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Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Encephalitis brain stem'. | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | DEXAMETHASONE | DrugsGivenReaction | CC BY-NC | 33435771 | 18,895,687 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Listeriosis'. | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | DEXAMETHASONE | DrugsGivenReaction | CC BY-NC | 33435771 | 18,895,687 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Respiratory failure'. | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | DEXAMETHASONE | DrugsGivenReaction | CC BY-NC | 33435771 | 18,866,141 | 2021-01 |
What was the administration route of drug 'DEXAMETHASONE'? | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY-NC | 33435771 | 18,895,687 | 2021-01 |
What was the dosage of drug 'DEXAMETHASONE'? | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | 10 mg (milligrams). | DrugDosage | CC BY-NC | 33435771 | 18,866,141 | 2021-01 |
What was the outcome of reaction 'Condition aggravated'? | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | Recovered with sequelae (consequent health issues) | ReactionOutcome | CC BY-NC | 33435771 | 18,866,141 | 2021-01 |
What was the outcome of reaction 'Encephalitis brain stem'? | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | Recovered with sequelae (consequent health issues) | ReactionOutcome | CC BY-NC | 33435771 | 18,866,141 | 2021-01 |
What was the outcome of reaction 'Listeriosis'? | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | Recovered with sequelae (consequent health issues) | ReactionOutcome | CC BY-NC | 33435771 | 18,866,141 | 2021-01 |
What was the outcome of reaction 'Respiratory failure'? | Severe invasive Listeria monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man: a case report and literature review.
Neurolisteriosis is a foodborne infection of the central nervous system that is easily misdiagnosed, especially in healthy adults with atypical symptoms. A 50-year-old man presented with a 3-day history of distortion of the oral commissure. Facial neuritis was diagnosed and treated with intravenous dexamethasone. His condition deteriorated rapidly, and he presented with a slow pharyngeal reflex, stiff neck, and signs of peripheral facial paralysis. Brain magnetic resonance imaging revealed multiple ring-enhanced foci in the brainstem. Routine and biochemical cerebrospinal fluid (CSF) analyses showed increased white blood cells and microproteins. Blood culture and high-throughput genome sequencing revealed Listeria monocytogenes DNA in the CSF. Ampicillin, amikacin, and meropenem were administered, and the patient was transferred from the intensive care unit to a standard medical ward after 2 months. The patient could walk and eat normally; however, he required intermittent mechanical ventilation at 11 months after discharge. Although L. monocytogenes meningitis is rare in healthy immunocompetent adults, it must be considered as a differential diagnosis, especially in adults whose conditions do not improve with cephalosporin antibiotic administration. L. monocytogenes rhombencephalitis mimics facial neuritis and develops quickly. Prompt diagnosis is essential for rapid initiation of antibiotic therapy to achieve the best outcome.
Introduction
Listeriosis is a rare disease, with a reported annual incidence of 4.4 per 1 million individuals,1 and typical symptoms include fever, body aches, and gastrointestinal symptoms such as diarrhea. Immunocompromised, elderly, and pregnant individuals, as well as newborns, are most susceptible. Listeria monocytogenes is a Gram-positive facultative intracellular bacillus, and its transmission occurs mainly through the consumption of contaminated food. L. monocytogenes causes one of the most life-threatening bacterial infections of the central nervous system (CNS). Manifestations include meningitis, meningoencephalitis, and rhombencephalitis, and it is the third most common cause of bacterial meningitis.2
L. monocytogenes encephalitis has high mortality and neurological sequelae rates, at 20% and 68%, respectively.3 At present, reports of L. monocytogenes meningitis in immunocompetent and healthy adults with atypical initial symptoms (similar to facial neuritis) are limited. Such cases are easily misdiagnosed, and use of steroid therapy can endanger the patient’s life. Therefore, we report a case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man, and we present a comprehensive review of the literature to summarize similar reports.
Case report
In January 2018, a 50-year-old man was admitted to our hospital with a 3-day history of distortion of the oral commissure. He had dizziness, headache, malaise, and vomiting beginning 3 days before admission. A shallow nasolabial sulcus on the left side, distortion of the oral commissure, and weakness in closing the left eye subsequently appeared. The patient had no diplopia, dysdipsia, hemiplegia, or limb numbness, and he was lucid and alert. Diagnoses of acute upper respiratory tract infection and peripheral facial neuritis had been considered at another hospital. The patient was previously diagnosed with tuberculosis at 4 years old; he reported that the tuberculosis had long been cured, and he had no history of immunodeficiency. He had no history of major trauma, toxic exposure, smoking, alcoholism, drug abuse, or hereditary disease. His family denied any history of unpasteurized buttermilk consumption.
A physical examination on admission revealed a blood pressure of 135/85 mmHg, temperature of 36.5°C, and pulse rate of 85 bpm. He had no signs of meningismus or other neurological irregularities. The patient’s white blood cell count (10.61 × 109/L), fasting blood glucose level (6.3 mmol/L), and glycosylated hemoglobin level (6.1%) were slightly increased, whereas his serum creatinine, cholesterol, C-reactive protein, creatine kinase, and procalcitonin levels were normal. The blood coagulation parameters were within normal limits. Tests for antibodies to human immunodeficiency virus and Treponema pallidum were negative. Emergency head computed tomography (CT) showed no apparent abnormalities (Figure 1a). Chest CT showed chronic-appearing fibrotic streaks in both lungs. Peripheral facial neuritis was diagnosed; the patient was admitted, and dexamethasone (10 mg/day) was administered intravenously (iv) for 3 days.
Figure 1. Computed tomography (CT), magnetic resonance imaging (MRI), and blood culture results. (a) Head CT showing no apparent abnormality after admission. (b) The patient’s condition worsened; however, head CT re-examination showed no apparent abnormality on the third day after admission. (c) T1-weighted image (WI) showing low-signal midbrain lesions (arrow). (d) T2-WI showing a hyperintense dorsal pontine lesion (arrow). (e) Diffusion WI showing no abnormal signal in the midbrain. (f) A high apparent diffusion coefficient was observed in the midbrain lesions (arrow). (g) Fluid-attenuated inversion recovery sequence showing hyperintense lesions (arrow) in the dorsal lower pons. (h–k) Gadolinium-enhanced MRI showing multiple ring-enhanced lesions in the (h) left midbrain (arrow), (i) medulla oblongata, (j) dorsal upper medulla oblongata (arrow), and (k) dorsal lower pons (arrow). (l) Listeria monocytogenes was cultured from peripheral blood. The tryptone soy blood agar plate produced round bacterial colonies with neat edges and central uplifting; the surfaces were smooth and whitish gray.
The patient then developed a headache, dysdipsia, malaise, and fever (39.2°C) 3 days after hospitalization, and a physical examination at that time showed neck stiffness and slowness of the pharyngeal reflex, along with signs of peripheral facial paralysis. The facial nerve, glossopharyngeal nerve, and meninges were considered affected; however, the nature of the lesion was unknown. No apparent abnormality was found in a head CT re-examination (Figure 1b). The patient’s condition worsened rapidly, and he developed somnolence, aphagia, and slurred speech. Brain magnetic resonance imaging (MRI) (Figure 1c–k) performed 4 days after admission showed multiple abnormal foci in the brainstem. The cerebrospinal fluid (CSF) opening pressure was 245 mmH2O. Routine CSF testing showed a markedly increased white blood cell count (8.35 × 108/L). Biochemical CSF examination showed a potassium concentration of 2.5 mmol/L, chloride concentration of 144 mmol/L, glucose concentration of 3.0 mmol/L, and microprotein concentration of 2.19 g/L. No organisms were observed on Gram, India ink, or acid-fast staining.
The patient was considered to have a tuberculous or bacterial intracranial infection and was transferred to the intensive care unit (ICU). He underwent physical cooling using an ice blanket and ice cap and was given anti-infective and antiviral medications (ceftriaxone, 2 g iv every 12 hours; ganciclovir, 0.25 g iv every 12 hours). Furthermore, he underwent intracranial decompression using mannitol and symptomatic and supportive treatment in the ICU. Further examination revealed negative tests for influenza A and B viral antigens and anti-Toxoplasma antibodies. Two days later, L. monocytogenes (Figure 1l) was isolated from the blood culture and was identified by time-of-flight mass spectrometry, but no abnormality was found in the CSF culture. Genetic identification of CSF pathogens by high-throughput genome sequencing found only L. monocytogenes (sequence number 210).
The patient’s condition quickly progressed to respiratory failure; thus, mechanical ventilation was initiated. Based on the sequencing and antimicrobial susceptibility testing results, anti-infectives (ampicillin, 1 g iv every 8 hours; amikacin, 0.4 g iv every 12 hours; and later, meropenem, 2 g iv every 8 hours) were administered, and the patient was transferred from the ICU to a standard medical ward after 2 months. The patient could walk and eat normally; however, damage to the respiratory center resulted in central respiratory insufficiency, and he required intermittent mechanical ventilation at the 11-month post-discharge follow-up visit. He was satisfied with his treatment and recovery.
Discussion
This case of severe, invasive L. monocytogenes rhombencephalitis mimicking facial neuritis in a healthy middle-aged man showed that the disease can be easily misdiagnosed in this population. Moreover, this case showed that observation of ring-enhanced brainstem lesions on MRI and high-throughput genome sequencing results are important for accurate diagnosis. Furthermore, this case showed that the choice of proper antimicrobials is key to a successful therapy.
L. monocytogenes is routinely described as an opportunistic bacterium, and it typically infects pregnant women, newborns, the immunocompromised, and older adults. As in healthy adults, it is very rare in healthy children4 beyond the neonatal period. The cause of L. monocytogenes encephalitis in healthy adults may differ from that in patients with immunodeficiency.
The main transmission route of L. monocytogenes infection is the digestive tract, from which it is absorbed into the peripheral blood. From the peripheral blood, it can access the CNS by crossing the blood–brain barrier.5 Most patients have gastrointestinal symptoms (Table 1);6–10 however, as in this case, they often do not have a clear history of contaminated food ingestion.
Table 1. Clinical features of healthy and immunocompetent young and middle-aged individuals with Listeria meningitis.
Author, year of publication Age (years) and sex Consumption of unpasteurized buttermilk Symptoms and signs Brain CT/MRI CSF/CSF cultures for LM Blood/CSF cultures for LM Specific antibiotic treatment for LM and outcome
Zhang et al. [6], 2012 34, M Not mentioned Fever, headache, nausea, and vomiting for 3 days Not mentioned Leukocytosis and high protein levels –/+ Initial treatment with vancomycin (1 g iv q12h) and ceftriaxone (2 g iv q12h); however, his condition deteriorated
Altered consciousness for 1 day Consequently, the patient was given a combination of ampicillin (4 g iv q8h) and amikacin (0.4 g iv daily), to which he responded well
Meningeal irritation sign (+) The patient remained in good clinical condition on follow-up
Callaghan et al. [7], 2012 35, F Not mentioned Headaches, nausea, vomiting, and malaise for 4 days, followed by hemianesthesia, facial weakness, nystagmus, and ataxia MRI showed multiple ring-enhancing lesions in the brainstem Leukocytosis and high protein levels −/− Amoxicillin (2 g six times daily) and gentamicin (80 mg iv three times daily) for 2 weeks, to which the patient responded well
CSF PCR: + She had recovered well on follow-up
Vrbiü et al. [8], 2013 18, M Not mentioned Fever, severe headache, and vomiting for 3 days CT showed diffuse cerebral edema Leucocytes ↑ and high proteins levels –/+ Initial treatment with vancomycin and ceftriaxone, substituted with meropenem (2 g iv q8h), had no clinical effect
Meningeal irritation sign (+) Subsequently, ampicillin (2 g iv q4h) was administered after LM was isolated; the patient recovered completely.
Décard et al. [9], 2017 31, F Yes Isolated right facial numbness, followed by dysphagia, nystagmus, diplopia, peripheral facial palsy, and hemiparesis MRI showed multiple ring-enhancing lesions in the brainstem Lymphocytic pleocytosis and slightly elevated protein levels +/+ Ampicillin, ceftriaxone, and acyclovir were initiated and substituted with ampicillin and gentamicin after culturing of LM, followed by combination treatment with ampicillin, rifampicin, linezolid, and cotrimoxazole
The patient had severe sequelae
Li et al. [10], 2019 37, M Not mentioned Fever for 2 days, with dysphoria, followed by coma and respiratory and circulatory failure CT showed swelling of the brain and hydrocephalus High CSF pressure, increased leucocytes, and normal protein levels +/− Vancomycin (0.5 g iv q8h) and meropenem (0.5 g iv q8h)
The patient died 2 weeks after admission
CSF, cerebrospinal fluid; CT, computed tomography; F, female; iv, intravenously; LM, Listeria monocytogenes; MRI, magnetic resonance imaging; M, male; PCR, polymerase chain reaction; q4h, every 4 hours; q8h, every 8 hours; q12h, every 12 hours; −, negative; +, positive.
Some patients with L. monocytogenes encephalitis show mild symptoms and have a good treatment response.7 However, the disease can be aggressive, as in the case of our patient.
L. monocytogenes rhombencephalitis typically has a biphasic course, with non-specific prodromal symptoms such as fever, malaise, fatigue, headache, nausea, and vomiting, followed by any combination of cranial nerve palsies, ataxia, hemiparesis, hypesthesia,2 and altered consciousness. L. monocytogenes rhombencephalitis develops quickly and is often complicated by sepsis and respiratory failure.11 Common clinical findings include fever (57%), headache (57%), and focal neurological signs (64%).11
L. monocytogenes rhombencephalitis should be differentiated from tuberculous meningitis; both have similar CSF and brain MRI abnormalities, but L. monocytogenes rhombencephalitis often presents with high fever and progresses rapidly. Listeria may be identified in CSF cultures and much more rarely in blood cultures. L. monocytogenes rhombencephalitis should be distinguished from cryptococcal meningitis and other types of bacterial encephalitis. MRI usually shows multiple ring-enhanced lesions in the brainstem,7,9 as was observed in this case. With its high resolution, high-throughput genome sequencing is a promising technique for pathogen identification.
In general, penicillin, ampicillin,12 and amoxicillin are effective treatments for L. monocytogenes infection, but some strains are resistant. Thus, effective antibacterial agents, such as trimethoprim-sulfamethoxazole,13 meropenem, linezolid, and aminoglycosides,12 should be selected with the guidance of an antimicrobial sensitivity test. Guidelines recommend early steroid therapy for facial neuritis;14 however, patients with neurolisteriosis receiving adjunctive dexamethasone have higher mortality,15 and this treatment could aggravate conditions in patients without effective antibiotic treatment, as in this case. The treatment course with effective antibiotics should be at least 21 days. Because L. monocytogenes has a natural resistance to cephalosporin antibiotics, L. monocytogenes should be considered when third-generation cephalosporins are not effective against bacterial encephalitis. The empirical treatment of bacterial meningitis should include agents effective against listeriosis.16 The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early.17 Younger, immunocompetent individuals with L. monocytogenes meningitis have favorable disease outcomes.8 However, approximately 60% of survivors develop neurological sequelae.17 Timely and effective antibacterial therapy is crucial to improving the prognosis.
We searched the PubMed database for the Medical Subject Heading terms “facial paralysis/cranial nerve injuries” and “Listeria monocytogenes/Listeria” in different combinations, but no case similar to ours was found. It is necessary to further study L. monocytogenes encephalitis with atypical initial symptoms to aid early identification.
In conclusion, although Listeria meningitis is rare in healthy, immunocompetent adults, it must be considered in the differential diagnosis, especially in those whose disease conditions do not improve with cephalosporin antibiotic treatment. L. monocytogenes rhombencephalitis develops quickly, and prompt diagnosis of L. monocytogenes encephalitis, which mimics facial neuritis, is essential so that adequate antibiotic treatment can be initiated and the best outcome is achieved.
Ethics Statement: The study design was approved by the ethics review board of the Third Affiliated Hospital of Shenzhen University (No. 2020-SZLH-LW-015). We obtained written consent for publication from the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iD: Liming Cao https://orcid.org/0000-0003-2836-9347 | Recovered with sequelae (consequent health issues) | ReactionOutcome | CC BY-NC | 33435771 | 18,866,141 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Disease progression'. | Failure of multiple surgical procedures and adjuvant chemotherapy in early-stage steroid-cell ovarian tumor treatment: a case report and literature review.
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary, accounting for less than 0.1% of all ovarian tumors. Not otherwise specified (NOS) tumors are the most common subtype. More than half of patients with SCTs-NOS show hyperandrogenic symptoms. The primary treatment for SCTs is surgery, as most cases are early-staged and benign. Because of the low incidence of metastatic disease, there is insufficient reliable information on the role of adjuvant therapy and the most effective treatment regimen. In this report, a rare case of a recurrent SCT-NOS in a 36-year-old female patient without endocrine symptoms is presented, highlighting the significance of appropriate pathological evaluation and immunohistochemical testing for the accurate diagnosis of this malignancy, particularly in the case of hormonally "silent" tumors. The metastatic tumor described here showed no response to four courses of adjuvant chemotherapy after several debulking surgeries. Based on the clinical findings, the neoplastic etiology should always be considered during the resection of ovarian tumors to prevent possible disease dissemination due to inappropriate surgical techniques.
Introduction
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary that account for less than 0.1% of all ovarian tumors. They are classified into three categories based on the cell origin: stromal luteoma, Leydig-cell tumor, and not otherwise specified (NOS). NOS tumors are the most common subtype and comprise the largest proportion of cases (60%), whereas stromal luteoma and Leydig-cell tumors each account for ∼20% of cases.1–3
A SCT of the ovary was first described in 1943 as a “virilizing lipoid cell tumor” by Gemma Barzilai in the Atlas of Ovarian Pathology.4 Later, the term “lipoid cell tumor” was replaced by “steroid-cell tumor.” This change was based on the argument that although all SCTs can produce steroid hormones, up to 25% of them contain little or no lipids.4 In 1979, the term “steroid-cell tumor, not otherwise specified,” was coined by Scully2 to indicate that the cell lineage from which the tumor arises is unknown. SCTs-NOS usually develop in reproductive-aged women with an average age of 43 years. The major symptoms are hirsutism and virilization caused by high levels of testosterone. The cornerstone of SCT-NOS treatment is surgery. However, as most tumors are diagnosed in the early clinical stage and do not recur or metastasize, there is limited information on their response to chemotherapy.1
In this report, a rare case of a recurrent SCT-NOS in a patient without endocrine symptoms is presented. The tumor showed no response to adjuvant chemotherapy after several debulking surgeries.
Case presentation
A 36-year-old Caucasian female patient, gravida 2 para 2, underwent laparoscopic right salpingo-oophorectomy in 2015 at a secondary-level hospital after the identification of a right ovarian tumor (the tumor was fragmented within the abdominal cavity, but it was unclear if it was within the endobag). The post-surgical pathological report revealed a Sertoli–Leydig-cell tumor. The patient was not referred to a clinical oncologist because radical removal was expected. Her past medical and surgical history was unremarkable, and she had no relevant family history. No signs of virilization or hirsutism were observed.
After 6 months, the patient was referred to the Gynecologic Department of our tertiary hospital because of metastases. During laparoscopy, multiple metastatic lesions were found in the pelvic peritoneum, on the diaphragm, and at the site of the former trocar; the left ovary was solid with suspected tumor spread. Metastatic lesions appeared as 3- to 4-mm subperitoneal fatty nodules, which were biopsied. Washings from the peritoneal cavity were negative. Unexpectedly, the histopathological diagnosis was a SCT-NOS. Laboratory analysis revealed normal values of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol. No hormonal assays were performed prior to surgery, as there were no clinical manifestations of any excessive hormone secretion. The ovarian tumor markers, including human epididymis protein 4, β-human chorionic gonadotropin, and α-fetoprotein, were within the normal range. Cancer antigen 125 (Ca125) was elevated to 48.8 kU/L (normal value up to 35 kU/L), but after 3 weeks, the level returned to normal (24.8 kU/L). Ten months after the first surgery, the patient underwent a total abdominal hysterectomy, left salpingo-oophorectomy, omentectomy, pelvic and diaphragmatic peritonectomy, and pelvic lymphadenectomy. Optimal cytoreductive surgery was performed. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization but no signs of nuclear atypia and a mitotic count of 5 per 10 high-power fields. No Reinke crystals, which are usually observed in Sertoli–Leydig-cell tumors, were identified. Immunohistochemistry revealed positive expression of inhibin A, calretinin, and synaptophysin but negative expression of pan-cytokeratin, CD68, epithelial membrane antigen, chromogranin A, and estrogen receptor/androgen receptor. The Ki-67 labeling index was up to 15% (Figure 1). The histopathologic features supported the diagnosis of a SCT-NOS.
Figure 1. Microscopic appearance of the SCT-NOS (H&E) in a 36-year-old Caucasian female patient (a) and positive staining of the tumor cells for inhibin A (b), calretinin (c), and Ki-67 (d) (20×)
H&E: hematoxylin and eosin.
After the surgery, the patient received six cycles of adjuvant carboplatin-paclitaxel chemotherapy. Ca125 was measured several times during the treatment period; its levels were within the normal range. At the end of the six courses of chemotherapy, a control computed tomography (CT) scan was performed, revealing the absence of disease progression.
After 3 months of follow-up, the CT scan showed disease progression in the left lateral abdominal flank; hence, we decided to perform secondary cytoreductive surgery. McBurney’s laparotomy was performed. During abdominal exploration, an infiltrative growing tumor was found at the site of the right former trocar that had grown in all layers of the abdomen and spread into the right flank. The laparotomy was extended in the cephalad direction. Metastases were also observed in the left superior diaphragmatic space and the previous left trocar area. Secondary optimal cytoreduction was performed to remove metastases from the right iliac region and abdominal lateral canals. The analysis of frozen sections demonstrated SCT metastases in fibrous adipose tissue. The patient experienced disease-free survival for only 6 months, after which the follow-up CT scan showed disease progression and a large incisional hernia at the incision site of the previous McBurney’s laparotomy. Serum tumor markers and hormone levels were within the normal range. During the subsequent laparotomy, micrometastases were observed on the aponeurosis with multiple small metastases on the small and large intestines, which could not be radically removed. The excision of injured subcutaneous tissue and incisional hernia repair with mesh were performed. The histological diagnosis was SCT metastasis to soft tissues. The patient was subjected to six cycles of second-line bleomycin, etoposide, and cisplatin (BEP) chemotherapy. On account of a dry cough, bleomycin has not been prescribed after the second cycle due to suspicion of pulmonary toxicity. The patient was put under surveillance every 3 months after the completion of chemotherapy. An abdominopelvic CT scan revealed the absence of disease progression, and no metastases were observed on the right side of the anterior abdominal wall or the bowel. Serum tumor markers and hormone levels were normal. After 1 year, disease progression was observed on the CT scan, which revealed increased foci in the anterior and lateral abdominal wall (>50%) and a small amount of ascites in the lateral flanks. Systemic treatment was recommended, and five cycles of liposomal doxorubicin were administered. Despite undergoing treatment, radiologic disease progression was observed after 4 months, as there was an ∼40% increase in tumor mass above the vaginal cuff on the left side with an increase in fluid volume in the pelvis. On the right abdominal wall, supra-umbilically, most small tumor foci had increased in size. The patient required re-operation. On the anterior abdominal wall, several necrotic metastases were found. In the abdominal cavity, there was a 10-cm ruptured tumor between the sigmoid colon and the vaginal cuff. Multiple 1- to 4-cm retroperitoneal metastases were found on the right internal iliac artery, right ureter, and pelvic veins. The right ureter was approximately 2 cm in diameter, with metastases completely fixed to the pelvic bones/fasciae. Few small metastases were noted on the peritoneum and mesentery. Because of the infiltration of the lateral pelvic walls, the tumors could not be radically resected; sigmoid colon resection was rendered ineffective because of the presence of residual tumor masses. Only bleeding tumors and metastases on the peritoneum and mesentery were non-radically removed. Histopathological verification revealed metastases of the SCT-NOS. At the pain management center, the patient was advised to start treatment with transdermal fentanyl patches for the treatment of intense abdominal pain. The patient was referred to a urologist because of right hydronephrosis, and a long-term ureteral stent was inserted. The multidisciplinary team decided to repeat the previous regimen of chemotherapy based on carboplatin and paclitaxel (six cycles). After completion of the chemotherapy, a CT scan was performed. The pelvic, vaginal cuff, and sigmoid colon masses were enlarged, indicating disease progression. Only palliative treatment was recommended. After 5 months, another CT scan was performed, and significant disease progression and noticeable bilateral hydronephrosis were detected, with the metastases enlarged by 80% (Figure 2). To manage the pain, the subcutaneous right lumbar mass was resected under regional anesthesia. The right ureteral stent was replaced, and a long-term stent was also inserted into the left ureter. Currently, the hormone levels and cancer biomarkers are within the normal reference ranges. Over the last 2 months, the patient was administered dendritic cell-based cancer vaccines; however, no effect has been reported. Because of the severe pain caused by the large-volume pelvic tumor, laparotomy was performed with palliative intention, revealing an approximately 15-cm metastasis on the rectosigmoid mesentery, a 3-cm metastasis on the sigmoid colon, and multiple 1- to 2-mm implants on the small intestines. All lesions were removed, and Hartmann’s operation was performed because of heavy bleeding from the mesorectum and rectosigmoid mesentery. The patient was discharged from the hospital on the fourth uneventful postoperative day and is currently receiving palliative care. The course of the disease is shown in Figure 3.
Figure 2. CT scan of the patient reported in this case showing 14.5 × 8.8-cm metastatic masses within the pelvis.
CT: computed tomography.
Figure 3. Treatment course of the patient described in this report.
BEP: bleomycin, etoposide, and cisplatin.
Discussion
Ovarian SCTs-NOS occur at any age (2.5–93 years), but they are usually observed in reproductive-aged women, with an average age of 43 years.5,6 More than half (56%–77%) of patients with an SCT-NOS show hyperandrogenic symptoms and signs of virilization, such as hirsutism, acne, deepened voice, clitoromegaly, amenorrhea, and infertility.1,7–9 Additionally, it can present with estrogenic manifestations (6%–23%), such as menorrhagia or postmenopausal bleeding. Some patients also develop endometrial cancer.10 Ovarian SCTs can secrete steroid hormones, such as progesterone, cortisol, and aldosterone, which may cause corresponding clinical symptoms. Hyperandrogenic tumors may also be associated with paraneoplastic manifestations, such as hypercalcemia, erythrocytosis, or ascites.11 However, about 25% of patients with SCTs may have atypical presentations, without any symptoms of virilization. In these cases, the diagnosis is usually made postoperatively after histopathological verification.12 In the patient reported in this case, no virilization symptoms were noted, and the tumor was hormonally “silent.” Additional symptoms in some patients may include abdominal distension due to ascites and palpable abdominal masses.13 After tumor dissemination, the patient reported here manifested severe abdominal pain with palpable abdominal masses.
Serum laboratory analyses typically show elevated levels of testosterone and androstenedione, indicating an ovarian origin of androgen release and normal DHEA-S levels, thereby excluding adrenal causes of hyperandrogenism.1 In the reported case, the hormone levels were within the reference range, even after considerable disease dissemination. There is no known specific tumor marker established for the preoperative diagnosis of SCTs-NOS. Tumor markers, such as CA-125 and α-fetoprotein, are generally within the normal range, and the data from the literature do not indicate whether elevated levels signify malignant potential. Some studies even claim that these markers could facilitate the differential diagnosis of ovarian adenocarcinoma.11,14 This correlates with the described case, as even after disease relapse, tumor markers remained within the normal range.
SCTs-NOS are unilateral in 94% of cases, large at diagnosis (range from 1.2 to 45 cm in the greatest dimension, with an average reported size of 8.4 cm), and typically solid and well-circumscribed. SCTs-NOS should be distinguished from other ovarian tumors and SCTs, in which the proliferation of steroid hormone-producing cells occurs as a secondary event. These include stromal luteomas, Leydig-cell tumors, luteinized thecomas, and pregnancy luteomas.15 SCTs-NOS are different from Leydig-cell tumors in terms of their deficiency in cytoplasmic Reinke crystals. In addition, Leydig-cell tumors are usually situated in hilar locations, and they are commonly associated with Leydig-cell hyperplasia.16,17 Stromal luteomas are confined to the ovarian stroma and frequently occur in association with stromal hyperthecosis.12 SCTs-NOS might have a fibromatous component, similar to that of thecomas, but this component accounts for only less than 10% of tumors.18 Pregnancy luteomas are more commonly multifocal (bilateral in one-third of cases), usually discovered at the time of cesarean section, and regress spontaneously after pregnancy.12
In addition to the microscopic features, immunohistochemistry is particularly helpful for proper diagnosis. The sensitivity of positive calretinin is 60% to 90%, whereas the sensitivity of inhibin reactivity ranges from 5% to 90%.3,19 SCTs-NOS are also commonly vimentin, Melan-A, and CD99 positive and variably AE1/3, CAM5.2, HMB45, and S100 positive.3 Currently, techniques are being developed to define SCTs-NOS pathologically by immunohistochemical staining of steroidogenic enzymes. In the future, validated enzymes could serve as markers for the differential diagnosis of hyperandrogenic ovarian conditions.20 It is worth noting that differential diagnosis is challenging, as the primary histopathological diagnosis of this case from the previous hospital indicated a Sertoli–Leydig-cell tumor. However, no Reinke crystals were found after careful evaluation of the metastases resected in our hospital. In this patient, the tumor showed strong and diffuse positivity for inhibin and calretinin.
Usually, these tumors are benign; however, 25% to 43% of SCTs are malignant, with 20% of cases found to exhibit metastases beyond the ovary. Metastatic lesions usually occur within the peritoneal cavity and rarely occur at distant sites.15 In one study, the clinical and pathological features of 63 SCTs-NOS were reviewed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated as probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was malignant. Despite the various chemotherapy and radiation regimens, 12 patients experienced recurrences, and 14 patients died from the disease, suggesting a poor prognosis if the tumor is at an advanced stage, large, or recurring. Five pathological features are considered the best correlates of malignant behavior: the presence of two or more mitotic figures per 10 high-power fields (92% malignant), necrosis (86% malignant), a diameter of 7 cm or greater (78% malignant), hemorrhage (77% malignant), and grade 2 or 3 nuclear atypia (64% malignant).6 In our patient, we were unable to evaluate these features because the primary tumor was not resected at our hospital. We assume that the rapid spread was related to possible tumor fragmentation within the abdominal cavity, as we are uncertain of the use of an endobag.
Because the incidence of SCTs-NOS is low, there are currently no established treatment protocols. Therefore, the tumors are treated similarly to stromal tumors depending on several factors, including the surgical stage, histological type, patient’s age, and history of childbirth. The primary treatment for SCTs is surgery. Ovarian SCTs are generally considered benign because they are often detected at an early clinical stage. Therefore, unilateral salpingo-oophorectomy or tumor removal is acceptable in reproductive-aged women.1 However, regular follow-up with measurement of serum testosterone levels is mandatory. As there is limited information on the mechanisms of these tumors, the optimal length of follow-up is yet to be determined.15 In addition, there have already been reported cases of spontaneous pregnancies after tumor removal, probably because of the decline in testosterone levels.21,22
Surgical treatments using total abdominal hysterectomy and bilateral salpingo-oophorectomy are an appropriate management option for postmenopausal patients and those who have completed childbearing. Endometrial sampling should be performed when fertility-sparing surgery is planned because many of these patients may have coexisting endometrial hyperplasia or even uterine adenocarcinoma that might affect the decision for performing a hysterectomy.1
Because of the limited incidence of metastatic disease, there is a lack of reliable information on the role of adjuvant therapy in SCTs-NOS. The adjuvant chemotherapy regimens currently recommended for treatment are as follows: BEP; cisplatin, doxorubicin, and cyclophosphamide; taxane and platinum; and bleomycin, vinblastine, and cisplatin.23–27 It was reported that intraperitoneal dissemination and liver metastases were completely removed with debulking surgery, radiofrequency ablation of the liver metastasis, and adjuvant BEP (follow-up, 43 months) in a patient with a recurrent SCT-NOS occurring 5 years after the initial surgery.28 In another study, the treatment protocol consisted of docetaxel and nedaplatin, and the patient survived for 2 years with multiple bone metastases.29 Another SCT-NOS case with progressive disease after surgical debulking was reported. Treatment with multi-agent chemotherapy failed, but the patient subsequently showed a robust response to gonadotropin-releasing hormone agonist (GnRHa) therapy. Therefore, although typically treated with surgery alone, GnRHa may be required when abnormal serum hormone levels persist, and there is suspicion of residual tumors, recurrences, or metastases. It is suggested that GnRHa treatment be considered prior to cytotoxic chemotherapy in cases with SCTs-NOS.30 In the case described above, a chemotherapeutic regimen with BEP was administered for the recurrent SCT-NOS. After three cycles of BEP, the chemotherapeutic agent was changed to paclitaxel and carboplatin following a stable disease course. After eight cycles of paclitaxel and carboplatin, chemotherapy was discontinued because of prolonged neutropenia and peripheral neuropathy. The CT scan showed clinically stable disease. As the immunohistochemical analysis of the primary tumor revealed positive cytoplasmic staining for the GnRH receptor, GnRHa therapy was attempted based on reference to previous case reports and the lack of other effective alternatives. After six cycles of GnRHa therapy, the CT scan confirmed significant tumor size reduction. Adverse events were not observed, and the patient’s hirsutism and virilization had improved. Therefore, the treatment was discontinued after the administration of six cycles. However, the testosterone level was increased 2 months after GnRHa discontinuation, and the CT scan revealed an increase in tumor size. Subsequently, GnRHa was re-administered, resulting in immediate normalization of the serum testosterone level and shrinkage of the recurrent tumor. Thus, GnRHa therapy was continued (22 months after the first GnRHa treatment with the absence of symptoms).31
In our patient, the SCT-NOS was completely insensitive to chemotherapy, despite undergoing several debulking surgeries. Removal of the multi-metastases resulted in only short-term improvement. Unfortunately, the dendritic cell-based vaccine recently administered for 2 months did not yield a positive outcome.
Conclusions
Based on our clinical experience of this case, we highlight the importance of ovarian tumor resection without direct fragmentation within the abdominal cavity, as the neoplastic etiology should always be taken into account. Pathological evaluation is essential for the diagnosis of this malignancy, and immunohistochemical testing also aids in the formulation of an accurate diagnosis, especially in a patient with an apparent absence of endocrine manifestations. Surgery is the main treatment method for SCTs-NOS, as most cases are benign, and there are no generalized conclusions on the response of malignant cases to therapies, such as chemotherapy or radiation therapy. According to recent studies, GnRHa therapy could serve as a feasible treatment method and is worthy of further research with large patient groups.
Acknowledgment
We acknowledge oncologists from the Department of Oncology and Chemotherapy of Vilnius University Hospital Santaros Clinics for their considerable contribution to the treatment of the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Ethics statement: In our country, it is not mandatory to receive written consent from the local Ethics Committee for a case report, as it does not strictly meet the criteria of research because it does not require investigation and is intended to develop information to be shared for medical or educational purposes. Written informed consent was obtained from the patient for the publication of this report and the accompanying images.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iDs: Danuta Vasilevska https://orcid.org/0000-0002-3136-7281
Ugnius Mickys https://orcid.org/0000-0001-7805-0727
Andrzej Semczuk https://orcid.org/0000-0001-9344-9437 | BLEOMYCIN SULFATE, CARBOPLATIN, CISPLATIN, ETOPOSIDE, PACLITAXEL | DrugsGivenReaction | CC BY-NC | 33435776 | 18,956,668 | 2021-01 |
What was the dosage of drug 'BLEOMYCIN SULFATE'? | Failure of multiple surgical procedures and adjuvant chemotherapy in early-stage steroid-cell ovarian tumor treatment: a case report and literature review.
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary, accounting for less than 0.1% of all ovarian tumors. Not otherwise specified (NOS) tumors are the most common subtype. More than half of patients with SCTs-NOS show hyperandrogenic symptoms. The primary treatment for SCTs is surgery, as most cases are early-staged and benign. Because of the low incidence of metastatic disease, there is insufficient reliable information on the role of adjuvant therapy and the most effective treatment regimen. In this report, a rare case of a recurrent SCT-NOS in a 36-year-old female patient without endocrine symptoms is presented, highlighting the significance of appropriate pathological evaluation and immunohistochemical testing for the accurate diagnosis of this malignancy, particularly in the case of hormonally "silent" tumors. The metastatic tumor described here showed no response to four courses of adjuvant chemotherapy after several debulking surgeries. Based on the clinical findings, the neoplastic etiology should always be considered during the resection of ovarian tumors to prevent possible disease dissemination due to inappropriate surgical techniques.
Introduction
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary that account for less than 0.1% of all ovarian tumors. They are classified into three categories based on the cell origin: stromal luteoma, Leydig-cell tumor, and not otherwise specified (NOS). NOS tumors are the most common subtype and comprise the largest proportion of cases (60%), whereas stromal luteoma and Leydig-cell tumors each account for ∼20% of cases.1–3
A SCT of the ovary was first described in 1943 as a “virilizing lipoid cell tumor” by Gemma Barzilai in the Atlas of Ovarian Pathology.4 Later, the term “lipoid cell tumor” was replaced by “steroid-cell tumor.” This change was based on the argument that although all SCTs can produce steroid hormones, up to 25% of them contain little or no lipids.4 In 1979, the term “steroid-cell tumor, not otherwise specified,” was coined by Scully2 to indicate that the cell lineage from which the tumor arises is unknown. SCTs-NOS usually develop in reproductive-aged women with an average age of 43 years. The major symptoms are hirsutism and virilization caused by high levels of testosterone. The cornerstone of SCT-NOS treatment is surgery. However, as most tumors are diagnosed in the early clinical stage and do not recur or metastasize, there is limited information on their response to chemotherapy.1
In this report, a rare case of a recurrent SCT-NOS in a patient without endocrine symptoms is presented. The tumor showed no response to adjuvant chemotherapy after several debulking surgeries.
Case presentation
A 36-year-old Caucasian female patient, gravida 2 para 2, underwent laparoscopic right salpingo-oophorectomy in 2015 at a secondary-level hospital after the identification of a right ovarian tumor (the tumor was fragmented within the abdominal cavity, but it was unclear if it was within the endobag). The post-surgical pathological report revealed a Sertoli–Leydig-cell tumor. The patient was not referred to a clinical oncologist because radical removal was expected. Her past medical and surgical history was unremarkable, and she had no relevant family history. No signs of virilization or hirsutism were observed.
After 6 months, the patient was referred to the Gynecologic Department of our tertiary hospital because of metastases. During laparoscopy, multiple metastatic lesions were found in the pelvic peritoneum, on the diaphragm, and at the site of the former trocar; the left ovary was solid with suspected tumor spread. Metastatic lesions appeared as 3- to 4-mm subperitoneal fatty nodules, which were biopsied. Washings from the peritoneal cavity were negative. Unexpectedly, the histopathological diagnosis was a SCT-NOS. Laboratory analysis revealed normal values of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol. No hormonal assays were performed prior to surgery, as there were no clinical manifestations of any excessive hormone secretion. The ovarian tumor markers, including human epididymis protein 4, β-human chorionic gonadotropin, and α-fetoprotein, were within the normal range. Cancer antigen 125 (Ca125) was elevated to 48.8 kU/L (normal value up to 35 kU/L), but after 3 weeks, the level returned to normal (24.8 kU/L). Ten months after the first surgery, the patient underwent a total abdominal hysterectomy, left salpingo-oophorectomy, omentectomy, pelvic and diaphragmatic peritonectomy, and pelvic lymphadenectomy. Optimal cytoreductive surgery was performed. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization but no signs of nuclear atypia and a mitotic count of 5 per 10 high-power fields. No Reinke crystals, which are usually observed in Sertoli–Leydig-cell tumors, were identified. Immunohistochemistry revealed positive expression of inhibin A, calretinin, and synaptophysin but negative expression of pan-cytokeratin, CD68, epithelial membrane antigen, chromogranin A, and estrogen receptor/androgen receptor. The Ki-67 labeling index was up to 15% (Figure 1). The histopathologic features supported the diagnosis of a SCT-NOS.
Figure 1. Microscopic appearance of the SCT-NOS (H&E) in a 36-year-old Caucasian female patient (a) and positive staining of the tumor cells for inhibin A (b), calretinin (c), and Ki-67 (d) (20×)
H&E: hematoxylin and eosin.
After the surgery, the patient received six cycles of adjuvant carboplatin-paclitaxel chemotherapy. Ca125 was measured several times during the treatment period; its levels were within the normal range. At the end of the six courses of chemotherapy, a control computed tomography (CT) scan was performed, revealing the absence of disease progression.
