'? | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | Transplacental | DrugAdministrationRoute | CC BY | 33313931 | 20,186,598 | 2021-07-01 |
What was the dosage of drug 'HYDROXYCHLOROQUINE SULFATE'? | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | (MATERNAL DOSE: 200 MG QD) | DrugDosageText | CC BY | 33313931 | 20,186,598 | 2021-07-01 |
What was the dosage of drug 'METHYLDOPA'? | Role of placental inflammatory mediators and growth factors in patients with rheumatic diseases with a focus on systemic sclerosis.
Pregnancy in SSc is burdened with an increased risk of obstetric complications. Little is known about the underlying placental alterations. This study aimed to better understand pathological changes and the role of inflammation in SSc placentas. Leucocyte infiltration, inflammatory mediators and atypical chemokine receptor 2 (ACKR2) expression in SSc placentas were compared with those in other rheumatic diseases (ORD) and healthy controls (HC).
A case-control study was conducted on eight pregnant SSc patients compared with 16 patients with ORD and 16 HC matched for gestational age. Clinical data were collected. Placentas were obtained for histopathological analysis and immunohistochemistry (CD3, CD20, CD11c, CD68, ACKR2). Samples from four SSc, eight ORD and eight HC were analysed by qPCR for ACKR2 expression and by multiplex assay for cytokines, chemokines and growth factors involved in angiogenesis and inflammation.
The number of placental CD3, CD68 and CD11 cells was significantly higher in patients affected by rheumatic diseases (SSc+ORD) compared with HC. Hepatocyte growth factor was significantly increased in the group of rheumatic diseases patients (SSc+ORD) compared with HC, while chemokine (C-C motif) ligand 5 (CCL5) was significantly higher in SSc patients compared with ORD and HC. CCL5 levels directly correlated with the number of all local inflammatory cells and higher levels were associated with histological villitis.
Inflammatory alterations characterize placentas from rheumatic disease patients and could predispose to obstetric complications in these subjects.
pmc Rheumatology key messages
Placental leukocytes are more numerous in rheumatic diseases, with a possible role in obstetric complications.
HGF placental levels are higher in rheumatic diseases than in controls and may promote placentation.
CCL5 expression is higher in SSc placentas and this supports its pathogenetic role.
Introduction
Patients with rheumatic diseases (RD), especially connective tissue diseases, are at increased risk of obstetric complications and have historically been advised against pregnancy. In recent years, contraindications have been revised in light of new knowledge of the pathogenesis of the complications and of therapies for their management [1–4].
Most studies examining fetal outcome and placental changes in RD concern SLE and APS. Preterm birth, intrauterine growth restriction (IUGR) and preeclampsia are frequent complications in SLE [5] and are associated with trophoblast alterations, villitis, vasculopathy and a high number of inflammatory cells [6, 7]. In APS the higher risk of abortion, stillbirth, IUGR and preterm birth [8] is associated with trophoblast alterations, infarction and a higher number of placental inflammatory cells [9, 10]. In chronic arthritis, a slightly increased risk of spontaneous abortion or preterm birth compared with healthy population has been described and a lot of studies have been performed with respect to therapy [11], but no histological analysis of the placenta has been conducted so far.
An Italian multicentre study showed that women with SSc have a higher than normal risk of IUGR, preterm delivery and very low birth weight babies [12]. In a case series of 13 SSc patients [13], five showed decidual vasculopathy, associated with fetal death in four cases. The vessels had increased number of perivascular macrophages, immunoglobulin deposits and CD4 lymphocytes compared with healthy controls. A study of three cases [14] described decidual vasculopathy, villous hypovascularity, stromal fibrosis, increased syncytiotrophoblast knotting and infarcts in the placentas of SSc patients compared with healthy controls. Immunohistochemical analysis revealed increased staining for VEGF, VEGF receptor 2, connective tissue growth factor and α-smooth muscle actin in myofibroblasts in SSc patients, as signs of altered vascular remodelling and fibrosis.
We are particularly interested in the role of the atypical chemokine receptor 2 (ACKR2), which does not signal in response to chemokines, but internalizes ligand and targets it for intracellular degradation, acting as a chemokine ‘scavenger’ [15]. It is highly expressed in trophoblasts and may be important in reducing the risk of inflammation-related miscarriage, minimizing inflammatory chemokine exchange between mother and fetus [16]. ACKR2 knock out mice have fetal loss if infused with antiphospholipid antibodies or lipopolysaccharides [17]. Furthermore, ACKR2 levels are higher in the peripheral blood mononuclear cells (PBMCs) of patients with SSc compared with healthy controls [18].
The aim of our study was to analyse the histopathological placental features of a cohort of SSc patients, with a focus on the role of inflammation in the pathogenesis of obstetric complications and to determine whether placental ACKR2 might have a role in it.
Methods
Patients
Patients attending the Rheumatology Unit of the IRCCS Policlinico San Matteo’s Foundation in Pavia, Italy, who fulfilled the 2013 European League Against Rheumatism/American College of Rheumatology classification criteria for SSc [19] and who consecutively became pregnant between 2013 and 2018, were enrolled in this prospective study. Pregnant patients with other RD (ORD) classified according to the current classification criteria [20–23] were enrolled as the first control group and healthy pregnant women followed at the Gynaecology and Obstetrics Unit formed the second control group (healthy controls, HC). Patients for comparison groups were consecutively enrolled if matched to SSc patients by age, body mass index and week of delivery, with a ratio of 1:2:2. Patients were followed up by the same physicians during pregnancy. Organ involvement was evaluated according to the presence of signs and symptoms of disease at the visits and imaging data. Pulmonary involvement was recorded if the chest X-ray, high resolution CT scan of the thorax, pulmonary function tests or echocardiography had previously given an indication of interstitial or vasculopathic lung disease. Laboratory tests, including autoantibodies, were evaluated using commercially available kits.
This study was carried out in accordance with the Declaration of Helsinki. The local ethics committee has approved the research protocol and all patients provided their written informed consent to use their placentas in the study.
Macroscopic and histopathological analysis
Placentas were weighed and underwent macroscopic examination. Full thickness samples were obtained, fixed in 10% buffered formalin and embedded in paraffin. Sections (3 µm) were stained with haematoxylin, eosin and Masson’s trichrome for histopathological examination according to the most recent guidelines [24] by an expert pathologist who was blind to sample classification.
Immunohistochemistry
Paraffin-embedded full thickness placental samples were sliced into 3 µm sections, dewaxed and heated in 0.01 M pH 6 sodium citrate buffer for antigen retrieval. After blocking endogenous peroxidase activity and non-specific binding, the sections were incubated overnight with the following primary antihuman antibodies: mouse monoclonal anti-CD3 (F7.2.38, 1:70, Dako, Glostrup, Denmark), mouse monoclonal anti-CD20 (L26, 1:126, Dako), mouse monoclonal anti-CD68 (PG-M1, 1:30, Dako), rabbit monoclonal anti-CD11c (EP1347Y, 1:500, Abcam, Cambridge, UK) and rabbit polyclonal anti-ACKR2 (1:400, Sigma-Aldrich, St Louis, MO, USA). Sections were then incubated with the appropriate chromogenic secondary antibody (ImmPRESS Polymer Detection Kit, anti-rabbit and anti-mouse, Vector Laboratories, Burlingame, CA, USA). The immunoreactivity was developed using 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories) as chromogen. Isotype-matched control antibodies were included as a negative control and tonsil sections as a positive control. The sections were observed under a light microscope (Olympus BX43, Olympus, Tokyo, Japan) and photographed by digital camera (DP22 using Olympus Cell Sense Entry 2.2 for imaging acquisition).
Analysis of immunostaining
The immunostaining for CD3, CD20, CD11c and CD68 was assessed as follow. Photographs were taken of 10 random fields (×40 magnification) along the sections and representative of all placental layers. Stained cells were counted by two blinded observers and normalized to the tissue area. The percentage of stained area was assessed in sections stained for ACKR2 and with Masson’s thrichrome. ImageJ 2.0 software was used to measure stained area and total area of tissue represented in the fields examined.
Real-time quantitative polymerase chain reaction
Random parenchymal biopsies were performed in half of the samples and stored in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C. To extract RNA, samples were lysed and homogenized in β-mercaptoethanol and RLT buffer by shaking with steel beads in a Tissue Lyser LT (Qiagen, Valencia, CA, USA). RNA was then extracted and purified from the fluid phase using the RNeasy Mini extraction kit (Qiagen). Purified RNA was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Fisher Scientific). Samples were tested in triplicate and qPCR for ACKR2 was performed as previously described [25]. ACKR2 transcript levels were normalized to TATA-binding protein. The samples were run on a QuantStudio 7 flex machine (Thermo Fisher Scientific).
Protein extraction and multiplex cytokine assay
Placental samples were suspended in tissue extraction buffer (homemade with 100 mM pH 7.4 Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors), homogenized and the concentration of total proteins in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein concentration in the samples was normalized to the sample with the lowest concentration. Samples were analysed as per protocol using a 30-Plex bioassay (Thermo Fisher Scientific) measuring interleukin (IL)-1β, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, G-CSF, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, CCL5, chemokine (C-X-C motif) ligand (CXCL)9, CXCL10, CCL11, VEGF, fibroblast growth factor, hepatocyte growth factor (HGF) and epidermal growth factor.