After 3 months of follow-up, the CT scan showed disease progression in the left lateral abdominal flank; hence, we decided to perform secondary cytoreductive surgery. McBurney’s laparotomy was performed. During abdominal exploration, an infiltrative growing tumor was found at the site of the right former trocar that had grown in all layers of the abdomen and spread into the right flank. The laparotomy was extended in the cephalad direction. Metastases were also observed in the left superior diaphragmatic space and the previous left trocar area. Secondary optimal cytoreduction was performed to remove metastases from the right iliac region and abdominal lateral canals. The analysis of frozen sections demonstrated SCT metastases in fibrous adipose tissue. The patient experienced disease-free survival for only 6 months, after which the follow-up CT scan showed disease progression and a large incisional hernia at the incision site of the previous McBurney’s laparotomy. Serum tumor markers and hormone levels were within the normal range. During the subsequent laparotomy, micrometastases were observed on the aponeurosis with multiple small metastases on the small and large intestines, which could not be radically removed. The excision of injured subcutaneous tissue and incisional hernia repair with mesh were performed. The histological diagnosis was SCT metastasis to soft tissues. The patient was subjected to six cycles of second-line bleomycin, etoposide, and cisplatin (BEP) chemotherapy. On account of a dry cough, bleomycin has not been prescribed after the second cycle due to suspicion of pulmonary toxicity. The patient was put under surveillance every 3 months after the completion of chemotherapy. An abdominopelvic CT scan revealed the absence of disease progression, and no metastases were observed on the right side of the anterior abdominal wall or the bowel. Serum tumor markers and hormone levels were normal. After 1 year, disease progression was observed on the CT scan, which revealed increased foci in the anterior and lateral abdominal wall (>50%) and a small amount of ascites in the lateral flanks. Systemic treatment was recommended, and five cycles of liposomal doxorubicin were administered. Despite undergoing treatment, radiologic disease progression was observed after 4 months, as there was an ∼40% increase in tumor mass above the vaginal cuff on the left side with an increase in fluid volume in the pelvis. On the right abdominal wall, supra-umbilically, most small tumor foci had increased in size. The patient required re-operation. On the anterior abdominal wall, several necrotic metastases were found. In the abdominal cavity, there was a 10-cm ruptured tumor between the sigmoid colon and the vaginal cuff. Multiple 1- to 4-cm retroperitoneal metastases were found on the right internal iliac artery, right ureter, and pelvic veins. The right ureter was approximately 2 cm in diameter, with metastases completely fixed to the pelvic bones/fasciae. Few small metastases were noted on the peritoneum and mesentery. Because of the infiltration of the lateral pelvic walls, the tumors could not be radically resected; sigmoid colon resection was rendered ineffective because of the presence of residual tumor masses. Only bleeding tumors and metastases on the peritoneum and mesentery were non-radically removed. Histopathological verification revealed metastases of the SCT-NOS. At the pain management center, the patient was advised to start treatment with transdermal fentanyl patches for the treatment of intense abdominal pain. The patient was referred to a urologist because of right hydronephrosis, and a long-term ureteral stent was inserted. The multidisciplinary team decided to repeat the previous regimen of chemotherapy based on carboplatin and paclitaxel (six cycles). After completion of the chemotherapy, a CT scan was performed. The pelvic, vaginal cuff, and sigmoid colon masses were enlarged, indicating disease progression. Only palliative treatment was recommended. After 5 months, another CT scan was performed, and significant disease progression and noticeable bilateral hydronephrosis were detected, with the metastases enlarged by 80% (Figure 2). To manage the pain, the subcutaneous right lumbar mass was resected under regional anesthesia. The right ureteral stent was replaced, and a long-term stent was also inserted into the left ureter. Currently, the hormone levels and cancer biomarkers are within the normal reference ranges. Over the last 2 months, the patient was administered dendritic cell-based cancer vaccines; however, no effect has been reported. Because of the severe pain caused by the large-volume pelvic tumor, laparotomy was performed with palliative intention, revealing an approximately 15-cm metastasis on the rectosigmoid mesentery, a 3-cm metastasis on the sigmoid colon, and multiple 1- to 2-mm implants on the small intestines. All lesions were removed, and Hartmann’s operation was performed because of heavy bleeding from the mesorectum and rectosigmoid mesentery. The patient was discharged from the hospital on the fourth uneventful postoperative day and is currently receiving palliative care. The course of the disease is shown in Figure 3.
Figure 2. CT scan of the patient reported in this case showing 14.5 × 8.8-cm metastatic masses within the pelvis.
CT: computed tomography.
Figure 3. Treatment course of the patient described in this report.
BEP: bleomycin, etoposide, and cisplatin.
Discussion
Ovarian SCTs-NOS occur at any age (2.5–93 years), but they are usually observed in reproductive-aged women, with an average age of 43 years.5,6 More than half (56%–77%) of patients with an SCT-NOS show hyperandrogenic symptoms and signs of virilization, such as hirsutism, acne, deepened voice, clitoromegaly, amenorrhea, and infertility.1,7–9 Additionally, it can present with estrogenic manifestations (6%–23%), such as menorrhagia or postmenopausal bleeding. Some patients also develop endometrial cancer.10 Ovarian SCTs can secrete steroid hormones, such as progesterone, cortisol, and aldosterone, which may cause corresponding clinical symptoms. Hyperandrogenic tumors may also be associated with paraneoplastic manifestations, such as hypercalcemia, erythrocytosis, or ascites.11 However, about 25% of patients with SCTs may have atypical presentations, without any symptoms of virilization. In these cases, the diagnosis is usually made postoperatively after histopathological verification.12 In the patient reported in this case, no virilization symptoms were noted, and the tumor was hormonally “silent.” Additional symptoms in some patients may include abdominal distension due to ascites and palpable abdominal masses.13 After tumor dissemination, the patient reported here manifested severe abdominal pain with palpable abdominal masses.
Serum laboratory analyses typically show elevated levels of testosterone and androstenedione, indicating an ovarian origin of androgen release and normal DHEA-S levels, thereby excluding adrenal causes of hyperandrogenism.1 In the reported case, the hormone levels were within the reference range, even after considerable disease dissemination. There is no known specific tumor marker established for the preoperative diagnosis of SCTs-NOS. Tumor markers, such as CA-125 and α-fetoprotein, are generally within the normal range, and the data from the literature do not indicate whether elevated levels signify malignant potential. Some studies even claim that these markers could facilitate the differential diagnosis of ovarian adenocarcinoma.11,14 This correlates with the described case, as even after disease relapse, tumor markers remained within the normal range.
SCTs-NOS are unilateral in 94% of cases, large at diagnosis (range from 1.2 to 45 cm in the greatest dimension, with an average reported size of 8.4 cm), and typically solid and well-circumscribed. SCTs-NOS should be distinguished from other ovarian tumors and SCTs, in which the proliferation of steroid hormone-producing cells occurs as a secondary event. These include stromal luteomas, Leydig-cell tumors, luteinized thecomas, and pregnancy luteomas.15 SCTs-NOS are different from Leydig-cell tumors in terms of their deficiency in cytoplasmic Reinke crystals. In addition, Leydig-cell tumors are usually situated in hilar locations, and they are commonly associated with Leydig-cell hyperplasia.16,17 Stromal luteomas are confined to the ovarian stroma and frequently occur in association with stromal hyperthecosis.12 SCTs-NOS might have a fibromatous component, similar to that of thecomas, but this component accounts for only less than 10% of tumors.18 Pregnancy luteomas are more commonly multifocal (bilateral in one-third of cases), usually discovered at the time of cesarean section, and regress spontaneously after pregnancy.12
In addition to the microscopic features, immunohistochemistry is particularly helpful for proper diagnosis. The sensitivity of positive calretinin is 60% to 90%, whereas the sensitivity of inhibin reactivity ranges from 5% to 90%.3,19 SCTs-NOS are also commonly vimentin, Melan-A, and CD99 positive and variably AE1/3, CAM5.2, HMB45, and S100 positive.3 Currently, techniques are being developed to define SCTs-NOS pathologically by immunohistochemical staining of steroidogenic enzymes. In the future, validated enzymes could serve as markers for the differential diagnosis of hyperandrogenic ovarian conditions.20 It is worth noting that differential diagnosis is challenging, as the primary histopathological diagnosis of this case from the previous hospital indicated a Sertoli–Leydig-cell tumor. However, no Reinke crystals were found after careful evaluation of the metastases resected in our hospital. In this patient, the tumor showed strong and diffuse positivity for inhibin and calretinin.
Usually, these tumors are benign; however, 25% to 43% of SCTs are malignant, with 20% of cases found to exhibit metastases beyond the ovary. Metastatic lesions usually occur within the peritoneal cavity and rarely occur at distant sites.15 In one study, the clinical and pathological features of 63 SCTs-NOS were reviewed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated as probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was malignant. Despite the various chemotherapy and radiation regimens, 12 patients experienced recurrences, and 14 patients died from the disease, suggesting a poor prognosis if the tumor is at an advanced stage, large, or recurring. Five pathological features are considered the best correlates of malignant behavior: the presence of two or more mitotic figures per 10 high-power fields (92% malignant), necrosis (86% malignant), a diameter of 7 cm or greater (78% malignant), hemorrhage (77% malignant), and grade 2 or 3 nuclear atypia (64% malignant).6 In our patient, we were unable to evaluate these features because the primary tumor was not resected at our hospital. We assume that the rapid spread was related to possible tumor fragmentation within the abdominal cavity, as we are uncertain of the use of an endobag.
Because the incidence of SCTs-NOS is low, there are currently no established treatment protocols. Therefore, the tumors are treated similarly to stromal tumors depending on several factors, including the surgical stage, histological type, patient’s age, and history of childbirth. The primary treatment for SCTs is surgery. Ovarian SCTs are generally considered benign because they are often detected at an early clinical stage. Therefore, unilateral salpingo-oophorectomy or tumor removal is acceptable in reproductive-aged women.1 However, regular follow-up with measurement of serum testosterone levels is mandatory. As there is limited information on the mechanisms of these tumors, the optimal length of follow-up is yet to be determined.15 In addition, there have already been reported cases of spontaneous pregnancies after tumor removal, probably because of the decline in testosterone levels.21,22
Surgical treatments using total abdominal hysterectomy and bilateral salpingo-oophorectomy are an appropriate management option for postmenopausal patients and those who have completed childbearing. Endometrial sampling should be performed when fertility-sparing surgery is planned because many of these patients may have coexisting endometrial hyperplasia or even uterine adenocarcinoma that might affect the decision for performing a hysterectomy.1
Because of the limited incidence of metastatic disease, there is a lack of reliable information on the role of adjuvant therapy in SCTs-NOS. The adjuvant chemotherapy regimens currently recommended for treatment are as follows: BEP; cisplatin, doxorubicin, and cyclophosphamide; taxane and platinum; and bleomycin, vinblastine, and cisplatin.23–27 It was reported that intraperitoneal dissemination and liver metastases were completely removed with debulking surgery, radiofrequency ablation of the liver metastasis, and adjuvant BEP (follow-up, 43 months) in a patient with a recurrent SCT-NOS occurring 5 years after the initial surgery.28 In another study, the treatment protocol consisted of docetaxel and nedaplatin, and the patient survived for 2 years with multiple bone metastases.29 Another SCT-NOS case with progressive disease after surgical debulking was reported. Treatment with multi-agent chemotherapy failed, but the patient subsequently showed a robust response to gonadotropin-releasing hormone agonist (GnRHa) therapy. Therefore, although typically treated with surgery alone, GnRHa may be required when abnormal serum hormone levels persist, and there is suspicion of residual tumors, recurrences, or metastases. It is suggested that GnRHa treatment be considered prior to cytotoxic chemotherapy in cases with SCTs-NOS.30 In the case described above, a chemotherapeutic regimen with BEP was administered for the recurrent SCT-NOS. After three cycles of BEP, the chemotherapeutic agent was changed to paclitaxel and carboplatin following a stable disease course. After eight cycles of paclitaxel and carboplatin, chemotherapy was discontinued because of prolonged neutropenia and peripheral neuropathy. The CT scan showed clinically stable disease. As the immunohistochemical analysis of the primary tumor revealed positive cytoplasmic staining for the GnRH receptor, GnRHa therapy was attempted based on reference to previous case reports and the lack of other effective alternatives. After six cycles of GnRHa therapy, the CT scan confirmed significant tumor size reduction. Adverse events were not observed, and the patient’s hirsutism and virilization had improved. Therefore, the treatment was discontinued after the administration of six cycles. However, the testosterone level was increased 2 months after GnRHa discontinuation, and the CT scan revealed an increase in tumor size. Subsequently, GnRHa was re-administered, resulting in immediate normalization of the serum testosterone level and shrinkage of the recurrent tumor. Thus, GnRHa therapy was continued (22 months after the first GnRHa treatment with the absence of symptoms).31
In our patient, the SCT-NOS was completely insensitive to chemotherapy, despite undergoing several debulking surgeries. Removal of the multi-metastases resulted in only short-term improvement. Unfortunately, the dendritic cell-based vaccine recently administered for 2 months did not yield a positive outcome.
Conclusions
Based on our clinical experience of this case, we highlight the importance of ovarian tumor resection without direct fragmentation within the abdominal cavity, as the neoplastic etiology should always be taken into account. Pathological evaluation is essential for the diagnosis of this malignancy, and immunohistochemical testing also aids in the formulation of an accurate diagnosis, especially in a patient with an apparent absence of endocrine manifestations. Surgery is the main treatment method for SCTs-NOS, as most cases are benign, and there are no generalized conclusions on the response of malignant cases to therapies, such as chemotherapy or radiation therapy. According to recent studies, GnRHa therapy could serve as a feasible treatment method and is worthy of further research with large patient groups.
Acknowledgment
We acknowledge oncologists from the Department of Oncology and Chemotherapy of Vilnius University Hospital Santaros Clinics for their considerable contribution to the treatment of the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Ethics statement: In our country, it is not mandatory to receive written consent from the local Ethics Committee for a case report, as it does not strictly meet the criteria of research because it does not require investigation and is intended to develop information to be shared for medical or educational purposes. Written informed consent was obtained from the patient for the publication of this report and the accompanying images.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iDs: Danuta Vasilevska https://orcid.org/0000-0002-3136-7281
Ugnius Mickys https://orcid.org/0000-0001-7805-0727
Andrzej Semczuk https://orcid.org/0000-0001-9344-9437 | 6 CYCLES , UNK | DrugDosageText | CC BY-NC | 33435776 | 18,956,668 | 2021-01 |
What was the dosage of drug 'CARBOPLATIN'? | Failure of multiple surgical procedures and adjuvant chemotherapy in early-stage steroid-cell ovarian tumor treatment: a case report and literature review.
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary, accounting for less than 0.1% of all ovarian tumors. Not otherwise specified (NOS) tumors are the most common subtype. More than half of patients with SCTs-NOS show hyperandrogenic symptoms. The primary treatment for SCTs is surgery, as most cases are early-staged and benign. Because of the low incidence of metastatic disease, there is insufficient reliable information on the role of adjuvant therapy and the most effective treatment regimen. In this report, a rare case of a recurrent SCT-NOS in a 36-year-old female patient without endocrine symptoms is presented, highlighting the significance of appropriate pathological evaluation and immunohistochemical testing for the accurate diagnosis of this malignancy, particularly in the case of hormonally "silent" tumors. The metastatic tumor described here showed no response to four courses of adjuvant chemotherapy after several debulking surgeries. Based on the clinical findings, the neoplastic etiology should always be considered during the resection of ovarian tumors to prevent possible disease dissemination due to inappropriate surgical techniques.
Introduction
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary that account for less than 0.1% of all ovarian tumors. They are classified into three categories based on the cell origin: stromal luteoma, Leydig-cell tumor, and not otherwise specified (NOS). NOS tumors are the most common subtype and comprise the largest proportion of cases (60%), whereas stromal luteoma and Leydig-cell tumors each account for ∼20% of cases.1–3
A SCT of the ovary was first described in 1943 as a “virilizing lipoid cell tumor” by Gemma Barzilai in the Atlas of Ovarian Pathology.4 Later, the term “lipoid cell tumor” was replaced by “steroid-cell tumor.” This change was based on the argument that although all SCTs can produce steroid hormones, up to 25% of them contain little or no lipids.4 In 1979, the term “steroid-cell tumor, not otherwise specified,” was coined by Scully2 to indicate that the cell lineage from which the tumor arises is unknown. SCTs-NOS usually develop in reproductive-aged women with an average age of 43 years. The major symptoms are hirsutism and virilization caused by high levels of testosterone. The cornerstone of SCT-NOS treatment is surgery. However, as most tumors are diagnosed in the early clinical stage and do not recur or metastasize, there is limited information on their response to chemotherapy.1
In this report, a rare case of a recurrent SCT-NOS in a patient without endocrine symptoms is presented. The tumor showed no response to adjuvant chemotherapy after several debulking surgeries.
Case presentation
A 36-year-old Caucasian female patient, gravida 2 para 2, underwent laparoscopic right salpingo-oophorectomy in 2015 at a secondary-level hospital after the identification of a right ovarian tumor (the tumor was fragmented within the abdominal cavity, but it was unclear if it was within the endobag). The post-surgical pathological report revealed a Sertoli–Leydig-cell tumor. The patient was not referred to a clinical oncologist because radical removal was expected. Her past medical and surgical history was unremarkable, and she had no relevant family history. No signs of virilization or hirsutism were observed.
After 6 months, the patient was referred to the Gynecologic Department of our tertiary hospital because of metastases. During laparoscopy, multiple metastatic lesions were found in the pelvic peritoneum, on the diaphragm, and at the site of the former trocar; the left ovary was solid with suspected tumor spread. Metastatic lesions appeared as 3- to 4-mm subperitoneal fatty nodules, which were biopsied. Washings from the peritoneal cavity were negative. Unexpectedly, the histopathological diagnosis was a SCT-NOS. Laboratory analysis revealed normal values of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol. No hormonal assays were performed prior to surgery, as there were no clinical manifestations of any excessive hormone secretion. The ovarian tumor markers, including human epididymis protein 4, β-human chorionic gonadotropin, and α-fetoprotein, were within the normal range. Cancer antigen 125 (Ca125) was elevated to 48.8 kU/L (normal value up to 35 kU/L), but after 3 weeks, the level returned to normal (24.8 kU/L). Ten months after the first surgery, the patient underwent a total abdominal hysterectomy, left salpingo-oophorectomy, omentectomy, pelvic and diaphragmatic peritonectomy, and pelvic lymphadenectomy. Optimal cytoreductive surgery was performed. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization but no signs of nuclear atypia and a mitotic count of 5 per 10 high-power fields. No Reinke crystals, which are usually observed in Sertoli–Leydig-cell tumors, were identified. Immunohistochemistry revealed positive expression of inhibin A, calretinin, and synaptophysin but negative expression of pan-cytokeratin, CD68, epithelial membrane antigen, chromogranin A, and estrogen receptor/androgen receptor. The Ki-67 labeling index was up to 15% (Figure 1). The histopathologic features supported the diagnosis of a SCT-NOS.
Figure 1. Microscopic appearance of the SCT-NOS (H&E) in a 36-year-old Caucasian female patient (a) and positive staining of the tumor cells for inhibin A (b), calretinin (c), and Ki-67 (d) (20×)
H&E: hematoxylin and eosin.
After the surgery, the patient received six cycles of adjuvant carboplatin-paclitaxel chemotherapy. Ca125 was measured several times during the treatment period; its levels were within the normal range. At the end of the six courses of chemotherapy, a control computed tomography (CT) scan was performed, revealing the absence of disease progression.
After 3 months of follow-up, the CT scan showed disease progression in the left lateral abdominal flank; hence, we decided to perform secondary cytoreductive surgery. McBurney’s laparotomy was performed. During abdominal exploration, an infiltrative growing tumor was found at the site of the right former trocar that had grown in all layers of the abdomen and spread into the right flank. The laparotomy was extended in the cephalad direction. Metastases were also observed in the left superior diaphragmatic space and the previous left trocar area. Secondary optimal cytoreduction was performed to remove metastases from the right iliac region and abdominal lateral canals. The analysis of frozen sections demonstrated SCT metastases in fibrous adipose tissue. The patient experienced disease-free survival for only 6 months, after which the follow-up CT scan showed disease progression and a large incisional hernia at the incision site of the previous McBurney’s laparotomy. Serum tumor markers and hormone levels were within the normal range. During the subsequent laparotomy, micrometastases were observed on the aponeurosis with multiple small metastases on the small and large intestines, which could not be radically removed. The excision of injured subcutaneous tissue and incisional hernia repair with mesh were performed. The histological diagnosis was SCT metastasis to soft tissues. The patient was subjected to six cycles of second-line bleomycin, etoposide, and cisplatin (BEP) chemotherapy. On account of a dry cough, bleomycin has not been prescribed after the second cycle due to suspicion of pulmonary toxicity. The patient was put under surveillance every 3 months after the completion of chemotherapy. An abdominopelvic CT scan revealed the absence of disease progression, and no metastases were observed on the right side of the anterior abdominal wall or the bowel. Serum tumor markers and hormone levels were normal. After 1 year, disease progression was observed on the CT scan, which revealed increased foci in the anterior and lateral abdominal wall (>50%) and a small amount of ascites in the lateral flanks. Systemic treatment was recommended, and five cycles of liposomal doxorubicin were administered. Despite undergoing treatment, radiologic disease progression was observed after 4 months, as there was an ∼40% increase in tumor mass above the vaginal cuff on the left side with an increase in fluid volume in the pelvis. On the right abdominal wall, supra-umbilically, most small tumor foci had increased in size. The patient required re-operation. On the anterior abdominal wall, several necrotic metastases were found. In the abdominal cavity, there was a 10-cm ruptured tumor between the sigmoid colon and the vaginal cuff. Multiple 1- to 4-cm retroperitoneal metastases were found on the right internal iliac artery, right ureter, and pelvic veins. The right ureter was approximately 2 cm in diameter, with metastases completely fixed to the pelvic bones/fasciae. Few small metastases were noted on the peritoneum and mesentery. Because of the infiltration of the lateral pelvic walls, the tumors could not be radically resected; sigmoid colon resection was rendered ineffective because of the presence of residual tumor masses. Only bleeding tumors and metastases on the peritoneum and mesentery were non-radically removed. Histopathological verification revealed metastases of the SCT-NOS. At the pain management center, the patient was advised to start treatment with transdermal fentanyl patches for the treatment of intense abdominal pain. The patient was referred to a urologist because of right hydronephrosis, and a long-term ureteral stent was inserted. The multidisciplinary team decided to repeat the previous regimen of chemotherapy based on carboplatin and paclitaxel (six cycles). After completion of the chemotherapy, a CT scan was performed. The pelvic, vaginal cuff, and sigmoid colon masses were enlarged, indicating disease progression. Only palliative treatment was recommended. After 5 months, another CT scan was performed, and significant disease progression and noticeable bilateral hydronephrosis were detected, with the metastases enlarged by 80% (Figure 2). To manage the pain, the subcutaneous right lumbar mass was resected under regional anesthesia. The right ureteral stent was replaced, and a long-term stent was also inserted into the left ureter. Currently, the hormone levels and cancer biomarkers are within the normal reference ranges. Over the last 2 months, the patient was administered dendritic cell-based cancer vaccines; however, no effect has been reported. Because of the severe pain caused by the large-volume pelvic tumor, laparotomy was performed with palliative intention, revealing an approximately 15-cm metastasis on the rectosigmoid mesentery, a 3-cm metastasis on the sigmoid colon, and multiple 1- to 2-mm implants on the small intestines. All lesions were removed, and Hartmann’s operation was performed because of heavy bleeding from the mesorectum and rectosigmoid mesentery. The patient was discharged from the hospital on the fourth uneventful postoperative day and is currently receiving palliative care. The course of the disease is shown in Figure 3.
Figure 2. CT scan of the patient reported in this case showing 14.5 × 8.8-cm metastatic masses within the pelvis.
CT: computed tomography.
Figure 3. Treatment course of the patient described in this report.
BEP: bleomycin, etoposide, and cisplatin.
Discussion
Ovarian SCTs-NOS occur at any age (2.5–93 years), but they are usually observed in reproductive-aged women, with an average age of 43 years.5,6 More than half (56%–77%) of patients with an SCT-NOS show hyperandrogenic symptoms and signs of virilization, such as hirsutism, acne, deepened voice, clitoromegaly, amenorrhea, and infertility.1,7–9 Additionally, it can present with estrogenic manifestations (6%–23%), such as menorrhagia or postmenopausal bleeding. Some patients also develop endometrial cancer.10 Ovarian SCTs can secrete steroid hormones, such as progesterone, cortisol, and aldosterone, which may cause corresponding clinical symptoms. Hyperandrogenic tumors may also be associated with paraneoplastic manifestations, such as hypercalcemia, erythrocytosis, or ascites.11 However, about 25% of patients with SCTs may have atypical presentations, without any symptoms of virilization. In these cases, the diagnosis is usually made postoperatively after histopathological verification.12 In the patient reported in this case, no virilization symptoms were noted, and the tumor was hormonally “silent.” Additional symptoms in some patients may include abdominal distension due to ascites and palpable abdominal masses.13 After tumor dissemination, the patient reported here manifested severe abdominal pain with palpable abdominal masses.
Serum laboratory analyses typically show elevated levels of testosterone and androstenedione, indicating an ovarian origin of androgen release and normal DHEA-S levels, thereby excluding adrenal causes of hyperandrogenism.1 In the reported case, the hormone levels were within the reference range, even after considerable disease dissemination. There is no known specific tumor marker established for the preoperative diagnosis of SCTs-NOS. Tumor markers, such as CA-125 and α-fetoprotein, are generally within the normal range, and the data from the literature do not indicate whether elevated levels signify malignant potential. Some studies even claim that these markers could facilitate the differential diagnosis of ovarian adenocarcinoma.11,14 This correlates with the described case, as even after disease relapse, tumor markers remained within the normal range.
SCTs-NOS are unilateral in 94% of cases, large at diagnosis (range from 1.2 to 45 cm in the greatest dimension, with an average reported size of 8.4 cm), and typically solid and well-circumscribed. SCTs-NOS should be distinguished from other ovarian tumors and SCTs, in which the proliferation of steroid hormone-producing cells occurs as a secondary event. These include stromal luteomas, Leydig-cell tumors, luteinized thecomas, and pregnancy luteomas.15 SCTs-NOS are different from Leydig-cell tumors in terms of their deficiency in cytoplasmic Reinke crystals. In addition, Leydig-cell tumors are usually situated in hilar locations, and they are commonly associated with Leydig-cell hyperplasia.16,17 Stromal luteomas are confined to the ovarian stroma and frequently occur in association with stromal hyperthecosis.12 SCTs-NOS might have a fibromatous component, similar to that of thecomas, but this component accounts for only less than 10% of tumors.18 Pregnancy luteomas are more commonly multifocal (bilateral in one-third of cases), usually discovered at the time of cesarean section, and regress spontaneously after pregnancy.12
In addition to the microscopic features, immunohistochemistry is particularly helpful for proper diagnosis. The sensitivity of positive calretinin is 60% to 90%, whereas the sensitivity of inhibin reactivity ranges from 5% to 90%.3,19 SCTs-NOS are also commonly vimentin, Melan-A, and CD99 positive and variably AE1/3, CAM5.2, HMB45, and S100 positive.3 Currently, techniques are being developed to define SCTs-NOS pathologically by immunohistochemical staining of steroidogenic enzymes. In the future, validated enzymes could serve as markers for the differential diagnosis of hyperandrogenic ovarian conditions.20 It is worth noting that differential diagnosis is challenging, as the primary histopathological diagnosis of this case from the previous hospital indicated a Sertoli–Leydig-cell tumor. However, no Reinke crystals were found after careful evaluation of the metastases resected in our hospital. In this patient, the tumor showed strong and diffuse positivity for inhibin and calretinin.
Usually, these tumors are benign; however, 25% to 43% of SCTs are malignant, with 20% of cases found to exhibit metastases beyond the ovary. Metastatic lesions usually occur within the peritoneal cavity and rarely occur at distant sites.15 In one study, the clinical and pathological features of 63 SCTs-NOS were reviewed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated as probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was malignant. Despite the various chemotherapy and radiation regimens, 12 patients experienced recurrences, and 14 patients died from the disease, suggesting a poor prognosis if the tumor is at an advanced stage, large, or recurring. Five pathological features are considered the best correlates of malignant behavior: the presence of two or more mitotic figures per 10 high-power fields (92% malignant), necrosis (86% malignant), a diameter of 7 cm or greater (78% malignant), hemorrhage (77% malignant), and grade 2 or 3 nuclear atypia (64% malignant).6 In our patient, we were unable to evaluate these features because the primary tumor was not resected at our hospital. We assume that the rapid spread was related to possible tumor fragmentation within the abdominal cavity, as we are uncertain of the use of an endobag.
Because the incidence of SCTs-NOS is low, there are currently no established treatment protocols. Therefore, the tumors are treated similarly to stromal tumors depending on several factors, including the surgical stage, histological type, patient’s age, and history of childbirth. The primary treatment for SCTs is surgery. Ovarian SCTs are generally considered benign because they are often detected at an early clinical stage. Therefore, unilateral salpingo-oophorectomy or tumor removal is acceptable in reproductive-aged women.1 However, regular follow-up with measurement of serum testosterone levels is mandatory. As there is limited information on the mechanisms of these tumors, the optimal length of follow-up is yet to be determined.15 In addition, there have already been reported cases of spontaneous pregnancies after tumor removal, probably because of the decline in testosterone levels.21,22
Surgical treatments using total abdominal hysterectomy and bilateral salpingo-oophorectomy are an appropriate management option for postmenopausal patients and those who have completed childbearing. Endometrial sampling should be performed when fertility-sparing surgery is planned because many of these patients may have coexisting endometrial hyperplasia or even uterine adenocarcinoma that might affect the decision for performing a hysterectomy.1
Because of the limited incidence of metastatic disease, there is a lack of reliable information on the role of adjuvant therapy in SCTs-NOS. The adjuvant chemotherapy regimens currently recommended for treatment are as follows: BEP; cisplatin, doxorubicin, and cyclophosphamide; taxane and platinum; and bleomycin, vinblastine, and cisplatin.23–27 It was reported that intraperitoneal dissemination and liver metastases were completely removed with debulking surgery, radiofrequency ablation of the liver metastasis, and adjuvant BEP (follow-up, 43 months) in a patient with a recurrent SCT-NOS occurring 5 years after the initial surgery.28 In another study, the treatment protocol consisted of docetaxel and nedaplatin, and the patient survived for 2 years with multiple bone metastases.29 Another SCT-NOS case with progressive disease after surgical debulking was reported. Treatment with multi-agent chemotherapy failed, but the patient subsequently showed a robust response to gonadotropin-releasing hormone agonist (GnRHa) therapy. Therefore, although typically treated with surgery alone, GnRHa may be required when abnormal serum hormone levels persist, and there is suspicion of residual tumors, recurrences, or metastases. It is suggested that GnRHa treatment be considered prior to cytotoxic chemotherapy in cases with SCTs-NOS.30 In the case described above, a chemotherapeutic regimen with BEP was administered for the recurrent SCT-NOS. After three cycles of BEP, the chemotherapeutic agent was changed to paclitaxel and carboplatin following a stable disease course. After eight cycles of paclitaxel and carboplatin, chemotherapy was discontinued because of prolonged neutropenia and peripheral neuropathy. The CT scan showed clinically stable disease. As the immunohistochemical analysis of the primary tumor revealed positive cytoplasmic staining for the GnRH receptor, GnRHa therapy was attempted based on reference to previous case reports and the lack of other effective alternatives. After six cycles of GnRHa therapy, the CT scan confirmed significant tumor size reduction. Adverse events were not observed, and the patient’s hirsutism and virilization had improved. Therefore, the treatment was discontinued after the administration of six cycles. However, the testosterone level was increased 2 months after GnRHa discontinuation, and the CT scan revealed an increase in tumor size. Subsequently, GnRHa was re-administered, resulting in immediate normalization of the serum testosterone level and shrinkage of the recurrent tumor. Thus, GnRHa therapy was continued (22 months after the first GnRHa treatment with the absence of symptoms).31
In our patient, the SCT-NOS was completely insensitive to chemotherapy, despite undergoing several debulking surgeries. Removal of the multi-metastases resulted in only short-term improvement. Unfortunately, the dendritic cell-based vaccine recently administered for 2 months did not yield a positive outcome.
Conclusions
Based on our clinical experience of this case, we highlight the importance of ovarian tumor resection without direct fragmentation within the abdominal cavity, as the neoplastic etiology should always be taken into account. Pathological evaluation is essential for the diagnosis of this malignancy, and immunohistochemical testing also aids in the formulation of an accurate diagnosis, especially in a patient with an apparent absence of endocrine manifestations. Surgery is the main treatment method for SCTs-NOS, as most cases are benign, and there are no generalized conclusions on the response of malignant cases to therapies, such as chemotherapy or radiation therapy. According to recent studies, GnRHa therapy could serve as a feasible treatment method and is worthy of further research with large patient groups.
Acknowledgment
We acknowledge oncologists from the Department of Oncology and Chemotherapy of Vilnius University Hospital Santaros Clinics for their considerable contribution to the treatment of the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Ethics statement: In our country, it is not mandatory to receive written consent from the local Ethics Committee for a case report, as it does not strictly meet the criteria of research because it does not require investigation and is intended to develop information to be shared for medical or educational purposes. Written informed consent was obtained from the patient for the publication of this report and the accompanying images.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iDs: Danuta Vasilevska https://orcid.org/0000-0002-3136-7281
Ugnius Mickys https://orcid.org/0000-0001-7805-0727
Andrzej Semczuk https://orcid.org/0000-0001-9344-9437 | 6 CYCLES , UNK | DrugDosageText | CC BY-NC | 33435776 | 18,956,668 | 2021-01 |
What was the dosage of drug 'CISPLATIN'? | Failure of multiple surgical procedures and adjuvant chemotherapy in early-stage steroid-cell ovarian tumor treatment: a case report and literature review.
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary, accounting for less than 0.1% of all ovarian tumors. Not otherwise specified (NOS) tumors are the most common subtype. More than half of patients with SCTs-NOS show hyperandrogenic symptoms. The primary treatment for SCTs is surgery, as most cases are early-staged and benign. Because of the low incidence of metastatic disease, there is insufficient reliable information on the role of adjuvant therapy and the most effective treatment regimen. In this report, a rare case of a recurrent SCT-NOS in a 36-year-old female patient without endocrine symptoms is presented, highlighting the significance of appropriate pathological evaluation and immunohistochemical testing for the accurate diagnosis of this malignancy, particularly in the case of hormonally "silent" tumors. The metastatic tumor described here showed no response to four courses of adjuvant chemotherapy after several debulking surgeries. Based on the clinical findings, the neoplastic etiology should always be considered during the resection of ovarian tumors to prevent possible disease dissemination due to inappropriate surgical techniques.
Introduction
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary that account for less than 0.1% of all ovarian tumors. They are classified into three categories based on the cell origin: stromal luteoma, Leydig-cell tumor, and not otherwise specified (NOS). NOS tumors are the most common subtype and comprise the largest proportion of cases (60%), whereas stromal luteoma and Leydig-cell tumors each account for ∼20% of cases.1–3
A SCT of the ovary was first described in 1943 as a “virilizing lipoid cell tumor” by Gemma Barzilai in the Atlas of Ovarian Pathology.4 Later, the term “lipoid cell tumor” was replaced by “steroid-cell tumor.” This change was based on the argument that although all SCTs can produce steroid hormones, up to 25% of them contain little or no lipids.4 In 1979, the term “steroid-cell tumor, not otherwise specified,” was coined by Scully2 to indicate that the cell lineage from which the tumor arises is unknown. SCTs-NOS usually develop in reproductive-aged women with an average age of 43 years. The major symptoms are hirsutism and virilization caused by high levels of testosterone. The cornerstone of SCT-NOS treatment is surgery. However, as most tumors are diagnosed in the early clinical stage and do not recur or metastasize, there is limited information on their response to chemotherapy.1
In this report, a rare case of a recurrent SCT-NOS in a patient without endocrine symptoms is presented. The tumor showed no response to adjuvant chemotherapy after several debulking surgeries.
Case presentation
A 36-year-old Caucasian female patient, gravida 2 para 2, underwent laparoscopic right salpingo-oophorectomy in 2015 at a secondary-level hospital after the identification of a right ovarian tumor (the tumor was fragmented within the abdominal cavity, but it was unclear if it was within the endobag). The post-surgical pathological report revealed a Sertoli–Leydig-cell tumor. The patient was not referred to a clinical oncologist because radical removal was expected. Her past medical and surgical history was unremarkable, and she had no relevant family history. No signs of virilization or hirsutism were observed.
After 6 months, the patient was referred to the Gynecologic Department of our tertiary hospital because of metastases. During laparoscopy, multiple metastatic lesions were found in the pelvic peritoneum, on the diaphragm, and at the site of the former trocar; the left ovary was solid with suspected tumor spread. Metastatic lesions appeared as 3- to 4-mm subperitoneal fatty nodules, which were biopsied. Washings from the peritoneal cavity were negative. Unexpectedly, the histopathological diagnosis was a SCT-NOS. Laboratory analysis revealed normal values of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol. No hormonal assays were performed prior to surgery, as there were no clinical manifestations of any excessive hormone secretion. The ovarian tumor markers, including human epididymis protein 4, β-human chorionic gonadotropin, and α-fetoprotein, were within the normal range. Cancer antigen 125 (Ca125) was elevated to 48.8 kU/L (normal value up to 35 kU/L), but after 3 weeks, the level returned to normal (24.8 kU/L). Ten months after the first surgery, the patient underwent a total abdominal hysterectomy, left salpingo-oophorectomy, omentectomy, pelvic and diaphragmatic peritonectomy, and pelvic lymphadenectomy. Optimal cytoreductive surgery was performed. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization but no signs of nuclear atypia and a mitotic count of 5 per 10 high-power fields. No Reinke crystals, which are usually observed in Sertoli–Leydig-cell tumors, were identified. Immunohistochemistry revealed positive expression of inhibin A, calretinin, and synaptophysin but negative expression of pan-cytokeratin, CD68, epithelial membrane antigen, chromogranin A, and estrogen receptor/androgen receptor. The Ki-67 labeling index was up to 15% (Figure 1). The histopathologic features supported the diagnosis of a SCT-NOS.
Figure 1. Microscopic appearance of the SCT-NOS (H&E) in a 36-year-old Caucasian female patient (a) and positive staining of the tumor cells for inhibin A (b), calretinin (c), and Ki-67 (d) (20×)
H&E: hematoxylin and eosin.
After the surgery, the patient received six cycles of adjuvant carboplatin-paclitaxel chemotherapy. Ca125 was measured several times during the treatment period; its levels were within the normal range. At the end of the six courses of chemotherapy, a control computed tomography (CT) scan was performed, revealing the absence of disease progression.
After 3 months of follow-up, the CT scan showed disease progression in the left lateral abdominal flank; hence, we decided to perform secondary cytoreductive surgery. McBurney’s laparotomy was performed. During abdominal exploration, an infiltrative growing tumor was found at the site of the right former trocar that had grown in all layers of the abdomen and spread into the right flank. The laparotomy was extended in the cephalad direction. Metastases were also observed in the left superior diaphragmatic space and the previous left trocar area. Secondary optimal cytoreduction was performed to remove metastases from the right iliac region and abdominal lateral canals. The analysis of frozen sections demonstrated SCT metastases in fibrous adipose tissue. The patient experienced disease-free survival for only 6 months, after which the follow-up CT scan showed disease progression and a large incisional hernia at the incision site of the previous McBurney’s laparotomy. Serum tumor markers and hormone levels were within the normal range. During the subsequent laparotomy, micrometastases were observed on the aponeurosis with multiple small metastases on the small and large intestines, which could not be radically removed. The excision of injured subcutaneous tissue and incisional hernia repair with mesh were performed. The histological diagnosis was SCT metastasis to soft tissues. The patient was subjected to six cycles of second-line bleomycin, etoposide, and cisplatin (BEP) chemotherapy. On account of a dry cough, bleomycin has not been prescribed after the second cycle due to suspicion of pulmonary toxicity. The patient was put under surveillance every 3 months after the completion of chemotherapy. An abdominopelvic CT scan revealed the absence of disease progression, and no metastases were observed on the right side of the anterior abdominal wall or the bowel. Serum tumor markers and hormone levels were normal. After 1 year, disease progression was observed on the CT scan, which revealed increased foci in the anterior and lateral abdominal wall (>50%) and a small amount of ascites in the lateral flanks. Systemic treatment was recommended, and five cycles of liposomal doxorubicin were administered. Despite undergoing treatment, radiologic disease progression was observed after 4 months, as there was an ∼40% increase in tumor mass above the vaginal cuff on the left side with an increase in fluid volume in the pelvis. On the right abdominal wall, supra-umbilically, most small tumor foci had increased in size. The patient required re-operation. On the anterior abdominal wall, several necrotic metastases were found. In the abdominal cavity, there was a 10-cm ruptured tumor between the sigmoid colon and the vaginal cuff. Multiple 1- to 4-cm retroperitoneal metastases were found on the right internal iliac artery, right ureter, and pelvic veins. The right ureter was approximately 2 cm in diameter, with metastases completely fixed to the pelvic bones/fasciae. Few small metastases were noted on the peritoneum and mesentery. Because of the infiltration of the lateral pelvic walls, the tumors could not be radically resected; sigmoid colon resection was rendered ineffective because of the presence of residual tumor masses. Only bleeding tumors and metastases on the peritoneum and mesentery were non-radically removed. Histopathological verification revealed metastases of the SCT-NOS. At the pain management center, the patient was advised to start treatment with transdermal fentanyl patches for the treatment of intense abdominal pain. The patient was referred to a urologist because of right hydronephrosis, and a long-term ureteral stent was inserted. The multidisciplinary team decided to repeat the previous regimen of chemotherapy based on carboplatin and paclitaxel (six cycles). After completion of the chemotherapy, a CT scan was performed. The pelvic, vaginal cuff, and sigmoid colon masses were enlarged, indicating disease progression. Only palliative treatment was recommended. After 5 months, another CT scan was performed, and significant disease progression and noticeable bilateral hydronephrosis were detected, with the metastases enlarged by 80% (Figure 2). To manage the pain, the subcutaneous right lumbar mass was resected under regional anesthesia. The right ureteral stent was replaced, and a long-term stent was also inserted into the left ureter. Currently, the hormone levels and cancer biomarkers are within the normal reference ranges. Over the last 2 months, the patient was administered dendritic cell-based cancer vaccines; however, no effect has been reported. Because of the severe pain caused by the large-volume pelvic tumor, laparotomy was performed with palliative intention, revealing an approximately 15-cm metastasis on the rectosigmoid mesentery, a 3-cm metastasis on the sigmoid colon, and multiple 1- to 2-mm implants on the small intestines. All lesions were removed, and Hartmann’s operation was performed because of heavy bleeding from the mesorectum and rectosigmoid mesentery. The patient was discharged from the hospital on the fourth uneventful postoperative day and is currently receiving palliative care. The course of the disease is shown in Figure 3.