Statistical analysis
For all statistical tests, non-parametric data were analysed using the Mann–Whitney U-test and parametric data using Student’s unpaired t-test. For multiple comparisons, a Kruskal–Wallis correction was applied to the test. To detect significant correlation between variables, Spearman’s correlation coefficient was used, where r = 1 denotes a perfect positive correlation and r = −1 a perfect negative correlation. P < 0.05 denotes significant differences. Comparisons were also made between each of the following groups: SSc, SLE, UCTD, defined connective tissue disease (SSc+SLE+Sjögren’s syndrome) patients; SSc patients with obstetric complications, SSc patients without obstetric complications, SSC+ORD patients with obstetric complications, SSc+ORD patients without obstetric complications, HC, all complicated pregnancies, patients who had preeclampsia, patients with poor fetal outcome (IUGR, small for gestational age, death), patients with preterm birth, patients with premature rupture of membranes, all non-complicated pregnancies.
Statistical analyses were performed using Prism 8.0.2 for Macintosh (GraphPad Software Inc., La Jolla, CA, USA).
Results
Main clinical features of the study patients
A total of eight patients affected by SSc, 16 with ORD and 16 HC were enrolled in the study. All SSc patients but one took low dose acetylsalicylic acid during pregnancy and one patient took prednisolone 4 mg daily in addition. Their main clinical and pregnancy-related features are shown in Table 1.
Table 1 Characteristics of SSc patients
Patient Disease subset Duration of disease, years mRSS Autoantibodies Internal organ involvement Therapy before/during (b/d) pregnancy BMI, kg/m2 Comorbidities and risk factors Age at conception, years Gestational week at delivery Obstetric complications Newborn weight, g Placental characteristics (descriptive)
1a lc-SSc 3 2 ACA None b and d: levothyroxine, ASA 21.5 Gestational hypothyroidism 34 38 + 6 PROM 3170 Dystrophic calcifications, mild acute chorioamnionitis
2a lc-SSc 1 4 ACA Gastrointestinal b: prostanoids.
d: MPD 4 mg
23.8 — 38 36 HELLP,
preterm birth
2660 Hypoxic hypervascularization, syncytial knots, decidual inflammation
3a dc-SSc 12 13 Anti-Scl70 None b: bosentan, prostanoids.
d: CCB, ASA
28.1 — 31 40 — 3175 Focal subchorial fibrin deposits
4a dc-SSc 1 3 Anti-Scl70 None b and d: ASA 22.6 — 33 40 Preeclampsia 3690 Mild acute chorioamnionitis, low grade chronic villitis, few dystrophic calcifications
5 dc-SSc 1 2 Anti-Scl70 None b and d: ASA 23.1 — 29 36 + 4 Preterm birth 2800 Dystrophic calcifications, mild acute chorioamnionitis, fibrin deposits
6 dc-SSc 4 6 Anti-Scl70 None b: prostanoids. d: levothyroxine, ASA 26 Hashimoto’s thyroiditis 29 40 — 3140 Rare avascular villi, perivillous and villous fibrin deposits, focal chronic villitis
7 dc-SSc 2 2 Anti-Scl70 None b: CCB
d: LMWH, ASA
25.5 — 35 26 Preterm birth, IUGR, HELLP 511, SGA Villous haemorrhage, areas of infarction, decidual arteriopathy, mural thrombi
8 dc-SSc 7 12 Anti-Scl70 Gastrointestinal, cardiopulmonary b: CCB
d: ASA
27 — 34 30 + 6 Preterm birth, neonatal death 1428 Chronic decidual inflammation, decidual arteriopathy
a Patient with both paraffin and frozen samples. ASA: acetylsalicylic acid 100 mg; CCB: calcium channel blockers; dc-SSc: diffuse cutaneous SSc; HELLP: haemolysis, elevated liver enzymes, low platelets; IUGR: intrauterine growth restriction; lc-SSc: limited cutaneous SSc; LMWH: low molecular weight heparin; MPD: metylprednisolone; mRSS: modified Rodnan skin score; PROM: premature rupture of membranes; SGA: small for gestational age.
The patients affected by ORD included 10 patients with UCTD, characterized by presence of autoantibodies and arthritis or cytopenias, none satisfying Very Early Diagnosis of SSc (VEDOSS) classification criteria, three with SLE, two with idiopathic juvenile arthritis and one patient with Sjögren’s syndrome (SjS). Their main clinical and pregnancy-related features are shown in Supplementary Tables 1 and 2, available at Rheumatology online.
Macroscopic and histopathological findings
No macroscopic placental differences (dimension, weight) were observed between groups. At histological examination, no significant difference (P > 0.05) was found between groups, or between the HC vs the SSc patients and vs the RD patients (SSc+ORD) regarding the presence of deciduitis, villitis, materno-fetal inflammation, placental abruption, vascular alterations or fibrin deposits. The latter was examined both by histopathological examination and by Masson’s trichrome staining. The histopathological data did not show any correlation or association with disease-related features as disease subset, disease duration, modified Rodnan skin score (mRSS) and organ involvement.
Inflammatory cells within placentas
The number of placental CD3 and CD11c+ cells found by immunohistochemistry was significantly higher in patients affected by RD (SSc+ORD) compared with HC. The SSc group alone did not statistically differ from the ORD group nor the HC, possibly due to a smaller sample size. The number of placental CD68+ cells was significantly higher in both the SSc and ORD groups compared with HC (Fig. 1). Patients with histological evidence of placental abruption had a higher number of placental CD68+ cells, regardless of diagnosis (Fig. 2).
Fig. 1 Immunohistochemical analysis
(A) CD3, CD11c and CD68 expression in placentas from patients with SSc, other rheumatic diseases (ORD) and healthy controls (HC). Scale bar: 20 μm. (B) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 in HC compared with rheumatic disease patients (ORD+SSc). (C) Median and 95% CI of CD3+, CD11c+ and CD68+ cell expression per mm2 of HC, ORD and SSc groups. *P <0.05, **P <0.001, ***P <0.0001
Fig. 2 Placental macrophages in relation to placental abruption
The number of CD68+ cells/mm2 in placentas from patients with and without placental abruption (pl. abrupt.). *P <0.05.
The number of CD20+ cells was not statistically different between the groups (Supplementary Fig. S1, available at Rheumatology online), even though there was a trend towards higher numbers in the SSc+ORD group (P = 0.058).
Similar results were obtained comparing SSc patients and HC: CD3+ (P < 0.05), CD11c+ (P < 0.05) and CD68+ (P < 0.01) cells were significantly higher in the first group, while CD20+ cell number was not different.
Placental ACKR2 expression and transcription
There was strong staining of ACKR2 in all sections and there was no difference in the stained area between the groups (SSc vs ORD vs HC, SSc vs HC, SSc+ORD vs HC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed very high transcript levels in all groups, without significant differences between them. Moreover, ACKR2 expression and transcription levels did not correlate with any clinical (disease subset, disease duration, mRSS, internal organ involvement) or obstetric variable (presence of complications, week of delivery, presence of histological alterations as above detailed).
ACKR2 transcript levels correlated with the percentage of stained area in immunohistochemistry (Supplementary Fig. S2, available at Rheumatology online), indicating concordance with protein expression.
Inflammatory mediators and growth factors in placenta
We measured levels of a broad range of inflammatory mediators and growth factors in the placentas from four SSc patients (two dc-SSc, two lc-SSc), eight patients affected by ORD and eight HC. Only those molecules showing significant differences between groups were considered in further analyses and in clinical correlates. Specifically, HGF was significantly increased in RD patients (SSc+ORD) compared with HC (P < 0.05) and CCL5 was significantly higher in SSc patients compared with ORD (P < 0.05) and with HC (P < 0.01) (Fig. 3).
Fig. 3 HGF and CCL5 placental levels
Levels of the hepatocyte growth factor (HGF) and of chemokine (C-C motif) ligand 5 (CCL5) in healthy controls (HC), patients with other rheumatic diseases (ORD) and SSc. *P <0.05, **P <0.01, ns: not significant.
When analysing SSc vs HC group, HGF levels were not different (P > 0.05), while CCL5 levels were significantly higher (P < 0.01).
HGF levels inversely correlated with the gestational week at delivery (Fig. 4A) and when the disease groups were analysed separately, a significant inverse correlation was seen in the rheumatic disease patients group (SSc+ORD), but not in HC (Fig. 4B and C). The same was detected for placental weight, which inversely correlated with HGF in patients affected by RD (Supplementary Fig. S3, available at Rheumatology online). Accordingly, HGF levels were higher in patients with preterm delivery, regardless of the diagnosis (Fig. 4D). Higher levels of placental CCL5 were associated with histological villitis (Fig. 4E).
Fig. 4 HGF/CCL5 levels and obstetric complications
(A) Considering all patients, levels of hepatocyte growth factor (HGF) inversely correlated with the gestational week at delivery (r = 0.47, P < 0.05). (B, C) The inverse correlation is maintained in the rheumatic diseases group [other rheumatic diseases (ORD)+SSc; r = 0.5, P < 0.05] (B), but not in the healthy controls (HC) group (P > 0.05) (C). (D) Differences in HGF levels in patients with and without preterm delivery. (E) Chemokine (C-C motif) ligand 5 (CCL5) levels in patients with and without histological villitis. *P < 0.05, **P < 0.01.
No clear associations were seen between HGF, CCL5 and disease-related clinical features (disease subset, disease duration, mRSS, internal organ involvement), while direct significant correlation was noted between CCL5 and the number of all inflammatory cells considered in immunohistochemistry. Moreover, the number of CD3+ cells directly correlated with the number of CD20+ and CD11c+ cells. The number of CD68+ cells directly correlated with the number of CD11c+ cells and with decidual HGF levels (see Table 2 for descriptive statistics presented as r-values).
Table 2 Correlations between the number of inflammatory cells, ACKR2 transcript, HGF and CCL5 levels
CD3
CD3 1 CD20
CD20 0.41** 1 CD11c
CD11c 0.37** 0.10 1 CD68
CD68 0.25 0.19 0.49*** 1 ACKR2
ACKR2 −0.01 0.1 −0.4 −0.5 1 HGF
HGF 0.1 −0.3 0.2 0.5** −0.2 1 CCL5
CCL5 0.3* 0.4** 0.4* 0.3* −0.2 0.02 1
Correlation coefficients obtained from Spearman tests. *P <0.05, **P <0.01, ***P <0.001. ACKR2: atypical chemokine receptor 2; CCL5: chemokine (C-C motif) ligand 5; HGF: hepatocyte growth factor.
Comparisons between distinct rheumatic diseases
As the ORD group included heterogeneous RD, we analysed if any differences in placental leukocytes, inflammatory mediators or growth factors, or ACKR2 levels could be detected among them. In particular, we considered SSc vs SLE patients, SSc vs UCTD patients, SLE vs UCTD patients and UCTD vs defined connective tissue disease (SSc+SLE+SjS). CD20+ cells were higher in placentas from defined connective tissue diseases compared with UCTD (P < 0.01). No other significant findings were observed between groups, except from a trend toward higher placental CCL5 levels (P = 0.06) in SSc compared with UCTD patients.
Comparisons between successful and complicated pregnancies
We investigated if any distinctive alteration could be found in patients with obstetric complications. Therefore, we considered sub-groups of patients and analysed if differences could be detected in placental CD3+, CD20+, CD11c+, CD68+ cells, ACKR2 expression and transcription, inflammatory mediators and growth factors. No significant findings have been detected, except from a trend toward higher placental CD68+ cells (P = 0.07) and HGF (P = 0.06) in preterm placentas and to higher CCL5 in patients with preeclampsia (P = 0.07).
Discussion
In this study we analysed how inflammation might play a role in obstetric complications that frequently occur in the pregnancies of patients affected by RD, in particular SSc. To our knowledge, this is the largest cohort thus far analysed in SSc.
We found that patients with RD had higher numbers of placental leukocytes, specifically T lymphocytes (CD3+ cells), antigen-presenting cells (APCs, CD11+ cells) and macrophages (CD68+ cells), compared with HC.
Our results are in line with and reinforce previous literature showing an increased number of placental leukocytes in these patients [6, 7, 9, 10]. This has been associated with obstetric complications such as IUGR, preeclampsia, fetal death and preterm delivery [26–28]. Placental macrophage infiltration might play a role in reducing trophoblastic invasion, in placental abruption [9, 29] and in preterm labour [30]. In addition, an association between high maternal serum and placental concentrations of M-CSF with IUGR [31] and preeclampsia [32] has been reported. Other evidence suggests that placental T cell infiltration and imbalance are important in the aetiopathogenesis of preeclampsia [33]. In our population a higher number of placental macrophages was associated with placental abruption and a trend towards higher CD68+ cells in preterm placentas was shown, regardless of diagnosis of rheumatic diseases. No other significant association was found between inflammatory cell numbers and obstetric complications, considering SSc patients, RD patients or all complicated pregnancies regardless of diagnosis. It is possible that with our small population we did not have enough statistical power to detect more subtle differences in other leucocyte populations among patients with and without obstetric complications (and in sub-groups of rheumatic diseases patients). Therefore, we can only speculate that SSc patients, and in general RD women, may be more predisposed to obstetric complications due to the development of placental inflammatory alterations.
The proangiogenic factor HGF [34] was higher in patients with RD (SSc+ORD) compared with HC. In placenta, HGF is produced by stromal cells of the villous mesenchyme and stimulates trophoblast invasion in the decidua [35]. Its levels are reduced in hypoxic conditions and in patients with preeclampsia [36]. It might be speculated that patients with RD need higher levels of HGF to promote trophoblast invasion and placentation. In support of this, in our population HGF levels inversely correlated with gestational week and placental weight in patients with RD but not in controls, suggesting an important role of this factor in women affected by autoimmune diseases, with higher levels in early stages when placenta is still developing and lower values in the end stages of pregnancy.
An important insight provided by our study concerns CCL5, which was significantly higher in placenta from patients with SSc compared with ORD and HC, with no difference between the latter two groups and which appeared to be related to villitis and to preeclampsia, regardless of rheumatic disease, although the latter association did not reach full statistical significance. These may indicate a disease specific role of this chemokine in SSc. CCL5 mediates trafficking and activation of several immune cells [37]. An association has been demonstrated between a specific polymorphism of the gene coding for CCL5 and susceptibility for SSc [38]. CCL5 has been implicated in the pathogenesis of perivascular inflammation, vascular dysfunction [39, 40], hepatic and renal fibrosis [41–43], and myocardial remodelling [44]. Furthermore, CCL5 is highly expressed in the skin of patients with SSc, while no expression has been found in the skin of controls [45]. Specifically, CCL5 is highly expressed in skin in early SSc, as are CCL2, CCL3, CCL4 and CX3CL1. In advanced stages CCL7 and CXCL10 predominate [46]. The early expression of CCL2, CCL3 and CCL5 is also observed in a mouse model of scleroderma, with a subsequent rapid reduction of CCL5 and maintained high expression of CCL2 and CCL3 [47]. Another study showed that CCL2, CCL3 and CCL5 were significantly higher in serum of patients with SSc than in controls and therapy with prostaglandins down-regulated CCL2 and CCL5, suggesting an effect of vasodilator therapy on inflammation in SSc [48]. Considering the placenta as a new organ, with possible gradual involvement by the disease, CCL5 could be a key regulator of the pathological process. Through its chemoattractive activity it could promote the formation of a placental inflammatory infiltrate, and in fact in this study we have shown a correlation between CCL5 levels and leukocytes infiltration. Moreover, it could be a key factor in the development of vascular alterations and, in the subsequent stages, of fibrosis.
We did not find different levels of transcription or expression of placental ACKR2 in SSc or ORD compared with HC. In a previous study, ACKR2 levels were higher in PBMCs of SSc patients compared with controls [18]. Furthermore, ACKR2 was elevated in PBMCs and synovial tissue of patients with inflammatory arthropathies [49]. In our population, PBMCs from pregnant patients were not always available and thus we could not perform a group analysis, but it would be interesting in future studies to compare PBMCs and placental levels of ACKR2. A possible explanation for similar ACKR2 levels in our groups could be that ACKR2 is strongly expressed in placenta and in our patients its immunomodulatory role was sufficient to control the inflammation induced by the inflammatory cells present in the tissue. In fact, the number of leukocytes, although higher in patients with RD, was not associated with obstetric complications, except from placental abruption. The only ACKR2 ligand found to be elevated was CCL5 in patients with SSc, underlining a prominent activity of this chemokine in these patients.
In conclusion, there is increased placental leucocyte infiltration in patients with RD and this may contribute to the risk of complications. High HGF levels could represent a protective mechanism for an adequate placentation. In SSc, CCL5 might be a key factor, with a role in chemotaxis, vascular remodelling and fibrosis development.
This could be considered a pilot study and a larger population of SSc and RD patients should be enrolled in order to improve statistical power, perhaps in a multicentric study. The detection of defined inflammatory alterations could help in understanding the pathogenesis of the poor outcomes affecting SSc and RD pregnancies. Moreover, an analysis of inflammatory features in relation to therapy could be performed, to detect if low dose corticosteroids, hydroxychloroquine, other immunosuppressants or anti-platelet agents could have a role in SSc and RD placental alterations and in specific obstetric complications. In addition, a comparison between alterations in placental tissue and peripheral blood could lead to the detection of serum markers predictive of higher-risk pregnancies, easy to detect at early stages, when a timely personalized pharmacological intervention may be performed to prevent complications.
Supplementary Material
keaa782_Supplementary_Data Click here for additional data file.
Acknowledgements
The authors wish to thank Ms Barbara Vitolo for her valuable support to this project, her precious contribution in sample collection, preparation and storage and for the critical review of the manuscript.
Funding: This work was supported by grants from a Wellcome Trust Investigation Award [Grant number 099251/Z/12/Z] and the UK Medical Research Council [Grant number MR/M019764/1].
Disclosure statement: H.J. was funded by the Chief Scientist Office during the conduct of the study. G.J.G. reports grants from the Wellcome Trust and from the Medical Research Council during the conduct of the study. There are no other interests to disclose.
Data availability statement
The data underlying this article are available in the article and in its online supplementary material.