Figure 2. CT scan of the patient reported in this case showing 14.5 × 8.8-cm metastatic masses within the pelvis.
CT: computed tomography.
Figure 3. Treatment course of the patient described in this report.
BEP: bleomycin, etoposide, and cisplatin.
Discussion
Ovarian SCTs-NOS occur at any age (2.5–93 years), but they are usually observed in reproductive-aged women, with an average age of 43 years.5,6 More than half (56%–77%) of patients with an SCT-NOS show hyperandrogenic symptoms and signs of virilization, such as hirsutism, acne, deepened voice, clitoromegaly, amenorrhea, and infertility.1,7–9 Additionally, it can present with estrogenic manifestations (6%–23%), such as menorrhagia or postmenopausal bleeding. Some patients also develop endometrial cancer.10 Ovarian SCTs can secrete steroid hormones, such as progesterone, cortisol, and aldosterone, which may cause corresponding clinical symptoms. Hyperandrogenic tumors may also be associated with paraneoplastic manifestations, such as hypercalcemia, erythrocytosis, or ascites.11 However, about 25% of patients with SCTs may have atypical presentations, without any symptoms of virilization. In these cases, the diagnosis is usually made postoperatively after histopathological verification.12 In the patient reported in this case, no virilization symptoms were noted, and the tumor was hormonally “silent.” Additional symptoms in some patients may include abdominal distension due to ascites and palpable abdominal masses.13 After tumor dissemination, the patient reported here manifested severe abdominal pain with palpable abdominal masses.
Serum laboratory analyses typically show elevated levels of testosterone and androstenedione, indicating an ovarian origin of androgen release and normal DHEA-S levels, thereby excluding adrenal causes of hyperandrogenism.1 In the reported case, the hormone levels were within the reference range, even after considerable disease dissemination. There is no known specific tumor marker established for the preoperative diagnosis of SCTs-NOS. Tumor markers, such as CA-125 and α-fetoprotein, are generally within the normal range, and the data from the literature do not indicate whether elevated levels signify malignant potential. Some studies even claim that these markers could facilitate the differential diagnosis of ovarian adenocarcinoma.11,14 This correlates with the described case, as even after disease relapse, tumor markers remained within the normal range.
SCTs-NOS are unilateral in 94% of cases, large at diagnosis (range from 1.2 to 45 cm in the greatest dimension, with an average reported size of 8.4 cm), and typically solid and well-circumscribed. SCTs-NOS should be distinguished from other ovarian tumors and SCTs, in which the proliferation of steroid hormone-producing cells occurs as a secondary event. These include stromal luteomas, Leydig-cell tumors, luteinized thecomas, and pregnancy luteomas.15 SCTs-NOS are different from Leydig-cell tumors in terms of their deficiency in cytoplasmic Reinke crystals. In addition, Leydig-cell tumors are usually situated in hilar locations, and they are commonly associated with Leydig-cell hyperplasia.16,17 Stromal luteomas are confined to the ovarian stroma and frequently occur in association with stromal hyperthecosis.12 SCTs-NOS might have a fibromatous component, similar to that of thecomas, but this component accounts for only less than 10% of tumors.18 Pregnancy luteomas are more commonly multifocal (bilateral in one-third of cases), usually discovered at the time of cesarean section, and regress spontaneously after pregnancy.12
In addition to the microscopic features, immunohistochemistry is particularly helpful for proper diagnosis. The sensitivity of positive calretinin is 60% to 90%, whereas the sensitivity of inhibin reactivity ranges from 5% to 90%.3,19 SCTs-NOS are also commonly vimentin, Melan-A, and CD99 positive and variably AE1/3, CAM5.2, HMB45, and S100 positive.3 Currently, techniques are being developed to define SCTs-NOS pathologically by immunohistochemical staining of steroidogenic enzymes. In the future, validated enzymes could serve as markers for the differential diagnosis of hyperandrogenic ovarian conditions.20 It is worth noting that differential diagnosis is challenging, as the primary histopathological diagnosis of this case from the previous hospital indicated a Sertoli–Leydig-cell tumor. However, no Reinke crystals were found after careful evaluation of the metastases resected in our hospital. In this patient, the tumor showed strong and diffuse positivity for inhibin and calretinin.
Usually, these tumors are benign; however, 25% to 43% of SCTs are malignant, with 20% of cases found to exhibit metastases beyond the ovary. Metastatic lesions usually occur within the peritoneal cavity and rarely occur at distant sites.15 In one study, the clinical and pathological features of 63 SCTs-NOS were reviewed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated as probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was malignant. Despite the various chemotherapy and radiation regimens, 12 patients experienced recurrences, and 14 patients died from the disease, suggesting a poor prognosis if the tumor is at an advanced stage, large, or recurring. Five pathological features are considered the best correlates of malignant behavior: the presence of two or more mitotic figures per 10 high-power fields (92% malignant), necrosis (86% malignant), a diameter of 7 cm or greater (78% malignant), hemorrhage (77% malignant), and grade 2 or 3 nuclear atypia (64% malignant).6 In our patient, we were unable to evaluate these features because the primary tumor was not resected at our hospital. We assume that the rapid spread was related to possible tumor fragmentation within the abdominal cavity, as we are uncertain of the use of an endobag.
Because the incidence of SCTs-NOS is low, there are currently no established treatment protocols. Therefore, the tumors are treated similarly to stromal tumors depending on several factors, including the surgical stage, histological type, patient’s age, and history of childbirth. The primary treatment for SCTs is surgery. Ovarian SCTs are generally considered benign because they are often detected at an early clinical stage. Therefore, unilateral salpingo-oophorectomy or tumor removal is acceptable in reproductive-aged women.1 However, regular follow-up with measurement of serum testosterone levels is mandatory. As there is limited information on the mechanisms of these tumors, the optimal length of follow-up is yet to be determined.15 In addition, there have already been reported cases of spontaneous pregnancies after tumor removal, probably because of the decline in testosterone levels.21,22
Surgical treatments using total abdominal hysterectomy and bilateral salpingo-oophorectomy are an appropriate management option for postmenopausal patients and those who have completed childbearing. Endometrial sampling should be performed when fertility-sparing surgery is planned because many of these patients may have coexisting endometrial hyperplasia or even uterine adenocarcinoma that might affect the decision for performing a hysterectomy.1
Because of the limited incidence of metastatic disease, there is a lack of reliable information on the role of adjuvant therapy in SCTs-NOS. The adjuvant chemotherapy regimens currently recommended for treatment are as follows: BEP; cisplatin, doxorubicin, and cyclophosphamide; taxane and platinum; and bleomycin, vinblastine, and cisplatin.23–27 It was reported that intraperitoneal dissemination and liver metastases were completely removed with debulking surgery, radiofrequency ablation of the liver metastasis, and adjuvant BEP (follow-up, 43 months) in a patient with a recurrent SCT-NOS occurring 5 years after the initial surgery.28 In another study, the treatment protocol consisted of docetaxel and nedaplatin, and the patient survived for 2 years with multiple bone metastases.29 Another SCT-NOS case with progressive disease after surgical debulking was reported. Treatment with multi-agent chemotherapy failed, but the patient subsequently showed a robust response to gonadotropin-releasing hormone agonist (GnRHa) therapy. Therefore, although typically treated with surgery alone, GnRHa may be required when abnormal serum hormone levels persist, and there is suspicion of residual tumors, recurrences, or metastases. It is suggested that GnRHa treatment be considered prior to cytotoxic chemotherapy in cases with SCTs-NOS.30 In the case described above, a chemotherapeutic regimen with BEP was administered for the recurrent SCT-NOS. After three cycles of BEP, the chemotherapeutic agent was changed to paclitaxel and carboplatin following a stable disease course. After eight cycles of paclitaxel and carboplatin, chemotherapy was discontinued because of prolonged neutropenia and peripheral neuropathy. The CT scan showed clinically stable disease. As the immunohistochemical analysis of the primary tumor revealed positive cytoplasmic staining for the GnRH receptor, GnRHa therapy was attempted based on reference to previous case reports and the lack of other effective alternatives. After six cycles of GnRHa therapy, the CT scan confirmed significant tumor size reduction. Adverse events were not observed, and the patient’s hirsutism and virilization had improved. Therefore, the treatment was discontinued after the administration of six cycles. However, the testosterone level was increased 2 months after GnRHa discontinuation, and the CT scan revealed an increase in tumor size. Subsequently, GnRHa was re-administered, resulting in immediate normalization of the serum testosterone level and shrinkage of the recurrent tumor. Thus, GnRHa therapy was continued (22 months after the first GnRHa treatment with the absence of symptoms).31
In our patient, the SCT-NOS was completely insensitive to chemotherapy, despite undergoing several debulking surgeries. Removal of the multi-metastases resulted in only short-term improvement. Unfortunately, the dendritic cell-based vaccine recently administered for 2 months did not yield a positive outcome.
Conclusions
Based on our clinical experience of this case, we highlight the importance of ovarian tumor resection without direct fragmentation within the abdominal cavity, as the neoplastic etiology should always be taken into account. Pathological evaluation is essential for the diagnosis of this malignancy, and immunohistochemical testing also aids in the formulation of an accurate diagnosis, especially in a patient with an apparent absence of endocrine manifestations. Surgery is the main treatment method for SCTs-NOS, as most cases are benign, and there are no generalized conclusions on the response of malignant cases to therapies, such as chemotherapy or radiation therapy. According to recent studies, GnRHa therapy could serve as a feasible treatment method and is worthy of further research with large patient groups.
Acknowledgment
We acknowledge oncologists from the Department of Oncology and Chemotherapy of Vilnius University Hospital Santaros Clinics for their considerable contribution to the treatment of the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Ethics statement: In our country, it is not mandatory to receive written consent from the local Ethics Committee for a case report, as it does not strictly meet the criteria of research because it does not require investigation and is intended to develop information to be shared for medical or educational purposes. Written informed consent was obtained from the patient for the publication of this report and the accompanying images.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iDs: Danuta Vasilevska https://orcid.org/0000-0002-3136-7281
Ugnius Mickys https://orcid.org/0000-0001-7805-0727
Andrzej Semczuk https://orcid.org/0000-0001-9344-9437 | 6 CYCLES , UNK | DrugDosageText | CC BY-NC | 33435776 | 18,956,668 | 2021-01 |
What was the dosage of drug 'ETOPOSIDE'? | Failure of multiple surgical procedures and adjuvant chemotherapy in early-stage steroid-cell ovarian tumor treatment: a case report and literature review.
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary, accounting for less than 0.1% of all ovarian tumors. Not otherwise specified (NOS) tumors are the most common subtype. More than half of patients with SCTs-NOS show hyperandrogenic symptoms. The primary treatment for SCTs is surgery, as most cases are early-staged and benign. Because of the low incidence of metastatic disease, there is insufficient reliable information on the role of adjuvant therapy and the most effective treatment regimen. In this report, a rare case of a recurrent SCT-NOS in a 36-year-old female patient without endocrine symptoms is presented, highlighting the significance of appropriate pathological evaluation and immunohistochemical testing for the accurate diagnosis of this malignancy, particularly in the case of hormonally "silent" tumors. The metastatic tumor described here showed no response to four courses of adjuvant chemotherapy after several debulking surgeries. Based on the clinical findings, the neoplastic etiology should always be considered during the resection of ovarian tumors to prevent possible disease dissemination due to inappropriate surgical techniques.
Introduction
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary that account for less than 0.1% of all ovarian tumors. They are classified into three categories based on the cell origin: stromal luteoma, Leydig-cell tumor, and not otherwise specified (NOS). NOS tumors are the most common subtype and comprise the largest proportion of cases (60%), whereas stromal luteoma and Leydig-cell tumors each account for ∼20% of cases.1–3
A SCT of the ovary was first described in 1943 as a “virilizing lipoid cell tumor” by Gemma Barzilai in the Atlas of Ovarian Pathology.4 Later, the term “lipoid cell tumor” was replaced by “steroid-cell tumor.” This change was based on the argument that although all SCTs can produce steroid hormones, up to 25% of them contain little or no lipids.4 In 1979, the term “steroid-cell tumor, not otherwise specified,” was coined by Scully2 to indicate that the cell lineage from which the tumor arises is unknown. SCTs-NOS usually develop in reproductive-aged women with an average age of 43 years. The major symptoms are hirsutism and virilization caused by high levels of testosterone. The cornerstone of SCT-NOS treatment is surgery. However, as most tumors are diagnosed in the early clinical stage and do not recur or metastasize, there is limited information on their response to chemotherapy.1
In this report, a rare case of a recurrent SCT-NOS in a patient without endocrine symptoms is presented. The tumor showed no response to adjuvant chemotherapy after several debulking surgeries.
Case presentation
A 36-year-old Caucasian female patient, gravida 2 para 2, underwent laparoscopic right salpingo-oophorectomy in 2015 at a secondary-level hospital after the identification of a right ovarian tumor (the tumor was fragmented within the abdominal cavity, but it was unclear if it was within the endobag). The post-surgical pathological report revealed a Sertoli–Leydig-cell tumor. The patient was not referred to a clinical oncologist because radical removal was expected. Her past medical and surgical history was unremarkable, and she had no relevant family history. No signs of virilization or hirsutism were observed.
After 6 months, the patient was referred to the Gynecologic Department of our tertiary hospital because of metastases. During laparoscopy, multiple metastatic lesions were found in the pelvic peritoneum, on the diaphragm, and at the site of the former trocar; the left ovary was solid with suspected tumor spread. Metastatic lesions appeared as 3- to 4-mm subperitoneal fatty nodules, which were biopsied. Washings from the peritoneal cavity were negative. Unexpectedly, the histopathological diagnosis was a SCT-NOS. Laboratory analysis revealed normal values of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol. No hormonal assays were performed prior to surgery, as there were no clinical manifestations of any excessive hormone secretion. The ovarian tumor markers, including human epididymis protein 4, β-human chorionic gonadotropin, and α-fetoprotein, were within the normal range. Cancer antigen 125 (Ca125) was elevated to 48.8 kU/L (normal value up to 35 kU/L), but after 3 weeks, the level returned to normal (24.8 kU/L). Ten months after the first surgery, the patient underwent a total abdominal hysterectomy, left salpingo-oophorectomy, omentectomy, pelvic and diaphragmatic peritonectomy, and pelvic lymphadenectomy. Optimal cytoreductive surgery was performed. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization but no signs of nuclear atypia and a mitotic count of 5 per 10 high-power fields. No Reinke crystals, which are usually observed in Sertoli–Leydig-cell tumors, were identified. Immunohistochemistry revealed positive expression of inhibin A, calretinin, and synaptophysin but negative expression of pan-cytokeratin, CD68, epithelial membrane antigen, chromogranin A, and estrogen receptor/androgen receptor. The Ki-67 labeling index was up to 15% (Figure 1). The histopathologic features supported the diagnosis of a SCT-NOS.
Figure 1. Microscopic appearance of the SCT-NOS (H&E) in a 36-year-old Caucasian female patient (a) and positive staining of the tumor cells for inhibin A (b), calretinin (c), and Ki-67 (d) (20×)
H&E: hematoxylin and eosin.
After the surgery, the patient received six cycles of adjuvant carboplatin-paclitaxel chemotherapy. Ca125 was measured several times during the treatment period; its levels were within the normal range. At the end of the six courses of chemotherapy, a control computed tomography (CT) scan was performed, revealing the absence of disease progression.
After 3 months of follow-up, the CT scan showed disease progression in the left lateral abdominal flank; hence, we decided to perform secondary cytoreductive surgery. McBurney’s laparotomy was performed. During abdominal exploration, an infiltrative growing tumor was found at the site of the right former trocar that had grown in all layers of the abdomen and spread into the right flank. The laparotomy was extended in the cephalad direction. Metastases were also observed in the left superior diaphragmatic space and the previous left trocar area. Secondary optimal cytoreduction was performed to remove metastases from the right iliac region and abdominal lateral canals. The analysis of frozen sections demonstrated SCT metastases in fibrous adipose tissue. The patient experienced disease-free survival for only 6 months, after which the follow-up CT scan showed disease progression and a large incisional hernia at the incision site of the previous McBurney’s laparotomy. Serum tumor markers and hormone levels were within the normal range. During the subsequent laparotomy, micrometastases were observed on the aponeurosis with multiple small metastases on the small and large intestines, which could not be radically removed. The excision of injured subcutaneous tissue and incisional hernia repair with mesh were performed. The histological diagnosis was SCT metastasis to soft tissues. The patient was subjected to six cycles of second-line bleomycin, etoposide, and cisplatin (BEP) chemotherapy. On account of a dry cough, bleomycin has not been prescribed after the second cycle due to suspicion of pulmonary toxicity. The patient was put under surveillance every 3 months after the completion of chemotherapy. An abdominopelvic CT scan revealed the absence of disease progression, and no metastases were observed on the right side of the anterior abdominal wall or the bowel. Serum tumor markers and hormone levels were normal. After 1 year, disease progression was observed on the CT scan, which revealed increased foci in the anterior and lateral abdominal wall (>50%) and a small amount of ascites in the lateral flanks. Systemic treatment was recommended, and five cycles of liposomal doxorubicin were administered. Despite undergoing treatment, radiologic disease progression was observed after 4 months, as there was an ∼40% increase in tumor mass above the vaginal cuff on the left side with an increase in fluid volume in the pelvis. On the right abdominal wall, supra-umbilically, most small tumor foci had increased in size. The patient required re-operation. On the anterior abdominal wall, several necrotic metastases were found. In the abdominal cavity, there was a 10-cm ruptured tumor between the sigmoid colon and the vaginal cuff. Multiple 1- to 4-cm retroperitoneal metastases were found on the right internal iliac artery, right ureter, and pelvic veins. The right ureter was approximately 2 cm in diameter, with metastases completely fixed to the pelvic bones/fasciae. Few small metastases were noted on the peritoneum and mesentery. Because of the infiltration of the lateral pelvic walls, the tumors could not be radically resected; sigmoid colon resection was rendered ineffective because of the presence of residual tumor masses. Only bleeding tumors and metastases on the peritoneum and mesentery were non-radically removed. Histopathological verification revealed metastases of the SCT-NOS. At the pain management center, the patient was advised to start treatment with transdermal fentanyl patches for the treatment of intense abdominal pain. The patient was referred to a urologist because of right hydronephrosis, and a long-term ureteral stent was inserted. The multidisciplinary team decided to repeat the previous regimen of chemotherapy based on carboplatin and paclitaxel (six cycles). After completion of the chemotherapy, a CT scan was performed. The pelvic, vaginal cuff, and sigmoid colon masses were enlarged, indicating disease progression. Only palliative treatment was recommended. After 5 months, another CT scan was performed, and significant disease progression and noticeable bilateral hydronephrosis were detected, with the metastases enlarged by 80% (Figure 2). To manage the pain, the subcutaneous right lumbar mass was resected under regional anesthesia. The right ureteral stent was replaced, and a long-term stent was also inserted into the left ureter. Currently, the hormone levels and cancer biomarkers are within the normal reference ranges. Over the last 2 months, the patient was administered dendritic cell-based cancer vaccines; however, no effect has been reported. Because of the severe pain caused by the large-volume pelvic tumor, laparotomy was performed with palliative intention, revealing an approximately 15-cm metastasis on the rectosigmoid mesentery, a 3-cm metastasis on the sigmoid colon, and multiple 1- to 2-mm implants on the small intestines. All lesions were removed, and Hartmann’s operation was performed because of heavy bleeding from the mesorectum and rectosigmoid mesentery. The patient was discharged from the hospital on the fourth uneventful postoperative day and is currently receiving palliative care. The course of the disease is shown in Figure 3.
Figure 2. CT scan of the patient reported in this case showing 14.5 × 8.8-cm metastatic masses within the pelvis.
CT: computed tomography.
Figure 3. Treatment course of the patient described in this report.
BEP: bleomycin, etoposide, and cisplatin.
Discussion
Ovarian SCTs-NOS occur at any age (2.5–93 years), but they are usually observed in reproductive-aged women, with an average age of 43 years.5,6 More than half (56%–77%) of patients with an SCT-NOS show hyperandrogenic symptoms and signs of virilization, such as hirsutism, acne, deepened voice, clitoromegaly, amenorrhea, and infertility.1,7–9 Additionally, it can present with estrogenic manifestations (6%–23%), such as menorrhagia or postmenopausal bleeding. Some patients also develop endometrial cancer.10 Ovarian SCTs can secrete steroid hormones, such as progesterone, cortisol, and aldosterone, which may cause corresponding clinical symptoms. Hyperandrogenic tumors may also be associated with paraneoplastic manifestations, such as hypercalcemia, erythrocytosis, or ascites.11 However, about 25% of patients with SCTs may have atypical presentations, without any symptoms of virilization. In these cases, the diagnosis is usually made postoperatively after histopathological verification.12 In the patient reported in this case, no virilization symptoms were noted, and the tumor was hormonally “silent.” Additional symptoms in some patients may include abdominal distension due to ascites and palpable abdominal masses.13 After tumor dissemination, the patient reported here manifested severe abdominal pain with palpable abdominal masses.
Serum laboratory analyses typically show elevated levels of testosterone and androstenedione, indicating an ovarian origin of androgen release and normal DHEA-S levels, thereby excluding adrenal causes of hyperandrogenism.1 In the reported case, the hormone levels were within the reference range, even after considerable disease dissemination. There is no known specific tumor marker established for the preoperative diagnosis of SCTs-NOS. Tumor markers, such as CA-125 and α-fetoprotein, are generally within the normal range, and the data from the literature do not indicate whether elevated levels signify malignant potential. Some studies even claim that these markers could facilitate the differential diagnosis of ovarian adenocarcinoma.11,14 This correlates with the described case, as even after disease relapse, tumor markers remained within the normal range.
SCTs-NOS are unilateral in 94% of cases, large at diagnosis (range from 1.2 to 45 cm in the greatest dimension, with an average reported size of 8.4 cm), and typically solid and well-circumscribed. SCTs-NOS should be distinguished from other ovarian tumors and SCTs, in which the proliferation of steroid hormone-producing cells occurs as a secondary event. These include stromal luteomas, Leydig-cell tumors, luteinized thecomas, and pregnancy luteomas.15 SCTs-NOS are different from Leydig-cell tumors in terms of their deficiency in cytoplasmic Reinke crystals. In addition, Leydig-cell tumors are usually situated in hilar locations, and they are commonly associated with Leydig-cell hyperplasia.16,17 Stromal luteomas are confined to the ovarian stroma and frequently occur in association with stromal hyperthecosis.12 SCTs-NOS might have a fibromatous component, similar to that of thecomas, but this component accounts for only less than 10% of tumors.18 Pregnancy luteomas are more commonly multifocal (bilateral in one-third of cases), usually discovered at the time of cesarean section, and regress spontaneously after pregnancy.12
In addition to the microscopic features, immunohistochemistry is particularly helpful for proper diagnosis. The sensitivity of positive calretinin is 60% to 90%, whereas the sensitivity of inhibin reactivity ranges from 5% to 90%.3,19 SCTs-NOS are also commonly vimentin, Melan-A, and CD99 positive and variably AE1/3, CAM5.2, HMB45, and S100 positive.3 Currently, techniques are being developed to define SCTs-NOS pathologically by immunohistochemical staining of steroidogenic enzymes. In the future, validated enzymes could serve as markers for the differential diagnosis of hyperandrogenic ovarian conditions.20 It is worth noting that differential diagnosis is challenging, as the primary histopathological diagnosis of this case from the previous hospital indicated a Sertoli–Leydig-cell tumor. However, no Reinke crystals were found after careful evaluation of the metastases resected in our hospital. In this patient, the tumor showed strong and diffuse positivity for inhibin and calretinin.
Usually, these tumors are benign; however, 25% to 43% of SCTs are malignant, with 20% of cases found to exhibit metastases beyond the ovary. Metastatic lesions usually occur within the peritoneal cavity and rarely occur at distant sites.15 In one study, the clinical and pathological features of 63 SCTs-NOS were reviewed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated as probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was malignant. Despite the various chemotherapy and radiation regimens, 12 patients experienced recurrences, and 14 patients died from the disease, suggesting a poor prognosis if the tumor is at an advanced stage, large, or recurring. Five pathological features are considered the best correlates of malignant behavior: the presence of two or more mitotic figures per 10 high-power fields (92% malignant), necrosis (86% malignant), a diameter of 7 cm or greater (78% malignant), hemorrhage (77% malignant), and grade 2 or 3 nuclear atypia (64% malignant).6 In our patient, we were unable to evaluate these features because the primary tumor was not resected at our hospital. We assume that the rapid spread was related to possible tumor fragmentation within the abdominal cavity, as we are uncertain of the use of an endobag.
Because the incidence of SCTs-NOS is low, there are currently no established treatment protocols. Therefore, the tumors are treated similarly to stromal tumors depending on several factors, including the surgical stage, histological type, patient’s age, and history of childbirth. The primary treatment for SCTs is surgery. Ovarian SCTs are generally considered benign because they are often detected at an early clinical stage. Therefore, unilateral salpingo-oophorectomy or tumor removal is acceptable in reproductive-aged women.1 However, regular follow-up with measurement of serum testosterone levels is mandatory. As there is limited information on the mechanisms of these tumors, the optimal length of follow-up is yet to be determined.15 In addition, there have already been reported cases of spontaneous pregnancies after tumor removal, probably because of the decline in testosterone levels.21,22
Surgical treatments using total abdominal hysterectomy and bilateral salpingo-oophorectomy are an appropriate management option for postmenopausal patients and those who have completed childbearing. Endometrial sampling should be performed when fertility-sparing surgery is planned because many of these patients may have coexisting endometrial hyperplasia or even uterine adenocarcinoma that might affect the decision for performing a hysterectomy.1
Because of the limited incidence of metastatic disease, there is a lack of reliable information on the role of adjuvant therapy in SCTs-NOS. The adjuvant chemotherapy regimens currently recommended for treatment are as follows: BEP; cisplatin, doxorubicin, and cyclophosphamide; taxane and platinum; and bleomycin, vinblastine, and cisplatin.23–27 It was reported that intraperitoneal dissemination and liver metastases were completely removed with debulking surgery, radiofrequency ablation of the liver metastasis, and adjuvant BEP (follow-up, 43 months) in a patient with a recurrent SCT-NOS occurring 5 years after the initial surgery.28 In another study, the treatment protocol consisted of docetaxel and nedaplatin, and the patient survived for 2 years with multiple bone metastases.29 Another SCT-NOS case with progressive disease after surgical debulking was reported. Treatment with multi-agent chemotherapy failed, but the patient subsequently showed a robust response to gonadotropin-releasing hormone agonist (GnRHa) therapy. Therefore, although typically treated with surgery alone, GnRHa may be required when abnormal serum hormone levels persist, and there is suspicion of residual tumors, recurrences, or metastases. It is suggested that GnRHa treatment be considered prior to cytotoxic chemotherapy in cases with SCTs-NOS.30 In the case described above, a chemotherapeutic regimen with BEP was administered for the recurrent SCT-NOS. After three cycles of BEP, the chemotherapeutic agent was changed to paclitaxel and carboplatin following a stable disease course. After eight cycles of paclitaxel and carboplatin, chemotherapy was discontinued because of prolonged neutropenia and peripheral neuropathy. The CT scan showed clinically stable disease. As the immunohistochemical analysis of the primary tumor revealed positive cytoplasmic staining for the GnRH receptor, GnRHa therapy was attempted based on reference to previous case reports and the lack of other effective alternatives. After six cycles of GnRHa therapy, the CT scan confirmed significant tumor size reduction. Adverse events were not observed, and the patient’s hirsutism and virilization had improved. Therefore, the treatment was discontinued after the administration of six cycles. However, the testosterone level was increased 2 months after GnRHa discontinuation, and the CT scan revealed an increase in tumor size. Subsequently, GnRHa was re-administered, resulting in immediate normalization of the serum testosterone level and shrinkage of the recurrent tumor. Thus, GnRHa therapy was continued (22 months after the first GnRHa treatment with the absence of symptoms).31
In our patient, the SCT-NOS was completely insensitive to chemotherapy, despite undergoing several debulking surgeries. Removal of the multi-metastases resulted in only short-term improvement. Unfortunately, the dendritic cell-based vaccine recently administered for 2 months did not yield a positive outcome.
Conclusions
Based on our clinical experience of this case, we highlight the importance of ovarian tumor resection without direct fragmentation within the abdominal cavity, as the neoplastic etiology should always be taken into account. Pathological evaluation is essential for the diagnosis of this malignancy, and immunohistochemical testing also aids in the formulation of an accurate diagnosis, especially in a patient with an apparent absence of endocrine manifestations. Surgery is the main treatment method for SCTs-NOS, as most cases are benign, and there are no generalized conclusions on the response of malignant cases to therapies, such as chemotherapy or radiation therapy. According to recent studies, GnRHa therapy could serve as a feasible treatment method and is worthy of further research with large patient groups.
Acknowledgment
We acknowledge oncologists from the Department of Oncology and Chemotherapy of Vilnius University Hospital Santaros Clinics for their considerable contribution to the treatment of the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Ethics statement: In our country, it is not mandatory to receive written consent from the local Ethics Committee for a case report, as it does not strictly meet the criteria of research because it does not require investigation and is intended to develop information to be shared for medical or educational purposes. Written informed consent was obtained from the patient for the publication of this report and the accompanying images.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iDs: Danuta Vasilevska https://orcid.org/0000-0002-3136-7281
Ugnius Mickys https://orcid.org/0000-0001-7805-0727
Andrzej Semczuk https://orcid.org/0000-0001-9344-9437 | 6 CYCLES , UNK | DrugDosageText | CC BY-NC | 33435776 | 18,956,668 | 2021-01 |
What was the dosage of drug 'PACLITAXEL'? | Failure of multiple surgical procedures and adjuvant chemotherapy in early-stage steroid-cell ovarian tumor treatment: a case report and literature review.
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary, accounting for less than 0.1% of all ovarian tumors. Not otherwise specified (NOS) tumors are the most common subtype. More than half of patients with SCTs-NOS show hyperandrogenic symptoms. The primary treatment for SCTs is surgery, as most cases are early-staged and benign. Because of the low incidence of metastatic disease, there is insufficient reliable information on the role of adjuvant therapy and the most effective treatment regimen. In this report, a rare case of a recurrent SCT-NOS in a 36-year-old female patient without endocrine symptoms is presented, highlighting the significance of appropriate pathological evaluation and immunohistochemical testing for the accurate diagnosis of this malignancy, particularly in the case of hormonally "silent" tumors. The metastatic tumor described here showed no response to four courses of adjuvant chemotherapy after several debulking surgeries. Based on the clinical findings, the neoplastic etiology should always be considered during the resection of ovarian tumors to prevent possible disease dissemination due to inappropriate surgical techniques.
Introduction
Ovarian steroid-cell tumors (SCTs) are a rare subgroup of sex-cord tumors of the ovary that account for less than 0.1% of all ovarian tumors. They are classified into three categories based on the cell origin: stromal luteoma, Leydig-cell tumor, and not otherwise specified (NOS). NOS tumors are the most common subtype and comprise the largest proportion of cases (60%), whereas stromal luteoma and Leydig-cell tumors each account for ∼20% of cases.1–3
A SCT of the ovary was first described in 1943 as a “virilizing lipoid cell tumor” by Gemma Barzilai in the Atlas of Ovarian Pathology.4 Later, the term “lipoid cell tumor” was replaced by “steroid-cell tumor.” This change was based on the argument that although all SCTs can produce steroid hormones, up to 25% of them contain little or no lipids.4 In 1979, the term “steroid-cell tumor, not otherwise specified,” was coined by Scully2 to indicate that the cell lineage from which the tumor arises is unknown. SCTs-NOS usually develop in reproductive-aged women with an average age of 43 years. The major symptoms are hirsutism and virilization caused by high levels of testosterone. The cornerstone of SCT-NOS treatment is surgery. However, as most tumors are diagnosed in the early clinical stage and do not recur or metastasize, there is limited information on their response to chemotherapy.1
In this report, a rare case of a recurrent SCT-NOS in a patient without endocrine symptoms is presented. The tumor showed no response to adjuvant chemotherapy after several debulking surgeries.
Case presentation
A 36-year-old Caucasian female patient, gravida 2 para 2, underwent laparoscopic right salpingo-oophorectomy in 2015 at a secondary-level hospital after the identification of a right ovarian tumor (the tumor was fragmented within the abdominal cavity, but it was unclear if it was within the endobag). The post-surgical pathological report revealed a Sertoli–Leydig-cell tumor. The patient was not referred to a clinical oncologist because radical removal was expected. Her past medical and surgical history was unremarkable, and she had no relevant family history. No signs of virilization or hirsutism were observed.
After 6 months, the patient was referred to the Gynecologic Department of our tertiary hospital because of metastases. During laparoscopy, multiple metastatic lesions were found in the pelvic peritoneum, on the diaphragm, and at the site of the former trocar; the left ovary was solid with suspected tumor spread. Metastatic lesions appeared as 3- to 4-mm subperitoneal fatty nodules, which were biopsied. Washings from the peritoneal cavity were negative. Unexpectedly, the histopathological diagnosis was a SCT-NOS. Laboratory analysis revealed normal values of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, progesterone, testosterone, dehydroepiandrosterone sulfate (DHEA-S), and cortisol. No hormonal assays were performed prior to surgery, as there were no clinical manifestations of any excessive hormone secretion. The ovarian tumor markers, including human epididymis protein 4, β-human chorionic gonadotropin, and α-fetoprotein, were within the normal range. Cancer antigen 125 (Ca125) was elevated to 48.8 kU/L (normal value up to 35 kU/L), but after 3 weeks, the level returned to normal (24.8 kU/L). Ten months after the first surgery, the patient underwent a total abdominal hysterectomy, left salpingo-oophorectomy, omentectomy, pelvic and diaphragmatic peritonectomy, and pelvic lymphadenectomy. Optimal cytoreductive surgery was performed. Microscopic findings exhibited diffuse tumor cells with abundant eosinophilic granular cytoplasm and vacuolization but no signs of nuclear atypia and a mitotic count of 5 per 10 high-power fields. No Reinke crystals, which are usually observed in Sertoli–Leydig-cell tumors, were identified. Immunohistochemistry revealed positive expression of inhibin A, calretinin, and synaptophysin but negative expression of pan-cytokeratin, CD68, epithelial membrane antigen, chromogranin A, and estrogen receptor/androgen receptor. The Ki-67 labeling index was up to 15% (Figure 1). The histopathologic features supported the diagnosis of a SCT-NOS.
Figure 1. Microscopic appearance of the SCT-NOS (H&E) in a 36-year-old Caucasian female patient (a) and positive staining of the tumor cells for inhibin A (b), calretinin (c), and Ki-67 (d) (20×)
H&E: hematoxylin and eosin.
After the surgery, the patient received six cycles of adjuvant carboplatin-paclitaxel chemotherapy. Ca125 was measured several times during the treatment period; its levels were within the normal range. At the end of the six courses of chemotherapy, a control computed tomography (CT) scan was performed, revealing the absence of disease progression.
After 3 months of follow-up, the CT scan showed disease progression in the left lateral abdominal flank; hence, we decided to perform secondary cytoreductive surgery. McBurney’s laparotomy was performed. During abdominal exploration, an infiltrative growing tumor was found at the site of the right former trocar that had grown in all layers of the abdomen and spread into the right flank. The laparotomy was extended in the cephalad direction. Metastases were also observed in the left superior diaphragmatic space and the previous left trocar area. Secondary optimal cytoreduction was performed to remove metastases from the right iliac region and abdominal lateral canals. The analysis of frozen sections demonstrated SCT metastases in fibrous adipose tissue. The patient experienced disease-free survival for only 6 months, after which the follow-up CT scan showed disease progression and a large incisional hernia at the incision site of the previous McBurney’s laparotomy. Serum tumor markers and hormone levels were within the normal range. During the subsequent laparotomy, micrometastases were observed on the aponeurosis with multiple small metastases on the small and large intestines, which could not be radically removed. The excision of injured subcutaneous tissue and incisional hernia repair with mesh were performed. The histological diagnosis was SCT metastasis to soft tissues. The patient was subjected to six cycles of second-line bleomycin, etoposide, and cisplatin (BEP) chemotherapy. On account of a dry cough, bleomycin has not been prescribed after the second cycle due to suspicion of pulmonary toxicity. The patient was put under surveillance every 3 months after the completion of chemotherapy. An abdominopelvic CT scan revealed the absence of disease progression, and no metastases were observed on the right side of the anterior abdominal wall or the bowel. Serum tumor markers and hormone levels were normal. After 1 year, disease progression was observed on the CT scan, which revealed increased foci in the anterior and lateral abdominal wall (>50%) and a small amount of ascites in the lateral flanks. Systemic treatment was recommended, and five cycles of liposomal doxorubicin were administered. Despite undergoing treatment, radiologic disease progression was observed after 4 months, as there was an ∼40% increase in tumor mass above the vaginal cuff on the left side with an increase in fluid volume in the pelvis. On the right abdominal wall, supra-umbilically, most small tumor foci had increased in size. The patient required re-operation. On the anterior abdominal wall, several necrotic metastases were found. In the abdominal cavity, there was a 10-cm ruptured tumor between the sigmoid colon and the vaginal cuff. Multiple 1- to 4-cm retroperitoneal metastases were found on the right internal iliac artery, right ureter, and pelvic veins. The right ureter was approximately 2 cm in diameter, with metastases completely fixed to the pelvic bones/fasciae. Few small metastases were noted on the peritoneum and mesentery. Because of the infiltration of the lateral pelvic walls, the tumors could not be radically resected; sigmoid colon resection was rendered ineffective because of the presence of residual tumor masses. Only bleeding tumors and metastases on the peritoneum and mesentery were non-radically removed. Histopathological verification revealed metastases of the SCT-NOS. At the pain management center, the patient was advised to start treatment with transdermal fentanyl patches for the treatment of intense abdominal pain. The patient was referred to a urologist because of right hydronephrosis, and a long-term ureteral stent was inserted. The multidisciplinary team decided to repeat the previous regimen of chemotherapy based on carboplatin and paclitaxel (six cycles). After completion of the chemotherapy, a CT scan was performed. The pelvic, vaginal cuff, and sigmoid colon masses were enlarged, indicating disease progression. Only palliative treatment was recommended. After 5 months, another CT scan was performed, and significant disease progression and noticeable bilateral hydronephrosis were detected, with the metastases enlarged by 80% (Figure 2). To manage the pain, the subcutaneous right lumbar mass was resected under regional anesthesia. The right ureteral stent was replaced, and a long-term stent was also inserted into the left ureter. Currently, the hormone levels and cancer biomarkers are within the normal reference ranges. Over the last 2 months, the patient was administered dendritic cell-based cancer vaccines; however, no effect has been reported. Because of the severe pain caused by the large-volume pelvic tumor, laparotomy was performed with palliative intention, revealing an approximately 15-cm metastasis on the rectosigmoid mesentery, a 3-cm metastasis on the sigmoid colon, and multiple 1- to 2-mm implants on the small intestines. All lesions were removed, and Hartmann’s operation was performed because of heavy bleeding from the mesorectum and rectosigmoid mesentery. The patient was discharged from the hospital on the fourth uneventful postoperative day and is currently receiving palliative care. The course of the disease is shown in Figure 3.
Figure 2. CT scan of the patient reported in this case showing 14.5 × 8.8-cm metastatic masses within the pelvis.
CT: computed tomography.
Figure 3. Treatment course of the patient described in this report.
BEP: bleomycin, etoposide, and cisplatin.
Discussion
Ovarian SCTs-NOS occur at any age (2.5–93 years), but they are usually observed in reproductive-aged women, with an average age of 43 years.5,6 More than half (56%–77%) of patients with an SCT-NOS show hyperandrogenic symptoms and signs of virilization, such as hirsutism, acne, deepened voice, clitoromegaly, amenorrhea, and infertility.1,7–9 Additionally, it can present with estrogenic manifestations (6%–23%), such as menorrhagia or postmenopausal bleeding. Some patients also develop endometrial cancer.10 Ovarian SCTs can secrete steroid hormones, such as progesterone, cortisol, and aldosterone, which may cause corresponding clinical symptoms. Hyperandrogenic tumors may also be associated with paraneoplastic manifestations, such as hypercalcemia, erythrocytosis, or ascites.11 However, about 25% of patients with SCTs may have atypical presentations, without any symptoms of virilization. In these cases, the diagnosis is usually made postoperatively after histopathological verification.12 In the patient reported in this case, no virilization symptoms were noted, and the tumor was hormonally “silent.” Additional symptoms in some patients may include abdominal distension due to ascites and palpable abdominal masses.13 After tumor dissemination, the patient reported here manifested severe abdominal pain with palpable abdominal masses.