Supplementary data
Supplementary data are available at Rheumatology online. | (MATERNAL DOSE: UNKNOWN) | DrugDosageText | CC BY | 33313931 | 20,186,598 | 2021-07-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Alanine aminotransferase increased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Aspartate aminotransferase increased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Blood lactate dehydrogenase increased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug-induced liver injury'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Gamma-glutamyltransferase increased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Glioblastoma multiforme'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hepatic enzyme increased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Intentional product misuse'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Leukopenia'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Monocyte count decreased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Overdose'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Product use in unapproved indication'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'White blood cell count decreased'. | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | ARTESUNATE, CLOBAZAM, DIVALPROEX SODIUM, LEVETIRACETAM, LORAZEPAM, ONDANSETRON, TEMOZOLOMIDE, VALPROIC ACID | DrugsGivenReaction | CC BY | 33313992 | 15,046,217 | 2021-06 |
What was the administration route of drug 'ARTESUNATE'? | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Oral | DrugAdministrationRoute | CC BY | 33313992 | 15,046,217 | 2021-06 |
What was the administration route of drug 'CLOBAZAM'? | Drug repurposing using transcriptome sequencing and virtual drug screening in a patient with glioblastoma.
Background Precision medicine and drug repurposing are attractive strategies, especially for tumors with worse prognosis. Glioblastoma is a highly malignant brain tumor with limited treatment options and short survival times. We identified novel BRAF (47-438del) and PIK3R1 (G376R) mutations in a glioblastoma patient by RNA-sequencing. Methods The protein expression of BRAF and PIK3R1 as well as the lack of EGFR expression as analyzed by immunohistochemistry corroborated RNA-sequencing data. The expression of additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) emphasized the aggressiveness of the tumor. Then, we screened a chemical library of > 1500 FDA-approved drugs and > 25,000 novel compounds in the ZINC database to find established drugs targeting BRAF47-438del and PIK3R1-G376R mutated proteins. Results Several compounds (including anthracyclines) bound with higher affinities than the control drugs (sorafenib and vemurafenib for BRAF and PI-103 and LY-294,002 for PIK3R1). Subsequent cytotoxicity analyses showed that anthracyclines might be suitable drug candidates. Aclarubicin revealed higher cytotoxicity than both sorafenib and vemurafenib, whereas idarubicin and daunorubicin revealed higher cytotoxicity than LY-294,002. Liposomal formulations of anthracyclines may be suitable to cross the blood brain barrier. Conclusions In conclusion, we identified novel small molecules via a drug repurposing approach that could be effectively used for personalized glioblastoma therapy especially for patients carrying BRAF47-438del and PIK3R1-G376R mutations.
Background
Half of the brain tumors represent diffuse gliomas, and the World Health Organization (WHO) provided classification criteria to predict the clinical behavior of these neoplasms [1]. Glioblastoma is classified as grade IV/IV gliomas with specific characteristics, including high cellularity, cellular pleomorphism, nuclear atypia and necrosis [2]. Glioblastoma has a dismal prognosis despite aggressive treatment [2, 3]. Various genetic mutations and aberration have been identified in glioblastoma patients, such as MGMT methylation [3, 4], BRAFG596A [5], BRAFV600E [5–7], PIK3R1G376R, PIK3R1D560Y, PIK3R1N564K mutations [8].
Mutations in proteins critical for cancer biology usually lead to uncontrolled cell growth, resistance to conventional chemotherapy and subsequently, therapy failure. In the past decade, the demand for effective novel agents to combat cancer has drawn the attention to specific molecular alterations in cancer cells as targets for therapy [9]. The concept of precision medicine implies the application of targeted drugs coupled with specific diagnostic assays in order to determine whether patients are likely to benefit from targeted therapy. The shift from classical, non-selective, cytotoxic chemotherapy to molecular targeted cancer drugs resulted in increased tumor response and patients’ survival rates [10–12].
Therapeutic monoclonal antibodies are considered a successful strategy for targeted cancer therapy. Antibodies have, however, several limitations, including targeting only cellular surface epitopes, high immunogenicity and high production costs [13]. Small molecule inhibitors possess advantages compared to antibodies, as they address both intracellular and surface proteins. A showcase example is the BCR-ABL inhibitor imatinib, which resulted in dramatic improvements in the survival of chronic myeloid leukemia patients. The same applies to small molecule inhibitors directed against targets in solid tumors, e.g. the epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib to treat non-small cell lung cancer and the vascular endothelial growth factor receptor (VEGFR) kinase inhibitor sorafenib against renal cancer [14–16]. In the case of EGFR mutations in the kinase domain of the receptor, it happens that erlotinib is no longer therapeutically effective as cancer cells develop resistance to erlotinib. Numerous mutations appear during tumor progression and cause resistance [17, 18]. Therefore, it is essential to find novel inhibitors, which target and inhibit tumor-related mutant proteins.
The sequencing of tumor genomes and transcriptomes is more and more routinely applied in clinical oncology. The concept of precision medicine by mutations identified by sequence analyses may serve as a basis to select treatment options specifically addressing these mutations. Since such mutations would exclusively occur in tumors but not in normal tissues, it is expected that targeted treatments ought to provoke no or only minimal side effects. However, the vast majority of the data acquired cannot be used for therapeutic purposes yet, as we still lack drugs addressing all relevant mutations. Also, tumors frequently consist of heterogeneous subpopulations with mutational profiles different from the main cell population. Resistance to a targeted drug develop if subpopulations without the treatment-specific mutation appear. This may foster the fatal outcome of malignant diseases. Hence, one wishes to have a larger arsenal of drugs at hand to combat otherwise drug-resistant subpopulations of tumors with mutations, for which no targeted drugs are available currently.
One approach to address this latter problem is to develop individualized tumor vaccination to target mutated tumor surface proteins of individual patients. With the advent of advanced vaccination technologies, it is possible to generate individual vaccines to treat each patient according to the tumor’s individual mutational profile [19, 20]. This approach is difficult to achieve for intracellularly located proteins with tumor-specific mutations since antibodies mainly address extracellular rather than intracellular epitopes in living cells (although endocytic antibody internalization may take place). Therefore, therapeutic strategies are required to address intracellular tumor proteins, which constitute a considerable – if not the largest – portion of the tumor proteome.
In an endeavor to device new strategies for precision medicine based on small molecules to attack mutated intracellular proteins, we developed a concept, which integrates transcriptomic data with virtual drug screening of chemical libraries.
A central element in this context may be the concept of drug repurposing. Drug repurposing is a promising strategy and based on the successful usage of already approved drugs, which were initially developed for other indications than cancer [21]. One of the advantages of these drugs is that they already passed clinical safety testing in clinical trials. Considerable investments in terms of money and manpower are being spent to generate clinical evidence of safety and tolerability of a new drug to fulfill the high standards of the drug-approving authorities. Hence, drug repurposing is attractive from economic as well as medical points of view. The identification of thalidomide against severe erythema nodosum leprosum and retinoic acid against acute promyelocytic leukemia are two successful examples of drug repositioning [22, 23]. Plerixafor was initially developed as HIV drug to block viral entry into the cell, but clinical trials failed. However, leukocytosis in peripheral blood CD34 hematopoietic stem cells led to repurposing of plerixafor as stem cell mobilizing drug [24]. Our group has recently reported on drug repurposing of the anti-malarial artesunate for cancer therapy [25], e.g. prostate carcinoma [26] and colorectal cancer [27] as well as for other diseases such as viral infections [28] and schistosomiasis [29]. In addition, we found that the anti-fungal niclosamide exerted cytotoxic activity towards multidrug-resistant leukemia [30].
In the present study, we identified novel mutations in a glioblastoma patient and first screened a chemical library of more than 1500 FDA-approved drugs to find established drugs, which target novel BRAF and PIK3R1 mutations. Furthermore, we screened more than 25,000 novel compounds from the ZINC database. Then, the cytotoxicity of the selected compounds was evaluated. Furthermore, the protein expression of BRAF, PIK3R1 and other cancer biomarker proteins in the brain tumor tissue obtained from the patient was analyzed by immunohistochemistry. Finally, we identified novel small molecules that effectively targeted cells carrying mutant BRAF and PIK3R1, implying their potential for personalized glioblastoma therapy.
Methods
Patient characteristics
The patient characteristics were recently described [31]. In brief, a 65-year old patient had been diagnosed with glioblastoma WHO grade IV on May 26th, 2014. Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016). The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063). Temozolomide-based radiochemotherapy had been applied according to the current standard of care [32]. Treatment led to stable disease until May 2017, when surgery of the progressive tumor became necessary.
Transcriptome sequencing of tumor and normal tissue was performed at the National Center for Tumor Diseases (NCT, Heidelberg, Germany), which confirmed the histological diagnosis of glioblastoma. In total, 65 nucleotide substitutions, three focal and 17 larger copy number changes, as well as MGMT promoter methylation was found.
Magnetic resonance imaging (MRI) and computer tomography (CT)
Tumor development at the glioblastoma patient within 1 year (before, during and after temozolomide treatment) can be observed from the MRI scans depicted in Fig. 1. Tumor shrinkage occurred after temozolomide treatment.Fig. 1 Computed tomography (CT) scans and magnetic resonance images (MRI) before, during and after the temozolomide treatment of the glioblastoma patient
In silico analyses
A library of FDA-approved drugs (> 1,500 compounds) (http://zinc15.docking.org/substances/subsets/fda/) was screened for established drugs binding with high affinity towards wildtype BRAF, BRAF47-438del and PIK3R1G376R. The PyRx software [33] was used for initial virtual drug screening. The 10 top-ranked compounds with the highest affinities were selected for further analyses. As a second step, the ZINC database library [34] was used (> 25,000 compounds from the “all clean subset with pH 7”) to identify novel non-approved investigational compounds. Again, the 10 top-ranked compounds with the highest affinities towards BRAF47-438del and the 10 top-ranked compounds with the highest affinities towards PIK3R1G376R were selected for further analyses. The selected candidate compounds were further subjected to molecular docking with AutoDock 4 [35]. Whole protein surfaces were taken into account for virtual screening and molecular docking. Three independent docking calculations were conducted, with 25,000,000 energy evaluations and 250 runs by using the Lamarckian Genetic Algorithm. The corresponding lowest binding energies (LBE) were obtained from the docking log files (dlg), and mean values ± SD were calculated. For visualization of docking results, AutoDock Tools and Visual Molecular Dynamics (VMD) were used (Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign) (http://www.ks.uiuc.edu/Research/vmd/). Two known BRAF inhibitors (sorafenib [36] and vemurafenib [37]), two known PIK3R1 inhibitors (PI-103 [38] and LY-294,002 [39]) were used as control drugs.
Immunohistochemistry
For immunohistochemical protein detection, we applied the UltraVision polymer detection method (kit from Thermo Fisher Scientific GmbH, Dreieich, Germany). The method was previously described in detail [26]. Briefly, formalin-fixed, and paraffin-embedded tumor tissue was incubated in a humidified chamber for 1 h at room temperature with primary antibodies against BRAF (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), PI3KR1 (1:100, clone U5, Life Technologies GmbH, Darmstadt, Germany), EGFR (1:50, clone H11, Life Technologies GmbH, Darmstadt, Germany), SRC (1:200, clone 1F11, Life Technologies GmbH, Darmstadt, Germany), AKT (1:100, clone 9Q7, Life Technologies GmbH, Darmstadt, Germany), mTOR (1:100, clone F11, Life Technologies Europe BV, Bleiswijk, Netherlands), NF-κB (1:100, polyclonal, Life Technologies GmbH, Darmstadt, Germany), or Ki-67 (ab16667, dilution 1:100, Abcam, Cambridge, UK). Afterwards, Primary Antibody Amplifier Quanto (Thermo Scientific) was applied for 10 min at room temperature, and HRP Polymer Quanto (Thermo Scientific) was applied for another 10 min. Protein expression was visualized by diaminobenzidine (DAB). Quanto chromogen (Thermo Scientific) was mixed with 1 ml DAB Quanto. The tissues were counterstained in hemalaun solution (Merck KGaA, Darmstadt, Germany). Standard histochemical staining was performed with hematoxylin-eosin (HE).
The immunostained slides were scanned by Panoramic Desk (3D Histotech Pannoramic digital slide scanner, Budapest, Ungary) and quantified by panoramic viewer software (NuclearQuant and MembraneQuant, 3D HISTECH) as previously described [26].
NCI cell lines
The panel of cell lines of the Developmental Therapeutics Program (National Cancer Institute, USA) consists of 7 brain tumor cell lines (SF-295, SF-539, SNB-19, SNB-75, SNB-78, U251, XF498) and of cell lines from other tumor origins (leukemia, melanoma as well as carcinoma of the breast, ovary, prostate, lung, colon, and kidney). The origin of the cell lines has been described [40]. Up to 70 cell lines have been tested per drug using a sulforhodamine assay [41]. Some of these drugs identified by our virtual drug screening approach have been tested in this panel of cell lines, and the log10IC50 values have been deposited at the NCI website (https://dtp.cancer.gov/) [42].
Cytotoxicity
Fifty per cent inhibitory concentrations (IC50) values for the selected compounds from virtual screening of FDA approved drugs towards the brain cancer cell line panel of the NCI cell lines were selected from the National Cancer Institute (NCI) database (http://dtp.nci.nih.gov). The cytotoxicity for the NCI cell line panel was assayed by the sulforhodamine B test, as previously described [43].
Results
Genotyping
The copy number variation plot with selected mutations and variations obtained from tumor genome sequencing of a glioblastoma patient is depicted in Fig. 2. Specific mutations have been identified in BRAF (BRAF47-438del, BRAF-TTYH3 fusion), PIK3R1, MAP3K4, MAP3K5, and PTK2. The promoter of MGMT was methylated. Additionally, several genes were completely deleted, i.e. ABCA13, ABCB4, CDK13, EGFR, EIF4H, and HGF).
Fig. 2 Copy number variation plot and the relevant mutations in the glioblastoma patient
Immunohistochemistry
In order to confirm the relevance of the results of RNA-sequencing, we performed immunohistochemistry for selected markers, i.e. BRAF, PIK3R1, and EGFR. In addition, signaling molecules in the EGFR downstream signaling cascade have been investigated, e.g. kinases (AKT, SRC) and transcription factors (mTOR, NF-κB). Furthermore, general tumor features have been investigated by hematoxilin-eosin staining to monitor the typical glioblastoma multiforme morphology and by the immunohistochemical detection of Ki-67 to estimate the proliferation rate of the tumor. As shown in Fig. 3, all tumor markers except EGFR revealed strong immunopositivity. The genotyping data revealed EGFR whole gene deletion and this is confirmed by EGFR staining. The protein expression of BRAF and PIK3R1, as well as the lack of EGFR expression, fit well to the data obtained by RNA-sequencing. The expression of the additional markers (AKT, SRC, mTOR, NF-κB, Ki-67) are clues for the aggressiveness of the tumor.
Fig. 3 Immunohistochemical detection of BRAF, PIK3R1, EGFR, AKT, SRC, mTOR, NF-kB, Bar, 50 µM. HE, hematoxylin-eosin staining; NC, negative control (w/o primary antibody)
In silico analyses
In order to search for novel treatment options based on individual mutational profiles of glioblastoma genomes, we used the mutations of this patient for further bioinformatical analyses. We focused on the BRAF47-438del and PIK3R1G376R mutations because genetic alterations in these two genes are frequently assumed as driver mutations with relevance for tumor development and progression. The other mutations in the MAP3K4, MSP3K5 and PTK2 genes were not further considered.
The BRAF47-438del is a novel mutation found in this patient for the first time. Therefore, we compared this novel mutation with the most common BRAFV600E mutation. The BRAF-TTYH3 fusion protein could not be used for virtual drug screening, as no crystal structure of this protein was available in the Protein Data Bank (https://www.rcsb.org/). Therefore, a reliable homology model of this fusion protein could not be generated on the basis of the single wildtype BRAF and TTYH3 proteins.