Serum laboratory analyses typically show elevated levels of testosterone and androstenedione, indicating an ovarian origin of androgen release and normal DHEA-S levels, thereby excluding adrenal causes of hyperandrogenism.1 In the reported case, the hormone levels were within the reference range, even after considerable disease dissemination. There is no known specific tumor marker established for the preoperative diagnosis of SCTs-NOS. Tumor markers, such as CA-125 and α-fetoprotein, are generally within the normal range, and the data from the literature do not indicate whether elevated levels signify malignant potential. Some studies even claim that these markers could facilitate the differential diagnosis of ovarian adenocarcinoma.11,14 This correlates with the described case, as even after disease relapse, tumor markers remained within the normal range.
SCTs-NOS are unilateral in 94% of cases, large at diagnosis (range from 1.2 to 45 cm in the greatest dimension, with an average reported size of 8.4 cm), and typically solid and well-circumscribed. SCTs-NOS should be distinguished from other ovarian tumors and SCTs, in which the proliferation of steroid hormone-producing cells occurs as a secondary event. These include stromal luteomas, Leydig-cell tumors, luteinized thecomas, and pregnancy luteomas.15 SCTs-NOS are different from Leydig-cell tumors in terms of their deficiency in cytoplasmic Reinke crystals. In addition, Leydig-cell tumors are usually situated in hilar locations, and they are commonly associated with Leydig-cell hyperplasia.16,17 Stromal luteomas are confined to the ovarian stroma and frequently occur in association with stromal hyperthecosis.12 SCTs-NOS might have a fibromatous component, similar to that of thecomas, but this component accounts for only less than 10% of tumors.18 Pregnancy luteomas are more commonly multifocal (bilateral in one-third of cases), usually discovered at the time of cesarean section, and regress spontaneously after pregnancy.12
In addition to the microscopic features, immunohistochemistry is particularly helpful for proper diagnosis. The sensitivity of positive calretinin is 60% to 90%, whereas the sensitivity of inhibin reactivity ranges from 5% to 90%.3,19 SCTs-NOS are also commonly vimentin, Melan-A, and CD99 positive and variably AE1/3, CAM5.2, HMB45, and S100 positive.3 Currently, techniques are being developed to define SCTs-NOS pathologically by immunohistochemical staining of steroidogenic enzymes. In the future, validated enzymes could serve as markers for the differential diagnosis of hyperandrogenic ovarian conditions.20 It is worth noting that differential diagnosis is challenging, as the primary histopathological diagnosis of this case from the previous hospital indicated a Sertoli–Leydig-cell tumor. However, no Reinke crystals were found after careful evaluation of the metastases resected in our hospital. In this patient, the tumor showed strong and diffuse positivity for inhibin and calretinin.
Usually, these tumors are benign; however, 25% to 43% of SCTs are malignant, with 20% of cases found to exhibit metastases beyond the ovary. Metastatic lesions usually occur within the peritoneal cavity and rarely occur at distant sites.15 In one study, the clinical and pathological features of 63 SCTs-NOS were reviewed. Follow-up data ranging from 1 to 19 years (average 5.2 years) in duration were available for 50 patients. In 24 cases, the tumor was designated as probably benign (no evidence of spread beyond the ovary within 3 or more years postoperatively). In 18 patients, the tumor was malignant. Despite the various chemotherapy and radiation regimens, 12 patients experienced recurrences, and 14 patients died from the disease, suggesting a poor prognosis if the tumor is at an advanced stage, large, or recurring. Five pathological features are considered the best correlates of malignant behavior: the presence of two or more mitotic figures per 10 high-power fields (92% malignant), necrosis (86% malignant), a diameter of 7 cm or greater (78% malignant), hemorrhage (77% malignant), and grade 2 or 3 nuclear atypia (64% malignant).6 In our patient, we were unable to evaluate these features because the primary tumor was not resected at our hospital. We assume that the rapid spread was related to possible tumor fragmentation within the abdominal cavity, as we are uncertain of the use of an endobag.
Because the incidence of SCTs-NOS is low, there are currently no established treatment protocols. Therefore, the tumors are treated similarly to stromal tumors depending on several factors, including the surgical stage, histological type, patient’s age, and history of childbirth. The primary treatment for SCTs is surgery. Ovarian SCTs are generally considered benign because they are often detected at an early clinical stage. Therefore, unilateral salpingo-oophorectomy or tumor removal is acceptable in reproductive-aged women.1 However, regular follow-up with measurement of serum testosterone levels is mandatory. As there is limited information on the mechanisms of these tumors, the optimal length of follow-up is yet to be determined.15 In addition, there have already been reported cases of spontaneous pregnancies after tumor removal, probably because of the decline in testosterone levels.21,22
Surgical treatments using total abdominal hysterectomy and bilateral salpingo-oophorectomy are an appropriate management option for postmenopausal patients and those who have completed childbearing. Endometrial sampling should be performed when fertility-sparing surgery is planned because many of these patients may have coexisting endometrial hyperplasia or even uterine adenocarcinoma that might affect the decision for performing a hysterectomy.1
Because of the limited incidence of metastatic disease, there is a lack of reliable information on the role of adjuvant therapy in SCTs-NOS. The adjuvant chemotherapy regimens currently recommended for treatment are as follows: BEP; cisplatin, doxorubicin, and cyclophosphamide; taxane and platinum; and bleomycin, vinblastine, and cisplatin.23–27 It was reported that intraperitoneal dissemination and liver metastases were completely removed with debulking surgery, radiofrequency ablation of the liver metastasis, and adjuvant BEP (follow-up, 43 months) in a patient with a recurrent SCT-NOS occurring 5 years after the initial surgery.28 In another study, the treatment protocol consisted of docetaxel and nedaplatin, and the patient survived for 2 years with multiple bone metastases.29 Another SCT-NOS case with progressive disease after surgical debulking was reported. Treatment with multi-agent chemotherapy failed, but the patient subsequently showed a robust response to gonadotropin-releasing hormone agonist (GnRHa) therapy. Therefore, although typically treated with surgery alone, GnRHa may be required when abnormal serum hormone levels persist, and there is suspicion of residual tumors, recurrences, or metastases. It is suggested that GnRHa treatment be considered prior to cytotoxic chemotherapy in cases with SCTs-NOS.30 In the case described above, a chemotherapeutic regimen with BEP was administered for the recurrent SCT-NOS. After three cycles of BEP, the chemotherapeutic agent was changed to paclitaxel and carboplatin following a stable disease course. After eight cycles of paclitaxel and carboplatin, chemotherapy was discontinued because of prolonged neutropenia and peripheral neuropathy. The CT scan showed clinically stable disease. As the immunohistochemical analysis of the primary tumor revealed positive cytoplasmic staining for the GnRH receptor, GnRHa therapy was attempted based on reference to previous case reports and the lack of other effective alternatives. After six cycles of GnRHa therapy, the CT scan confirmed significant tumor size reduction. Adverse events were not observed, and the patient’s hirsutism and virilization had improved. Therefore, the treatment was discontinued after the administration of six cycles. However, the testosterone level was increased 2 months after GnRHa discontinuation, and the CT scan revealed an increase in tumor size. Subsequently, GnRHa was re-administered, resulting in immediate normalization of the serum testosterone level and shrinkage of the recurrent tumor. Thus, GnRHa therapy was continued (22 months after the first GnRHa treatment with the absence of symptoms).31
In our patient, the SCT-NOS was completely insensitive to chemotherapy, despite undergoing several debulking surgeries. Removal of the multi-metastases resulted in only short-term improvement. Unfortunately, the dendritic cell-based vaccine recently administered for 2 months did not yield a positive outcome.
Conclusions
Based on our clinical experience of this case, we highlight the importance of ovarian tumor resection without direct fragmentation within the abdominal cavity, as the neoplastic etiology should always be taken into account. Pathological evaluation is essential for the diagnosis of this malignancy, and immunohistochemical testing also aids in the formulation of an accurate diagnosis, especially in a patient with an apparent absence of endocrine manifestations. Surgery is the main treatment method for SCTs-NOS, as most cases are benign, and there are no generalized conclusions on the response of malignant cases to therapies, such as chemotherapy or radiation therapy. According to recent studies, GnRHa therapy could serve as a feasible treatment method and is worthy of further research with large patient groups.
Acknowledgment
We acknowledge oncologists from the Department of Oncology and Chemotherapy of Vilnius University Hospital Santaros Clinics for their considerable contribution to the treatment of the patient.
Declaration of conflicting interest: The authors declare that there is no conflict of interest.
Ethics statement: In our country, it is not mandatory to receive written consent from the local Ethics Committee for a case report, as it does not strictly meet the criteria of research because it does not require investigation and is intended to develop information to be shared for medical or educational purposes. Written informed consent was obtained from the patient for the publication of this report and the accompanying images.
Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
ORCID iDs: Danuta Vasilevska https://orcid.org/0000-0002-3136-7281
Ugnius Mickys https://orcid.org/0000-0001-7805-0727
Andrzej Semczuk https://orcid.org/0000-0001-9344-9437 | 6 CYCLES , UNK | DrugDosageText | CC BY-NC | 33435776 | 18,956,668 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective for unapproved indication'. | The clinical and immunological features of the post-extracorporeal shock wave lithotripsy anti-glomerular basement membrane disease.
BACKGROUND
Extracorporeal shock wave lithotripsy (ESWL) is a noninvasive modality to treat urolithiasis, with complications including tissue damage and hematoma of kidney parenchyma. Anti-glomerular basement membrane (GBM) disease is suggested to be a rare complication of ESWL since it was reported in several cases to occur after ESWL. However, the clinical and immunological features of the ESWL-associated anti-GBM disease have not been fully investigated so far.
METHODS
Here, we present the clinical, pathological, and immunological characteristics of three patients with the post-ESWL anti-GBM disease in our hospital. Anti-GBM disease occurred within a median of 22 months after ESWL treatment. It presented with similar clinical features to the classic anti-GBM disease, including fever, gross hematuria, and rapidly progressive glomerulonephritis (RPGN) with poor renal prognosis. Sera from all patients recognized the α3(IV)NC1 in GBM, but with IgG2 and IgG4 as the dominant IgG subclasses.
CONCLUSIONS
Although further exploration is required to prove the causal relationship in this rare condition, our study reminds physicians that patients developing acute renal insufficiency after ESWL should lead to the suspicion of anti-GBM disease and in-time diagnosis and treatment.
Introduction
Anti-glomerular basement membrane (GBM) disease or Goodpasture (GP) disease is a rare but fulminant and fatal disorder. It is widely accepted as a prototypical autoimmune disease induced by autoantibodies targeting the glomerular and alveolar basement membrane [1,2]. The well-defined autoantigen is localized to the non-collagen domain (NC1) of the α3 chain of type IV collagen [α3(IV)NC1] in GBM [3]. In addition, the autoantibodies have been proven to recognize the other 4 α chains (α1, 2, 4, and 5) within type IV collagen [4,5]. Two conformational epitopes, EA and EB, were mapped inside α3(IV)NC1 [6] and sequestered within the GBM in healthy individuals [7]. It is still unknown how the immune tolerance would be breached and epitopes exposed. One of the accepted mechanisms is that factors such as smoking [8], hydrocarbon exposure [9], infection [10], or preceding glomerular diseases [11–13] would damage the basement membrane and unmask the epitopes [14]. Rare cases have been reported about anti-GBM diseases associated with urinary obstruction [15–18] or nephrectomy [19].
Extracorporeal shock wave lithotripsy (ESWL) is a common and noninvasive modality to treat urolithiasis, disintegrating stones by shock waves. Although generally safe [20], it could cause short-term complications as renal colic or obstruction by stone fragments, tissue damage or hematoma, and even temporary reduction of glomerular filtration rate [21]. Renal histopathological findings of human and animal models shortly after ESWL included focal damage to vascular endothelium, nephron, renal tubules, and interstitia [20,22–24]. In 1990, Guerin et al. first reported a 67-year-old male with rapidly progressive renal disfunction 7 months after ESWL. His serum anti-GBM antibody was positive, while the serum before ESWL was not [25]. Since then, there have been several case reports about the post-ESWL anti-GBM disease, proposing anti-GBM disease as a rare complication of ESWL [26–29]. However, the causal relationship between the two has not been elucidated yet, nor the antigen spectrum or IgG subclasses of the autoantibodies in this rare entity.
In this study, we identified three patients with the post-ESWL anti-GBM disease after retrospectively reviewing 166 consecutive patients diagnosed with the anti-GBM disease in Peking University First Hospital from January 1, 2010 to January 31, 2020. We analyzed their clinical, pathological, and immunological features and reviewed five previously reported cases, aiming to draw a more comprehensive picture and bring insight into possible etiologies of this rare condition.
Case report
Case 1
A 36-year-old male was otherwise healthy except for undergoing one session of ESWL for kidney stones 24 months ago. Kidney function (represented as serum creatinine) was normal. Twenty-four months later, he presented with fever, cough, and hemoptysis for 5 days. He was anemic with hemoglobin 75 g/L and serum creatinine was 691 μmol/L (eGFR, 8 mL/min/1.73m2). Two days later, he developed gross hematuria and serum creatinine rose to 884 μmol/L (eGFR, 6 mL/min/1.73m2), at which time he was referred to our hospital. He was a long-term cigarette smoker and had gasoline exposure 15 days ago.
On admission, he was pale and febrile with no lymphadenopathy. Breath sounds were weak in both lungs on auscultation. Serum creatinine rapidly increased to 1279 μmol/L (eGFR, 4 mL/min/1.73m2) with oliguria and hemoglobin decreased to 49 g/L. Chest radiography suggested bilateral pulmonary hemorrhage. The circulating anti-GBM antibody was positive, with the titer >200 RU/mL (Euroimmune ELISA kit, normal range <20 RU/mL), while the serum anti-neutrophil cytoplasmic antibody (ANCA) was undetected (Table 1).
Table 1. Clinical, pathological, and immunological features of patients with post-ESWL anti-GBM disease in our hospital.
Patient 1 Patient 2 Patient 3
Sex/age, y M/36 M/50 F/74
Hydrocarbon exposure (Y/N) Y N N
Smoking (Y/N) Y Y N
Prodromal infection (Y/N) Y N Y
Fever (Y/N) Y Y Y
Pulmonary hemorrhage (Y/N) Y N N
Gross hematuria (Y/N) Y Y Y
Oliguria/Anuria (Y/N) Y N Y
Hemoglobin on diagnosis, g/L 49.0 102.0 63.0
Serum albumin, g/L 30.0 27.1 29.0
Urinary protein, g/24h 2.0 1.6 0.6
Nephrotic syndrome (Y/N) N N N
Serum creatinine on diagnosis, μmol/L 1279 364 1066
eGFR on diagnosis, mL/min/1.73m2 4.0 15.0 3.0
Anti-GBM antibodies on diagnosis, RU/mL >200 183 196
Positive ANCA (Y/N) N N N
Treatment PE/MP/Pred/CTX PE/MP/Pred MP/Pred/CTX
Anti-GBM antibody on discharge, RU/mL 33 51 102
Serum creatinine on discharge, μmol/L 425 353 618
eGFR on discharge, mL/min/1.73 m2 14.0 16.3 5.3
Dialysis-dependent on discharge (Y/N) N Y Y
Duration of follow-up, m 3 60 48
Dialysis-dependent at last follow-up (Y/N) N Y (PD) Y (HD)
Death at last follow-up (Y/N) (cause of death) N N Y (Esophagus cancer)
Stone location kidney ureter kidney
Total ESWL number 1 1 1
ESWL-to-onset interval, m 24 22 10
Anti-α1(IV)NC1 antibody (ref range, <0.04) 0.06 0.89 0.33
Anti-α2(IV)NC1 antibody (ref range, <0.05) 0.01 0.83 0.12
Anti-α3(IV)NC1 antibody (ref range, <0.06) 2.11 2.08 2.15
Anti-α4(IV)NC1 antibody (ref range, <0.58) 0.27 1.43 0.70
Anti-α5(IV)NC1 antibody (ref range, <0.02) 0.00 0.48 0.10
Anti-α3EA antibody (ref range, <0.07) 1.39 1.67 1.61
Anti-α3EB antibody (ref range, <0.15) 0.59 1.54 1.35
Anti-α3(IV)NC1 IgG1 (ref range, <0.02) 0.68 0.57 0.73
Anti-α3(IV)NC1 IgG2 (ref range, <0.07) 1.51 1.32 1.25
Anti-α3(IV)NC1 IgG3 (ref range, <0.01) 0.53 0.49 0.63
Anti-α3(IV)NC1 IgG4 (ref range, <0.01) 2.34 0.56 0.43
M: male; F: female; eGFR: estimated glomerular filtration rate; GBM: glomerular basement membrane; ANCA: anti-neutrophil cytoplasmic antibody; PE: plasma exchange; MP: methylprednisolone pulse; Pred: prednisone; CTX: cyclophosphamide; PD: peritoneal dialysis; HD: hemodialysis; ESWL: extracorporeal shock wave lithotripsy; IgG: immunoglobulin G; ref: reference; Y: yes; N: no.
Anti-GBM disease was diagnosed based on the renal-pulmonary involvement and positive serum anti-GBM antibody. The patient was prompted to plasma exchange, intravenous methylprednisolone pulse treatment, cyclophosphamide, and hemodialysis. After 17 sessions of plasma exchange, he became afebrile without hemoptysis. His serum anti-GBM antibody decreased to 33 RU/ml and serum creatine to 425 μmol/L (eGFR, 14 mL/min/1.73m2). Repeated chest radiography indicated the absorption of his hemorrhage. He was dialysis-independent on discharge and a 3-month follow-up.
Sera from this patient were tested for antigen spectrum using recombinant human α1–5(IV)NC1 chains and chimeric proteins containing epitopes EA and EB on α3(IV)NC1 (Supplementary materials and methods). The patient's serum recognized α1 and α3(IV)NC1 domains, and both epitopes EA and EB of α3(IV)NC1. The immunoglobulin G (IgG) subclass distribution against α3(IV)NC1 was also tested for the sera. All 4 subclasses of IgG were detected, with the dominance of IgG2 and IgG4 (Table 1; Figure S1).
Figure 1. Renal pathology of patient 3 with post-extracorporeal shock wave lithotripsy (ESWL) anti-GBM disease showed cellular crescent formation in a glomerulus by periodic acid-silver methenamine and Masson trichrome stain on light microscopy (400×).
Case 2
A 50-year-old male undertook one session of ESWL for ureter stones with normal kidney function 22 months ago. He was a 20-year cigarette smoker but had quit smoking for 10 years. Twenty-two months later he was referred to our hospital for fever, gross hematuria, and rapidly progressive renal dysfunction. He had suffered from intermittent fever for 2 months without other indicating symptoms and it could not be resolved by antibiotics. Fifteen days prior to admission, he developed gross hematuria and the serum creatinine progressed from 160 μmol/L (eGFR, 40 mL/min/1.73m2) to 504 μmol/L (eGFR, 11 mL/min/1.73m2). No stones were found by renal ultrasound.
After admission, he was diagnosed with anti-GBM disease due to positive serum anti-GBM antibody (183 RU/mL). His serum ANCA was tested negative. He received 7 sessions of plasma exchange, intravenous methylprednisolone pulse treatment, and hemodialysis. His condition improved and hematuria disappeared afterward. Serum creatinine decreased to 353 μmol/L (eGFR, 16 mL/min/1.73m2) and anti-GBM antibody titer to 51 RU/mL on discharge. The patient was dialysis-dependent during a 60-month follow-up (Table 1).
The patient's sera reacted with all five α chains and both EA and EB epitopes of α3(IV)NC1. All 4 IgG subclasses to α3(IV)NC1 were detected with the dominance of IgG2 (Table 1; Figure S1).
Case 3
A 74-year-old female received one session of ESWL for kidney stones 10 months ago when the renal function was normal. Ten months after ESWL, she complained of fever and gross hematuria with urinary irritation symptoms for 2 weeks. No leukocytosis was found, but her urinalysis revealed white and red blood cells. Initial workups showed elevated C-reactive protein (96 mg/L) and normal serum creatinine (87 μmol/L; eGFR, 55 mL/min/1.73m2). Renal ultrasound showed no stones. She was diagnosed with urinary tract infection and treated by intravenous antibiotics, after which she reported no symptom improvement but developed oliguria and bilateral edema of lower extremities. Her serum creatinine deteriorated to 1066 μmol/L (eGFR, 3 mL/min/1.73m2) within 9 days. Then she was referred to our hospital.
On admission, physical examination revealed a sick and pale woman with no rales on auscultation. Serum anti-GBM antibody was positive (196 RU/mL) while the serum ANCA was not. Renal biopsy revealed severe necrosis of glomerular capillary walls and cellular/cellular-fibrous crescents in all 32 glomeruli on light microscopy (Figure 1), with no electron-dense deposits on electron microscopy. The immunofluorescence microscopy was negative on a sclerotic glomerulus. She was treated by methylprednisolone pulse and cyclophosphamide. Plasma exchange was not performed, given the poor prognosis based on renal biopsy. Her renal function did not recover and she remained dialysis-dependent. The patient died of esophagus cancer 2 years after discharge.
The patient's sera recognized all five α chains and both EA and EB epitopes of α3(IV)NC1. As for circulating IgG to α3(IV)NC1, all four subclasses were detected with the dominance of IgG2 (Table 1; Figure S1).
Discussion
In this study, we described three patients with anti-GBM disease occurring within 2 years after ESWL. The incidence of such cases among all anti-GBM patients from our hospital in the past 10 years was 1.81% (3/166). Their clinical manifestations, pathological features, and antigen spectrum were similar to the classic anti-GBM disease. All three patients presented with fever, gross hematuria, and rapidly progressive glomerulonephritis (RPGN) with poor renal prognosis. One of them suffered from pulmonary hemorrhage. The one available renal biopsy revealed 100% crescent formation in the glomeruli. All three patients recognized a3(IV)NC1 and EA/EB epitopes. None were positive for anti-neutrophil cytoplasmic antibody (ANCA).
Post-ESWL anti-GBM disease has been rarely reported. After searching in PubMed using keywords ‘anti-GBM’, ‘Goodpasture’, ‘ESWL’, and ‘lithotripsy’, we found five previous cases of the post-ESWL anti-GBM disease with full text in English, reviewed in Table 2. Among the five patients, three of them were male and two were female, with a median age of 67 years old (range, 32–72 years). Human leukocyte antigen (HLA) phenotyping was performed in all five patients, with four of them expressing susceptible serotypes for anti-GBM disease [30]. All previous cases presented with shorter ESWL-to-onset intervals (1 week to 7 months) than ours, possibly due to more ESWL treatment numbers. Renal damage by ESWL appears to be cumulative in animal experiments and human, proportional to the application frequency and the number of treatments [31–33]. Their clinical manifestations, renal pathologies, and prognosis were similar to ours. Specifically, fever occurred in all previous reports and ours. However, only a few of them (3/8, 37.5%) were reported to bear prodromal infections and none in either group were positive for ANCA. Fever was a common feature in anti-GBM disease patients, as shown in our previous study in a 140-patient cohort, and 78.7% of febrile patients had infections [34]. The infection rate in post-ESWL anti-GBM disease patients seemed to be lower, which might indicate distinct disease triggers other than prodromal infections in these patients.
Table 2. Clinical and pathological data of previously reported post-ESWL anti-GBM disease cases.
1 2 3 4 5
Authors Cranfield et al. Sellin et al. Xenocostas et al. Iwamoto et al. Guerin et al.
Publication year 2015 2005 1999 1998 1990
Sex/age, y F/67 M/32 M/72 F/37 M/67
Hydrocarbon exposure (Y/N) NA NA NA NA NA
Smoking (Y/N) NA NA NA NA Y
Prodromal infection (Y/N) N N Y N N
Fever (Y/N) Y Y Y Y Y
Pulmonary hemorrhage(Y/N) Y N N N N
Gross hematuria (Y/N) NA Y N Y NA
Serum creatinine on diagnosis, μmol/L 1179 919 NA 1114 1074
Positive anti-GBM antibody(Y/N) Y Y Y Y Y
Positive ANCA (Y/N) N NA N N N
HLA phenotype DR4, DQ6 DRB1*11 & 13 DR15 DR2 DR2
Treatment PE/MP/Pred/CTX Pred/CTX NA PE/MP/Pred/CTX NA
Dialysis-dependent (Y/N) Y Y Y Y Y
Renal biopsy (Y/N) NA Y Y Y Y
Immunofluorescence NA Linear IgG deposits along GBM Linear IgG deposits along GBM Linear IgG and C3 deposits along GBM Linear IgG and C3 deposits along GBM
Light microscopy NA Crescent formation in 23/25 glomeruli Crescent formation in nearly all glomeruli Cellular crescents in all glomeruli Cellular crescents in all glomeruli
Electron-dense deposits on electron microscopy NA NA N N NA
Stone location NA Infundibulum Left renal pelvis Right kidney Left kidney
Stone component NA Calcium oxalate NA NA NA
Total ESWL number 2 within 4 weeks 3 within 4 months 1 NA 2 within 10 days
ESWL-to-onset interval 1 week 5 months 3 months 3 months 7 months
Shock number 3200 NA 4000 1000 NA
Energy data 75 kPa NA NA 17 kV NA
Renal function at last ESWL NA NA Normal Normal Normal
Anti-GBM antibody before ESWL NA NA NA Negative Negative
F: female; M: male; GBM: glomerular basement membrane; ANCA: anti-neutrophil cytoplasmic antibody; HLA: human leukocyte antigen; PE: plasma exchange; MP: methylprednisolone pulse; Pred: prednisone; CTX: cyclophosphamide; IgG: immunoglobulin G; C3: complement 3; ESWL: extracorporeal shock wave lithotripsy; ref: reference; Y: yes; N: no; NA: not available.
Further studies are still needed to elucidate the effect of ESWL on the initiation of anti-GBM disease. However, there has been evidence about the collagen cleaving effect of ultrasound since early 1980s when researchers tried to isolate and define the components of GBM by physical methods. Glomeruli were sequestered and sonicated to separate the GBM, which then released split products of collagen with antigenicity [35], indicating ultrasound could cause local damage of the GBM. There was also evidence of transient nephrotic-range proteinuria immediately after ESWL in a patient cohort [36] and mesangial proliferative glomerulopathy after ESWL in experimental animal models of pig [37]. Moreover, shock wave lithotripsy is utilized in pancreatic and large common bile duct stones or sialolithiasis, but no cases of anti-GBM disease have been reported in either of these conditions yet, suggesting ESWL for urinary stones may affect kidneys more directly. Therefore, ESWL could possibly damage and expose the antigen inside the GBM via direct damage by shock waves or secondary damage by immune-complex induced by ESWL debris [37].
Anti-GBM IgG subclass distribution is associated with disease severity [38]. The IgG1 and IgG3 dominated the IgG subclasses in patients with severe renal impairment, while IgG2 and IgG4 were associated with milder renal damage [38]. In post-ESWL anti-GBM disease patients from this study, the IgG2 and IgG4 were the dominant subclasses against α3(IV)NC1. However, these patients presented with severe renal damage and poor prognosis. The IgG subclass switching after B cell activation follows the sequence of IgG3→IgG1→IgG2→IgG4. It has been suggested that IgG4 production results from chronic or repetitive antigenic stimulation [39,40]. Moreover, low-level natural anti-GBM autoantibodies existed in healthy human sera, predominantly of IgG2 and IgG4 [41]. We speculated that ESWL might expose GBM autoantigens and induce IgG autoantibodies chronically during the ESWL-to-onset interval, allowing them to complete subclass switching. The autoantibodies may accumulate beyond a threshold to disturb the immune tolerance in healthy individuals and provoke the pathogenic autoimmunity.
Anti-GBM diseases were also reported scarcely to associate with obstructive uropathy due to urinary malignancy, neurogenic bladder, or ureteral stenosis [15–18]. The anti-GBM antibody level and renal function seemed to be parallel with treatment efficacy of hydronephrosis [18]. Intact NC1 hexamer could be detected in the serum and be secreted in the urine of healthy individuals [17,42]. Rabbits immunized with their own urinary concentrate developed anti-GBM glomerulonephritis [43]. It was suggested that urinary α3(IV)NC1 under urinary obstruction might enter the renal interstitia, dissociate under acidic pH changed by inflammatory infiltrate (such as infection) and act as an immunogen [17]. These studies provide another theory for the induction of anti-GBM disease associated with urinary stones, including patients treated by ESWL.
The limitations of our study lay in at least two points. Firstly, the number of cases is too small to draw the causal relationship between ESWL and anti-GBM disease. Moreover, we cannot rule out the possibility that individuals with anti-GBM disease or with susceptible HLA phenotypes may bear a higher risk of urinary obstructions, which lead to the production of anti-GBM antibodies. A prospective cohort study in patients undergoing ESWL might be required to further elucidate the relationship between the procedure and anti-GBM disease. Secondly, not all patients had detailed ESWL information (stone component, shock wave frequency, and energy), kidney biopsy, and HLA phenotypes for us to draw a full picture.
In summary, the anti-GBM disease could happen within weeks to months after ESWL treatment and present with similar clinical features, antigen spectrum, and prognosis to classic anti-GBM disease. IgG2 and IgG4 were the two dominant subclasses against α3(IV)NC1. Although the causal relationship between ESWL and anti-GBM disease still needs further exploration, our study here may act as a reminder for physicians that patients developing acute renal insufficiency after ESWL should lead to the suspicion of anti-GBM disease and in-time diagnosis and treatment.
Supplementary Material
Supplemental Material
Click here for additional data file.
Geolocation information
City: Beijing, Latitude: 39.9289, Longitude: 116.3883.
Ethical policy and institutional review board statement
Written informed consents were obtained from the patients for publication of the three cases and any accompanying images. The experiments in this study complied with the Declaration of Helsinki and were approved by the ethics committee of Peking University First Hospital.
Disclosure statement
No potential conflict of interest was reported by the author(s). | CYCLOPHOSPHAMIDE, METHYLPREDNISOLONE, PREDNISONE | DrugsGivenReaction | CC BY | 33435789 | 20,046,560 | 2021-12 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'. | The clinical and immunological features of the post-extracorporeal shock wave lithotripsy anti-glomerular basement membrane disease.
BACKGROUND
Extracorporeal shock wave lithotripsy (ESWL) is a noninvasive modality to treat urolithiasis, with complications including tissue damage and hematoma of kidney parenchyma. Anti-glomerular basement membrane (GBM) disease is suggested to be a rare complication of ESWL since it was reported in several cases to occur after ESWL. However, the clinical and immunological features of the ESWL-associated anti-GBM disease have not been fully investigated so far.
METHODS
Here, we present the clinical, pathological, and immunological characteristics of three patients with the post-ESWL anti-GBM disease in our hospital. Anti-GBM disease occurred within a median of 22 months after ESWL treatment. It presented with similar clinical features to the classic anti-GBM disease, including fever, gross hematuria, and rapidly progressive glomerulonephritis (RPGN) with poor renal prognosis. Sera from all patients recognized the α3(IV)NC1 in GBM, but with IgG2 and IgG4 as the dominant IgG subclasses.
CONCLUSIONS
Although further exploration is required to prove the causal relationship in this rare condition, our study reminds physicians that patients developing acute renal insufficiency after ESWL should lead to the suspicion of anti-GBM disease and in-time diagnosis and treatment.
Introduction
Anti-glomerular basement membrane (GBM) disease or Goodpasture (GP) disease is a rare but fulminant and fatal disorder. It is widely accepted as a prototypical autoimmune disease induced by autoantibodies targeting the glomerular and alveolar basement membrane [1,2]. The well-defined autoantigen is localized to the non-collagen domain (NC1) of the α3 chain of type IV collagen [α3(IV)NC1] in GBM [3]. In addition, the autoantibodies have been proven to recognize the other 4 α chains (α1, 2, 4, and 5) within type IV collagen [4,5]. Two conformational epitopes, EA and EB, were mapped inside α3(IV)NC1 [6] and sequestered within the GBM in healthy individuals [7]. It is still unknown how the immune tolerance would be breached and epitopes exposed. One of the accepted mechanisms is that factors such as smoking [8], hydrocarbon exposure [9], infection [10], or preceding glomerular diseases [11–13] would damage the basement membrane and unmask the epitopes [14]. Rare cases have been reported about anti-GBM diseases associated with urinary obstruction [15–18] or nephrectomy [19].
Extracorporeal shock wave lithotripsy (ESWL) is a common and noninvasive modality to treat urolithiasis, disintegrating stones by shock waves. Although generally safe [20], it could cause short-term complications as renal colic or obstruction by stone fragments, tissue damage or hematoma, and even temporary reduction of glomerular filtration rate [21]. Renal histopathological findings of human and animal models shortly after ESWL included focal damage to vascular endothelium, nephron, renal tubules, and interstitia [20,22–24]. In 1990, Guerin et al. first reported a 67-year-old male with rapidly progressive renal disfunction 7 months after ESWL. His serum anti-GBM antibody was positive, while the serum before ESWL was not [25]. Since then, there have been several case reports about the post-ESWL anti-GBM disease, proposing anti-GBM disease as a rare complication of ESWL [26–29]. However, the causal relationship between the two has not been elucidated yet, nor the antigen spectrum or IgG subclasses of the autoantibodies in this rare entity.
In this study, we identified three patients with the post-ESWL anti-GBM disease after retrospectively reviewing 166 consecutive patients diagnosed with the anti-GBM disease in Peking University First Hospital from January 1, 2010 to January 31, 2020. We analyzed their clinical, pathological, and immunological features and reviewed five previously reported cases, aiming to draw a more comprehensive picture and bring insight into possible etiologies of this rare condition.
Case report
Case 1
A 36-year-old male was otherwise healthy except for undergoing one session of ESWL for kidney stones 24 months ago. Kidney function (represented as serum creatinine) was normal. Twenty-four months later, he presented with fever, cough, and hemoptysis for 5 days. He was anemic with hemoglobin 75 g/L and serum creatinine was 691 μmol/L (eGFR, 8 mL/min/1.73m2). Two days later, he developed gross hematuria and serum creatinine rose to 884 μmol/L (eGFR, 6 mL/min/1.73m2), at which time he was referred to our hospital. He was a long-term cigarette smoker and had gasoline exposure 15 days ago.
On admission, he was pale and febrile with no lymphadenopathy. Breath sounds were weak in both lungs on auscultation. Serum creatinine rapidly increased to 1279 μmol/L (eGFR, 4 mL/min/1.73m2) with oliguria and hemoglobin decreased to 49 g/L. Chest radiography suggested bilateral pulmonary hemorrhage. The circulating anti-GBM antibody was positive, with the titer >200 RU/mL (Euroimmune ELISA kit, normal range <20 RU/mL), while the serum anti-neutrophil cytoplasmic antibody (ANCA) was undetected (Table 1).
Table 1. Clinical, pathological, and immunological features of patients with post-ESWL anti-GBM disease in our hospital.
Patient 1 Patient 2 Patient 3
Sex/age, y M/36 M/50 F/74
Hydrocarbon exposure (Y/N) Y N N
Smoking (Y/N) Y Y N
Prodromal infection (Y/N) Y N Y
Fever (Y/N) Y Y Y
Pulmonary hemorrhage (Y/N) Y N N
Gross hematuria (Y/N) Y Y Y
Oliguria/Anuria (Y/N) Y N Y
Hemoglobin on diagnosis, g/L 49.0 102.0 63.0
Serum albumin, g/L 30.0 27.1 29.0
Urinary protein, g/24h 2.0 1.6 0.6
Nephrotic syndrome (Y/N) N N N
Serum creatinine on diagnosis, μmol/L 1279 364 1066
eGFR on diagnosis, mL/min/1.73m2 4.0 15.0 3.0
Anti-GBM antibodies on diagnosis, RU/mL >200 183 196
Positive ANCA (Y/N) N N N
Treatment PE/MP/Pred/CTX PE/MP/Pred MP/Pred/CTX
Anti-GBM antibody on discharge, RU/mL 33 51 102
Serum creatinine on discharge, μmol/L 425 353 618
eGFR on discharge, mL/min/1.73 m2 14.0 16.3 5.3
Dialysis-dependent on discharge (Y/N) N Y Y
Duration of follow-up, m 3 60 48
Dialysis-dependent at last follow-up (Y/N) N Y (PD) Y (HD)
Death at last follow-up (Y/N) (cause of death) N N Y (Esophagus cancer)
Stone location kidney ureter kidney
Total ESWL number 1 1 1
ESWL-to-onset interval, m 24 22 10
Anti-α1(IV)NC1 antibody (ref range, <0.04) 0.06 0.89 0.33
Anti-α2(IV)NC1 antibody (ref range, <0.05) 0.01 0.83 0.12
Anti-α3(IV)NC1 antibody (ref range, <0.06) 2.11 2.08 2.15
Anti-α4(IV)NC1 antibody (ref range, <0.58) 0.27 1.43 0.70
Anti-α5(IV)NC1 antibody (ref range, <0.02) 0.00 0.48 0.10
Anti-α3EA antibody (ref range, <0.07) 1.39 1.67 1.61
Anti-α3EB antibody (ref range, <0.15) 0.59 1.54 1.35
Anti-α3(IV)NC1 IgG1 (ref range, <0.02) 0.68 0.57 0.73
Anti-α3(IV)NC1 IgG2 (ref range, <0.07) 1.51 1.32 1.25
Anti-α3(IV)NC1 IgG3 (ref range, <0.01) 0.53 0.49 0.63
Anti-α3(IV)NC1 IgG4 (ref range, <0.01) 2.34 0.56 0.43
M: male; F: female; eGFR: estimated glomerular filtration rate; GBM: glomerular basement membrane; ANCA: anti-neutrophil cytoplasmic antibody; PE: plasma exchange; MP: methylprednisolone pulse; Pred: prednisone; CTX: cyclophosphamide; PD: peritoneal dialysis; HD: hemodialysis; ESWL: extracorporeal shock wave lithotripsy; IgG: immunoglobulin G; ref: reference; Y: yes; N: no.
Anti-GBM disease was diagnosed based on the renal-pulmonary involvement and positive serum anti-GBM antibody. The patient was prompted to plasma exchange, intravenous methylprednisolone pulse treatment, cyclophosphamide, and hemodialysis. After 17 sessions of plasma exchange, he became afebrile without hemoptysis. His serum anti-GBM antibody decreased to 33 RU/ml and serum creatine to 425 μmol/L (eGFR, 14 mL/min/1.73m2). Repeated chest radiography indicated the absorption of his hemorrhage. He was dialysis-independent on discharge and a 3-month follow-up.
Sera from this patient were tested for antigen spectrum using recombinant human α1–5(IV)NC1 chains and chimeric proteins containing epitopes EA and EB on α3(IV)NC1 (Supplementary materials and methods). The patient's serum recognized α1 and α3(IV)NC1 domains, and both epitopes EA and EB of α3(IV)NC1. The immunoglobulin G (IgG) subclass distribution against α3(IV)NC1 was also tested for the sera. All 4 subclasses of IgG were detected, with the dominance of IgG2 and IgG4 (Table 1; Figure S1).
Figure 1. Renal pathology of patient 3 with post-extracorporeal shock wave lithotripsy (ESWL) anti-GBM disease showed cellular crescent formation in a glomerulus by periodic acid-silver methenamine and Masson trichrome stain on light microscopy (400×).
Case 2
A 50-year-old male undertook one session of ESWL for ureter stones with normal kidney function 22 months ago. He was a 20-year cigarette smoker but had quit smoking for 10 years. Twenty-two months later he was referred to our hospital for fever, gross hematuria, and rapidly progressive renal dysfunction. He had suffered from intermittent fever for 2 months without other indicating symptoms and it could not be resolved by antibiotics. Fifteen days prior to admission, he developed gross hematuria and the serum creatinine progressed from 160 μmol/L (eGFR, 40 mL/min/1.73m2) to 504 μmol/L (eGFR, 11 mL/min/1.73m2). No stones were found by renal ultrasound.
After admission, he was diagnosed with anti-GBM disease due to positive serum anti-GBM antibody (183 RU/mL). His serum ANCA was tested negative. He received 7 sessions of plasma exchange, intravenous methylprednisolone pulse treatment, and hemodialysis. His condition improved and hematuria disappeared afterward. Serum creatinine decreased to 353 μmol/L (eGFR, 16 mL/min/1.73m2) and anti-GBM antibody titer to 51 RU/mL on discharge. The patient was dialysis-dependent during a 60-month follow-up (Table 1).
The patient's sera reacted with all five α chains and both EA and EB epitopes of α3(IV)NC1. All 4 IgG subclasses to α3(IV)NC1 were detected with the dominance of IgG2 (Table 1; Figure S1).
Case 3
A 74-year-old female received one session of ESWL for kidney stones 10 months ago when the renal function was normal. Ten months after ESWL, she complained of fever and gross hematuria with urinary irritation symptoms for 2 weeks. No leukocytosis was found, but her urinalysis revealed white and red blood cells. Initial workups showed elevated C-reactive protein (96 mg/L) and normal serum creatinine (87 μmol/L; eGFR, 55 mL/min/1.73m2). Renal ultrasound showed no stones. She was diagnosed with urinary tract infection and treated by intravenous antibiotics, after which she reported no symptom improvement but developed oliguria and bilateral edema of lower extremities. Her serum creatinine deteriorated to 1066 μmol/L (eGFR, 3 mL/min/1.73m2) within 9 days. Then she was referred to our hospital.