Using PyRx and AutoDock 4.2, virtual screening of the library of FDA-approved drugs revealed drugs with high affinities towards BRAF47-438del and PIK3R1G376R (Table 1). The docking poses of top 10 compounds as well as of vemurafenib and sorafenib (control drugs for BRAF), LY-294,002 and PI-103 (control drugs for PIK3R1) are visualized in Fig. 4. With regard to the PyRx screening results, all 10 compounds revealed a stronger affinity to BRAF47-438del than vemurafenib and sorafenib. Concerning PIK3R1G376R, the top 10 compounds revealed stronger interaction than LY-294,002. All top 10 compounds except tubocurarine, metocurine and idarubicin possess higher affinity than PI-103. Concerning the BRAF47-438del AutoDock results, STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol revealed stronger interaction than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also have stronger affinity than vemurafenib. Within the compounds revealed by PIK3R1G376R docking results, all top 10 compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002. The established anti-BRAF drug sorafenib bound with slightly less affinity to BRAF47-438del (-9.39 ± 0.09 vs. -9.57 ± 0.22 kcal/mol) than to the corresponding wildtype protein as observed in molecular docking analyses. The established anti-PIK3R1 inhibitors; LY-294,002 and PI-103 revealed stronger binding to PIK3R1G376R mutant protein than wildtype protein (-6.47 ± < 0.01 vs. -5.19 ± 0.02 for LY-294,002 and − 7.22 ± 0.01 vs -5.76 ± 0.06 kcal/mol for PI-103). Accordingly, it was possible to identify drugs that bind stronger to BRAF47-438del than sorafenib and vemurafenib and drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103, all of those can specifically target mutant proteins.Table 1 Top 10 out of > 1500 FDA-approved established drugs with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
Tubocurarine -11.0 -8.61 ± 0.01 0.48 ± < 0.01 Met1, Ala3, Ser5, Gly11, Ala12, Glu13, Gly15, Leu18, Gly21, Asp22, Glu26
STK396645 -10.9 -13.78 ± 0.45 0.0001 ± < 0.001 Lys288, Ala296, Lys298, Arg299, Lys291, Cys293, Pro294, Lys295, Leu329, Pro330
Celecoxib -10.7 -8.94 ± 0.01 0.280 ± 0.004 Ser340, Arg343, Glu153, Phe156, Met158, Leu161, Thr259, Asn292, Glu338
Aclarubicin -10.6 -8.39 ± 0.81 1.09 ± 0.88 Ser222, Tyr368, Ser75, Phe76, Ala105, Asn108, Asp184, Lys186, Phe203, Gly204, Leu205, Ala206, Thr207, Gln220, Ala370, Val373
Pimozide -10.4 -10.55 ± 0.27 0.02 ± 0.01 Gly223, Lys186, Leu221, Ser222, Gly223, Ser224, Ile225, Met228, Leu262, Arg270, Ile363, Ala365, Ala370, Phe371, Val373, His374
Folidan -10.3 -10.98 ± 0.30 0.009 ± 0.004 Lys288, Ser291, Lys298, Arg299, Asn292, Cys293, Pro294, Lys295, Arg334, Ser335
Acetophenone -10.2 -10.48 ± 0.04 0.02 ± 0.02 Ser340, Arg343, Phe156, Glu157, Met158, Leu161, Met258, Gln261, Leu286, Asn292, Glu338, Pro339, Leu341
LS-194,959 -10.2 -13.34 ± 0.21 0.0002 ± < 0.001 Lys288, Ser291, Lys295, Lys298, Arg299, Cys293, Pro294, Ala296
Danazol -10.2 -9.41 ± < 0.01 0.126 ± 0.0004 His150, Glu153, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Leu341, Asn342
Triamterene -10.1 -7.69 ± 0.01 2.29 ± 0.01 Leu149, His150, Met258, Glu153, Phe156, Glu157, Met158, Leu161, Asn292, Glu338, Leu341
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
LS-194,959 -9.9 -8.07 ± 0.05 1.22 ± 0.10 Arg40, Lys79, Leu80, Ile81, Lys82, Tyr116
STK396645 -9.7 -9.66 ± 0.16 0.08 ± 0.02 Asp37, Lys130, Ile142, Met282, Thr285, Gln286, Lys287, Gly288, Val289, Arg290
Carminomycin -9.3 -7.36 ± 0.51 5.21 ± 4.75 Glu143, Gly146, Leu149, His150, Leu284, Thr285, Val289, Gln291
Albamycin -9.3 -5.97 ± 0.07 42.39 ± 4.96 Lys275, Thr285, Trp35, Ile38, Glu42, Lys46, Val128, Gln132, Asp278, Gln279, Leu281, Met282,
Gliquidone -9.2 -7.38 ± 0.59 5.56 ± 5.67 Trp35, Lys46, Thr50, Ala51, Thr54, Tyr126, Pro127, Val128, Lys130, Gln132, Gln133, Gln279, Met282
Fazadon -9.2 -7.73 ± 0.06 2.15 ± 0.19 Glu143, Gly146, Leu149, Asn153, Leu284, Val289, Lys293
Daunorubicin -9.1 -6.40 ± 0.67 27.83 ± 19.78 Asn153, Gly146, Leu149, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Tubocurarine -9.0 -6.72 ± 0.02 11.76 ± 0.38 Leu149, Glu42, Arg277, Leu281, Leu284, Thr285, Gln291, Lys293
Metocurine -8.9 -7.30 ± 0.01 4.48 ± 0.03 Gly146, Leu149, His150, Asn153, Arg277, Leu281, Leu284, Val289, Gln291
Idarubicin -8.8 -7.32 ± 0.76 7.61 ± 9.51 Trp35, Asp37, Glu42, Lys46, Leu281, Met282, Thr285
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 4 Docking poses of the top 10 ranked FDA-approved established drugs on the BRAF47-438del and PIK3R1G376R mutant proteins
Furthermore, 10 investigational non-approved compounds from the ZINC database were identified for BRAF47-438del and PIK3R1G376R mutated proteins (Table 2). Residues forming hydrogen bonds were labeled in bold. Docking poses are depicted in Fig. 5. With regards to the PyRx results, all compounds revealed stronger interaction than the control compounds. BRAF47-438del AutoDock results pointed out that all compounds interact stronger than sorafenib. ZINC09339473, ZINC22798105, ZINC33068262, ZINC00691692, ZINC08667624, ZINC33068261, ZINC09219428, ZINC31840966 interact stronger than both vemurafenib and sorafenib. PIK3R1G376R AutoDock results revealed that all compounds except ZINC02690584 interact stronger than both PI-103 and LY-294,002 (Table 3).Table 2 Top 10 out of > 25,000 non-approved investigational compounds from the ZINC database with highest binding affinities towards mutant proteins (LBE = lowest binding energy, kcal/mol, pKi = predicted inhibition constant, µM)
LBE
BRAF47-438del PyRx AutoDock pKi Interacting residues
ZINC09339473 -12.3 -11.30 ± 0.28 0.006 ± 0.003 Phe156, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Glu338, Ser340, Asn342, Arg343, Ala344
ZINC10578766 -12.3 -10.16 ± 0.47 0.043 ± 0.033 Leu149, His150, Glu153, Thr154, Met258, Thr259, Gln261, Arg290, Glu338, Ser340, Leu341, Asn342
ZINC22798105 -12.2 -10.32 ± 0.24 0.03 ± 0.01 Ser5, Gly6, Gly9, Ala12, Gly15, Ala17, Leu18, Gly21, Asp22, Glu24, Glu26
ZINC33068262 -11.8 -11.39 ± 0.36 0.005 ± 0.003 Leu149, Met258, Thr259, Gln261, Leu286, Val289, Arg290, Asn292, Glu338, Pro339, Ser340, Arg343, Ala344
ZINC00691692 -11.8 -10.87 ± 0.21 0.014 ± 0.006 Leu161, Met258, Thr259, Gln261, Ser265, Leu286, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Arg343
ZINC08667624 -11.8 -12.35 ± 0.23 0.001 ± < 0.001 Glu153, Phe156, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Glu338, Pro339, Ser340, Leu341, Arg343
ZINC33068261 -11.7 -11.41 ± 0.10 0.004 ± 0.001 Leu149, His150, Glu153, Phe156, Leu161, Met258, Thr259, Gly260, Gln261, Arg290, Glu338, Pro339, Ser340
ZINC09219428 -11.7 -10.19 ± 0.39 0.039 ± 0.027 His150, Glu153, Leu161, Met258, Thr259, Gln261, Arg290, Asn292, Cys293, Glu338, Ser340, Leu341, Asn342, Arg343
ZINC21793973 -11.6 -9.66 ± 0.01 0.083 ± 0.002 Ser222, Gly223, Ile225, Leu226, Met228, Ile233, Leu262, Ile267, Arg270, Ile273, Ile363, Ala370, Phe371, Val373
ZINC31840966 -11.6 -10.81 ± 0.17 0.012 ± 0.004 Leu149, His150, Met158, Leu161, Met258, Thr259, Gln261, Asn292, Cys293, Glu338, Ser340, Leu341, Asn 342, Arg343, Ala344
sorafenib -8.5 -9.39 ± 0.09 0.13 ± 0.02 Leu49, Gly50, Arg51, Arg52, Asp57, Trp58, Ile60, Gln64, Trp84, Met125
vemurafenib -7.8 -10.17 ± 0.16 0.04 ± 0.01 Leu149, His150, Glu153, Phe156, Met258, Thr259, Gly260, Gln261, Arg290, Asn292, Pro339, Ser340, Asn342, Arg343
PIK3R1G376R PyRx AutoDock pKi Interacting residues
ZINC12583338 -10.8 -8.73 ± 0.24 0.42 ± 0.15 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Val289, Gln291, Lys293
ZINC13828412 -10.3 9.27 ± < 0.01 0.16 ± < 0.01 Ile142, Glu143, Gly146, Leu149, His150, Asn153, Leu281, Leu284, Thr285, Val289, Arg290, Gln291
ZINC08854569 -10.1 -8.08 ± 0.20 1.25 ± 0.45 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Gln291
ZINC01801780 -10 -7.36 ± 0.01 4.00 ± 0.01 Glu143, Glu146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291
ZINC02277300 -10 -7.66 ± 0.01 2.41 ± 0.02 Ile142, Glu143, Gly146, Leu281, Leu284, Val289, Gln291, Lys293
ZINC09358971 -9.9 -7.85 ± 0.04 1.77 ± 0.12 Glu143, Gly146, Leu149, Leu284, Thr285, Val289, Gln291, Lys293
ZINC02690584 -9.8 -6.73 ± 0.01 11.59 ± 0.22 Gly146, Leu149, Leu284, Thr285, Val289, Lys292
ZINC09153343 -9.8 -8.04 ± 0.12 1.29 ± 0.27 Ile142, Glu143, Gly146, Lys147, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13730374 -9.8 -9.02 ± 0.01 0.24 ± < 0.01 Ile142, Glu143, Gly146, Leu149, Leu281, Leu284, Thr285, Val289, Gln291, Lys293
ZINC13828408 -9.8 -7.87 ± 0.01 1.69 ± 0.02 Ile142, Glu143, Gly146, Leu149, His150, Leu284, Val289, Gln291, Lys293
PI-103 -9.0 -7.22 ± 0.01 5.10 ± 0.08 Ile142, Gly146, His150, Leu281, Leu284, Val289, Gln291, Lys293
LY-294,002 -8.1 -6.47 ± < 0.01 18.07 ± 0.02 Ile142, Glu143, Gly146, Leu284, Val289, Lys293
Fig. 5 Docking poses of the top 10 ranked non-approved investigational compounds of the ZINC database to the BRAF47-438del and PIK3R1G376R mutant proteins
Table 3 Disease indications and modes of action of FDA-approved drugs identified by virtual drug screening
Drug Disease/application Mode of action Origin Potentially recommendable for therapy
BRAF47-438del:
Tubocurarine Arrow poison (main component of curare) Non-depolarizing muscle relaxant; competitive inhibitor of acetylcholine receptors at the postsynaptic membrane; inhibition of ligand-driven natrium ion channels. Condrodendron tomentosum No
STK396645 No
Celecoxib Degenerative joint diseases (arthrosis), rheumatoid arthritis; Morbus Bechterew (Spondylitis ankylosans) Non-steroidal anti-rheumatic; selective COX2 inhibitor; inhibition of prostaglandin synthesis Synthetic Yes
Aclarubicin Cancer Anthracycline; histone eviction from chromatin upon DNA intercalation Strepomyces galilaeus Yes
Pimozide Chronic schizophrenic psychoses Antipsychotic; postsynaptic inhibition of dopamin receptors and thereby stimulation of presynaptic dopamin release; inhibition of acidic sphingomyelinase Synthetic Yes
Folidan (Calcium folinate, Leucovorin) Thrombocytopenia, neutropenia, anemia; Folic acid source to prevent depletion by folic acid antagonists; counteracts toxicity by folic acid antagonists Synthetic Yes
Acetophenone chemical precursor for the synthesis of flagnaces and pharmaceuticals Hypnotic, anticonvulsant, sedative Ingredient of essential oils (labdanum, castoreum, Stirlingia latifolia) No
LS-194,959 Cancer Inhibitor of CDK2 synthetic No
Danazol Endometriosis Testosterone derivative; inhibitor of gonatropine release (FSH, LH); interruption of telomere erosion Semisynthetic Yes
Triamterene Hypertonia; chronic heart insufficiency Natrium-sparing diuretic; inhibition of aldosteron-dependent epithelial natrium channels in late distal tubuli Synthetic Yes
PIK3R1G376R:
LS-194,959 see above No
STK396645 see above No
Carminomycin Cancer Anthracycline; DNA intercalation; DNA topoisomerase II inhibitor Actinomadura carminata Yes
Novobiocin Bacerial infections Aminocoumarin antibiotic; inhibition of bacterial gyrase, subunit GyrB Streptomyces niveus Yes
Gliquidone Diabetes mellitus Sulyonylurea; inhibition of natrium channels and depolarization of B-cells leading to opening of current-regulated potassium channels. The potassium influx depletes insulin from storage vesicles and increases insulin release into the blood. Synthetic Yes
Fazadon (fazadinium bromide) Anaesthetic in otorhinolaryngological surgery Non-depolarizing muscle relaxant; antagonist of acetylcholine through neuromuscular blockage Synthetic Yes
Daunorubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor; generation of cytotoxic superoxide. And hydroxyl-radicals Streptomyces peucetius and S. coeruleorubidus Yes
Tubocurare see above
Metocurine Convulsive conditions: anesthetic adjuvant Non-depolarizing muscle relaxant; antagonist of acetylcholine by competitive binding to cholinergic receptors at the motor-end plate Synthetic Yes
Idarubicin Acute leukemia Anthracycline; DNA intercalaction; DNA topoisomerase II inhibitor Semisynthetic Yes
Cytotoxicity
Virtual drug screening revealed that anthracyclines, among other drugs, might target the mutated BRAF and PIK3R1 proteins. Since these drugs are not clinically used for the treatment of glioblastoma multiforme, we explored whether these compounds are able to kill glioblastoma cells in vitro with comparable efficacy than cell lines from other tumor origin. For this reason, we screened the NCI database for test results of the compounds by our virtual drug screening approach. The log10IC50 values of several brain tumor cell lines for the corresponding compounds included into this database are shown in Fig. 6. For comparison, the log10IC50 mean values for each of the test compounds of 46–63 other tumor cell lines have been calculated. The following observations were made: (1) the anthracyclines (daunorubicin, idarubicin, aclacinomycin) were the most cytotoxic compounds in this panel of drugs; (2) Pimazole also showed considerable cytotoxicity against tumor cells, although this is a psychoactive drug used for the treatment of chronic schizophrenic psychoses; (3) The cytotoxicity of the compounds tested were not significantly different between glioblastoma cell lines and all other tumor cell lines, indicating that these compounds might be effective in glioblastoma patients, given that appropriate formulations will be chosen to cross the blood-brain barrier. Aclarubicin revealed cytotoxicity (log10IC50) in the range of -7.2 to -7.6, higher than both sorafenib (in the range of -5.6 to -5.8) and vemurafenib (in the range of -5.3 to -5.6) (Fig. 6a). Idarubicin and daunorubicin (in the range of -7.4 to -7.9) revealed higher cytotoxicity than LY-294,002 (in the range of -4.7 to -5.6) (Fig. 6b).Fig. 6 Cytotoxicity of the available top 10 ranked FDA-approved established drugs (a BRAF-47-438del screening, b PIK3R1-G376R screening) and the known inhibitors towards brain cancer cell lines of the NCI (Bethesda, USA) panel. Shown are log10IC50 values (M)
Discussion
Since many years, MRI/CT imaging is routinely used for monitoring treatment success. However, imaging cannot deliver hints, which salvage therapy could be applied if standard therapy fails. A novel concept in precision medicine is to sequence tumor transcriptomes to identify patient-specific mutations, which can be then addressed by targeted therapeutics [44].
In the present investigation, we explored the possibility to use transcriptomic information for virtual drug screening, in order to indicate novel treatment options for individual patients. For this purpose, we used the mutational profile of a glioblastoma patient as an example to prove the feasibility of this approach. We first focused on screening of FDA-approved drugs, since repurposing of drugs initially approved for other diseases than cancer provided interesting treatment alternatives in many cases. The mutation profile obtained by genotyping of the patient’s tumor has several implications for therapy:The BRAF inhibitors vemurafenib and sorafenib may be less efficient to inhibit mutated BRAF proteins than wildtype BRAF.
Promoter-methylation of the MGMT gene indicates that MGMT protein expression may be downregulated. As MGMT represents a resistance factor for temozolomide [45], this drug may be exquisitely efficient to treat this patient. Indeed, the treatment history of the patient demonstrated a very good response to the temozolomide-based radiochemotherapy protocol.
The EGFR deletion indicated that EGFR-directed drugs (such as erlotinib, gefitinib and others) might not be effective.
Small molecules addressing the other mutations are not available yet, which limits the therapeutic options in this patient.
The HGF gene deletion might be relevant for toxic side effects. HGF (hepatocyte growth factor) is known to protect from drug-induced hepatotoxicity [46–48]. In fact, this patient experienced hepatotoxicity by radiochemotherapy with temozolomide and compassionate use of artesunate and Chinese herbs [31]. It can be speculated that the observed hepatotoxicity might be explained at least in part by the HGF deletion.
To validate the RNA-sequencing data, we performed immunohistochemical analyses. The strong immunostaining of BRAF and PI3KR1 indicated that the primary antibodies used for immunohistochemistry detect not only wildtype but also mutated forms of these proteins and that both proteins were expressed at high levels in this glioblastoma case. Accordingly, immuno-positive staining of tissue slices by these antibodies is probably not the suitable indicator for the usage of the compounds newly identified, whereas sequencing of tissue is more appropriate for the detection of the new mutations.
Furthermore, the sequencing results showed that the EGFR gene was deleted, which was confirmed by immunohistochemistry since the tumor was EGFR-negative in immunohistochemical staining. Even if EGFR is not expressed in this tumor, the question arises, whether downstream signaling pathways are still operative, which might then not be linked to EGFR but to other signaling molecules. We observed strong expressions of signaling kinases (SRC, AKT) and transcription factors (mTOR, NF-κB). Hence, these signaling molecules may still be susceptible to specific small molecule inhibitors against SRC, AKT, mTOR, or NF-κB) [49–52].
A limitation of the tumor sequencing approach is that therapeutic antibodies and small molecules are approved only for some of the major tumor-related proteins, but the vast majority of mutations cannot be therapeutically addressed as of yet. A premise to exploit the full potential of precision medicine would be that the therapeutic options keep pace with the diagnostic power and that ideally hundreds of drugs are available to inhibit the numerous tumor-related mutated proteins. An approach to reach this goal may be for instance tumor vaccination based on mutanomic profiling [20]. Comparable approaches are difficult to realize for small molecules, because the clinical approval of novel drugs is laborious and cost-intensive, and it can take years to bring a new drug to the market.
Therefore, it may be feasible to search for drugs, which have been already approved for other diseases and conditions and which also show activity against cancer by inhibiting tumor-related proteins. This approach has been termed drug repurposing and recently led to astonishing treatment successes [53–55].