On admission, physical examination revealed a sick and pale woman with no rales on auscultation. Serum anti-GBM antibody was positive (196 RU/mL) while the serum ANCA was not. Renal biopsy revealed severe necrosis of glomerular capillary walls and cellular/cellular-fibrous crescents in all 32 glomeruli on light microscopy (Figure 1), with no electron-dense deposits on electron microscopy. The immunofluorescence microscopy was negative on a sclerotic glomerulus. She was treated by methylprednisolone pulse and cyclophosphamide. Plasma exchange was not performed, given the poor prognosis based on renal biopsy. Her renal function did not recover and she remained dialysis-dependent. The patient died of esophagus cancer 2 years after discharge.
The patient's sera recognized all five α chains and both EA and EB epitopes of α3(IV)NC1. As for circulating IgG to α3(IV)NC1, all four subclasses were detected with the dominance of IgG2 (Table 1; Figure S1).
Discussion
In this study, we described three patients with anti-GBM disease occurring within 2 years after ESWL. The incidence of such cases among all anti-GBM patients from our hospital in the past 10 years was 1.81% (3/166). Their clinical manifestations, pathological features, and antigen spectrum were similar to the classic anti-GBM disease. All three patients presented with fever, gross hematuria, and rapidly progressive glomerulonephritis (RPGN) with poor renal prognosis. One of them suffered from pulmonary hemorrhage. The one available renal biopsy revealed 100% crescent formation in the glomeruli. All three patients recognized a3(IV)NC1 and EA/EB epitopes. None were positive for anti-neutrophil cytoplasmic antibody (ANCA).
Post-ESWL anti-GBM disease has been rarely reported. After searching in PubMed using keywords ‘anti-GBM’, ‘Goodpasture’, ‘ESWL’, and ‘lithotripsy’, we found five previous cases of the post-ESWL anti-GBM disease with full text in English, reviewed in Table 2. Among the five patients, three of them were male and two were female, with a median age of 67 years old (range, 32–72 years). Human leukocyte antigen (HLA) phenotyping was performed in all five patients, with four of them expressing susceptible serotypes for anti-GBM disease [30]. All previous cases presented with shorter ESWL-to-onset intervals (1 week to 7 months) than ours, possibly due to more ESWL treatment numbers. Renal damage by ESWL appears to be cumulative in animal experiments and human, proportional to the application frequency and the number of treatments [31–33]. Their clinical manifestations, renal pathologies, and prognosis were similar to ours. Specifically, fever occurred in all previous reports and ours. However, only a few of them (3/8, 37.5%) were reported to bear prodromal infections and none in either group were positive for ANCA. Fever was a common feature in anti-GBM disease patients, as shown in our previous study in a 140-patient cohort, and 78.7% of febrile patients had infections [34]. The infection rate in post-ESWL anti-GBM disease patients seemed to be lower, which might indicate distinct disease triggers other than prodromal infections in these patients.
Table 2. Clinical and pathological data of previously reported post-ESWL anti-GBM disease cases.
1 2 3 4 5
Authors Cranfield et al. Sellin et al. Xenocostas et al. Iwamoto et al. Guerin et al.
Publication year 2015 2005 1999 1998 1990
Sex/age, y F/67 M/32 M/72 F/37 M/67
Hydrocarbon exposure (Y/N) NA NA NA NA NA
Smoking (Y/N) NA NA NA NA Y
Prodromal infection (Y/N) N N Y N N
Fever (Y/N) Y Y Y Y Y
Pulmonary hemorrhage(Y/N) Y N N N N
Gross hematuria (Y/N) NA Y N Y NA
Serum creatinine on diagnosis, μmol/L 1179 919 NA 1114 1074
Positive anti-GBM antibody(Y/N) Y Y Y Y Y
Positive ANCA (Y/N) N NA N N N
HLA phenotype DR4, DQ6 DRB1*11 & 13 DR15 DR2 DR2
Treatment PE/MP/Pred/CTX Pred/CTX NA PE/MP/Pred/CTX NA
Dialysis-dependent (Y/N) Y Y Y Y Y
Renal biopsy (Y/N) NA Y Y Y Y
Immunofluorescence NA Linear IgG deposits along GBM Linear IgG deposits along GBM Linear IgG and C3 deposits along GBM Linear IgG and C3 deposits along GBM
Light microscopy NA Crescent formation in 23/25 glomeruli Crescent formation in nearly all glomeruli Cellular crescents in all glomeruli Cellular crescents in all glomeruli
Electron-dense deposits on electron microscopy NA NA N N NA
Stone location NA Infundibulum Left renal pelvis Right kidney Left kidney
Stone component NA Calcium oxalate NA NA NA
Total ESWL number 2 within 4 weeks 3 within 4 months 1 NA 2 within 10 days
ESWL-to-onset interval 1 week 5 months 3 months 3 months 7 months
Shock number 3200 NA 4000 1000 NA
Energy data 75 kPa NA NA 17 kV NA
Renal function at last ESWL NA NA Normal Normal Normal
Anti-GBM antibody before ESWL NA NA NA Negative Negative
F: female; M: male; GBM: glomerular basement membrane; ANCA: anti-neutrophil cytoplasmic antibody; HLA: human leukocyte antigen; PE: plasma exchange; MP: methylprednisolone pulse; Pred: prednisone; CTX: cyclophosphamide; IgG: immunoglobulin G; C3: complement 3; ESWL: extracorporeal shock wave lithotripsy; ref: reference; Y: yes; N: no; NA: not available.
Further studies are still needed to elucidate the effect of ESWL on the initiation of anti-GBM disease. However, there has been evidence about the collagen cleaving effect of ultrasound since early 1980s when researchers tried to isolate and define the components of GBM by physical methods. Glomeruli were sequestered and sonicated to separate the GBM, which then released split products of collagen with antigenicity [35], indicating ultrasound could cause local damage of the GBM. There was also evidence of transient nephrotic-range proteinuria immediately after ESWL in a patient cohort [36] and mesangial proliferative glomerulopathy after ESWL in experimental animal models of pig [37]. Moreover, shock wave lithotripsy is utilized in pancreatic and large common bile duct stones or sialolithiasis, but no cases of anti-GBM disease have been reported in either of these conditions yet, suggesting ESWL for urinary stones may affect kidneys more directly. Therefore, ESWL could possibly damage and expose the antigen inside the GBM via direct damage by shock waves or secondary damage by immune-complex induced by ESWL debris [37].
Anti-GBM IgG subclass distribution is associated with disease severity [38]. The IgG1 and IgG3 dominated the IgG subclasses in patients with severe renal impairment, while IgG2 and IgG4 were associated with milder renal damage [38]. In post-ESWL anti-GBM disease patients from this study, the IgG2 and IgG4 were the dominant subclasses against α3(IV)NC1. However, these patients presented with severe renal damage and poor prognosis. The IgG subclass switching after B cell activation follows the sequence of IgG3→IgG1→IgG2→IgG4. It has been suggested that IgG4 production results from chronic or repetitive antigenic stimulation [39,40]. Moreover, low-level natural anti-GBM autoantibodies existed in healthy human sera, predominantly of IgG2 and IgG4 [41]. We speculated that ESWL might expose GBM autoantigens and induce IgG autoantibodies chronically during the ESWL-to-onset interval, allowing them to complete subclass switching. The autoantibodies may accumulate beyond a threshold to disturb the immune tolerance in healthy individuals and provoke the pathogenic autoimmunity.
Anti-GBM diseases were also reported scarcely to associate with obstructive uropathy due to urinary malignancy, neurogenic bladder, or ureteral stenosis [15–18]. The anti-GBM antibody level and renal function seemed to be parallel with treatment efficacy of hydronephrosis [18]. Intact NC1 hexamer could be detected in the serum and be secreted in the urine of healthy individuals [17,42]. Rabbits immunized with their own urinary concentrate developed anti-GBM glomerulonephritis [43]. It was suggested that urinary α3(IV)NC1 under urinary obstruction might enter the renal interstitia, dissociate under acidic pH changed by inflammatory infiltrate (such as infection) and act as an immunogen [17]. These studies provide another theory for the induction of anti-GBM disease associated with urinary stones, including patients treated by ESWL.
The limitations of our study lay in at least two points. Firstly, the number of cases is too small to draw the causal relationship between ESWL and anti-GBM disease. Moreover, we cannot rule out the possibility that individuals with anti-GBM disease or with susceptible HLA phenotypes may bear a higher risk of urinary obstructions, which lead to the production of anti-GBM antibodies. A prospective cohort study in patients undergoing ESWL might be required to further elucidate the relationship between the procedure and anti-GBM disease. Secondly, not all patients had detailed ESWL information (stone component, shock wave frequency, and energy), kidney biopsy, and HLA phenotypes for us to draw a full picture.
In summary, the anti-GBM disease could happen within weeks to months after ESWL treatment and present with similar clinical features, antigen spectrum, and prognosis to classic anti-GBM disease. IgG2 and IgG4 were the two dominant subclasses against α3(IV)NC1. Although the causal relationship between ESWL and anti-GBM disease still needs further exploration, our study here may act as a reminder for physicians that patients developing acute renal insufficiency after ESWL should lead to the suspicion of anti-GBM disease and in-time diagnosis and treatment.
Supplementary Material
Supplemental Material
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Geolocation information
City: Beijing, Latitude: 39.9289, Longitude: 116.3883.
Ethical policy and institutional review board statement
Written informed consents were obtained from the patients for publication of the three cases and any accompanying images. The experiments in this study complied with the Declaration of Helsinki and were approved by the ethics committee of Peking University First Hospital.
Disclosure statement
No potential conflict of interest was reported by the author(s). | CYCLOPHOSPHAMIDE, METHYLPREDNISOLONE, PREDNISONE | DrugsGivenReaction | CC BY | 33435789 | 20,046,560 | 2021-12 |
What was the dosage of drug 'METHYLPREDNISOLONE'? | The clinical and immunological features of the post-extracorporeal shock wave lithotripsy anti-glomerular basement membrane disease.
BACKGROUND
Extracorporeal shock wave lithotripsy (ESWL) is a noninvasive modality to treat urolithiasis, with complications including tissue damage and hematoma of kidney parenchyma. Anti-glomerular basement membrane (GBM) disease is suggested to be a rare complication of ESWL since it was reported in several cases to occur after ESWL. However, the clinical and immunological features of the ESWL-associated anti-GBM disease have not been fully investigated so far.
METHODS
Here, we present the clinical, pathological, and immunological characteristics of three patients with the post-ESWL anti-GBM disease in our hospital. Anti-GBM disease occurred within a median of 22 months after ESWL treatment. It presented with similar clinical features to the classic anti-GBM disease, including fever, gross hematuria, and rapidly progressive glomerulonephritis (RPGN) with poor renal prognosis. Sera from all patients recognized the α3(IV)NC1 in GBM, but with IgG2 and IgG4 as the dominant IgG subclasses.
CONCLUSIONS
Although further exploration is required to prove the causal relationship in this rare condition, our study reminds physicians that patients developing acute renal insufficiency after ESWL should lead to the suspicion of anti-GBM disease and in-time diagnosis and treatment.
Introduction
Anti-glomerular basement membrane (GBM) disease or Goodpasture (GP) disease is a rare but fulminant and fatal disorder. It is widely accepted as a prototypical autoimmune disease induced by autoantibodies targeting the glomerular and alveolar basement membrane [1,2]. The well-defined autoantigen is localized to the non-collagen domain (NC1) of the α3 chain of type IV collagen [α3(IV)NC1] in GBM [3]. In addition, the autoantibodies have been proven to recognize the other 4 α chains (α1, 2, 4, and 5) within type IV collagen [4,5]. Two conformational epitopes, EA and EB, were mapped inside α3(IV)NC1 [6] and sequestered within the GBM in healthy individuals [7]. It is still unknown how the immune tolerance would be breached and epitopes exposed. One of the accepted mechanisms is that factors such as smoking [8], hydrocarbon exposure [9], infection [10], or preceding glomerular diseases [11–13] would damage the basement membrane and unmask the epitopes [14]. Rare cases have been reported about anti-GBM diseases associated with urinary obstruction [15–18] or nephrectomy [19].
Extracorporeal shock wave lithotripsy (ESWL) is a common and noninvasive modality to treat urolithiasis, disintegrating stones by shock waves. Although generally safe [20], it could cause short-term complications as renal colic or obstruction by stone fragments, tissue damage or hematoma, and even temporary reduction of glomerular filtration rate [21]. Renal histopathological findings of human and animal models shortly after ESWL included focal damage to vascular endothelium, nephron, renal tubules, and interstitia [20,22–24]. In 1990, Guerin et al. first reported a 67-year-old male with rapidly progressive renal disfunction 7 months after ESWL. His serum anti-GBM antibody was positive, while the serum before ESWL was not [25]. Since then, there have been several case reports about the post-ESWL anti-GBM disease, proposing anti-GBM disease as a rare complication of ESWL [26–29]. However, the causal relationship between the two has not been elucidated yet, nor the antigen spectrum or IgG subclasses of the autoantibodies in this rare entity.
In this study, we identified three patients with the post-ESWL anti-GBM disease after retrospectively reviewing 166 consecutive patients diagnosed with the anti-GBM disease in Peking University First Hospital from January 1, 2010 to January 31, 2020. We analyzed their clinical, pathological, and immunological features and reviewed five previously reported cases, aiming to draw a more comprehensive picture and bring insight into possible etiologies of this rare condition.
Case report
Case 1
A 36-year-old male was otherwise healthy except for undergoing one session of ESWL for kidney stones 24 months ago. Kidney function (represented as serum creatinine) was normal. Twenty-four months later, he presented with fever, cough, and hemoptysis for 5 days. He was anemic with hemoglobin 75 g/L and serum creatinine was 691 μmol/L (eGFR, 8 mL/min/1.73m2). Two days later, he developed gross hematuria and serum creatinine rose to 884 μmol/L (eGFR, 6 mL/min/1.73m2), at which time he was referred to our hospital. He was a long-term cigarette smoker and had gasoline exposure 15 days ago.
On admission, he was pale and febrile with no lymphadenopathy. Breath sounds were weak in both lungs on auscultation. Serum creatinine rapidly increased to 1279 μmol/L (eGFR, 4 mL/min/1.73m2) with oliguria and hemoglobin decreased to 49 g/L. Chest radiography suggested bilateral pulmonary hemorrhage. The circulating anti-GBM antibody was positive, with the titer >200 RU/mL (Euroimmune ELISA kit, normal range <20 RU/mL), while the serum anti-neutrophil cytoplasmic antibody (ANCA) was undetected (Table 1).
Table 1. Clinical, pathological, and immunological features of patients with post-ESWL anti-GBM disease in our hospital.
Patient 1 Patient 2 Patient 3
Sex/age, y M/36 M/50 F/74
Hydrocarbon exposure (Y/N) Y N N
Smoking (Y/N) Y Y N
Prodromal infection (Y/N) Y N Y
Fever (Y/N) Y Y Y
Pulmonary hemorrhage (Y/N) Y N N
Gross hematuria (Y/N) Y Y Y
Oliguria/Anuria (Y/N) Y N Y
Hemoglobin on diagnosis, g/L 49.0 102.0 63.0
Serum albumin, g/L 30.0 27.1 29.0
Urinary protein, g/24h 2.0 1.6 0.6
Nephrotic syndrome (Y/N) N N N
Serum creatinine on diagnosis, μmol/L 1279 364 1066
eGFR on diagnosis, mL/min/1.73m2 4.0 15.0 3.0
Anti-GBM antibodies on diagnosis, RU/mL >200 183 196
Positive ANCA (Y/N) N N N
Treatment PE/MP/Pred/CTX PE/MP/Pred MP/Pred/CTX
Anti-GBM antibody on discharge, RU/mL 33 51 102
Serum creatinine on discharge, μmol/L 425 353 618
eGFR on discharge, mL/min/1.73 m2 14.0 16.3 5.3
Dialysis-dependent on discharge (Y/N) N Y Y
Duration of follow-up, m 3 60 48
Dialysis-dependent at last follow-up (Y/N) N Y (PD) Y (HD)
Death at last follow-up (Y/N) (cause of death) N N Y (Esophagus cancer)
Stone location kidney ureter kidney
Total ESWL number 1 1 1
ESWL-to-onset interval, m 24 22 10
Anti-α1(IV)NC1 antibody (ref range, <0.04) 0.06 0.89 0.33
Anti-α2(IV)NC1 antibody (ref range, <0.05) 0.01 0.83 0.12
Anti-α3(IV)NC1 antibody (ref range, <0.06) 2.11 2.08 2.15
Anti-α4(IV)NC1 antibody (ref range, <0.58) 0.27 1.43 0.70
Anti-α5(IV)NC1 antibody (ref range, <0.02) 0.00 0.48 0.10
Anti-α3EA antibody (ref range, <0.07) 1.39 1.67 1.61
Anti-α3EB antibody (ref range, <0.15) 0.59 1.54 1.35
Anti-α3(IV)NC1 IgG1 (ref range, <0.02) 0.68 0.57 0.73
Anti-α3(IV)NC1 IgG2 (ref range, <0.07) 1.51 1.32 1.25
Anti-α3(IV)NC1 IgG3 (ref range, <0.01) 0.53 0.49 0.63
Anti-α3(IV)NC1 IgG4 (ref range, <0.01) 2.34 0.56 0.43
M: male; F: female; eGFR: estimated glomerular filtration rate; GBM: glomerular basement membrane; ANCA: anti-neutrophil cytoplasmic antibody; PE: plasma exchange; MP: methylprednisolone pulse; Pred: prednisone; CTX: cyclophosphamide; PD: peritoneal dialysis; HD: hemodialysis; ESWL: extracorporeal shock wave lithotripsy; IgG: immunoglobulin G; ref: reference; Y: yes; N: no.
Anti-GBM disease was diagnosed based on the renal-pulmonary involvement and positive serum anti-GBM antibody. The patient was prompted to plasma exchange, intravenous methylprednisolone pulse treatment, cyclophosphamide, and hemodialysis. After 17 sessions of plasma exchange, he became afebrile without hemoptysis. His serum anti-GBM antibody decreased to 33 RU/ml and serum creatine to 425 μmol/L (eGFR, 14 mL/min/1.73m2). Repeated chest radiography indicated the absorption of his hemorrhage. He was dialysis-independent on discharge and a 3-month follow-up.
Sera from this patient were tested for antigen spectrum using recombinant human α1–5(IV)NC1 chains and chimeric proteins containing epitopes EA and EB on α3(IV)NC1 (Supplementary materials and methods). The patient's serum recognized α1 and α3(IV)NC1 domains, and both epitopes EA and EB of α3(IV)NC1. The immunoglobulin G (IgG) subclass distribution against α3(IV)NC1 was also tested for the sera. All 4 subclasses of IgG were detected, with the dominance of IgG2 and IgG4 (Table 1; Figure S1).
Figure 1. Renal pathology of patient 3 with post-extracorporeal shock wave lithotripsy (ESWL) anti-GBM disease showed cellular crescent formation in a glomerulus by periodic acid-silver methenamine and Masson trichrome stain on light microscopy (400×).
Case 2
A 50-year-old male undertook one session of ESWL for ureter stones with normal kidney function 22 months ago. He was a 20-year cigarette smoker but had quit smoking for 10 years. Twenty-two months later he was referred to our hospital for fever, gross hematuria, and rapidly progressive renal dysfunction. He had suffered from intermittent fever for 2 months without other indicating symptoms and it could not be resolved by antibiotics. Fifteen days prior to admission, he developed gross hematuria and the serum creatinine progressed from 160 μmol/L (eGFR, 40 mL/min/1.73m2) to 504 μmol/L (eGFR, 11 mL/min/1.73m2). No stones were found by renal ultrasound.
After admission, he was diagnosed with anti-GBM disease due to positive serum anti-GBM antibody (183 RU/mL). His serum ANCA was tested negative. He received 7 sessions of plasma exchange, intravenous methylprednisolone pulse treatment, and hemodialysis. His condition improved and hematuria disappeared afterward. Serum creatinine decreased to 353 μmol/L (eGFR, 16 mL/min/1.73m2) and anti-GBM antibody titer to 51 RU/mL on discharge. The patient was dialysis-dependent during a 60-month follow-up (Table 1).
The patient's sera reacted with all five α chains and both EA and EB epitopes of α3(IV)NC1. All 4 IgG subclasses to α3(IV)NC1 were detected with the dominance of IgG2 (Table 1; Figure S1).
Case 3
A 74-year-old female received one session of ESWL for kidney stones 10 months ago when the renal function was normal. Ten months after ESWL, she complained of fever and gross hematuria with urinary irritation symptoms for 2 weeks. No leukocytosis was found, but her urinalysis revealed white and red blood cells. Initial workups showed elevated C-reactive protein (96 mg/L) and normal serum creatinine (87 μmol/L; eGFR, 55 mL/min/1.73m2). Renal ultrasound showed no stones. She was diagnosed with urinary tract infection and treated by intravenous antibiotics, after which she reported no symptom improvement but developed oliguria and bilateral edema of lower extremities. Her serum creatinine deteriorated to 1066 μmol/L (eGFR, 3 mL/min/1.73m2) within 9 days. Then she was referred to our hospital.
On admission, physical examination revealed a sick and pale woman with no rales on auscultation. Serum anti-GBM antibody was positive (196 RU/mL) while the serum ANCA was not. Renal biopsy revealed severe necrosis of glomerular capillary walls and cellular/cellular-fibrous crescents in all 32 glomeruli on light microscopy (Figure 1), with no electron-dense deposits on electron microscopy. The immunofluorescence microscopy was negative on a sclerotic glomerulus. She was treated by methylprednisolone pulse and cyclophosphamide. Plasma exchange was not performed, given the poor prognosis based on renal biopsy. Her renal function did not recover and she remained dialysis-dependent. The patient died of esophagus cancer 2 years after discharge.
The patient's sera recognized all five α chains and both EA and EB epitopes of α3(IV)NC1. As for circulating IgG to α3(IV)NC1, all four subclasses were detected with the dominance of IgG2 (Table 1; Figure S1).
Discussion
In this study, we described three patients with anti-GBM disease occurring within 2 years after ESWL. The incidence of such cases among all anti-GBM patients from our hospital in the past 10 years was 1.81% (3/166). Their clinical manifestations, pathological features, and antigen spectrum were similar to the classic anti-GBM disease. All three patients presented with fever, gross hematuria, and rapidly progressive glomerulonephritis (RPGN) with poor renal prognosis. One of them suffered from pulmonary hemorrhage. The one available renal biopsy revealed 100% crescent formation in the glomeruli. All three patients recognized a3(IV)NC1 and EA/EB epitopes. None were positive for anti-neutrophil cytoplasmic antibody (ANCA).
Post-ESWL anti-GBM disease has been rarely reported. After searching in PubMed using keywords ‘anti-GBM’, ‘Goodpasture’, ‘ESWL’, and ‘lithotripsy’, we found five previous cases of the post-ESWL anti-GBM disease with full text in English, reviewed in Table 2. Among the five patients, three of them were male and two were female, with a median age of 67 years old (range, 32–72 years). Human leukocyte antigen (HLA) phenotyping was performed in all five patients, with four of them expressing susceptible serotypes for anti-GBM disease [30]. All previous cases presented with shorter ESWL-to-onset intervals (1 week to 7 months) than ours, possibly due to more ESWL treatment numbers. Renal damage by ESWL appears to be cumulative in animal experiments and human, proportional to the application frequency and the number of treatments [31–33]. Their clinical manifestations, renal pathologies, and prognosis were similar to ours. Specifically, fever occurred in all previous reports and ours. However, only a few of them (3/8, 37.5%) were reported to bear prodromal infections and none in either group were positive for ANCA. Fever was a common feature in anti-GBM disease patients, as shown in our previous study in a 140-patient cohort, and 78.7% of febrile patients had infections [34]. The infection rate in post-ESWL anti-GBM disease patients seemed to be lower, which might indicate distinct disease triggers other than prodromal infections in these patients.
Table 2. Clinical and pathological data of previously reported post-ESWL anti-GBM disease cases.
1 2 3 4 5
Authors Cranfield et al. Sellin et al. Xenocostas et al. Iwamoto et al. Guerin et al.
Publication year 2015 2005 1999 1998 1990
Sex/age, y F/67 M/32 M/72 F/37 M/67
Hydrocarbon exposure (Y/N) NA NA NA NA NA
Smoking (Y/N) NA NA NA NA Y
Prodromal infection (Y/N) N N Y N N
Fever (Y/N) Y Y Y Y Y
Pulmonary hemorrhage(Y/N) Y N N N N
Gross hematuria (Y/N) NA Y N Y NA
Serum creatinine on diagnosis, μmol/L 1179 919 NA 1114 1074
Positive anti-GBM antibody(Y/N) Y Y Y Y Y
Positive ANCA (Y/N) N NA N N N
HLA phenotype DR4, DQ6 DRB1*11 & 13 DR15 DR2 DR2
Treatment PE/MP/Pred/CTX Pred/CTX NA PE/MP/Pred/CTX NA
Dialysis-dependent (Y/N) Y Y Y Y Y
Renal biopsy (Y/N) NA Y Y Y Y
Immunofluorescence NA Linear IgG deposits along GBM Linear IgG deposits along GBM Linear IgG and C3 deposits along GBM Linear IgG and C3 deposits along GBM
Light microscopy NA Crescent formation in 23/25 glomeruli Crescent formation in nearly all glomeruli Cellular crescents in all glomeruli Cellular crescents in all glomeruli
Electron-dense deposits on electron microscopy NA NA N N NA
Stone location NA Infundibulum Left renal pelvis Right kidney Left kidney
Stone component NA Calcium oxalate NA NA NA
Total ESWL number 2 within 4 weeks 3 within 4 months 1 NA 2 within 10 days
ESWL-to-onset interval 1 week 5 months 3 months 3 months 7 months
Shock number 3200 NA 4000 1000 NA
Energy data 75 kPa NA NA 17 kV NA
Renal function at last ESWL NA NA Normal Normal Normal
Anti-GBM antibody before ESWL NA NA NA Negative Negative
F: female; M: male; GBM: glomerular basement membrane; ANCA: anti-neutrophil cytoplasmic antibody; HLA: human leukocyte antigen; PE: plasma exchange; MP: methylprednisolone pulse; Pred: prednisone; CTX: cyclophosphamide; IgG: immunoglobulin G; C3: complement 3; ESWL: extracorporeal shock wave lithotripsy; ref: reference; Y: yes; N: no; NA: not available.
Further studies are still needed to elucidate the effect of ESWL on the initiation of anti-GBM disease. However, there has been evidence about the collagen cleaving effect of ultrasound since early 1980s when researchers tried to isolate and define the components of GBM by physical methods. Glomeruli were sequestered and sonicated to separate the GBM, which then released split products of collagen with antigenicity [35], indicating ultrasound could cause local damage of the GBM. There was also evidence of transient nephrotic-range proteinuria immediately after ESWL in a patient cohort [36] and mesangial proliferative glomerulopathy after ESWL in experimental animal models of pig [37]. Moreover, shock wave lithotripsy is utilized in pancreatic and large common bile duct stones or sialolithiasis, but no cases of anti-GBM disease have been reported in either of these conditions yet, suggesting ESWL for urinary stones may affect kidneys more directly. Therefore, ESWL could possibly damage and expose the antigen inside the GBM via direct damage by shock waves or secondary damage by immune-complex induced by ESWL debris [37].
Anti-GBM IgG subclass distribution is associated with disease severity [38]. The IgG1 and IgG3 dominated the IgG subclasses in patients with severe renal impairment, while IgG2 and IgG4 were associated with milder renal damage [38]. In post-ESWL anti-GBM disease patients from this study, the IgG2 and IgG4 were the dominant subclasses against α3(IV)NC1. However, these patients presented with severe renal damage and poor prognosis. The IgG subclass switching after B cell activation follows the sequence of IgG3→IgG1→IgG2→IgG4. It has been suggested that IgG4 production results from chronic or repetitive antigenic stimulation [39,40]. Moreover, low-level natural anti-GBM autoantibodies existed in healthy human sera, predominantly of IgG2 and IgG4 [41]. We speculated that ESWL might expose GBM autoantigens and induce IgG autoantibodies chronically during the ESWL-to-onset interval, allowing them to complete subclass switching. The autoantibodies may accumulate beyond a threshold to disturb the immune tolerance in healthy individuals and provoke the pathogenic autoimmunity.
Anti-GBM diseases were also reported scarcely to associate with obstructive uropathy due to urinary malignancy, neurogenic bladder, or ureteral stenosis [15–18]. The anti-GBM antibody level and renal function seemed to be parallel with treatment efficacy of hydronephrosis [18]. Intact NC1 hexamer could be detected in the serum and be secreted in the urine of healthy individuals [17,42]. Rabbits immunized with their own urinary concentrate developed anti-GBM glomerulonephritis [43]. It was suggested that urinary α3(IV)NC1 under urinary obstruction might enter the renal interstitia, dissociate under acidic pH changed by inflammatory infiltrate (such as infection) and act as an immunogen [17]. These studies provide another theory for the induction of anti-GBM disease associated with urinary stones, including patients treated by ESWL.
The limitations of our study lay in at least two points. Firstly, the number of cases is too small to draw the causal relationship between ESWL and anti-GBM disease. Moreover, we cannot rule out the possibility that individuals with anti-GBM disease or with susceptible HLA phenotypes may bear a higher risk of urinary obstructions, which lead to the production of anti-GBM antibodies. A prospective cohort study in patients undergoing ESWL might be required to further elucidate the relationship between the procedure and anti-GBM disease. Secondly, not all patients had detailed ESWL information (stone component, shock wave frequency, and energy), kidney biopsy, and HLA phenotypes for us to draw a full picture.
In summary, the anti-GBM disease could happen within weeks to months after ESWL treatment and present with similar clinical features, antigen spectrum, and prognosis to classic anti-GBM disease. IgG2 and IgG4 were the two dominant subclasses against α3(IV)NC1. Although the causal relationship between ESWL and anti-GBM disease still needs further exploration, our study here may act as a reminder for physicians that patients developing acute renal insufficiency after ESWL should lead to the suspicion of anti-GBM disease and in-time diagnosis and treatment.
Supplementary Material
Supplemental Material
Click here for additional data file.
Geolocation information
City: Beijing, Latitude: 39.9289, Longitude: 116.3883.
Ethical policy and institutional review board statement
Written informed consents were obtained from the patients for publication of the three cases and any accompanying images. The experiments in this study complied with the Declaration of Helsinki and were approved by the ethics committee of Peking University First Hospital.
Disclosure statement
No potential conflict of interest was reported by the author(s). | UNK (PULSE) | DrugDosageText | CC BY | 33435789 | 20,046,560 | 2021-12 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hypoxia'. | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | ALBUTEROL, AZITHROMYCIN ANHYDROUS, CISATRACURIUM, FENTANYL, FLUTICASONE PROPIONATE\SALMETEROL XINAFOATE, MIDAZOLAM, OSELTAMIVIR, PROPOFOL | DrugsGivenReaction | CC BY | 33435939 | 18,784,956 | 2021-01-12 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Metabolic acidosis'. | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | ALBUTEROL, AZITHROMYCIN ANHYDROUS, CISATRACURIUM, FENTANYL, FLUTICASONE PROPIONATE\SALMETEROL XINAFOATE, MIDAZOLAM, OSELTAMIVIR, PROPOFOL | DrugsGivenReaction | CC BY | 33435939 | 18,784,956 | 2021-01-12 |
What was the administration route of drug 'ALBUTEROL'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | Respiratory (inhalation) | DrugAdministrationRoute | CC BY | 33435939 | 18,784,956 | 2021-01-12 |
What was the administration route of drug 'CALCIUM CHLORIDE\POTASSIUM CHLORIDE\SODIUM CHLORIDE\SODIUM LACTATE'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY | 33435939 | 18,862,444 | 2021-01-12 |
What was the administration route of drug 'MAGNESIUM SULFATE'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY | 33435939 | 18,862,444 | 2021-01-12 |
What was the dosage of drug 'ALBUTEROL SULFATE'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | 100MCG/PUFF (4?8 PUFFS EVERY 15?30 MIN) (TOTAL 315 PUFFS) | DrugDosageText | CC BY | 33435939 | 18,823,040 | 2021-01-12 |
What was the dosage of drug 'CALCIUM CHLORIDE\POTASSIUM CHLORIDE\SODIUM CHLORIDE\SODIUM LACTATE'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | 4.5 LITRES DURING THE FIRST 48 HOURS | DrugDosageText | CC BY | 33435939 | 18,862,444 | 2021-01-12 |
What was the outcome of reaction 'Hypoxia'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | Recovered | ReactionOutcome | CC BY | 33435939 | 18,784,956 | 2021-01-12 |
What was the outcome of reaction 'Lactic acidosis'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | Recovered | ReactionOutcome | CC BY | 33435939 | 18,862,444 | 2021-01-12 |
What was the outcome of reaction 'Toxicity to various agents'? | Salbutamol-induced lactic acidosis in status asthmaticus survivor.
BACKGROUND
Salbutamol-induced lactic acidosis is a rare presentation that could manifest in specific clinical context as acute asthmatic attack treatment. An increase of glycolysis pathway leading to pyruvate escalation is the mechanism of hyperlactatemia in β2-adrenergic agonist drug.
METHODS
A 40-year-old man who had poor-controlled asthma, presented with progressive dyspnea with coryza symptom for 6 days. He was intubated and admitted into medical intensive care unit due to deteriorated respiratory symptom. Severe asthmatic attack was diagnosed and approximate 1.5 canisters of salbutamol inhaler was administrated within 24 h of admission. Initial severe acidosis consisted of acute respiratory acidosis from ventilation-perfusion mismatch and acute metabolic acidosis resulting from bronchospasm and hypoxia-related lactic acidosis, respectively. The lactate level was normalized in 6 h after hypoxemia and ventilation correction. Given the lactate level re-elevated into a peak of 4.6 mmol/L without signs of tissue hypoxia nor other possible etiologies, the salbutamol toxicity was suspected and the inhaler was discontinued that contributed to rapid lactate clearance. The patient was safely discharged on the 6th day of admission.
CONCLUSIONS
The re-elevation of serum lactate in status asthmaticus patient who had been administrated with the vast amount of β2-adrenergic agonist should be considered for salbutamol-induced lactic acidosis and promptly discontinued especially when there were no common potentials.
Background
Salbutamol-induced lactic acidosis is an unusual presentation. It could be diagnosed in patients who require the large amount of β2-adrenergic agonist in short period as status asthmaticus treatment. Although this salbutamol inhaler is a common drug rescuing airway obstructive problem, there has unexpectedly serious adverse effect—lactic acidosis when it is excessively applied. However, there have no definite diagnostic criteria, exclusion of other potential etiologies is essentially required. The pathophysiology of salbutamol-induced lactic acidosis is an increase of glycolysis pathway resulting in pyruvate and lactate escalation [1–5].
Case presentation
A 40-year-old man was admitted to medical intensive care unit with acute hypercapnic respiratory failure due to status asthmaticus. His past medical history was poor-controlled asthma that intermittently used only a short-acting bronchodilator for 10 years. He was 12-pack-year smoker which quit over 10 years. He denied alcohol consumption nor recreational drug use. He presented with coryza, myalgia and low grade of fever for 6 days. On the day of admission, he developed difficulty of breathing, respiratory rate of 44 /minute. His blood pressure was 184/122 mm Hg and tachycardia of 120 /minute. An initial oxygen saturation was 85% at room air. On examination, he had poor air-entry with biphasic wheezing throughout the lung’s field. After intubation, he was deeply sedated and paralyzed in order to being controlled with a proper setting of mechanical ventilation. Arterial blood gas revealed acute respiratory acidosis with PH of 6.98, PaO2 of 90 mm Hg (under fraction of inspired oxygen-FiO2; 0.6) and PaCO2 of 119.5 mm Hg. The chest radiograph and initial serum investigations were unremarkable. Nasal swab for respiratory viral test was positive for Enterovirus/Rhinovirus. The essential investigations and dose of sedative drugs are shown in Table 1. During the admission, patient’s mean arterial pressure was over 65 mmHg without inotrope’s support. The salbutamol inhaler (100 mcg/puff) was administrated via ventilator’s inspiratory circuit, 4–8 puffs every 15–30 min. Furthermore, salmeterol/fluticasone propionate MDI (25 mcg/250 mcg) 4 inhalations every 12 h and four inhalations of tiotropium bromide soft mist inhaler (2.5 mcg) were used as inhaled controllers. Not only intravenous steroid was used for exacerbation regimen, but two grams of magnesium sulphate infusion was also given. Ceftriazone, azithromycin and oseltamivir were empirically started and discontinued when nasal swab, sputum culture and blood cultures revealed none of other co-infections. We did not use an aminophylline nor inhaled anesthetic agent due to unavailability. The ventilator setting was volume-controlled mode, 5 ml/kg of ideal body weight, 5 cm H2O of positive end expired pressure (PEEP), 0.6 of FiO2 and 60–100 L/min of decelerating flow. After 6 h of treatment, patient’s ventilation and oxygenation showed significant improvement contributing to lactate normalization (from 4.4 to 0.7 mmol/L). However, at 24 h of admission, his lactate level had re-elevated to a peak of 4.6 mmol/L contrary to the improvement of PaO2, PaCO2, patient’s symptoms and mechanical ventilator’s setting. There was a minimal rising of creatinine, potassium and creatinine phosphokinase (CPK) which was less likely to make a diagnosis of rhabdomyolysis. Furthermore, none of liver function test, urine analysis, lipid profiles and electrocardiogram’s finding had illustrated abnormality. Four and a half Litres of lactated Ringer’s solution were infusing for fluid resuscitation and maintenance during the first 48 h. The urine output throughout 48 h of admission was 45–180 ml/h with positive accumulative balance of 3.2 L. The salbutamol inhaler was discontinued while patient’s clinical status improved, a total dose of salbutamol was 31,500 mcg (315 puffs). The lactate level had rapidly decreased into normal range within 12 h of discontinuation and the patient was extubated safely at day 5 of the admission.Table 1 Laboratory investigations, ventilator settings and sedative drugs
0 h 6 h 12 h 24 h 32 h 48 h 54 h
Arterial blood gas
PH 6.98 7.29 7.24 7.26 7.42 7.43 7.45
PaO2 (mm Hg) 90 91.9 119.2 95.6 61.9 79.2 76
PaCO2 (mm Hg) 119.5 55.6 48 52 44 42.8 38.4
HCO3 (mmol/L) 20 22 16 12 22 25 25
Serum lactate (mmol/L) 4.4 0.7 2.3 4.6 1.3 0.9 0.7
Creatinine phosphokinase (U/L) – – 1,877 2,007 – 1,454 880
Creatinine (mg/dL) 0.87 – 1.16 – – 1.06 0.92
Potassium (mmol/L) 3.8 5.3 5.4 4.4 4 3.9 4.2
Ventilator settings
Mode VCV VCV VCV VCV VCV PSV PSV
Tidal volume (ml/kg) 5.5 5.5 4.3 4.3 6 6–7 6–7
Tidal volume (ml) 380–390 380–390 300 300 420 420–490 420–490
PEEP (cm H2O) 5 5 5 5 5 5 5
Respiratory rate (/min) 20–25 20–25 14 14 14 14 14
Flow (L/min) 60 60 70–100 70–100 60 – –
FiO2 0.6 0.4 0.4 0.4 0.3 0.3 0.3
Drugs
Propofol (mg/h) – 200 100 100 60 off –
Fentanyl (mcg/h) 50 80 50 80 80 80 50
Midazolam (mg/h) 4 5 6 6 4 off –
Cisatracurium (mcg/kg/min) – 2.2 2.2 off – – –
Salbutamol inhaler (puffs) N/A N/A N/A Total 315 → off – – –
VCV volume controlled ventilation, PSV pressure support ventilation, PEEP positive end-expiratory pressure
Discussion and conclusion
Status asthmaticus is a severe stage of asthma exacerbation which required multimodalities of treatment such as high dose of bronchodilators, intubation with high mechanical ventilator’s setting and deep sedation. To effectively manage peripheral airway obstruction, salbutamol inhaler is a drug of choice for ameliorating bronchospasm but it remains uncertainty of maximum dose especially using through mechanical ventilator. The initial reduction of lactate level in the first 6 h as a result of improvement of hypoxemia and bronchospasm. In arterial blood gas analysis, the initial presentation was a combination of acute respiratory acidosis and metabolic acidosis which respiratory cause was a major contribution. When the bronchospasm had been improved, the overall PH was rising. However, the recovery of airway disease was not complete, in addition to the re-worsening of lactic acidosis predisposing to ongoing acidosis. Our case demonstrated an explicit point of lactate level’s rebound after normalization, there were none of other potential etiologies of lactate’s re-elevation such as persistent hypotension, uncontrolled viral infection, hospital-acquired infection, abnormal lactate clearance relating to liver or renal dysfunction. The medication-induced lactatemia as type B (non-hypoperfusion cause) should be considered in this case which is reported in several mechanisms of lactate production [5–7] However, the potential drugs in our case were salbutamol inhaler and propofol infusion, which propofol is unlikely presented without propofol infusion syndrome (PRIS). PRIS generally consists of rhadomyolysis, abnormal lipid profiles and cardiac dysfunction, the uncoupling oxidative phosphorylation is the mechanism of PRIS-related hyperlactatemia [8–10]. Furthermore, lactated Ringer’s solution does not seem to increase circulating lactate concentration unless infusing large volumes (180 ml/kg/h) [11, 12].