In the present investigation, we attempted to utilize the wealth of information of RNA-sequencing data obtained from tumor genotyping to identify already FDA-approved drugs that bound with high affinity to mutated proteins in a glioblastoma patient. A database of > 1,500 FDA-approved drugs was first screened by PyRx. The results obtained were then refined by molecular docking with AutoDock 4.2. Furthermore, we used the novel BRAF47-438del mutation and the known PIK3R1G376R mutation as models to prove the feasibility of this approach. It is reasonable to search for drugs that stronger bind to BRAF47-438del than sorafenib and vemurafenib and to search for drugs that stronger bind to PIK3R1G376R than LY-294,002 and PI-103 to identify compounds that can specifically target mutant proteins. The BRAF47-438del docking results pointed out that STK396645, pimozide, folidan, acetophenone, LS-194,959, danazol bound stronger than sorafenib. STK396645, pimozide, folidan, acetophenone, LS-194,959 also had stronger affinities than vemurafenib. Considering the PIK3R1G376R results, all compounds except albamycin, daunorubicin, tubocurarine had higher affinities than PI-103, whereas all compounds except albamycin, daunorubicin revealed stronger binding than LY-294,002.
Virtual drug screening unraveled a panel of drugs that bound with high affinity to BRAF47-438del or PI3KR1G376R. These drugs were from diverse chemical classes and had different pharmacological activities. Strong poisons such as tubocurarine appeared in the screening, which cannot be considered for treatment. Remarkably, anthracyclines were also among the top-ranking drugs, which are known for their strong anticancer activity (daunorubicin, idarubicin, aclarubicin, carminomycin). Further drugs identified by virtual drug screening revealed anti-inflammatory (celecoxib), psycho-active (pimozide, acetophenone), muscle relaxant (fazadon, metocurine), diuretic (triamterene) antidiabetic (gliquidone), antibiotic (novobiocin), anti-endometriotic (danazol) or other pharmacological activities. The wide variety of drugs can be taken as a clue for the potential of repurposing drugs with diverse pharmacological modes of action for cancer therapy. The question arises, which of these drugs may be useful to treat the glioblastoma of the patient presented in our study. The decision may depend not only on the known side effects of these drugs but also on whether they are able to cross the blood-brain barrier (BBB). While this is well known for psychoactive drugs, anthracyclines are known substrates of the ATP-binding cassette (ABC) transporter, P-glycoprotein, which is an important constituent of the BBB. Anthracyclines such as daunorubicin, idarubicin and others are usually not used for the treatment of glioblastoma, since sufficient amounts cannot cross the BBB to exert considerable therapeutic anticancer effects in the brain [56]. This problem may, however, be tackled by novel nanotechnologically engineered anthracycline-releasing devices to overcome the BBB [57–59]. Since several anthracyclines appeared as top-ranked drugs to bind with high affinity to BRAF47-438del and PI3KR1G376R, it is worth reconsidering them for glioblastoma therapy, if appropriate nanotechnological formulations allow BBB-crossing. This problem may, however, be overcome, if appropriate nanotechnological formulations will be applied. There are daunorubicin liposomes preparations available on the market [60], and anthracycline liposomes have been shown to cross the blood-brain barrier [61–63].
Of course, anthracyclines cannot be viewed as mono-specific drugs addressing the two mutations in BRAF and PI3KR1 in an exclusive manner. As classical drugs of natural origin, they can be reconsidered to act in a multi-specific fashion. Anthracyclines are known to intercalate into DNA, to inhibit DNA topoisomerase II, and to generate cytotoxic superoxide- and hydroxyl radicals. Several other on- and off-target effects can be assumed, and binding to mutated BRAF and PI3KR1 proteins may belong to these still unknown modes of action of anthracyclines.
Having the multi-specific nature of many drugs in mind, it is reasonable to investigate their cytotoxic potential in cell lines with diverse mutations. Therefore, we compared the cytotoxicity of the drugs identified by our virtual drug screening approach in the panel of brain tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) and compared their activity with those of cell lines from other tumor origins. These results may deliver additional information, whether these drugs - initially developed for the treatment of other diseases - are suitable for drug repurposing in glioblastoma therapy. The data obtained with the NCI panel of cell lines pointed out aclarubicin, daunorubicin and idarubicin for glioblastoma treatment since they revealed higher cytotoxicity than the known inhibitors (sorafenib, vemurafenib and LY-294,002). Although the standard drug for glioblastoma treatment is temozolomide, anthracyclines also revealed strong cytotoxicity in vitro against brain tumor cell lines.
Pimozide is a psychoactive drug that does cross the BBB. Since this drug also revealed cytotoxicity towards brain tumor cell lines in vitro, its repurposing for clinical glioblastoma treatment may also be considered.
At the time point, when we obtained these results, the glioblastoma was already at a progressive state and the patient decided not to undergo any further drug treatment anymore. Unfortunately, the patient died before we could recommend an individualized treatment with a liposome-based anthracycline. Hence, a final proof of the clinical success of the concept presented in his paper could not be given. Nevertheless, our study represents a preclinical feasibility approach and a method to identify possible treatment candidates in cases that lack therapeutic options, which is a scenario frequently occurring in clinical practice.
We suggest that further investigations using the tumor genomes of patients should be made to predict active drugs and to observe whether treatment of patients with these drugs really leads to clinically significant response rates.
In general, repurposing of already approved drugs may not deliver more drugs for the individual treatment of cancer patients due to the multiplicity of tumor-related mutations. Therefore, we applied a second approach and used virtual drug screening to screen a chemical library of > 25,000 non-approved investigational compounds. We analyzed whether novel compounds can be identified that also bind to the BRAF and PI3KR1 mutations with high affinities. Indeed, most of the selected compounds revealed stronger binding than the known inhibitors. For instance, all of the selected compounds bound stronger to BRAF47-438del mutant protein than sorafenib and selected compounds except ZINC21793973 bound stronger than vemurafenib. The identified substances may be used as a starting point for further drug development to improve glioblastoma therapy in the future.
Despite the attractiveness of the virtual drug screening approach based on tumor sequencing data, there are also some limitations of this approach that have to be discussed:Virtual drug screening of both FDA-approved drug as well as non-approved investigational compounds may result in the identification of a certain rate of false positive candidates [64, 65]. Therefore, results from virtual drug screening should be verified by in vitro or in vivo experiments.
The sequencing of tumor genomes and transcriptomes delivers information on both relevant driver as well as irrelevant passenger mutations regarding tumor development and progression. Hence, careful visual inspection of the sequencing data is advisable to make rational decisions, which mutated proteins are most suitable for virtual drug screening to find suitable candidate drugs with the therapeutic potential to significantly and sustainably improve tumor response to treatment and to prolong the survival time of patients.
Conclusions
Systematic genome-dependent repurposing may reveal putative drug candidates for the further development of precision medicine in cancer and other gene-dependent diseases. A combined concept consisting of multiple techniques, i.e. tumor transcriptomics, diverse virtual drug screening methods, chemical libraries for drug repurposing and in vitro testing may help to make beneficial predictions on small molecules to inhibit distinct mutated proteins and to improve cancer therapy for each individual patient. We term the concept presented here “ReSeqtion” (Repurposing of drugs by genome Sequencing and bioinformatic calculation).
Author contributions
M.E.M.S., O.K. A.Y. and K.M. performed the bioinformatical in silico analyses. M.E.M.S. performed the immunohistochemical staining. F.W. and F.A.G. were the treating physicians. H.J.G. read and revised the manuscript. T.E. designed the concept and wrote the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Data availability
The data are available upon reasonable request.
Compliance with ethical standards
Ethical approval
The patient was enrolled in the INTRAGO trial [32], which was registered at clinictrials.gov, no. NCT02104882 (registration date: 03/26/2014) (https://clinicaltrials.gov/ct2/show/NCT02104882). INTRAGO was approved by the local ethics committee (Medical Ethics Commission II of the Faculty of Mannheim, University of Heidelberg 2013-548S-MA) and the Federal Office of Radiation Protection (Z 5-2246/2-2013-063).
Informed consent
Informed written consent had been given by the patient that the data are allowed to be scientifically used and published (dated: January 26, 2016).
Conflict of interest
All authors declare there is no conflict of interest. However, patent application #EP18211231 “A method to determine agents for personalized use” is disclosed.
Abbreviations
AKT AKT serine/threonine kinase 1
BBB blood brain barrier
BRAF B-Raf proto-oncogene, serine/threonine kinase
CD34 cluster of differentiation marker 34
CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats/CRISPR-associated 3
CT computer tomography
EGFR epidermal growth factor receptor
FDA Food and Drug Administration
HGF hepatocyte growth factor
LBE lowest binding energy
MAP3K4/5 Mitogen-activated protein kinase kinase kinase 4/5
MGMT O-6-methylguanine-DNA methyltransferase
MRI magnetic resonance imaging
NCI National Cancer Institute
NF-κB nuclear factor-kappa B
PIK3R1 Phosphoinositide-3-kinase regulatory subunit 1
PTK2 Protein tyrosine kinase 2
RMSD root mean square deviation
SRC SRC proto-oncogene, non-receptor tyrosine kinase
TTYH1 Tweety family member 1
VEGFR vascular endothelial growth factor receptor
VMD Visual Molecular Dynamics
WHO World Health Organization
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Oral | DrugAdministrationRoute | CC BY | 33313992 | 15,046,217 | 2021-06 |
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