Given there are no diagnostic criteria of salbutamol-induced lactic acidosis, drug levels nor determined dose, deliberate clinical evaluation and exclusion are essential in this context. There was a retrospective cohort that reported the 1200 mcg of albuterol causing lactic acidosis in a couple hours whereas our case was used in the higher amount [13]. As a systematic review, the lactate level of drug-induced hyperlactatemia was reported in wide range and 1.9 days was a median time of lactate clearance [4]. In our case illustrated the suspected salbutamol-induced lactic acidosis that showed the using dose (31,500 mcg), the duration of onset (within 24 h), the peak of drug-induced lactate (4.6 mmol/L) and the clearance of drug-induced lactate (8 h). This observational information may be useful for guiding diagnosis. After salbutamol inhaler discontinuation, the lactate level decreased rapidly, so salbutamol-induced lactic acidosis was diagnosed. Although there are some mechanisms reported that glucocorticoid enhances the efficacy of β2-adrenergic agent which may or may not worsen lactic acidosis, the benefit of glucocorticoid in asthmatic attack treatment is obvious [14]. For further study, the implementation of either drug toxicity level or minimum accumulative amount to confirm the diagnosis is essentially required.
Salbutamol-induced lactic acidosis is a rare presentation, potentially occurred when severe asthmatic attack diagnosed. The increase of glycolysis and pyruvate production without poor perfusion state is the main mechanism of lactate production in β2-adrenergic agents. Exclusion of other potential etiologies of lactic acidosis is necessary, but salbutamol-induced lactic acidosis should also be aware in this context.
Abbreviations
VCVVolume controlled ventilation
PSVPressure support ventilation
PEEPPositive end-expiratory pressure
FiO2Fraction of inspired oxygen
CPKCreatinine phosphokinase
PRISPropofol infusion syndrome
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Division of Pulmonary and Critical Care Medicine, Department of Medicine, Chulalongkorn University, Bangkok, Thailand and Medical Intensive Care II unit.
Authors’ contributions
VP and TS designed and drafted this manuscript. SB and PS researched related articles, drafted and revised manuscript. All authors interpreted and revised the final approval of the manuscript to be published. All authors contributed equally in the preparation of this manuscript.
Funding
This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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All data are available in the manuscript [and its supplementary information files].
Ethics approval and consent to participate
Not applicable.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Competing Interests
The authors declare that they have no competing interests. | Recovered | ReactionOutcome | CC BY | 33435939 | 18,872,759 | 2021-01-12 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Clostridium difficile infection'. | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | CANNABIDIOL, CLINDAMYCIN, CYCLOPHOSPHAMIDE, DOXORUBICIN HYDROCHLORIDE, DRONABINOL, GLUTAMINE, IRINOTECAN, PALBOCICLIB, PAZOPANIB, PREXASERTIB, RIBOFLAVIN, TEMOZOLOMIDE, TEMSIROLIMUS, VINCRISTINE, VINORELBINE TARTRATE, VORINOSTAT | DrugsGivenReaction | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Headache'. | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | CANNABIDIOL, CLINDAMYCIN, CYCLOPHOSPHAMIDE, DOXORUBICIN HYDROCHLORIDE, DRONABINOL, GLUTAMINE, IRINOTECAN, PALBOCICLIB, PAZOPANIB, PREXASERTIB, RIBOFLAVIN, TEMOZOLOMIDE, TEMSIROLIMUS, VINCRISTINE, VINORELBINE TARTRATE, VORINOSTAT | DrugsGivenReaction | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Mucosal inflammation'. | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | CANNABIDIOL, CLINDAMYCIN, CYCLOPHOSPHAMIDE, DOXORUBICIN HYDROCHLORIDE, DRONABINOL, GLUTAMINE, IRINOTECAN, PALBOCICLIB, PAZOPANIB, PREXASERTIB, RIBOFLAVIN, TEMOZOLOMIDE, TEMSIROLIMUS, VINCRISTINE, VINORELBINE TARTRATE, VORINOSTAT | DrugsGivenReaction | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Neuropathy peripheral'. | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | CANNABIDIOL, CLINDAMYCIN, CYCLOPHOSPHAMIDE, DOXORUBICIN HYDROCHLORIDE, DRONABINOL, GLUTAMINE, IRINOTECAN, PALBOCICLIB, PAZOPANIB, PREXASERTIB, RIBOFLAVIN, TEMOZOLOMIDE, TEMSIROLIMUS, VINCRISTINE, VINORELBINE TARTRATE, VORINOSTAT | DrugsGivenReaction | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'CANNABIDIOL'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'CLINDAMYCIN'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'CYCLOPHOSPHAMIDE'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'DOXORUBICIN HYDROCHLORIDE'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'DRONABINOL'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'GLUTAMINE'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'IRINOTECAN'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'PALBOCICLIB'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'PAZOPANIB'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'PREXASERTIB'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'RIBOFLAVIN'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'TEMOZOLOMIDE'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'TEMSIROLIMUS'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'VINCRISTINE'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'VINORELBINE TARTRATE'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
What was the dosage of drug 'VORINOSTAT'? | Refractory alveolar rhabdomyosarcoma in an 11-year-old male.
Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.
INTRODUCTION
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma occurring in children with two major subtypes, embryonal and alveolar (Rudzinski et al. 2017). Embryonal rhabdomyosarcoma (ERMS) tends to have a more favorable prognosis and is mainly seen in the orbit, head, neck, and retroperitoneum (Rudzinski et al. 2017). Alveolar rhabdomyosarcoma (ARMS), on the other hand, has a less favorable prognosis and often involves the trunk and extremities (Rudzinski et al. 2017). Although PAX3:FOXO1 or PAX7:FOXO1 chimeric oncogenes are found in the majority of ARMS cases, 15% of rhabdomyosarcomas with available fusion data are negative for FOXO1 rearrangement (Rudzinski et al. 2017). These cases are prognostically similar to embryonal rhabdomyosarcoma, which dictates their risk-based treatment (Rudzinski et al. 2017). In general, fusion status serves as a better predictor of prognosis than histology (Rudzinski et al. 2017). The 5-yr event-free survival (EFS) for fusion-negative ARMS is 29% (Rudzinski et al. 2017). Despite advancements in uncovering the clinical and mutational profiles of the myriad RMS subtypes, such as recurrent TP53 tumor suppressor gene loss of function in PAX fusion-negative (PFN) RMS, survival rates have not seen similar significant improvement (Breneman et al. 2003; Williams et al. 2004; Davis and Keller 2012; Malempati and Hawkins 2012; Rudzinski et al. 2017).
Several predisposition syndromes such as neurofibromatosis, Costello syndrome, and Li–Fraumeni syndrome have been linked to sarcomas (Farid and Ngeow 2016). Li–Fraumeni syndrome (LFS) is an autosomal dominant disorder commonly characterized by heterozygous germline mutations in TP53. LFS predisposes patients to a wide range of malignancies, including sarcomas, which account for 25%–35% of all LFS-associated tumors and 1%–10% of RMS (Olivier et al. 2003; Gonzalez et al. 2009; Palmero et al. 2010; Ognjanovic et al. 2012). Data suggest that overall survival outcomes were improved with early detection of tumors through surveillance of patients with TP53 mutations (Ballinger et al. 2015). However, detection of LFS can be challenging for clinicians. Presentation of clinical characteristics, surveillance strategies, and therapeutic interventions can all differ between patients given the wide range of malignancies associated with LFS. Most TP53 mutations associated with LFS are missense mutations, which may or may not significantly affect protein function (Olivier et al. 2003). The ambiguity of classifying these mutations as benign or deleterious can make clinicians reluctant to diagnose patients with LFS given the psychological ramifications that can come with diagnosis and active surveillance thereafter. The International Agency for Research on Cancer (IARC) TP53 Database has compiled TP53 mutations reported in published literature and can be used for further risk assessment (Hainaut et al. 1997). In the context of rhabdomyosarcoma, TP53 mutations have been associated with increased risk of second neoplastic malignancy (Pondrom et al. 2020) and associated with worsened overall survival (Casey et al. 2020), suggesting TP53 plays an important role in clinical progression and TP53 status should be investigated in the course of clinical decision making.
Advanced stage or metastatic patients often receive intensified treatments, which historically show a 3%–4% mortality rate from treatment-related toxicities (Crist et al. 2001). Molecularly targeted therapies provide a promising approach for treatment of RMS patients as these therapies have the potential to reduce drug-induced toxicities compared to traditional chemotherapies. Here, we present a case of an 11-yr-old male with fusion-negative ARMS who underwent standard of care followed by exhaustive precision medicine approaches to his progressive disease and a functional genomics characterization at end of life. Drug screening in conjunction with genetic analysis has provided insight to the biological status of the index case and potentially promising drug candidates for future preclinical and clinical studies.
RESULTS
Clinical Presentation
An 11-yr-old Caucasian male presented with a tender left upper arm mass. No recent injury was noted, but the patient had a history of prior supracondylar fracture of the distal humerus. Approximately 3 wk later, a magnetic resonance imaging (MRI) of the humerus revealed a lesion within the left upper arm measuring ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension within the triceps region of the left upper arm, possibly arising from the medial head of the triceps, with edema and enhancement down to the attachment of the mid-humeral shaft (Fig. 1). The differential diagnosis included soft-tissue fibroma, myxomatous tumor, or neuromuscular mass. Soft-tissue sarcoma was not excluded. Two weeks after presentation, a follow-up X ray of the upper arm mass found that the humerus appeared to be normal without focal bony abnormality, cortical disruption, or periostitis (Supplemental Figs. 1 and 2). Prominent soft tissue was found along the medial aspect of the distal portion of the upper arm corresponding in location to the known mass. The mass was also slightly seen dorsally on the internally rotated view. No calcifications or ossifications were identified in the soft tissues.
Figure 1. Orthopedic MRI of the humerus. An ovoid lesion (red asterisk) was found within the triceps region of the left upper arm utilizing a 1.5-Tesla magnet with the following sequences: axial T2 turbo spin echo (TSE) with fat saturation, axial T2 TSE pulse, axial T1 combination, and axial T1 TSE after 4.6 mL of gadolinium (+C) (from top to bottom, left). The following sequences were also obtained: coronal T1 TSE + C with fat saturation, coronal T1 TSE with fat saturation, coronal short-TI inversion recovery (STIR) combination, and sagittal T1 TSE + C with fat saturation (from top to bottom, right). The lesion measured ∼2.6 × 2 cm in the axial plane and ∼3.5 cm in the craniocaudal dimension (top, left and top, right). The lesion demonstrated T1 hypointensity and heterogeneous, but T2 hyperintensity without significant fat saturation. Within the ovoid region, there are two punctate foci of STIR hypointensity (29/19) and T1 hypointensity.
Four weeks after presentation, the mass had grown dramatically in size with increased pain. A biopsy of the primary left upper, posterior arm mass revealed a small round blue cell malignancy (Fig. 2). Histology was described as a poorly differentiated malignant neoplasm arranged in loosely cohesive sheets of primitive round to polygonal cells with interruptions by few fibrous septa and scattered large vessels. Desmin was reported to be diffusively positive, tumor cell myogenin was positive (∼60%), and cytokeratin was found negative. PTEN, BAF47 (INI1/SMARCB1), and BRG1 (SMARCA4) were positive. Focal areas of lower cellular cohesion created a vague papillary appearance. The individual tumor cells were reported to be pleomorphic, ranging from intermediate to large. The majority of cells had scant cytoplasm; however, rare cells had a moderate amount of densely eosinophilic cytoplasm with possible cross-striations (rhabdomyoblasts). Multiple scattered multinucleated giant cells were present which also contained deeply eosinophilic cytoplasm. Random cells had enlarged hyperchromatic and anaplastic nuclei. Numerous apoptoses and mitoses were seen. Rare, atypical mitotic figures were noted. The patient was diagnosed with a malignant neoplasm of connective and other soft tissue of the upper limb, including shoulder. Tumor morphology suggested an ARMS tumor. However, fluorescence in situ hybridization (FISH) studies were negative for the classic FOXO1 t(2,13) or t(1:13) rearrangements and did not support a diagnosis of ARMS. Bilateral bone marrow biopsies were performed, and were negative for small round blue cell morphology.
Figure 2. Histology slides from alveolar rhabdomyosarcoma case. Hematoxylin and eosin (H&E) staining of the left forearm (A), right lower lobe of the lung (B), pleural fluid (C) showing a metastatic small round blue cell malignancy, 200× magnification of right lower lobe of the lung (D), 400× magnification of right lower lobe of the lung (E), and 200× magnification of myogenin staining (F). Scale bars, 300 µM.
Five days after the biopsy, a follow-up postresection multiplanar multisequential MRI scan of the left humerus was performed with and without intravenous contrast. The MRI revealed a heterogeneous mass much larger than the previous scan, measuring 5.2 × 6 × 16.8 cm (AP × TR × CC). At the more inferior aspect of the mass, a central area of low signal intensity was found on the T1 weight sequence with peripheral rim enhancement measuring 8 × 2.4 cm that might represent a central cystic component (possibly representing central necrosis or fluid collection given the recent surgery). The mass abutted the ulnar neurovascular bundle. Nonspecific, small left axillary lymph nodes were noted. Staging investigations through a chest computed tomography (CT) scan revealed no lung metastasis.
Twelve days after diagnosis, the lymph nodes were biopsied and sent for histological analysis. The patient was found to have a metastatic neoplasm consistent with rhabdomyosarcoma in 4 out of 31 lymph nodes. The patient was classified as having stage III, intermediate risk group III RMS, N1, M0 according to the Children's Oncology Group (COG) protocol. The patient began treatment with protocol ARST0531 consisting of 42 wk of vincristine, adriamycin, cyclophosphamide (VAC) and alternating with vincristine and irinotecan (VI). Radiation was started 6 wk after diagnosis and given for a month and a half for local control (5040 cGy in 28fx at 180cGy/fx using 3DCRT). Complications of treatment included mucositis, Clostridioides difficile infection, headache, and exacerbated neuropathy.
Sixteen months after diagnosis (6 mo after chemotherapy was completed), chest CT showed new lung masses, raising concerns of metastatic recurrence. No arm pain was noted. On the positron emission tomography (PET)-CT scan, the fluorine-18-deoxyglucose (FDG) signal in the right lower lung lobe mass was most consistent with progressive metastatic disease. A new left lower lung lobe nodule further raised concern of metastatic disease, despite lack of discernable FDG activity in the left lower lobe. Lobectomy of the right lower lobe revealed a small round blue cell tumor, consistent with metastatic rhabdomyosarcoma (1.6 cm) with vascular invasion and positive bronchovascular margin. Clear margins were not obtained. The patient was diagnosed with relapsed stage IV ARMS.
Eighteen and a half months after diagnosis (two and a half months after relapse), the patient began reinduction chemotherapy with vinorelbine, cyclophosphamide, and temsirolimus (ARST-0921). Independent of ARST-0921, the patient also supplemented with glutamine and vitamin B2, as well as CBD/THC to control pain and anxiety. There was no overlap in supplement regimens. Three months after starting reinduction chemotherapy, the patient completed 28 d of radiation to the right lung. Relapse treatment was given over a period of 7.5 mo. At the end of relapse treatment, a full body MRI and local CT showed stable disease. Possible nodules in the left lung and a mass in the lower left forearm were noted.
Two months after completing relapse treatment (28 mo after initial diagnosis), the patient enrolled in clinical trial ADVL1515 and started treatment with prexasertib, a CHEK1 inhibitor. Radiographic scans one month after enrollment in ADVL1515 showed new cancerous growth in the arms and lungs. Prexasertib treatment was stopped and the patient enrolled in clinical trial ADVL1312 and began treatment with MK-1775, a WEE1 inhibitor. After one month of MK-1775 treatment, radiographic scans showed continued progression, and therapy was stopped. The patient had also enrolled in the Pediatric MATCH trial (APEC1621), but no targetable mutations were found from genomic sequencing.
Thirty months after initial diagnosis the patient began daily oral pazopanib, a 5-d course of temozolomide, and radiotherapy to the left forearm and lungs for local control. A biopsy was performed for clinical trial eligibility determination and development of a patient-derived xenograft at Champions Oncology. One month later (31 mo after initial diagnosis) the patient was determined to be eligible for clinical trial NCT-02162732, a genomically guided therapy trial for children with cancer. The patient began treatment with vorinostat and palbociclib and supplemented with myrrh oil. The patient was also treated with weekly thoracentesis.
Two weeks later, the patient presented to the ER with shortness of breath and chest pain. The necrotic tumor in the left arm was opened and debrided and the patient received i.v. clindamycin. A drain was placed for the patient's malignant pleural effusion with loculation. A week later (32 mo after initial diagnosis), the patient reported a moderate increase in symptoms attributed to his disease over a 24-h period. Doppler ultrasound showed acute occlusive venous thrombosis involving the right internal jugular vein, right subclavian vein, right axillary vein, and right cephalic vein, with additional nonocclusive thrombus in the right basilic vein (superior vena cava syndrome syndrome). CT chest scans showed near complete opacification of the right hemithorax (presumed malignant), extensive thrombosis, and an increased left lower lobe lesion measuring 3.9 cm. Pulmonary fluid increased with drainage reaching ∼1.5 L/day, and the patient required increased support and spent more time on a bilevel positive airway pressure (BiPAP) machine. Medical treatment was not felt to be a viable option given the patient's significant bleeding risk. Vorinostat and palbociclib, as well as supportive care, were discontinued by parental request. The patient was taken off BiPAP and passed away at the age of 14, ∼33 mo after initial diagnosis. The discharge diagnosis was progressive hypoxic respiratory failure, malignant pleural effusion, and relapsed rhabdomyosarcoma.
Genomic Analysis
Cytogenetics revealed 46,XY[20], normal male karyotype. Previous testing by OncoPlex identified a TP53 p.V172F (Tier 2A) mutation with associated loss of heterozygosity, suggesting that both copies of the gene are expected to be inactivated in the tumor tissue. FGFR1 amplification (Tier 2A/4A, log2 tumor/normal read ratio = 0.96 by whole-exome sequencing [WES]) and MYCL amplification (Tier 5A, log2 tumor/normal read ratio = 1.91 by WES) were both present. Targeted sequencing (Ambry Genetics) confirmed that the patient shared the same TP53 mutation (V172F) as his mother. Two previous generations also had early-onset breast cancer on the mother's side (maternal grandmother and great grandmother).
To identify further genomic alterations and potential therapeutic targets, we sequenced and analyzed DNA and RNA isolated from a tumor tissue sample from the left arm, as well as DNA from patient-matched normal tissue. Tissue for sequencing experiments were provided postmortem. After filtering for nonsynonymous mutations not identified in the database of single-nucleotide polymorphisms (Sherry et al. 2001), 109 mutations were found. No somatic mutations were found in genes commonly mutated in PFN tumors, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, two germline mutations were found in TP53, including the same variant found in the patient's mother (c.514G > T p.Val172Phe) (Table 1), which is considered likely pathogenic for hereditary cancer-predisposing syndrome (ClinVar ID: 428909). Another missense variant in TP53 (c.215C > G p.Pro72Arg) was found to alter drug response in several neoplasms but found to not be associated with Li–Fraumeni syndrome 1 (ClinVar ID: 12351). TP53 mutations have previously been associated with response to the majority (six of eight) therapeutic regimens administered to the patient (Table 2). No somatic mutations were found in genes previously reported to be recurrently mutated in RMS (Shern et al. 2014); however, germline protein coding mutations were found in FGFR4 (c.28G > A p.Val10Ile; c.407C > T p.Pro136Leu) and nonsense mediated decay variants were found in NF1 (c.37T > C p.Ter13Arg).
Table 1. Selected variants of known biological interest (aligned to GRCh38 reference genome)
Gene name Source Chr Transcript ID HGVS DNA reference HGVS protein reference dbSNP COSMIC ClinVar ID Genotype TPM CNV log ratio Clinical significance
TP53 Germline 17 ENST00000269305.8 c.514G > T p.Val172Phea rs1131691043 COSM3378354 428909 Het 11.7 −0.33 Likely pathogenic (hereditary cancer-predisposing syndrome)
COSM3378355
COSM354839
COSM354840
COSM354841
COSM354842
COSM44240
TP53 Germline 17 ENST00000269305.8 c.215C > G p.Pro72Arg rs1042522 COSM250061 12351 Het 11.7 −0.33 Drug response (Li–Fraumeni syndrome 1)
COSM3766190
COSM3766191
COSM3766192
COSM3766193
(Het) Heterozygous.
aMutation was also observed in the Oncoplex Panel.
Table 2. Possible treatment-related genomic features
Protocol/clinical trial Therapeutic regimen Molecular target Genomic features
ARST0531 VAC (vincristine, adriamycin, cyclophosphamide) + alternating vincristine and irinotecan (VI) N/A Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783), irinotecan (TP53, PMID25567130)
ARST091 Vinorelbine, cyclophosphamide, temsirolimus mTOR Cyclophosphamide (TP53: PMID17388661, PMID16243804, PMID26438783)
ADVL1515 Prexasertib CHEK1 N/A
ADVL1312 MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
ADVL1312 Pazopanib Tyrosine kinases, VEGF TP53: PMID26646755, PMID25669829
ADVL1312 Temozolomide Alkylating agent TP53: PMID21730979, PMID24248532
NCT02162732 Vorinostat HDAC inhibitor (class I and class II) TP53: PMID26009011, PMID25669829
NCT02162732 Palbociclib (supplemented with myrrh oil); weekly thoracentesis CDK4/CDK6 inhibitor N/A
In addition to the germline variants found in TP53, a germline disruptive in-frame insertion variant was found in FAM83D (c.345 347dupGGC p.Ala116dup), along with copy-number gain in FAM83D (log2 ratio 0.7). Further, FAM83D is overexpressed (23.5-fold, 2.84 TPM vs. 0.1207 TPM) compared to median expression of the GTEx normal skeletal muscle tissue cohort (n = 803). The clinical significance of the FAM83D mutation (c.345 347dupGGC p.Ala116dup) is at present unknown; however, FAM83D overexpression occurs frequently in hepatocellular carcinoma, ovarian cancer, and metastatic lung adenocarcinomas (Inamura et al. 2007; Ramakrishna et al. 2010; Liao et al. 2015; Wang et al. 2015) and correlated with significantly decreased overall survival and metastatic relapse-free survival in breast cancer (Wang et al. 2013). Further, elevated FAM83D was found to correlate with TP53 mutations and genome instability (Walian et al. 2016).
Other somatic mutations were identified, including KDM2B and HCAR1. Both had truncating mutations and copy-number loss (log2 ratio < −0.4). Several genes located on Chromosome 1 were found to be mutated, amplified, and have transcripts per million (TPM) of >100 or <10 (Fig. 3). Using RNA isolated from the tumor of the left arm, 10 gene fusions were identified using the STAR-Fusion analysis pipeline, with many fused genes located on Chromosome 1 (Fig. 3; Supplemental Table 1). Notably, RNA sequencing data from the tumor sample procured following recurrence did not identify any of the typical gene fusion events associated with alveolar rhabdomyosarcoma, including PAX3:FOXO1, PAX7:FOXO1, PAX3:NCOA1, PAX3:NCOA2, and PAX3:FOXO4. A second gene fusion analysis pipeline FusionCatcher also did not find evidence to support the presence of the ARMS-associated gene fusions (Supplemental Table 2). The lack of evidence of PAX3:FOXO1 in the patient's tumor at diagnosis (confirmed by FISH analysis) and lack of any evidence to support any ARMS-associated fusion in the recurrent tumor suggest the patient was unlikely to bear typical ARMS-associated gene fusions throughout the course of disease.
Figure 3. Circos plot. A Circos plot was generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Exome data was analyzed for somatic point mutation, indel, and copy-number variation data, as denoted by the inner and middle rings, respectively. Gene expression from RNA sequencing data was plotted on the outer ring. Genes listed in blue bear germline mutations, whereas genes listed in red bear somatic mutations.
The presence of clustered fusion events and 10 switches in copy-number status suggests Chromosome 1 may be chromothripsis-positive with high confidence event on Chromosome 1 (Bolkestein et al. 2020; Cortés-Ciriano et al. 2020). Notably, the lesions appear over a small region of the genome, suggesting the clustered variants may be classified as a kataegis event. FGFR4, CCND2, and IGF2 were found to be the top genes overexpressed in PFN RMS cases in a previous study (Shern et al. 2014) and were also found to be overexpressed (12-fold, 16-fold, and 153-fold overexpression, respectively) in the patient's tumor compared to a panel of normal skeletal muscle tissue samples (n = 803) from the Genotype-Tissue Expression (GTEx) project (Consortium 2013). A complete list of gene mutations and associated gene expression and copy-number variation data is provided in Supplemental Table 3 and Supplemental Table 4.
Given the relative rarity of fusion-negative ARMS cases among the RMS clinical milieu, we investigated genomic similarities between the index case and other clinical RMS samples using a previously published hierarchical clustering dendrogram approach (Supplemental Fig. 3; Ricker et al. 2020). Similar to previous findings regarding genomic similarities between ERMS and fusion-negative ARMS (Williamson et al. 2010), CF-00449 clusters among ERMS samples, as do the majority of other fusion-negative ARMS samples. Taken together, the histopathology designation of ARMS by multiple pathologists, the lack of evidence of fusions from FISH analysis and RNA sequencing analysis, and the genomic similarity to ERMS more so than ARMS strongly support the diagnosis of CF-00449 as a fusion-negative ARMS.
Functional Analyses
To identify molecular compounds that would inhibit tumor growth, we conducted a chemical screen consisting of a panel of 60 agents from a clinically relevant drug portfolio on cells harvested from the patient's pleural fluid (Table 3; Supplemental Fig. 4). Several drugs were highly active, including panobinostat (IC50 = 6 nM, pan HDAC inhibitor), brefeldin A (IC50 = 21 nM, ATPase inhibitor), CUDC-907 (IC50 = 99 nM, HDAC1/2/3/10 + PI3Kα inhibitor), YM155 (IC50 = 9 nM, Survivin inhibitor), INK128 (IC50 = 5 nM, mTORC1/2), MK-1775 (IC50 = 79 nM, WEE1), thapsigargin (IC50 = 9 nM, SERCA), BMS-754807 (IC50 = 66 nM, IGF1R + AURK inhibitor), JQ1(IC50 = 92 nM, BET (BRD4) inhibitor), and mithramycin A (IC50 = 89 nM, SP1 inhibitor). TP53 mutations were associated with response in multiple high and moderate sensitivity agents (Table 4). Chemical screening was performed using cells isolated pleural effusion fluid while the patient was still alive, and did not influence clinical decision making.
Table 3. Drug screen validation using cells from pleural fluid
Table 4. Drug activity genomic-related features
Drug Inhibitor Genomic-related features
High activity Panobinostat pan HDAC N/A
Brefeldin A ATPase N/A
CUDC-907 HDAC 1,2,3,10, PI3Kα N/A
YM155 Survivin inhibitor N/A
INK128 mTORC1/2 N/A
MK-1775 WEE1 TP53: PMID27601554, PMID27196784, PMID21992793
Thapsigargin SERCA N/A
BMS-754807 IGF1R, AURK IGF2: PMID19996272
JQ1 BET (BRD4) N/A
Mithramycin A SP1 N/A
Moderate activity GSK126 EZH2 N/A
CUDC-305 HSP90 N/A
AZD5363 AKT1–3 TP53: PMID24694055, PMID17145876, PMID17455259
ABT-263 BCL-2, BCL-XL, BCL-W TP53: PMID24694055
I-BET-762 (BET) BRD1–4 N/A
Dinaciclib CDKs N/A
Entinostat HDAC 1,3 N/A
Crizotinib c-MET, ALK TP53: PMID24694055
BKM120 PI3K, mTOR N/A
SGI-1776 PIM 1/2/3 kinase N/A
Volasertib PLK 1/2/3 N/A
UNC0642 G9a/GLP N/A
prima1 TP53 N/A
AZD 8931 EGFR, HER 2/3 N/A
JIB4 JMJD N/A
The patient received the nonselective class I/II histone deacetylase (HDAC) inhibitor, vorinostat, with limited response. Although HDAC inhibitors panobinostat, CUDC-907, and entinostat were found to impair growth of tumor cells in vitro, the HDAC inhibitor CUDC-101 did not demonstrate significant activity in vitro. Panobinostat is a pan-HDAC inhibitor with activity against all HDAC enzymes, whereas CUDC-907 inhibits class I-III HDAC genes plus phosphoinositide 3-kinase α (PI3K- α), and entinostat inhibits classes I and III. Meanwhile, CUDC-101 inhibits class I/II HDACs as well as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER). The discrepancy of response to tested HDAC inhibitors can potentially be explained by differences in their mechanism of action (Li and Seto 2016). These results suggest that inhibition of class I and II HDACs may not be sufficient for tumor suppression of PFN ARMS and targeting of other HDAC classes may have a more significant contribution to tumor survival. Further supporting this notion, panobinostat and entinostat were found to have different consequences in PAX3:FOXO1 ARMS (Bharathy et al. 2018).
The WEE1 inhibitor, MK-1775, which had high potency in vitro (IC50 = 79 nM), was administered to the patient under trial ADVL1312 and did not prevent progression. Prexasertib (LY2606368) was also used to treat the patient under trial ADVL1515 but did not suppress the patient's tumor growth, consistent with the limited in vitro activity of prexasertib (IC50 > 10 µM).
Clinically, temsirolimus (mTOR inhibitor) in combination with vinorelbine and cyclophosphamide resulted in stable disease with potential tumor growth. INK128 (mTORC1/mTORC2 inhibitor) and BKM120 (IC50 = 198 nM, mTOR and PI3K inhibitor) both had high activity in tumor cells, whereas sirolimus (also an mTOR inhibitor) did not have activity in tumor cells in vitro. BKM120 activity suggests that tumor growth and progression may be dependent on multiple pathways in PFN ARMS.
Additionally, agents associated with alteration of TP53 expression also demonstrated in vitro efficacy, including prima-1 (IC50 = 135 nM) and CUDC-305 (IC50 = 137 nM, HSP90 inhibitor). Previous studies have found that prima-1 reactivates TP53, which induces high levels of apoptosis, and has demonstrated tumor reduction with no apparent toxicity in p53 mutant tumor xenografts in SCID mice (Bykov et al. 2002). Despite efforts in elucidating the mechanism of TP53 reactivation by prima-1, the mechanism remains largely unknown. Several factors, including hypoxic conditions and proteins present within and surrounding the cell, can affect the sensitivity of cancer cells to prima-1 (Rieber and Strasberg-Rieber 2012; Peng et al. 2013). Similarly, TP53 expression can be altered by HSP90 inhibitors (such as CUDC-305) and thus could potentially be beneficial for TP53 mutant patients (Lin et al. 2008).
DISCUSSION
In this study, we have presented a case of an 11-yr-old male with fusion negative ARMS bearing germline TP53 mutations, one of which (c.514G > T, p.Val172Phe) is a hereditary mutation likely predisposing to LFS. The index case patient presented with a tender left upper arm mass and no injury was initially noted. Tissue injury is widely reported to be a preceding factor in sarcoma development, with ∼13% of patients with accidental injury to the extremities at risk of developing sarcoma (Olsson and Wagner 2017), likely as a result of activation of satellite cells to generate muscle precursor cells to repair the skeletal muscle (Shadrach and Wagers 2011). We suspect that the prior supracondylar fracture of the distal humerus was a preceding factor that potentially promoted sarcoma development in conjunction with germline TP53 mutations.
Our genomic findings were consistent with previous genetic findings on PFN ARMS cases, including overexpression of FGFR4, CCND2, and IGF2 and significantly higher rates of nonsynonymous somatic mutations than the PAX-gene fusion positive tumors (PFP) (17.8 vs. 6.4, P = 0.0002) (Shern et al. 2014). Whole-exome sequencing identified 109 nonsynonymous mutations for this case (∼3.6 mutations/MB), most consistent with a PFN genotype diagnosed at a later age. No somatic mutations in genes commonly mutated in PFN tumors were found in this case, including TP53, HRAS, KRAS, NRAS, FGFR4, PIK3CA, NF1, FBXW7, and BCOR (Stratton et al. 1989; Taylor et al. 2000, 2009; Shern et al. 2014). However, missense mutations were found in both TP53 and FGFR4 in the germline, as well as a nonsense-mediated decay mutation in NF1.
The biological significance of the FGFR4 germline variants and their contribution to sarcoma initiation, growth, and progression are largely unknown. Several previous studies have focused on the role of FGFR4 in RMS as a regulator of myogenic differentiation and muscle regeneration after injury. Somatic mutations in FGFR4 have been shown to contribute to the growth and metastasis of RMS, higher expression of FGFR4 correlates with poor survival, and FGFR4 suppresses phospho-Akt in RMS (Taylor et al. 2009). Germline variants in FGFR4 may play a similar role in promoting the invasion and metastasis of RMS.
The index case patient was treated with several targeted therapies, including temsirolimus (mTOR inhibitor), prexasertib (CHEK1 inhibitor), MK01775 (WEE1 inhibitor), pazopanib (multikinase inhibitor specific for VEGFR, PDGFR, c-KIT and FGFR), vorinostat (HDAC inhibitor), and palbociclib (CDK4/6 inhibitor). Functional testing of same or similar agents resulted in mixed concordance between in vitro response and clinical response. Patient tumor cells were highly sensitive to MK-1775, whereas clinical use resulted in continued disease progression. Efficacy of WEE1 inhibition may have differed from the cells that were harvested from intrapleural fluid because of host factors (e.g., drug metabolism) (HogenEsch and Nikitin 2012). HDAC inhibitor vorinostat was also provided to the patient with limited response, although multiple HDAC inhibitors (panobinostat, CUDC-907, and entinostat) were sensitive in vitro, whereas one HDAC inhibitor (CUDC-101) was not sensitive in vitro. CHEK1 inhibitor prexasertib was also used to treat the patient but did not suppress the patient's tumor growth, reflecting in vitro response of prexasertib. mTOR inhibitor temsirolimus (mTOR inhibitor) plus vinorelbine and cyclophosphamide stabilized the patient's disease. Two mTOR-associated agents (INK128 and BKM120) caused reduction in cell viability in vitro, whereas one mTOR agent (sirolimus) had limited in vitro efficacy.
TP53 interacting agents (prima-1 directly and CUDC-305 operating through HSP90) were also promising in vitro. Treatment with prima-1 refolds TP53 in mutated cells into the wild-type conformation and restores sequence-specific DNA binding (Zhang et al. 2018). Other therapies that restore TP53 function, such as reactivating p53 and inducing tumor apoptosis (RITA), may merit investigation for PFN ARMS. Additionally, repression of TP53 is thought to be controlled by the TP53-p21-DREAM-E2F/CHR pathway, which regulates FAM83D (Engeland 2018). Future studies should aim to further understand how germline variants in FAM83D coupled with TP53 germline mutations contribute to sarcoma initiation, growth, and progression. Understanding the biological and clinical significance of FAM83D could have predictive and prognostic implications across several cancer types and may be relevant for the index case patient, given FAM83D aberrations present in patient's germline.
In summary, we have reported a PFN ARMS case with an inherited likely pathogenic germline TP53 mutation. The patient was resistant to several targeted therapies, demonstrating a need for improving our understanding of the etiology and pathogenesis of PFN ARMS and discovering effective therapeutic approaches with limited treatment-related toxicities. In this case study, several novel mutations were reported and potential therapeutic interventions were identified for further study to motivate preclinical and clinical studies that aim to improve survival for fusion-negative RMS.
METHODS
Primary Cell Culture
Approximately 2 L of pleural fluid was collected from a chest drain catheter and immediately chilled on ice. Twenty-four hours after initial collection, the fluid was transferred to 50-mL conical vials and centrifuged at 1200 rpm for 10 min. Resulting cell pellets were broken up with Gibco RPMI 1640 media and checked for viability with 0.4% trypan blue stain, then counted on a Life Technologies Countess II cell counter. Three million viable cells were used for immediate drug screening. Remaining cells were added to 15-cm tissue culture plates containing Gibco RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The plates were placed in a water-jacketed 37°C incubator with 5% CO2. After 72 h, the media was changed and the remaining cellular debris was removed. The semiadherent cell culture continued to expand for a minimum of 18 passages. Every two to five passages, stocks were collected in cryovials containing an FBS/10% DMSO cryopreservation media and then slow cooled and stored in liquid nitrogen for future use.
Drug Screen
Cells from culture were counted, resuspended in media, and plated into white-walled 384-well plates containing prediluted known concentrations of various drugs. After 72 h, an equal volume of Cell-Titer Glo 2.0 (G9243, Promega) was added to each well. The cells were then incubated at room temperature while rocking in the dark for 15 min. Cell viability was measured by luminescence with the BioTek Synergy HT plate reader (BioTek). EC50 values were calculated using GraphPad Prism.
Whole-Exome Sequencing
Tumor and matched normal exome sequencing data were analyzed for the presence of somatic point mutation, somatic functional and structural mutations, potential germline mutations, polynucleotide insertions and deletions, and gene copy-number variation. Somatic mutations, variations, and indels were called using Genome Analysis Toolkit (GATK) Version 4.0 with strict calling criterion (Tumor logarithm, of odd [TLOD] scores above 6.3) with the GRCh38 human reference genome. Gene copy-number variations were identified using SAMtools and VarScan2 quantified as a log ratio of tumor copy to normal copy. Regions with a log ratio > 0.40 were called as gained, regions with a log ratio < −0.40 were called as lost. Genes overlapping gained or lost regions by at least 15% of the gene's genomic region were called as gained or lost, respectively. Sequencing coverage is provided in Supplemental Table 5.
RNA Sequencing
RNA sequencing data was analyzed for gene expression and gene fusion events. Transcriptome data was aligned to STAR-derived human transcriptome from GRCh38 human reference genome. Normalized gene expression was quantified using RSEM. Nontumor gene expression data was not provided, thus region-specific, skeletal muscle tissue, gene expression data was accessed from the GTEx project to serve as a population normal and to identify under expressed and overexpressed genes. Gene fusion events were identified using STAR-Fusion (Haas et al. 2019) and FusionCatcher (Nicorici et al. 2014) to identify transcriptome reads around the junction of fused genes and identify probable gene fusion events. Both fusion analyses were also performed using the GRCh38 human reference genome. Sequencing coverage is provided in Supplemental Table 5.
Hierarchical Clustering
The hierarchical clustering analysis approach was published previously (Ricker et al. 2020). Briefly, CF-00449 sequencing data was clustered among a cohort of RMS samples collected from multiple sources. Unsupervised hierarchical clustering was performed using average-linkage clustering and Euclidean distance metric. Hierarchical clustering analysis was performed in RStudio Version 3.6.2.
Circos Plots
Circos plots to visualize the collection of genomics and transcriptomics data were generated using tumor DNA exome, normal DNA exome, and tumor RNA sequencing data. Genes are identified as bearing variants, having increased gene copy-number, and being overexpressed. Also identified in blue are genes involved in identified gene fusion events, and genes identified as being of interest because of overarching genetic and transcriptomic features in the tumor samples. (See Fig. 3.)
ADDITIONAL INFORMATION
Data Deposition and Access
All raw data was deposited to our CuReFAST database. DNA and RNA sequencing data from the patient's sample are available through the European Genome-Phenome Archive (EGA) under accession ID EGAS00001004828. For hierarchical clustering analysis, data from the Gene Expression Omnibus (GSM758578, GSE138269, GSM984615), EGA (EGAS00001003981), and dbGaP (accession number phs001121.v1.p1) were used. Champions Oncology data were received from the Champions TumorGraft database. Genomics data from PDX models from The Jackson Laboratory are available through the Mouse Tumor Biology Database (http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).
Ethics Statement
The genetic studies and publication of clinical details was approved by the Children's Cancer Therapy Development Institute Institutional Review Board (IRB) and with full written informed consent obtained from parents of the proband.
Acknowledgments
We are tremendously grateful for all the patients who kindly donated their tumor tissue as part of our CuReFAST initiative.
Author Contributions
C.K. and C.A.R. designed the study. C.K., C.A.R., A.W., A.M., E.R.R., M.L., and N.E.B. collected and analyzed the data. G.S., R.G.I., and W.S. contributed materials. C.K., C.A.R., A.D.W., and N.E.B. wrote the manuscript.
Funding
This work was supported through Building Blocks to a Cure (1:1 match) crowdfunding campaign on Consano (https://consano.org/projects/building-blocks-to-a-cure-11-match/), the Children's Cancer Project, and the Sam Day Foundation.
Competing Interest Statement
The authors have declared no competing interest.
Supplementary Material
Supplemental Material
[Supplemental material is available for this article.] | UNKNOWN | DrugDosageText | CC BY-NC | 33436392 | 19,875,540 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Immune reconstitution inflammatory syndrome'. | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | CARBAMAZEPINE, FESOTERODINE, LAMOTRIGINE, LEVETIRACETAM, NATALIZUMAB, PHENOBARBITAL, PYRIDOXINE, VITAMIN D NOS | DrugsGivenReaction | CC BY | 33437143 | 18,783,981 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Progressive multifocal leukoencephalopathy'. | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | CARBAMAZEPINE, FESOTERODINE, LAMOTRIGINE, LEVETIRACETAM, NATALIZUMAB, PHENOBARBITAL, PYRIDOXINE, VITAMIN D NOS | DrugsGivenReaction | CC BY | 33437143 | 18,783,981 | 2021 |
What was the administration route of drug 'NATALIZUMAB'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY | 33437143 | 18,783,981 | 2021 |
What was the dosage of drug 'CARBAMAZEPINE'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 700 MG/ DAY | DrugDosageText | CC BY | 33437143 | 18,783,981 | 2021 |
What was the dosage of drug 'LAMOTRIGINE'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 600 MG/ DAY | DrugDosageText | CC BY | 33437143 | 18,783,981 | 2021 |
What was the dosage of drug 'LEVETIRACETAM'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 4 G/DAY | DrugDosageText | CC BY | 33437143 | 18,783,981 | 2021 |
What was the dosage of drug 'NATALIZUMAB'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 107 INFUSIONS | DrugDosageText | CC BY | 33437143 | 18,783,981 | 2021 |
What was the dosage of drug 'PHENOBARBITAL'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 105 MG/ DAY | DrugDosageText | CC BY | 33437143 | 18,783,981 | 2021 |
What was the dosage of drug 'VITAMIN D NOS'? | Natalizumab-associated progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS): "a case report from Ireland with review of literature, clinical pitfalls and future direction".
Progressive multifocal leukoencephalopathy (PML) is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS). PML is a serious complication of MS treatment which is most commonly related to natalizumab.
We report clinical course of progressive multifocal leukoencephalopathy (PML) in a 40-year-old man who was on treatment for highly active relapsing-remitting multiple sclerosis with natalizumab (Nz). He was treated with steroids, cidofovir, and mirtazapine and went on to develop long-term disability. The case describes the evolution of PML from diagnosis up till 5 months with changes on sequential brain scans and clinical symptoms in our patient.
Patients who are on natalizumab should be aware and consented for the risk of PML. They should be periodically re-assessed for their relative PML risk. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Background
PML is a serious demyelinating disorder that is caused by John Cunningham virus (JCV) due to its reactivation in certain immune states in different disease conditions. It is exclusively a disease of immunosuppressed individuals, and various associations include malignancy, HIV infection, organ transplantation, and autoimmune disorders [1–3].
The antibodies to JCV are prevalent in almost 86% of adults after an initial asymptomatic infection with JCV in childhood [4]. The latent virus usually stays in the kidneys and lymphoid organs, but cellular immunosuppression can cause its reactivation. Viral replication under immunomodulation leads to production of neurotropic variants that can replicate in glial cells [1].
PML should be suspected in patients with new neurological symptoms or deterioration who are on immunomodulation. The diagnostic modalities that help establish the diagnosis include MRI-brain and cerebrospinal fluid analysis for the presence of JCV DNA by PCR.
Pembrolizumab and nivolumab are newer checkpoint inhibitors that have shown some good outcomes in small selected number of PML patients although larger studies are needed to establish their clinical efficacy [5–7].
PML is one of the most serious treatment-related complications that is encountered in patients with multiple sclerosis (MS) [8]. It poses a significant risk of long-term disability and mortality.
Natalizumab has been conventionally associated with PML in MS patients; however, other disease-modifying therapies which pose PML risk include rituximab, fingolimod, dimethyl-fumarate [9–15].
Case presentation
We report a case of a 40-year-old man who presented with 7 days history of visual disturbance, leg weakness, pain, and unsteadiness. He reported seeing “spots” in his visual fields, double vision, and some retro-ocular pain. He described burning pain in his right thigh and proximal weakness of his right leg which restricted his daily activities. However, he was fully independent on presentation.
He had a medical history of relapsing-remitting multiple sclerosis for the last 15 years for which he was currently on monthly natalizumab for the last 9 years and received a total of 107 infusions over this period. Past disease-modifying therapies for multiple sclerosis (MS) included beta interferon and glatiramer acetate which were switched due to recurrent relapses. His serology for John Cunningham virus (JCV) was positive since 2012 as shown in Fig. 1. Fig. 1 JCV serology index (2012–2014)
He also had epilepsy for last 10 years, with a seizure semiology of partial with secondary generalized tonic-clonic seizures. He was on 4 anti-epileptics: levetiracetam 4gm/day, lamotrigine 600 mg/day, carbamazepine 700 mg/day, phenobarbitone 105 mg/day. The other regular medications included fesoterodine 8 mg, pyridoxine 100 mg, and vitamin D 2400 units daily.
His pertinent examination findings were horizontal nystagmus with diplopia in central and right gaze without any restriction in the eye movements. The right leg had reduced power grade in hip adduction, abduction, and flexion 4/5. His reflexes in bilateral lower limbs were 3+ with bilateral extensor plantar responses. There was past pointing on the right finger to nose test.
It was suspected that his current symptoms may be due to MS relapse. He was started on intravenous methyl prednisolone 1 g/day, and MRI brain was done as shown in Fig. 2 (day 1). The MRI brain prompted further investigation due to the atypical location and radiological features of the lesion including a lumbar puncture and CSF analysis for JCV-DNA PCR. Results received 3 days later showed a JCV viral load of 6620 copies/ml. Another lumbar puncture was done to further confirm the results 5 days that showed a viral load of 48,300 copies/ml. Fig. 2 Showing MRI brain axial sequences FLAIR/T2/T1 and T1 with contrast at various stages. Day 1, new non-enhancing cerebellar hemisphere T2 hyperintensities (larger on the right). The new ill-defined extensive T2 hyperintensity in the right middle cerebellar peduncle extending into the right cerebellar white matter is felt concerning for PML. Day 16, no pathological enhancement identified. The previously noted new high signal in the right cerebellar peduncle extending into the right cerebellar hemisphere is slightly more extensive than on the prior scan but no enhancement. Day 48, progression of right cerebellar and brainstem high signal and enhancement. Progression of the presumed PML in the right middle cerebellar peduncle extending into the right cerebellar hemisphere and now extending to the right side of the pons. Day 86, area of abnormal T2 high signal within the right middle cerebellar peduncle extending into the pons and right cerebellum is again demonstrated. There is increased T2 signal at the periphery of the lesion within the right cerebellar hemisphere, and involvement of the pons is also significantly increased, now extending to involve the central pons. New area of T2 low signal within the medial left cerebellar hemisphere and in the left cerebellum adjacent to the inferior left cerebellar peduncle faint peripheral enhancement in the area of increased FLAIR signal in the right cerebellar hemisphere. Faint enhancement within the peripheral aspect of the right cerebellar lesion suggestive of PML-IRIS. MRI findings are consistent with progression of disease with progressive involvement of the pons and new involvement of the left cerebellar hemisphere. Day 125, significant progression of high signal abnormality in the posterior fossa as described, now extending to the left of the midline associated with extensive patchy enhancement. The appearances would now be more in keeping with PML-IRIS. (These MRI-images are acquired through 1.5 T Machine)
He was started on mirtazapine at 60 mg daily doses. Cidofovir 5 mg/kg per dose, two doses 1 week apart followed by fortnightly doses a total of 6 doses over 12 weeks were administered. Mefloquine was considered but due to the potential of causing seizures in our patient was not commenced.
The clinical course is summarized in Table 1 over a 6 months period from PML onset to his transfer to a rehabilitation facility. Over this course, he deteriorated from being functionally independent to being wheel chair bound. There was a major deterioration in weeks 7–15 before his clinical condition stabilized and later improved with a major morbidity. Table 1 Disease course from onset to the final outcome
Hospital course Event Clinical features Disease evolution on MRI brain Treatment Outcomes
Day 1 Visual disturbance, leg weakness, pain, and unsteadiness • Right-sided hip weakness
• Ataxia
• Diplopia and nystagmus
PMLx related changes in cerebellum Methyl prednisolone 5 days, cidofovir, mirtazapine, probenecid deterioration
Day 16 Headaches, worsening blurry vision, and diplopia • Symptoms improved next 2 days
• Mobility slightly improved
PML-related changes slightly more extensive No changes made Stable
Day 48 Prolonged generalized tonic-clonic seizure which lasted almost 50-min • Decreased consciousness and right-sided Todd’s paresis.
• Mechanical ventilation, ICU admission
Progression of PML Loaded with phenobarbitone, methyl prednisolone 5 days, and oral taper over next 2 weeks Deterioration
Day 86 Worsened swallow, weak cough reflex, and difficulty clearing secretions, requiring suctioning • Significant dysarthria
• Right hand weakness and impaired coordination
• Gradually worsening swallow and gait, now using wheelchair
• Bladder dysfunction
Progression of PML changes and features suggestive of worsening PML- and onset of IRISxx Started on 5/7 course of IV immunoglobulins, antibiotics for recurrent aspirations, PEG tube inserted for feeding Deterioration
Day 125 Seizure, eye flickering, and unresponsiveness, self-aborted less than a minute • Nausea and vomiting
• Dysarthria started improving
• Truncal ataxia, completely wheel chair bound
Significant progression of PML and IRIS IV methyl prednisolone 1 g for 5 days given followed by oral prednisolone taper over 2 weeks Improvement
Day 158 Continued to improve mobility, still limited to wheelchair but sitting balance significantly improved • Speech became clearer
• Truncal ataxia improved
• Swallowing improved
MRI not done Transferred for long-term rehabilitation, stable at this point Improvement
xProgressive multifocal leukoencephalopathy
xxImmune reconstitution inflammatory syndrome
Discussion
The risk of PML in patients who are on natalizumab have been linked to the number of infusions and the presence of anti-JCV in the serum [16]. The risk has been estimated to be less than 0.07 in 1000 patients who are anti-JCV negative, 1.7% in those who were anti-JCV positive, and 2.7% in those who were anti-JCV positive with previous immunosuppression use over a 6-year cumulative period (72 infusions of natalizumab) [16].
The current national guidelines in Ireland for checking JCV status in serum on patients who are to be started on natalizumab include baseline antibody test before commencing treatment and in those who are negative to be repeated 6-monthly [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf]. Patients who are anti-JCV positive with a titer < 1.5 units, the serological tests are repeated 6-monthly. However, in those with a titer more than 1.5, no further serological testing is recommended [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
Neuroimaging with MRI brain plain is recommended within 3 months before commencing treatment and within 6 months after being on natalizumab. Annual MRI brain are repeated in JCV negative patients, 6-monthly in those with a JCV titer of < 1.5, and 4-monthly in those with a JCV > 1.5 [https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management-programme/ndmp-natalizumab-protocol.pdf].
The definite diagnosis requires brain biopsy [19]. The neuroimaging findings coupled with a positive cerebrospinal fluid for JCV-DNA PCR presence is also diagnostic [18]. The characteristic MRI brain features include hyperintense lesions on FLAIR and T2 sequences and hypointense on T1 with or without contrast enhancements and minimal oedema [17].
Immune reconstitution inflammatory syndrome (IRIS) occurs in around 70% of patients who are treated with plasma exchange (PLEX) [18]. Case series of 42 patients with PML due to Nz reported early immunologic rebound may be accelerated due to PLEX which has a poor prognostic value in survival [19]. High-dose steroids are used for counteracting the severe immune response which can pose a serious risk and poor prognosis [20]. For these reasons, we in our patient decided not to use plasma exchange because of his highly active disease activity in the past and likelihood of an accelerated immune response and a poorer outcome.
Cidofovir has been linked to mitigating IRIS rather affecting the JCV directly that has been thought to benefit these patients [21]. Mirtazapine through its effect on serotonergic 5HT2A sites on glial cells which are linked to JCV pathogenesis might also have some benefit [22].
Typically, those with a fatal outcome related to natalizumab-associated PML-IRIS die within 6 months [23]
Conclusion
PML is a serious complication of MS treatment which is most commonly related to natalizumab. Clinical features, neuroimaging findings, and cerebrospinal fluid are required to diagnose the condition. Patients who are on natalizumab should be aware and consented for the risk of PML. They should periodically be re-assessed for their relative PML risk; explanation and consenting in those who are on natalizumab with a higher risk should be taken into account for their MS management. There is a growing body of evidence that suggests switching patients from natalizumab who have a higher risk of PML to other safer treatment options.
Future guidelines relating to natalizumab in MS should consider taking into account the following: Assessment of the cumulative risk of PML beyond 6 years and risk stratification for those with a high titer of JCV in serum beyond 1.5 and more than 6 years on natalizumab.
Careful consideration in selecting safer MS therapies in patients who are at a higher risk of PML and potential risks of MS relapse during switching therapies.
Reinstitution and selection of MS treatment in those with PML and high MS activity.
Abbreviations
PML Progressive multifocal leukoencephalopathy
Nz Natalizumab
DNA Deoxyribonucleic acid
PCR Polymerase chain reaction
IRIS Immune reconstitution inflammatory syndrome
MS Multiple sclerosis
JCV John Cunningham virus
Acknowledgements
We acknowledge the continuous support and encouragement of our medicine department at Sligo University Hospital.
Authors’ contributions
SM, GM, HJA, SK, and KM were involved in writing of manuscript, diagnosing, and treating the patient. Rest of the authors AW and GMP were involved in care and treatment of the patient. All authors have read and approved the manuscript.
Funding
All authors declare receiving no funding for this case report.
Availability of data and materials
All the data available has been shared in this case report.
Ethics approval and consent to participate
Ethical approval from ERB at Sligo University Hospital is not applicable in case reports, and written consent was obtained.
Consent for publication
Written and informed consent is obtained for this case report from the patient.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 2400 UNITS/DAY | DrugDosageText | CC BY | 33437143 | 18,783,981 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'. | A case of resected pulmonary lymphomatoid granulomatosis.
Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder and was incorporated into the WHO classification of Tumours of the Lung, Pleura, Thymus and Heart in 2015. LYG is known to be associated with the host's immune function, and can be caused by some immunosuppressive agents, including methotrexate. A woman in her sixties with an 18-year history of methotrexate treatment for rheumatoid arthritis visited our hospital after detection of an abnormal chest shadow on her radiograph. She had been having anemia and a slight fever. Computed tomography (CT) revealed a 2.9-cm sized nodule in her left lung and hilar adenopathy, which suggested a primary lung carcinoma or an inflammatory lesion. We performed a left upper lobectomy with lymph node dissection for the purpose of diagnosis and treatment. Pathologic findings revealed that the tumor was a grade 3 LYG based on the number of EBV-positive B cells. The patient was treated with two chemotherapy regimens including R-CHOP at another hospital, and survived for four years after resection without recurrence in the lung. It is rare to find a case resected LYG, and the clinical or pathological findings of our case are expected to be extremely helpful in studying this disease and improving the understanding of this disease.
1 Introduction
Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder driven by the Epstein-Barr virus (EBV) and often develops as a methotrexate-associated lymphoproliferative disorder. LYG involves extra-nodal sites including the lungs, skin, central nervous system, liver, and kidneys. When it affects a single lung nodule, it could be difficult to distinguish from other lung nodules, including primary lung carcinoma or other granulomatous lesions in the lung on imaging. We should carefully survey patients’ symptoms or prescription history to make an accurate diagnosis of LYG.
2 Case presentation
A woman in her sixties, found out to have anemia, was referred to our hospital. She had been suffering from rheumatoid arthritis for 18 years and was on methotrexate prescribed by her doctor. No source of bleeding was identified by endoscopic examination. On a chest radiograph taken a month after her first visit, an abnormal shadow in her left lung was noted. She revisited our hospital and was admitted in the department of respiratory medicine for a detailed examination. On admission, she had a slight fever of 37 °C and complained of drenching night sweats. Physical examination revealed the following: body temperature, 36.3 °C (37.6 °C at night); heart rate, 85 beats per minute; blood pressure, 142/96 mmHg; and no rale, was noted. She had never smoked. Laboratory data included: RBC, 4.40 × 106/μL; hemoglobin, 11.3 g/dL; lactate dehydrogenase (LDH) (reference value), 251 (120–240) IU/L; C-reactive protein, 3.51 g/dL; soluble interleukin-2 (IL-2) receptor (reference value), 1320 (145–516) U/mL. Her chest X-ray and computed tomography (CT) showed a solid 2.9 × 2.7-cm sized nodule in the middle of her left upper lobe (Fig. 1). The tumor size increased within a few months. Ipsilateral hilar adenopathy was also seen. We performed bronchoscopy with both exfoliative and lavage cytology, and neither of them indicated malignant findings, lung carcinoma or other malignancies were suggested based on these imaging findings. Although it may not have been malignant, some inflammatory diseases such as mycosis or granuloma could have caused her symptoms. Given the appearance, we made a decision to perform surgery with excision on her lung and performed left upper lobectomy with lymph node dissection for the purposes of diagnosis and treatment. The resected specimen was an irregular, solid, white nodule with extensive yellow necrotic areas in the middle of the lobe (Fig. 2). Histopathological examination showed marked obstruction of vessels with extensive fibrosis around vessels. Severe angiocentric and angiodestructive infiltration of lymphocytes including reactive T-cells were also seen. There were variable numbers of larger atypical cells with large nucleoli, similar to Hodgkin cells, and some multi-nucleated giant cells around the fibrosis (Fig. 3). Immunohistochemical findings showed that the Hodgkin-like cells were positive for CD30, and also for LMP1, a marker for EBV. EBV was also confirmed by in situ hybridization (Fig. 4). Other immunohistochemical staining showed that the atypical cells were positive for CD20 and CD79a focally and negative for EML4-ALK fusion protein. The tumor was diagnosed as grade 3 lymphomatoid granulomatosis. Two of the 55 lymph nodes, one in subaortic (#5) and the other in lober (#12) lymph node, also had the same lesion with extensive necrosis and eosinophilic infiltration. Even after surgery, she had been suffering from a slight fever, loss of appetite, weight loss, and pleural/peritoneal effusion. An increase in the soluble IL-2 receptor level of 1320 U/mL was also seen. She was transferred to the hematology department of another hospital to receive chemotherapy treatment. She was administered two courses of R–CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) therapy; however, she abandoned it because of a poor response seen on the swollen lymph nodes throughout the body and soluble IL-2 receptor level, and myelosuppression caused by the agents. Alternatively, she received 20 courses of molecular-target therapy with brentuximab vedotin, an antibody-drug targeting CD30-positive Hodgkin lymphoma and systemic anaplastic large cell lymphoma, although it was not approved as a treatment of DLBCL. She has been carefully followed up at the hospital, and no recurrence in the lung has been noticed four years after the resection.Fig. 1 Chest radiography (A) and CT (B) images revealing a solid mass with the tendency to increase in the left upper lobe. The enlarged hilar lymph node is also seen.
Fig. 1Fig. 2 Partly lobulated white nodule observed in the resected lobe.
Fig. 2Fig. 3 Histopathological examination revealing central necrosis (A), a proliferation of spindle-shaped cells (B), angiocentric and angiodestructive lymphoid infiltration (C), atypical cells with an irregular nucleus, and multinucleated giant cells (D).
Fig. 3Fig. 4 Atypical cells positive for LMP1, a marker for EBV (A). More than fifty cells are positive for EBV, as assessed by in situ hybridization, per high-power field (B).
Fig. 4
3 Discussion
Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency that mainly occurs in patients with RA who are treated with long-term methotrexate therapy [1,2]. MTX-LPD was initially reported by Ellman in 1991 [3], was increasingly reported as MTX became a common therapy for RA, and has been listed as one of the serious complications in the MTX treatment guidelines in Japan. The LPD incidence ratio of RA patients is two to four fold in the control healthy population irrespective of MTX treatment [4]. MTX-LPD is categorized as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) according to the 2008 and WHO classification [5]. The pathological features of MTX-LPD vary; diffuse large B-cell lymphoma (DLBCL) type is most predominant (35–60%) followed by Hodgkin lymphoma (HL) type (12–25%) [4]. The positive rate for EBV is about 40%, especially high (70%) in HL type [4]. Making a diagnosis of MTX-LPD only by its histological findings is supposed to be extremely difficult considering that there are various histological types of this disease. Unlike other lymphoproliferative disorders, MTX-LPD often involves extra-nodal organs, including the lung, skin, pharynx/tonsil, and soft tissues. Approximately 40–70% of patients with MTX-LPD regress after MTX withdrawal [1], so the first step is the withdrawal of MTX [5]. Meanwhile, relapse/regrowth events (RRE) after regression are seen in some cases of MTX-LPD. Tokuhira et al. reported that MTX-LPD presents three patterns after methotrexate withdrawal to clarify the clinical management of MTX-LPD [5]. A regressive group (R-G) includes patients with LPD regression after immunosuppressive drugs (ISDs) withdrawal without RRE, a relapse/regrowth group (R/R-G) includes patients with LPD regression after ISD withdrawal with RRE, and a persistent group (P-G) includes patients with persistent LPD after ISD withdrawal. Patients in R/R-G and P-G require additional chemotherapy. They also reported that the serum C-reactive protein (CRP), serum soluble IL-2 receptor, and LDH were increased in the P-G group, whereas only CRP and soluble IL-2 receptor were increased in the R/R-G group. In our case, not only swollen lymph nodes but also the level of soluble IL-2 receptor did not regress after MTX withdrawal. This indicates that the patient belongs to the P-G group and requires chemotherapy based on diffuse large B-cell lymphoma (DLBCL), which is the commonest type of MTX-LPD [11].
Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus-driven lymphoproliferative disorder [6] that has unique histopathologic and clinical features [7]. LYG was initially incorporated into the World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues in 2001 [8] and has been categorized as a distinct mature B-cell neoplasm in the revised version in 2016 [9]. LYG, characterized by angiocentric and angiodestructive features, typically affects middle-aged adults in the fourth to sixth decades of life, commonly involves the lungs (70%) and involves multiple organs, including the central nervous system (40%), skin (34%), kidney (19%), liver (17%), spleen (10%), lymph nodes (6%), and others (19%) [7,10]. The most common radiographic feature of the lung lesion of LYG is multiple nodules, occurring in approximately 80% of all cases [11,12]. Single lung nodules rarely appear in LYG patients and are used to discriminate other diseases, such as lung cancer, primary pulmonary lymphoma, and other granulomatous lung lesions such as sarcoidosis, tuberculous or non-tuberculous mycobacterial infection, and mycosis.
In our case of MTX-induced LYG, the patient had a history of long-term MTX therapy, and a growing single lung nodule and hilar lymph node were observed on preoperative CT findings. She was initially suspected to have lung cancer. Her clinical findings with slight fever or the history of methotrexate intake could help establish an accurate diagnosis of the disease. The postoperative CT findings showed multiple swollen lymph nodes. Furthermore, pathological findings also showed obvious infiltration of tumor cells in the hilar/mediastinal lymph nodes. These features were also typical for MTX-induced LYG.
The chemotherapeutic regimen for LYG, including MTX-LPG/MTX-DLBCL, has not been standardized as it is a rare disease. Chaves et al. reported that approximately three-quarters of patients with LYG (grade 3 in 45%) treated with rituximab-based therapies, mainly R–CHOP achieved a response [13]. Another study reported that the overall response rate in patients to combination chemotherapy with DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) in patients with high-grade LYG was 77% with 41% complete response [2,14]. In our case, the main lung lesion and hilar/mediastinal lymph nodes were removed at the time of surgery; however, serum soluble IL-2 receptor did not respond to R–CHOP therapy. The patient consequently required another chemotherapy regimen and had survived without recurrence.
In conclusion, LYG is not a common disease to thoracic surgeons, but we should consider it as a differential diagnosis in patients with a single lung nodule or multiple lung nodules and a prescription history of immunosuppressive agents, including methotrexate. The clinical and pathological findings of our case are expected to help acquire an easier diagnosis and a deeper understanding of LYG as one of MTX-LPD.
Declaration of competing interest
All authors declare no potential conflict of interest. | BRENTUXIMAB VEDOTIN, CYCLOPHOSPHAMIDE, DOXORUBICIN, METHOTREXATE, PREDNISOLONE, RITUXIMAB, VINCRISTINE | DrugsGivenReaction | CC BY-NC-ND | 33437613 | 19,358,394 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Therapeutic product effect incomplete'. | A case of resected pulmonary lymphomatoid granulomatosis.
Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder and was incorporated into the WHO classification of Tumours of the Lung, Pleura, Thymus and Heart in 2015. LYG is known to be associated with the host's immune function, and can be caused by some immunosuppressive agents, including methotrexate. A woman in her sixties with an 18-year history of methotrexate treatment for rheumatoid arthritis visited our hospital after detection of an abnormal chest shadow on her radiograph. She had been having anemia and a slight fever. Computed tomography (CT) revealed a 2.9-cm sized nodule in her left lung and hilar adenopathy, which suggested a primary lung carcinoma or an inflammatory lesion. We performed a left upper lobectomy with lymph node dissection for the purpose of diagnosis and treatment. Pathologic findings revealed that the tumor was a grade 3 LYG based on the number of EBV-positive B cells. The patient was treated with two chemotherapy regimens including R-CHOP at another hospital, and survived for four years after resection without recurrence in the lung. It is rare to find a case resected LYG, and the clinical or pathological findings of our case are expected to be extremely helpful in studying this disease and improving the understanding of this disease.
1 Introduction
Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder driven by the Epstein-Barr virus (EBV) and often develops as a methotrexate-associated lymphoproliferative disorder. LYG involves extra-nodal sites including the lungs, skin, central nervous system, liver, and kidneys. When it affects a single lung nodule, it could be difficult to distinguish from other lung nodules, including primary lung carcinoma or other granulomatous lesions in the lung on imaging. We should carefully survey patients’ symptoms or prescription history to make an accurate diagnosis of LYG.
2 Case presentation
A woman in her sixties, found out to have anemia, was referred to our hospital. She had been suffering from rheumatoid arthritis for 18 years and was on methotrexate prescribed by her doctor. No source of bleeding was identified by endoscopic examination. On a chest radiograph taken a month after her first visit, an abnormal shadow in her left lung was noted. She revisited our hospital and was admitted in the department of respiratory medicine for a detailed examination. On admission, she had a slight fever of 37 °C and complained of drenching night sweats. Physical examination revealed the following: body temperature, 36.3 °C (37.6 °C at night); heart rate, 85 beats per minute; blood pressure, 142/96 mmHg; and no rale, was noted. She had never smoked. Laboratory data included: RBC, 4.40 × 106/μL; hemoglobin, 11.3 g/dL; lactate dehydrogenase (LDH) (reference value), 251 (120–240) IU/L; C-reactive protein, 3.51 g/dL; soluble interleukin-2 (IL-2) receptor (reference value), 1320 (145–516) U/mL. Her chest X-ray and computed tomography (CT) showed a solid 2.9 × 2.7-cm sized nodule in the middle of her left upper lobe (Fig. 1). The tumor size increased within a few months. Ipsilateral hilar adenopathy was also seen. We performed bronchoscopy with both exfoliative and lavage cytology, and neither of them indicated malignant findings, lung carcinoma or other malignancies were suggested based on these imaging findings. Although it may not have been malignant, some inflammatory diseases such as mycosis or granuloma could have caused her symptoms. Given the appearance, we made a decision to perform surgery with excision on her lung and performed left upper lobectomy with lymph node dissection for the purposes of diagnosis and treatment. The resected specimen was an irregular, solid, white nodule with extensive yellow necrotic areas in the middle of the lobe (Fig. 2). Histopathological examination showed marked obstruction of vessels with extensive fibrosis around vessels. Severe angiocentric and angiodestructive infiltration of lymphocytes including reactive T-cells were also seen. There were variable numbers of larger atypical cells with large nucleoli, similar to Hodgkin cells, and some multi-nucleated giant cells around the fibrosis (Fig. 3). Immunohistochemical findings showed that the Hodgkin-like cells were positive for CD30, and also for LMP1, a marker for EBV. EBV was also confirmed by in situ hybridization (Fig. 4). Other immunohistochemical staining showed that the atypical cells were positive for CD20 and CD79a focally and negative for EML4-ALK fusion protein. The tumor was diagnosed as grade 3 lymphomatoid granulomatosis. Two of the 55 lymph nodes, one in subaortic (#5) and the other in lober (#12) lymph node, also had the same lesion with extensive necrosis and eosinophilic infiltration. Even after surgery, she had been suffering from a slight fever, loss of appetite, weight loss, and pleural/peritoneal effusion. An increase in the soluble IL-2 receptor level of 1320 U/mL was also seen. She was transferred to the hematology department of another hospital to receive chemotherapy treatment. She was administered two courses of R–CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) therapy; however, she abandoned it because of a poor response seen on the swollen lymph nodes throughout the body and soluble IL-2 receptor level, and myelosuppression caused by the agents. Alternatively, she received 20 courses of molecular-target therapy with brentuximab vedotin, an antibody-drug targeting CD30-positive Hodgkin lymphoma and systemic anaplastic large cell lymphoma, although it was not approved as a treatment of DLBCL. She has been carefully followed up at the hospital, and no recurrence in the lung has been noticed four years after the resection.Fig. 1 Chest radiography (A) and CT (B) images revealing a solid mass with the tendency to increase in the left upper lobe. The enlarged hilar lymph node is also seen.
Fig. 1Fig. 2 Partly lobulated white nodule observed in the resected lobe.
Fig. 2Fig. 3 Histopathological examination revealing central necrosis (A), a proliferation of spindle-shaped cells (B), angiocentric and angiodestructive lymphoid infiltration (C), atypical cells with an irregular nucleus, and multinucleated giant cells (D).
Fig. 3Fig. 4 Atypical cells positive for LMP1, a marker for EBV (A). More than fifty cells are positive for EBV, as assessed by in situ hybridization, per high-power field (B).
Fig. 4
3 Discussion
Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency that mainly occurs in patients with RA who are treated with long-term methotrexate therapy [1,2]. MTX-LPD was initially reported by Ellman in 1991 [3], was increasingly reported as MTX became a common therapy for RA, and has been listed as one of the serious complications in the MTX treatment guidelines in Japan. The LPD incidence ratio of RA patients is two to four fold in the control healthy population irrespective of MTX treatment [4]. MTX-LPD is categorized as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) according to the 2008 and WHO classification [5]. The pathological features of MTX-LPD vary; diffuse large B-cell lymphoma (DLBCL) type is most predominant (35–60%) followed by Hodgkin lymphoma (HL) type (12–25%) [4]. The positive rate for EBV is about 40%, especially high (70%) in HL type [4]. Making a diagnosis of MTX-LPD only by its histological findings is supposed to be extremely difficult considering that there are various histological types of this disease. Unlike other lymphoproliferative disorders, MTX-LPD often involves extra-nodal organs, including the lung, skin, pharynx/tonsil, and soft tissues. Approximately 40–70% of patients with MTX-LPD regress after MTX withdrawal [1], so the first step is the withdrawal of MTX [5]. Meanwhile, relapse/regrowth events (RRE) after regression are seen in some cases of MTX-LPD. Tokuhira et al. reported that MTX-LPD presents three patterns after methotrexate withdrawal to clarify the clinical management of MTX-LPD [5]. A regressive group (R-G) includes patients with LPD regression after immunosuppressive drugs (ISDs) withdrawal without RRE, a relapse/regrowth group (R/R-G) includes patients with LPD regression after ISD withdrawal with RRE, and a persistent group (P-G) includes patients with persistent LPD after ISD withdrawal. Patients in R/R-G and P-G require additional chemotherapy. They also reported that the serum C-reactive protein (CRP), serum soluble IL-2 receptor, and LDH were increased in the P-G group, whereas only CRP and soluble IL-2 receptor were increased in the R/R-G group. In our case, not only swollen lymph nodes but also the level of soluble IL-2 receptor did not regress after MTX withdrawal. This indicates that the patient belongs to the P-G group and requires chemotherapy based on diffuse large B-cell lymphoma (DLBCL), which is the commonest type of MTX-LPD [11].
Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus-driven lymphoproliferative disorder [6] that has unique histopathologic and clinical features [7]. LYG was initially incorporated into the World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues in 2001 [8] and has been categorized as a distinct mature B-cell neoplasm in the revised version in 2016 [9]. LYG, characterized by angiocentric and angiodestructive features, typically affects middle-aged adults in the fourth to sixth decades of life, commonly involves the lungs (70%) and involves multiple organs, including the central nervous system (40%), skin (34%), kidney (19%), liver (17%), spleen (10%), lymph nodes (6%), and others (19%) [7,10]. The most common radiographic feature of the lung lesion of LYG is multiple nodules, occurring in approximately 80% of all cases [11,12]. Single lung nodules rarely appear in LYG patients and are used to discriminate other diseases, such as lung cancer, primary pulmonary lymphoma, and other granulomatous lung lesions such as sarcoidosis, tuberculous or non-tuberculous mycobacterial infection, and mycosis.
In our case of MTX-induced LYG, the patient had a history of long-term MTX therapy, and a growing single lung nodule and hilar lymph node were observed on preoperative CT findings. She was initially suspected to have lung cancer. Her clinical findings with slight fever or the history of methotrexate intake could help establish an accurate diagnosis of the disease. The postoperative CT findings showed multiple swollen lymph nodes. Furthermore, pathological findings also showed obvious infiltration of tumor cells in the hilar/mediastinal lymph nodes. These features were also typical for MTX-induced LYG.
The chemotherapeutic regimen for LYG, including MTX-LPG/MTX-DLBCL, has not been standardized as it is a rare disease. Chaves et al. reported that approximately three-quarters of patients with LYG (grade 3 in 45%) treated with rituximab-based therapies, mainly R–CHOP achieved a response [13]. Another study reported that the overall response rate in patients to combination chemotherapy with DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) in patients with high-grade LYG was 77% with 41% complete response [2,14]. In our case, the main lung lesion and hilar/mediastinal lymph nodes were removed at the time of surgery; however, serum soluble IL-2 receptor did not respond to R–CHOP therapy. The patient consequently required another chemotherapy regimen and had survived without recurrence.
In conclusion, LYG is not a common disease to thoracic surgeons, but we should consider it as a differential diagnosis in patients with a single lung nodule or multiple lung nodules and a prescription history of immunosuppressive agents, including methotrexate. The clinical and pathological findings of our case are expected to help acquire an easier diagnosis and a deeper understanding of LYG as one of MTX-LPD.
Declaration of competing interest
All authors declare no potential conflict of interest. | CYCLOPHOSPHAMIDE, DOXORUBICIN, METHOTREXATE, PREDNISONE, RITUXIMAB, VINCRISTINE | DrugsGivenReaction | CC BY-NC-ND | 33437613 | 19,357,373 | 2021 |
What was the dosage of drug 'BRENTUXIMAB VEDOTIN'? | A case of resected pulmonary lymphomatoid granulomatosis.
Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder and was incorporated into the WHO classification of Tumours of the Lung, Pleura, Thymus and Heart in 2015. LYG is known to be associated with the host's immune function, and can be caused by some immunosuppressive agents, including methotrexate. A woman in her sixties with an 18-year history of methotrexate treatment for rheumatoid arthritis visited our hospital after detection of an abnormal chest shadow on her radiograph. She had been having anemia and a slight fever. Computed tomography (CT) revealed a 2.9-cm sized nodule in her left lung and hilar adenopathy, which suggested a primary lung carcinoma or an inflammatory lesion. We performed a left upper lobectomy with lymph node dissection for the purpose of diagnosis and treatment. Pathologic findings revealed that the tumor was a grade 3 LYG based on the number of EBV-positive B cells. The patient was treated with two chemotherapy regimens including R-CHOP at another hospital, and survived for four years after resection without recurrence in the lung. It is rare to find a case resected LYG, and the clinical or pathological findings of our case are expected to be extremely helpful in studying this disease and improving the understanding of this disease.
1 Introduction
Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder driven by the Epstein-Barr virus (EBV) and often develops as a methotrexate-associated lymphoproliferative disorder. LYG involves extra-nodal sites including the lungs, skin, central nervous system, liver, and kidneys. When it affects a single lung nodule, it could be difficult to distinguish from other lung nodules, including primary lung carcinoma or other granulomatous lesions in the lung on imaging. We should carefully survey patients’ symptoms or prescription history to make an accurate diagnosis of LYG.
2 Case presentation
A woman in her sixties, found out to have anemia, was referred to our hospital. She had been suffering from rheumatoid arthritis for 18 years and was on methotrexate prescribed by her doctor. No source of bleeding was identified by endoscopic examination. On a chest radiograph taken a month after her first visit, an abnormal shadow in her left lung was noted. She revisited our hospital and was admitted in the department of respiratory medicine for a detailed examination. On admission, she had a slight fever of 37 °C and complained of drenching night sweats. Physical examination revealed the following: body temperature, 36.3 °C (37.6 °C at night); heart rate, 85 beats per minute; blood pressure, 142/96 mmHg; and no rale, was noted. She had never smoked. Laboratory data included: RBC, 4.40 × 106/μL; hemoglobin, 11.3 g/dL; lactate dehydrogenase (LDH) (reference value), 251 (120–240) IU/L; C-reactive protein, 3.51 g/dL; soluble interleukin-2 (IL-2) receptor (reference value), 1320 (145–516) U/mL. Her chest X-ray and computed tomography (CT) showed a solid 2.9 × 2.7-cm sized nodule in the middle of her left upper lobe (Fig. 1). The tumor size increased within a few months. Ipsilateral hilar adenopathy was also seen. We performed bronchoscopy with both exfoliative and lavage cytology, and neither of them indicated malignant findings, lung carcinoma or other malignancies were suggested based on these imaging findings. Although it may not have been malignant, some inflammatory diseases such as mycosis or granuloma could have caused her symptoms. Given the appearance, we made a decision to perform surgery with excision on her lung and performed left upper lobectomy with lymph node dissection for the purposes of diagnosis and treatment. The resected specimen was an irregular, solid, white nodule with extensive yellow necrotic areas in the middle of the lobe (Fig. 2). Histopathological examination showed marked obstruction of vessels with extensive fibrosis around vessels. Severe angiocentric and angiodestructive infiltration of lymphocytes including reactive T-cells were also seen. There were variable numbers of larger atypical cells with large nucleoli, similar to Hodgkin cells, and some multi-nucleated giant cells around the fibrosis (Fig. 3). Immunohistochemical findings showed that the Hodgkin-like cells were positive for CD30, and also for LMP1, a marker for EBV. EBV was also confirmed by in situ hybridization (Fig. 4). Other immunohistochemical staining showed that the atypical cells were positive for CD20 and CD79a focally and negative for EML4-ALK fusion protein. The tumor was diagnosed as grade 3 lymphomatoid granulomatosis. Two of the 55 lymph nodes, one in subaortic (#5) and the other in lober (#12) lymph node, also had the same lesion with extensive necrosis and eosinophilic infiltration. Even after surgery, she had been suffering from a slight fever, loss of appetite, weight loss, and pleural/peritoneal effusion. An increase in the soluble IL-2 receptor level of 1320 U/mL was also seen. She was transferred to the hematology department of another hospital to receive chemotherapy treatment. She was administered two courses of R–CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) therapy; however, she abandoned it because of a poor response seen on the swollen lymph nodes throughout the body and soluble IL-2 receptor level, and myelosuppression caused by the agents. Alternatively, she received 20 courses of molecular-target therapy with brentuximab vedotin, an antibody-drug targeting CD30-positive Hodgkin lymphoma and systemic anaplastic large cell lymphoma, although it was not approved as a treatment of DLBCL. She has been carefully followed up at the hospital, and no recurrence in the lung has been noticed four years after the resection.Fig. 1 Chest radiography (A) and CT (B) images revealing a solid mass with the tendency to increase in the left upper lobe. The enlarged hilar lymph node is also seen.
Fig. 1Fig. 2 Partly lobulated white nodule observed in the resected lobe.
Fig. 2Fig. 3 Histopathological examination revealing central necrosis (A), a proliferation of spindle-shaped cells (B), angiocentric and angiodestructive lymphoid infiltration (C), atypical cells with an irregular nucleus, and multinucleated giant cells (D).
Fig. 3Fig. 4 Atypical cells positive for LMP1, a marker for EBV (A). More than fifty cells are positive for EBV, as assessed by in situ hybridization, per high-power field (B).
Fig. 4
3 Discussion
Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency that mainly occurs in patients with RA who are treated with long-term methotrexate therapy [1,2]. MTX-LPD was initially reported by Ellman in 1991 [3], was increasingly reported as MTX became a common therapy for RA, and has been listed as one of the serious complications in the MTX treatment guidelines in Japan. The LPD incidence ratio of RA patients is two to four fold in the control healthy population irrespective of MTX treatment [4]. MTX-LPD is categorized as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) according to the 2008 and WHO classification [5]. The pathological features of MTX-LPD vary; diffuse large B-cell lymphoma (DLBCL) type is most predominant (35–60%) followed by Hodgkin lymphoma (HL) type (12–25%) [4]. The positive rate for EBV is about 40%, especially high (70%) in HL type [4]. Making a diagnosis of MTX-LPD only by its histological findings is supposed to be extremely difficult considering that there are various histological types of this disease. Unlike other lymphoproliferative disorders, MTX-LPD often involves extra-nodal organs, including the lung, skin, pharynx/tonsil, and soft tissues. Approximately 40–70% of patients with MTX-LPD regress after MTX withdrawal [1], so the first step is the withdrawal of MTX [5]. Meanwhile, relapse/regrowth events (RRE) after regression are seen in some cases of MTX-LPD. Tokuhira et al. reported that MTX-LPD presents three patterns after methotrexate withdrawal to clarify the clinical management of MTX-LPD [5]. A regressive group (R-G) includes patients with LPD regression after immunosuppressive drugs (ISDs) withdrawal without RRE, a relapse/regrowth group (R/R-G) includes patients with LPD regression after ISD withdrawal with RRE, and a persistent group (P-G) includes patients with persistent LPD after ISD withdrawal. Patients in R/R-G and P-G require additional chemotherapy. They also reported that the serum C-reactive protein (CRP), serum soluble IL-2 receptor, and LDH were increased in the P-G group, whereas only CRP and soluble IL-2 receptor were increased in the R/R-G group. In our case, not only swollen lymph nodes but also the level of soluble IL-2 receptor did not regress after MTX withdrawal. This indicates that the patient belongs to the P-G group and requires chemotherapy based on diffuse large B-cell lymphoma (DLBCL), which is the commonest type of MTX-LPD [11].
Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus-driven lymphoproliferative disorder [6] that has unique histopathologic and clinical features [7]. LYG was initially incorporated into the World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues in 2001 [8] and has been categorized as a distinct mature B-cell neoplasm in the revised version in 2016 [9]. LYG, characterized by angiocentric and angiodestructive features, typically affects middle-aged adults in the fourth to sixth decades of life, commonly involves the lungs (70%) and involves multiple organs, including the central nervous system (40%), skin (34%), kidney (19%), liver (17%), spleen (10%), lymph nodes (6%), and others (19%) [7,10]. The most common radiographic feature of the lung lesion of LYG is multiple nodules, occurring in approximately 80% of all cases [11,12]. Single lung nodules rarely appear in LYG patients and are used to discriminate other diseases, such as lung cancer, primary pulmonary lymphoma, and other granulomatous lung lesions such as sarcoidosis, tuberculous or non-tuberculous mycobacterial infection, and mycosis.
In our case of MTX-induced LYG, the patient had a history of long-term MTX therapy, and a growing single lung nodule and hilar lymph node were observed on preoperative CT findings. She was initially suspected to have lung cancer. Her clinical findings with slight fever or the history of methotrexate intake could help establish an accurate diagnosis of the disease. The postoperative CT findings showed multiple swollen lymph nodes. Furthermore, pathological findings also showed obvious infiltration of tumor cells in the hilar/mediastinal lymph nodes. These features were also typical for MTX-induced LYG.
The chemotherapeutic regimen for LYG, including MTX-LPG/MTX-DLBCL, has not been standardized as it is a rare disease. Chaves et al. reported that approximately three-quarters of patients with LYG (grade 3 in 45%) treated with rituximab-based therapies, mainly R–CHOP achieved a response [13]. Another study reported that the overall response rate in patients to combination chemotherapy with DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) in patients with high-grade LYG was 77% with 41% complete response [2,14]. In our case, the main lung lesion and hilar/mediastinal lymph nodes were removed at the time of surgery; however, serum soluble IL-2 receptor did not respond to R–CHOP therapy. The patient consequently required another chemotherapy regimen and had survived without recurrence.
In conclusion, LYG is not a common disease to thoracic surgeons, but we should consider it as a differential diagnosis in patients with a single lung nodule or multiple lung nodules and a prescription history of immunosuppressive agents, including methotrexate. The clinical and pathological findings of our case are expected to help acquire an easier diagnosis and a deeper understanding of LYG as one of MTX-LPD.
Declaration of competing interest
All authors declare no potential conflict of interest. | UNK, 20 COURSES | DrugDosageText | CC BY-NC-ND | 33437613 | 19,358,394 | 2021 |
What was the dosage of drug 'DOXORUBICIN HYDROCHLORIDE'? | A case of resected pulmonary lymphomatoid granulomatosis.
Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder and was incorporated into the WHO classification of Tumours of the Lung, Pleura, Thymus and Heart in 2015. LYG is known to be associated with the host's immune function, and can be caused by some immunosuppressive agents, including methotrexate. A woman in her sixties with an 18-year history of methotrexate treatment for rheumatoid arthritis visited our hospital after detection of an abnormal chest shadow on her radiograph. She had been having anemia and a slight fever. Computed tomography (CT) revealed a 2.9-cm sized nodule in her left lung and hilar adenopathy, which suggested a primary lung carcinoma or an inflammatory lesion. We performed a left upper lobectomy with lymph node dissection for the purpose of diagnosis and treatment. Pathologic findings revealed that the tumor was a grade 3 LYG based on the number of EBV-positive B cells. The patient was treated with two chemotherapy regimens including R-CHOP at another hospital, and survived for four years after resection without recurrence in the lung. It is rare to find a case resected LYG, and the clinical or pathological findings of our case are expected to be extremely helpful in studying this disease and improving the understanding of this disease.
1 Introduction
Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder driven by the Epstein-Barr virus (EBV) and often develops as a methotrexate-associated lymphoproliferative disorder. LYG involves extra-nodal sites including the lungs, skin, central nervous system, liver, and kidneys. When it affects a single lung nodule, it could be difficult to distinguish from other lung nodules, including primary lung carcinoma or other granulomatous lesions in the lung on imaging. We should carefully survey patients’ symptoms or prescription history to make an accurate diagnosis of LYG.
2 Case presentation
A woman in her sixties, found out to have anemia, was referred to our hospital. She had been suffering from rheumatoid arthritis for 18 years and was on methotrexate prescribed by her doctor. No source of bleeding was identified by endoscopic examination. On a chest radiograph taken a month after her first visit, an abnormal shadow in her left lung was noted. She revisited our hospital and was admitted in the department of respiratory medicine for a detailed examination. On admission, she had a slight fever of 37 °C and complained of drenching night sweats. Physical examination revealed the following: body temperature, 36.3 °C (37.6 °C at night); heart rate, 85 beats per minute; blood pressure, 142/96 mmHg; and no rale, was noted. She had never smoked. Laboratory data included: RBC, 4.40 × 106/μL; hemoglobin, 11.3 g/dL; lactate dehydrogenase (LDH) (reference value), 251 (120–240) IU/L; C-reactive protein, 3.51 g/dL; soluble interleukin-2 (IL-2) receptor (reference value), 1320 (145–516) U/mL. Her chest X-ray and computed tomography (CT) showed a solid 2.9 × 2.7-cm sized nodule in the middle of her left upper lobe (Fig. 1). The tumor size increased within a few months. Ipsilateral hilar adenopathy was also seen. We performed bronchoscopy with both exfoliative and lavage cytology, and neither of them indicated malignant findings, lung carcinoma or other malignancies were suggested based on these imaging findings. Although it may not have been malignant, some inflammatory diseases such as mycosis or granuloma could have caused her symptoms. Given the appearance, we made a decision to perform surgery with excision on her lung and performed left upper lobectomy with lymph node dissection for the purposes of diagnosis and treatment. The resected specimen was an irregular, solid, white nodule with extensive yellow necrotic areas in the middle of the lobe (Fig. 2). Histopathological examination showed marked obstruction of vessels with extensive fibrosis around vessels. Severe angiocentric and angiodestructive infiltration of lymphocytes including reactive T-cells were also seen. There were variable numbers of larger atypical cells with large nucleoli, similar to Hodgkin cells, and some multi-nucleated giant cells around the fibrosis (Fig. 3). Immunohistochemical findings showed that the Hodgkin-like cells were positive for CD30, and also for LMP1, a marker for EBV. EBV was also confirmed by in situ hybridization (Fig. 4). Other immunohistochemical staining showed that the atypical cells were positive for CD20 and CD79a focally and negative for EML4-ALK fusion protein. The tumor was diagnosed as grade 3 lymphomatoid granulomatosis. Two of the 55 lymph nodes, one in subaortic (#5) and the other in lober (#12) lymph node, also had the same lesion with extensive necrosis and eosinophilic infiltration. Even after surgery, she had been suffering from a slight fever, loss of appetite, weight loss, and pleural/peritoneal effusion. An increase in the soluble IL-2 receptor level of 1320 U/mL was also seen. She was transferred to the hematology department of another hospital to receive chemotherapy treatment. She was administered two courses of R–CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) therapy; however, she abandoned it because of a poor response seen on the swollen lymph nodes throughout the body and soluble IL-2 receptor level, and myelosuppression caused by the agents. Alternatively, she received 20 courses of molecular-target therapy with brentuximab vedotin, an antibody-drug targeting CD30-positive Hodgkin lymphoma and systemic anaplastic large cell lymphoma, although it was not approved as a treatment of DLBCL. She has been carefully followed up at the hospital, and no recurrence in the lung has been noticed four years after the resection.Fig. 1 Chest radiography (A) and CT (B) images revealing a solid mass with the tendency to increase in the left upper lobe. The enlarged hilar lymph node is also seen.
Fig. 1Fig. 2 Partly lobulated white nodule observed in the resected lobe.
Fig. 2Fig. 3 Histopathological examination revealing central necrosis (A), a proliferation of spindle-shaped cells (B), angiocentric and angiodestructive lymphoid infiltration (C), atypical cells with an irregular nucleus, and multinucleated giant cells (D).
Fig. 3Fig. 4 Atypical cells positive for LMP1, a marker for EBV (A). More than fifty cells are positive for EBV, as assessed by in situ hybridization, per high-power field (B).
Fig. 4
3 Discussion
Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency that mainly occurs in patients with RA who are treated with long-term methotrexate therapy [1,2]. MTX-LPD was initially reported by Ellman in 1991 [3], was increasingly reported as MTX became a common therapy for RA, and has been listed as one of the serious complications in the MTX treatment guidelines in Japan. The LPD incidence ratio of RA patients is two to four fold in the control healthy population irrespective of MTX treatment [4]. MTX-LPD is categorized as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) according to the 2008 and WHO classification [5]. The pathological features of MTX-LPD vary; diffuse large B-cell lymphoma (DLBCL) type is most predominant (35–60%) followed by Hodgkin lymphoma (HL) type (12–25%) [4]. The positive rate for EBV is about 40%, especially high (70%) in HL type [4]. Making a diagnosis of MTX-LPD only by its histological findings is supposed to be extremely difficult considering that there are various histological types of this disease. Unlike other lymphoproliferative disorders, MTX-LPD often involves extra-nodal organs, including the lung, skin, pharynx/tonsil, and soft tissues. Approximately 40–70% of patients with MTX-LPD regress after MTX withdrawal [1], so the first step is the withdrawal of MTX [5]. Meanwhile, relapse/regrowth events (RRE) after regression are seen in some cases of MTX-LPD. Tokuhira et al. reported that MTX-LPD presents three patterns after methotrexate withdrawal to clarify the clinical management of MTX-LPD [5]. A regressive group (R-G) includes patients with LPD regression after immunosuppressive drugs (ISDs) withdrawal without RRE, a relapse/regrowth group (R/R-G) includes patients with LPD regression after ISD withdrawal with RRE, and a persistent group (P-G) includes patients with persistent LPD after ISD withdrawal. Patients in R/R-G and P-G require additional chemotherapy. They also reported that the serum C-reactive protein (CRP), serum soluble IL-2 receptor, and LDH were increased in the P-G group, whereas only CRP and soluble IL-2 receptor were increased in the R/R-G group. In our case, not only swollen lymph nodes but also the level of soluble IL-2 receptor did not regress after MTX withdrawal. This indicates that the patient belongs to the P-G group and requires chemotherapy based on diffuse large B-cell lymphoma (DLBCL), which is the commonest type of MTX-LPD [11].
Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus-driven lymphoproliferative disorder [6] that has unique histopathologic and clinical features [7]. LYG was initially incorporated into the World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues in 2001 [8] and has been categorized as a distinct mature B-cell neoplasm in the revised version in 2016 [9]. LYG, characterized by angiocentric and angiodestructive features, typically affects middle-aged adults in the fourth to sixth decades of life, commonly involves the lungs (70%) and involves multiple organs, including the central nervous system (40%), skin (34%), kidney (19%), liver (17%), spleen (10%), lymph nodes (6%), and others (19%) [7,10]. The most common radiographic feature of the lung lesion of LYG is multiple nodules, occurring in approximately 80% of all cases [11,12]. Single lung nodules rarely appear in LYG patients and are used to discriminate other diseases, such as lung cancer, primary pulmonary lymphoma, and other granulomatous lung lesions such as sarcoidosis, tuberculous or non-tuberculous mycobacterial infection, and mycosis.
In our case of MTX-induced LYG, the patient had a history of long-term MTX therapy, and a growing single lung nodule and hilar lymph node were observed on preoperative CT findings. She was initially suspected to have lung cancer. Her clinical findings with slight fever or the history of methotrexate intake could help establish an accurate diagnosis of the disease. The postoperative CT findings showed multiple swollen lymph nodes. Furthermore, pathological findings also showed obvious infiltration of tumor cells in the hilar/mediastinal lymph nodes. These features were also typical for MTX-induced LYG.
The chemotherapeutic regimen for LYG, including MTX-LPG/MTX-DLBCL, has not been standardized as it is a rare disease. Chaves et al. reported that approximately three-quarters of patients with LYG (grade 3 in 45%) treated with rituximab-based therapies, mainly R–CHOP achieved a response [13]. Another study reported that the overall response rate in patients to combination chemotherapy with DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) in patients with high-grade LYG was 77% with 41% complete response [2,14]. In our case, the main lung lesion and hilar/mediastinal lymph nodes were removed at the time of surgery; however, serum soluble IL-2 receptor did not respond to R–CHOP therapy. The patient consequently required another chemotherapy regimen and had survived without recurrence.
In conclusion, LYG is not a common disease to thoracic surgeons, but we should consider it as a differential diagnosis in patients with a single lung nodule or multiple lung nodules and a prescription history of immunosuppressive agents, including methotrexate. The clinical and pathological findings of our case are expected to help acquire an easier diagnosis and a deeper understanding of LYG as one of MTX-LPD.
Declaration of competing interest
All authors declare no potential conflict of interest. | UNK, CYCLIC; ADMINISTERED TWO COURSES | DrugDosageText | CC BY-NC-ND | 33437613 | 19,389,586 | 2021 |
What was the dosage of drug 'PREDNISOLONE'? | A case of resected pulmonary lymphomatoid granulomatosis.
Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder and was incorporated into the WHO classification of Tumours of the Lung, Pleura, Thymus and Heart in 2015. LYG is known to be associated with the host's immune function, and can be caused by some immunosuppressive agents, including methotrexate. A woman in her sixties with an 18-year history of methotrexate treatment for rheumatoid arthritis visited our hospital after detection of an abnormal chest shadow on her radiograph. She had been having anemia and a slight fever. Computed tomography (CT) revealed a 2.9-cm sized nodule in her left lung and hilar adenopathy, which suggested a primary lung carcinoma or an inflammatory lesion. We performed a left upper lobectomy with lymph node dissection for the purpose of diagnosis and treatment. Pathologic findings revealed that the tumor was a grade 3 LYG based on the number of EBV-positive B cells. The patient was treated with two chemotherapy regimens including R-CHOP at another hospital, and survived for four years after resection without recurrence in the lung. It is rare to find a case resected LYG, and the clinical or pathological findings of our case are expected to be extremely helpful in studying this disease and improving the understanding of this disease.
1 Introduction
Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder driven by the Epstein-Barr virus (EBV) and often develops as a methotrexate-associated lymphoproliferative disorder. LYG involves extra-nodal sites including the lungs, skin, central nervous system, liver, and kidneys. When it affects a single lung nodule, it could be difficult to distinguish from other lung nodules, including primary lung carcinoma or other granulomatous lesions in the lung on imaging. We should carefully survey patients’ symptoms or prescription history to make an accurate diagnosis of LYG.
2 Case presentation
A woman in her sixties, found out to have anemia, was referred to our hospital. She had been suffering from rheumatoid arthritis for 18 years and was on methotrexate prescribed by her doctor. No source of bleeding was identified by endoscopic examination. On a chest radiograph taken a month after her first visit, an abnormal shadow in her left lung was noted. She revisited our hospital and was admitted in the department of respiratory medicine for a detailed examination. On admission, she had a slight fever of 37 °C and complained of drenching night sweats. Physical examination revealed the following: body temperature, 36.3 °C (37.6 °C at night); heart rate, 85 beats per minute; blood pressure, 142/96 mmHg; and no rale, was noted. She had never smoked. Laboratory data included: RBC, 4.40 × 106/μL; hemoglobin, 11.3 g/dL; lactate dehydrogenase (LDH) (reference value), 251 (120–240) IU/L; C-reactive protein, 3.51 g/dL; soluble interleukin-2 (IL-2) receptor (reference value), 1320 (145–516) U/mL. Her chest X-ray and computed tomography (CT) showed a solid 2.9 × 2.7-cm sized nodule in the middle of her left upper lobe (Fig. 1). The tumor size increased within a few months. Ipsilateral hilar adenopathy was also seen. We performed bronchoscopy with both exfoliative and lavage cytology, and neither of them indicated malignant findings, lung carcinoma or other malignancies were suggested based on these imaging findings. Although it may not have been malignant, some inflammatory diseases such as mycosis or granuloma could have caused her symptoms. Given the appearance, we made a decision to perform surgery with excision on her lung and performed left upper lobectomy with lymph node dissection for the purposes of diagnosis and treatment. The resected specimen was an irregular, solid, white nodule with extensive yellow necrotic areas in the middle of the lobe (Fig. 2). Histopathological examination showed marked obstruction of vessels with extensive fibrosis around vessels. Severe angiocentric and angiodestructive infiltration of lymphocytes including reactive T-cells were also seen. There were variable numbers of larger atypical cells with large nucleoli, similar to Hodgkin cells, and some multi-nucleated giant cells around the fibrosis (Fig. 3). Immunohistochemical findings showed that the Hodgkin-like cells were positive for CD30, and also for LMP1, a marker for EBV. EBV was also confirmed by in situ hybridization (Fig. 4). Other immunohistochemical staining showed that the atypical cells were positive for CD20 and CD79a focally and negative for EML4-ALK fusion protein. The tumor was diagnosed as grade 3 lymphomatoid granulomatosis. Two of the 55 lymph nodes, one in subaortic (#5) and the other in lober (#12) lymph node, also had the same lesion with extensive necrosis and eosinophilic infiltration. Even after surgery, she had been suffering from a slight fever, loss of appetite, weight loss, and pleural/peritoneal effusion. An increase in the soluble IL-2 receptor level of 1320 U/mL was also seen. She was transferred to the hematology department of another hospital to receive chemotherapy treatment. She was administered two courses of R–CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) therapy; however, she abandoned it because of a poor response seen on the swollen lymph nodes throughout the body and soluble IL-2 receptor level, and myelosuppression caused by the agents. Alternatively, she received 20 courses of molecular-target therapy with brentuximab vedotin, an antibody-drug targeting CD30-positive Hodgkin lymphoma and systemic anaplastic large cell lymphoma, although it was not approved as a treatment of DLBCL. She has been carefully followed up at the hospital, and no recurrence in the lung has been noticed four years after the resection.Fig. 1 Chest radiography (A) and CT (B) images revealing a solid mass with the tendency to increase in the left upper lobe. The enlarged hilar lymph node is also seen.
Fig. 1Fig. 2 Partly lobulated white nodule observed in the resected lobe.
Fig. 2Fig. 3 Histopathological examination revealing central necrosis (A), a proliferation of spindle-shaped cells (B), angiocentric and angiodestructive lymphoid infiltration (C), atypical cells with an irregular nucleus, and multinucleated giant cells (D).
Fig. 3Fig. 4 Atypical cells positive for LMP1, a marker for EBV (A). More than fifty cells are positive for EBV, as assessed by in situ hybridization, per high-power field (B).
Fig. 4
3 Discussion
Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency that mainly occurs in patients with RA who are treated with long-term methotrexate therapy [1,2]. MTX-LPD was initially reported by Ellman in 1991 [3], was increasingly reported as MTX became a common therapy for RA, and has been listed as one of the serious complications in the MTX treatment guidelines in Japan. The LPD incidence ratio of RA patients is two to four fold in the control healthy population irrespective of MTX treatment [4]. MTX-LPD is categorized as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) according to the 2008 and WHO classification [5]. The pathological features of MTX-LPD vary; diffuse large B-cell lymphoma (DLBCL) type is most predominant (35–60%) followed by Hodgkin lymphoma (HL) type (12–25%) [4]. The positive rate for EBV is about 40%, especially high (70%) in HL type [4]. Making a diagnosis of MTX-LPD only by its histological findings is supposed to be extremely difficult considering that there are various histological types of this disease. Unlike other lymphoproliferative disorders, MTX-LPD often involves extra-nodal organs, including the lung, skin, pharynx/tonsil, and soft tissues. Approximately 40–70% of patients with MTX-LPD regress after MTX withdrawal [1], so the first step is the withdrawal of MTX [5]. Meanwhile, relapse/regrowth events (RRE) after regression are seen in some cases of MTX-LPD. Tokuhira et al. reported that MTX-LPD presents three patterns after methotrexate withdrawal to clarify the clinical management of MTX-LPD [5]. A regressive group (R-G) includes patients with LPD regression after immunosuppressive drugs (ISDs) withdrawal without RRE, a relapse/regrowth group (R/R-G) includes patients with LPD regression after ISD withdrawal with RRE, and a persistent group (P-G) includes patients with persistent LPD after ISD withdrawal. Patients in R/R-G and P-G require additional chemotherapy. They also reported that the serum C-reactive protein (CRP), serum soluble IL-2 receptor, and LDH were increased in the P-G group, whereas only CRP and soluble IL-2 receptor were increased in the R/R-G group. In our case, not only swollen lymph nodes but also the level of soluble IL-2 receptor did not regress after MTX withdrawal. This indicates that the patient belongs to the P-G group and requires chemotherapy based on diffuse large B-cell lymphoma (DLBCL), which is the commonest type of MTX-LPD [11].
Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus-driven lymphoproliferative disorder [6] that has unique histopathologic and clinical features [7]. LYG was initially incorporated into the World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues in 2001 [8] and has been categorized as a distinct mature B-cell neoplasm in the revised version in 2016 [9]. LYG, characterized by angiocentric and angiodestructive features, typically affects middle-aged adults in the fourth to sixth decades of life, commonly involves the lungs (70%) and involves multiple organs, including the central nervous system (40%), skin (34%), kidney (19%), liver (17%), spleen (10%), lymph nodes (6%), and others (19%) [7,10]. The most common radiographic feature of the lung lesion of LYG is multiple nodules, occurring in approximately 80% of all cases [11,12]. Single lung nodules rarely appear in LYG patients and are used to discriminate other diseases, such as lung cancer, primary pulmonary lymphoma, and other granulomatous lung lesions such as sarcoidosis, tuberculous or non-tuberculous mycobacterial infection, and mycosis.
In our case of MTX-induced LYG, the patient had a history of long-term MTX therapy, and a growing single lung nodule and hilar lymph node were observed on preoperative CT findings. She was initially suspected to have lung cancer. Her clinical findings with slight fever or the history of methotrexate intake could help establish an accurate diagnosis of the disease. The postoperative CT findings showed multiple swollen lymph nodes. Furthermore, pathological findings also showed obvious infiltration of tumor cells in the hilar/mediastinal lymph nodes. These features were also typical for MTX-induced LYG.
The chemotherapeutic regimen for LYG, including MTX-LPG/MTX-DLBCL, has not been standardized as it is a rare disease. Chaves et al. reported that approximately three-quarters of patients with LYG (grade 3 in 45%) treated with rituximab-based therapies, mainly R–CHOP achieved a response [13]. Another study reported that the overall response rate in patients to combination chemotherapy with DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) in patients with high-grade LYG was 77% with 41% complete response [2,14]. In our case, the main lung lesion and hilar/mediastinal lymph nodes were removed at the time of surgery; however, serum soluble IL-2 receptor did not respond to R–CHOP therapy. The patient consequently required another chemotherapy regimen and had survived without recurrence.
In conclusion, LYG is not a common disease to thoracic surgeons, but we should consider it as a differential diagnosis in patients with a single lung nodule or multiple lung nodules and a prescription history of immunosuppressive agents, including methotrexate. The clinical and pathological findings of our case are expected to help acquire an easier diagnosis and a deeper understanding of LYG as one of MTX-LPD.
Declaration of competing interest
All authors declare no potential conflict of interest. | UNK, 2 COURSES OF (R?CHOP REGIMEN) | DrugDosageText | CC BY-NC-ND | 33437613 | 19,358,394 | 2021 |
What was the outcome of reaction 'Angiocentric lymphoma'? | A case of resected pulmonary lymphomatoid granulomatosis.
Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder and was incorporated into the WHO classification of Tumours of the Lung, Pleura, Thymus and Heart in 2015. LYG is known to be associated with the host's immune function, and can be caused by some immunosuppressive agents, including methotrexate. A woman in her sixties with an 18-year history of methotrexate treatment for rheumatoid arthritis visited our hospital after detection of an abnormal chest shadow on her radiograph. She had been having anemia and a slight fever. Computed tomography (CT) revealed a 2.9-cm sized nodule in her left lung and hilar adenopathy, which suggested a primary lung carcinoma or an inflammatory lesion. We performed a left upper lobectomy with lymph node dissection for the purpose of diagnosis and treatment. Pathologic findings revealed that the tumor was a grade 3 LYG based on the number of EBV-positive B cells. The patient was treated with two chemotherapy regimens including R-CHOP at another hospital, and survived for four years after resection without recurrence in the lung. It is rare to find a case resected LYG, and the clinical or pathological findings of our case are expected to be extremely helpful in studying this disease and improving the understanding of this disease.
1 Introduction
Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder driven by the Epstein-Barr virus (EBV) and often develops as a methotrexate-associated lymphoproliferative disorder. LYG involves extra-nodal sites including the lungs, skin, central nervous system, liver, and kidneys. When it affects a single lung nodule, it could be difficult to distinguish from other lung nodules, including primary lung carcinoma or other granulomatous lesions in the lung on imaging. We should carefully survey patients’ symptoms or prescription history to make an accurate diagnosis of LYG.
2 Case presentation
A woman in her sixties, found out to have anemia, was referred to our hospital. She had been suffering from rheumatoid arthritis for 18 years and was on methotrexate prescribed by her doctor. No source of bleeding was identified by endoscopic examination. On a chest radiograph taken a month after her first visit, an abnormal shadow in her left lung was noted. She revisited our hospital and was admitted in the department of respiratory medicine for a detailed examination. On admission, she had a slight fever of 37 °C and complained of drenching night sweats. Physical examination revealed the following: body temperature, 36.3 °C (37.6 °C at night); heart rate, 85 beats per minute; blood pressure, 142/96 mmHg; and no rale, was noted. She had never smoked. Laboratory data included: RBC, 4.40 × 106/μL; hemoglobin, 11.3 g/dL; lactate dehydrogenase (LDH) (reference value), 251 (120–240) IU/L; C-reactive protein, 3.51 g/dL; soluble interleukin-2 (IL-2) receptor (reference value), 1320 (145–516) U/mL. Her chest X-ray and computed tomography (CT) showed a solid 2.9 × 2.7-cm sized nodule in the middle of her left upper lobe (Fig. 1). The tumor size increased within a few months. Ipsilateral hilar adenopathy was also seen. We performed bronchoscopy with both exfoliative and lavage cytology, and neither of them indicated malignant findings, lung carcinoma or other malignancies were suggested based on these imaging findings. Although it may not have been malignant, some inflammatory diseases such as mycosis or granuloma could have caused her symptoms. Given the appearance, we made a decision to perform surgery with excision on her lung and performed left upper lobectomy with lymph node dissection for the purposes of diagnosis and treatment. The resected specimen was an irregular, solid, white nodule with extensive yellow necrotic areas in the middle of the lobe (Fig. 2). Histopathological examination showed marked obstruction of vessels with extensive fibrosis around vessels. Severe angiocentric and angiodestructive infiltration of lymphocytes including reactive T-cells were also seen. There were variable numbers of larger atypical cells with large nucleoli, similar to Hodgkin cells, and some multi-nucleated giant cells around the fibrosis (Fig. 3). Immunohistochemical findings showed that the Hodgkin-like cells were positive for CD30, and also for LMP1, a marker for EBV. EBV was also confirmed by in situ hybridization (Fig. 4). Other immunohistochemical staining showed that the atypical cells were positive for CD20 and CD79a focally and negative for EML4-ALK fusion protein. The tumor was diagnosed as grade 3 lymphomatoid granulomatosis. Two of the 55 lymph nodes, one in subaortic (#5) and the other in lober (#12) lymph node, also had the same lesion with extensive necrosis and eosinophilic infiltration. Even after surgery, she had been suffering from a slight fever, loss of appetite, weight loss, and pleural/peritoneal effusion. An increase in the soluble IL-2 receptor level of 1320 U/mL was also seen. She was transferred to the hematology department of another hospital to receive chemotherapy treatment. She was administered two courses of R–CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab) therapy; however, she abandoned it because of a poor response seen on the swollen lymph nodes throughout the body and soluble IL-2 receptor level, and myelosuppression caused by the agents. Alternatively, she received 20 courses of molecular-target therapy with brentuximab vedotin, an antibody-drug targeting CD30-positive Hodgkin lymphoma and systemic anaplastic large cell lymphoma, although it was not approved as a treatment of DLBCL. She has been carefully followed up at the hospital, and no recurrence in the lung has been noticed four years after the resection.Fig. 1 Chest radiography (A) and CT (B) images revealing a solid mass with the tendency to increase in the left upper lobe. The enlarged hilar lymph node is also seen.
Fig. 1Fig. 2 Partly lobulated white nodule observed in the resected lobe.
Fig. 2Fig. 3 Histopathological examination revealing central necrosis (A), a proliferation of spindle-shaped cells (B), angiocentric and angiodestructive lymphoid infiltration (C), atypical cells with an irregular nucleus, and multinucleated giant cells (D).
Fig. 3Fig. 4 Atypical cells positive for LMP1, a marker for EBV (A). More than fifty cells are positive for EBV, as assessed by in situ hybridization, per high-power field (B).
Fig. 4
3 Discussion
Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency that mainly occurs in patients with RA who are treated with long-term methotrexate therapy [1,2]. MTX-LPD was initially reported by Ellman in 1991 [3], was increasingly reported as MTX became a common therapy for RA, and has been listed as one of the serious complications in the MTX treatment guidelines in Japan. The LPD incidence ratio of RA patients is two to four fold in the control healthy population irrespective of MTX treatment [4]. MTX-LPD is categorized as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) according to the 2008 and WHO classification [5]. The pathological features of MTX-LPD vary; diffuse large B-cell lymphoma (DLBCL) type is most predominant (35–60%) followed by Hodgkin lymphoma (HL) type (12–25%) [4]. The positive rate for EBV is about 40%, especially high (70%) in HL type [4]. Making a diagnosis of MTX-LPD only by its histological findings is supposed to be extremely difficult considering that there are various histological types of this disease. Unlike other lymphoproliferative disorders, MTX-LPD often involves extra-nodal organs, including the lung, skin, pharynx/tonsil, and soft tissues. Approximately 40–70% of patients with MTX-LPD regress after MTX withdrawal [1], so the first step is the withdrawal of MTX [5]. Meanwhile, relapse/regrowth events (RRE) after regression are seen in some cases of MTX-LPD. Tokuhira et al. reported that MTX-LPD presents three patterns after methotrexate withdrawal to clarify the clinical management of MTX-LPD [5]. A regressive group (R-G) includes patients with LPD regression after immunosuppressive drugs (ISDs) withdrawal without RRE, a relapse/regrowth group (R/R-G) includes patients with LPD regression after ISD withdrawal with RRE, and a persistent group (P-G) includes patients with persistent LPD after ISD withdrawal. Patients in R/R-G and P-G require additional chemotherapy. They also reported that the serum C-reactive protein (CRP), serum soluble IL-2 receptor, and LDH were increased in the P-G group, whereas only CRP and soluble IL-2 receptor were increased in the R/R-G group. In our case, not only swollen lymph nodes but also the level of soluble IL-2 receptor did not regress after MTX withdrawal. This indicates that the patient belongs to the P-G group and requires chemotherapy based on diffuse large B-cell lymphoma (DLBCL), which is the commonest type of MTX-LPD [11].
Lymphomatoid granulomatosis (LYG) is an Epstein-Barr virus-driven lymphoproliferative disorder [6] that has unique histopathologic and clinical features [7]. LYG was initially incorporated into the World Health Organization classification of Tumours of Haematopoietic and Lymphoid Tissues in 2001 [8] and has been categorized as a distinct mature B-cell neoplasm in the revised version in 2016 [9]. LYG, characterized by angiocentric and angiodestructive features, typically affects middle-aged adults in the fourth to sixth decades of life, commonly involves the lungs (70%) and involves multiple organs, including the central nervous system (40%), skin (34%), kidney (19%), liver (17%), spleen (10%), lymph nodes (6%), and others (19%) [7,10]. The most common radiographic feature of the lung lesion of LYG is multiple nodules, occurring in approximately 80% of all cases [11,12]. Single lung nodules rarely appear in LYG patients and are used to discriminate other diseases, such as lung cancer, primary pulmonary lymphoma, and other granulomatous lung lesions such as sarcoidosis, tuberculous or non-tuberculous mycobacterial infection, and mycosis.
In our case of MTX-induced LYG, the patient had a history of long-term MTX therapy, and a growing single lung nodule and hilar lymph node were observed on preoperative CT findings. She was initially suspected to have lung cancer. Her clinical findings with slight fever or the history of methotrexate intake could help establish an accurate diagnosis of the disease. The postoperative CT findings showed multiple swollen lymph nodes. Furthermore, pathological findings also showed obvious infiltration of tumor cells in the hilar/mediastinal lymph nodes. These features were also typical for MTX-induced LYG.
The chemotherapeutic regimen for LYG, including MTX-LPG/MTX-DLBCL, has not been standardized as it is a rare disease. Chaves et al. reported that approximately three-quarters of patients with LYG (grade 3 in 45%) treated with rituximab-based therapies, mainly R–CHOP achieved a response [13]. Another study reported that the overall response rate in patients to combination chemotherapy with DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) in patients with high-grade LYG was 77% with 41% complete response [2,14]. In our case, the main lung lesion and hilar/mediastinal lymph nodes were removed at the time of surgery; however, serum soluble IL-2 receptor did not respond to R–CHOP therapy. The patient consequently required another chemotherapy regimen and had survived without recurrence.
In conclusion, LYG is not a common disease to thoracic surgeons, but we should consider it as a differential diagnosis in patients with a single lung nodule or multiple lung nodules and a prescription history of immunosuppressive agents, including methotrexate. The clinical and pathological findings of our case are expected to help acquire an easier diagnosis and a deeper understanding of LYG as one of MTX-LPD.
Declaration of competing interest
All authors declare no potential conflict of interest. | Recovering | ReactionOutcome | CC BY-NC-ND | 33437613 | 19,363,501 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chest discomfort'. | Variable clinical features of patients with Fabry disease and outcome of enzyme replacement therapy.
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme α-galactosidase A due to mutations in the GLA gene. This leads to an accumulation of globotriaosylceramide (GL-3) in many tissues, which results in progressive damage to the kidneys, heart, and nervous system. We present the molecular and clinical characteristics and long-term outcomes of FD patients from a multidisciplinary clinic at the University of California, Irvine treated with agalsidase beta enzyme replacement therapy (ERT) for 2-20 years. This cohort comprised 24 adults (11 males, 13 females) and two male children (median age 45; range 10-68 years). Of the 26 patients in this cohort, 20 were on ERT (12 males, 8 females). We describe one novel variant not previously reported in the literature in a patient with features of 'classic' FD. The vast majority of patients in this cohort presented with symptoms of 'classic' FD including peripheral neuropathic pain, some form of cardiac involvement, angiokeratomas, corneal verticillata, hypohidrosis, tinnitus, and gastrointestinal symptoms, primarily abdominal pain. The majority of males had clinically evident renal involvement. An annual eGFR reduction of -1.88 mL/min/1.73 m2/yr during the course of ERT was seen in this cohort. The most common renal presentation was proteinuria, and one individual required a renal transplant. Other common findings were pulmonary involvement, lymphedema, hearing loss, and significantly, three patients had strokes. Notably, there was a high prevalence of endocrine dysfunction and low bone mineral density, including several with osteoporosis. While enzyme replacement therapy (ERT) cleared plasma GL-3 in this cohort, there was limited improvement in renal function or health-related quality of life based on the patient-reported SF-36 Health Survey. Physical functioning significantly declined over the course of ERT treatment, which may be, in part, due to the late initiation of ERT in several patients. Further delineation of the phenotypic and genotypic spectrum in patients with FD and the long-term outcome of ERT will help improve management and treatment options for this disease.
1 Introduction
Fabry Disease (FD, OMIM#301500) is an inherited, X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficient or absent α-galactosidase A enzyme [1]. This consequently leads to the progressive deposition of globotriaosylceramide (GL-3) in several tissues throughout the body, notably in endothelial cells, smooth muscle cells, podocytes, and cardiomyocytes [2]. The absence of this enzyme may lead to multi-organ involvement including progressive renal disease, cardiac disease, cerebrovascular disease, neuropathic pain, corneal verticillata, angiokeratomas, tinnitus, hearing loss, pulmonary involvement, and gastrointestinal complaints, including abdominal pain, constipation, and diarrhea. FD is pan-ethnic and the second most prevalent lysosomal storage disorder with an estimated incidence of 1 in 40,000 to 117,000 males worldwide, though newborn screening initiatives have revealed that the incidence is much higher [[3], [4], [5], [6]]. This is likely due to the fact that early studies referred to the incidence of ‘classic’ FD, while newborn screening may identify individuals on the full phenotypic spectrum. Symptoms typically present early during childhood or adolescence. Affected males with little or absent α-galactosidase A activity (<1% of mean normal) may present with clinical features of ‘classic’ FD. Mutations that result in residual enzyme activity typically cause a ‘later-onset’ form of the disease with variable manifestations. Heterozygous females typically present with milder symptoms and later-onset and less commonly have ‘classic’ FD due to residual enzyme activity and X-chromosome inactivation pattern.
Management and guidelines of a newly diagnosed individual with FD includes documentation of symptoms, consideration of enzyme replacement therapy (ERT), and monitoring of disease progression [7,8]. ERT is provided to supplement or replace α-galactosidase A, and to slow the rate of disease progression. Thus, early identification of affected individuals is important in order to initiate ERT as early as possible [[8], [9], [10]]. Clinical evaluation during childhood is recommended if a familial mutation is known [11]. In the absence of a family history, a timely diagnosis of FD may be difficult due to the variation and unpredictability of clinical manifestations that can often be misdiagnosed as childhood growing pains. The addition of FD to newborn screening programs worldwide has allowed for the early detection of the disorder [12].
There are two types of enzyme replacement therapy: agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme); however only agalsidase beta is an approved ERT by the US Food and Drug Administration (FDA) [13,14]. There is some discussion on the most appropriate infusion as well as the efficacy and safety of the infusions, but studies have shown no adverse effects in switching therapies or an increased benefit of using one over the other [15,16]. There is one oral pharmacological chaperone therapy, migalastat (Galafold), that has been approved by the FDA [17]. This treatment is only available for adults with FD who have amenable gene variants that lead to misfolded α-galactosidase A enzyme that can be stabilized by migalastat. Once stabilized, protein folding is improved and trafficking of the α-galactosidase A enzyme to the lysosome is restored. Other treatments currently being investigated in clinical trials include plant-based ERT, gene therapy, and substrate reducing therapies.
We describe a cohort of 24 adults and two children with FD, 13 males and 13 females, who range in age from 10 to 68 years (mean age 46.1 ± 16.3). The majority of participants were on agalsidase beta ERT (20/26; 12 males, 8 females). Each participant demonstrated different response rates of symptoms and disease progression with ERT, including two individuals who developed adverse infusion reactions.
The aim of this study is to present the variable molecular and clinical features in a cohort of FD patients on ERT, treatment outcomes across organ systems, and highlight patients with previously unreported variants, to help improve treatment options and patient care.
2 Methods
2.1 Participants
This cohort comprised 13 male and 13 female patients, including two male children. All participants had a confirmed molecular diagnosis of FD and most also had recorded enzyme activity levels measured. GLA variants are listed using transcript NM_000169.2. These patients were followed over the course of a one to 12 year period in an outpatient multidisciplinary clinic at the University of California, Irvine. Earlier data were obtained from review of past medical records and the Fabry Registry, if available. Written IRB informed consent approved by UC Irvine (#2008–6631) was obtained from each participant, and all procedures were performed according to The Code of Ethics of the World Medical Association.
2.2 Clinical evaluations
Clinical surveillance visits were conducted every six months and included evaluations by a clinical geneticist, a cardiologist, and a nephrologist. The standard of care for patients with renal and cardiac disease from FD did not vary when compared to the management of other etiologies. At each visit, the following components were documented:• Quality of life surveys, including a Brief Pain Inventory (BPI) and the SF-36 Health Survey. These provided a scale for pain and represented an attempt to document how pain has influenced the quality of life of each individual. The SF-36 Health survey measures physical function, social function, physical role, emotional role, mental health, energy, pain, and general health perception. Scores ranged from 0 (worst) to 100 (best).
• The development or change in the status of clinical features specific to FD including cardiac, renal, pulmonary, and nervous system involvement, in addition to angiokeratomas, acroparesthesia, GI health, hyper or hypohidrosis, hearing loss and tinnitus, lymphedema, and corneal whorling.
• Plasma and urine GL-3 levels and plasma Lyso-GL-3 to assess overall glycolipid burden.
• Renal status and disease progression were monitored by labs: serum creatinine levels to measure the mean estimated glomerular filtration rate (eGFR), in addition to urine creatinine, urine protein, and urine albumin/micro-albumin to measure proteinuria and albuminuria. Renal biopsy was only performed if clinically indicated.
Evaluations performed at less frequent intervals:• Cardiac status was determined by echocardiography (with strain), electrocardiograms, and holter monitor screened at annual intervals in males and less frequently in females and children. Cardiac MRI was conducted every 2–3 years or as needed.
• Brain imaging was performed by MRI, MRA, and CT scan.
• Ophthalmologic exams were performed for evaluation of corneal whorls, vessel tortuosity, and lenticular opacities.
• Audiometry was performed for evaluation of sensorineural hearing loss and tinnitus, as needed.
• Dual-energy X-ray absorptiometry (DXA) & the World Health Organization (WHO) classification of bone mineral density were used to classify severity. T-scores between −1.0 and −2.5 SD were categorized as osteopenia and T-scores below −2.5 SD were categorized as osteoporosis.
2.3 ERT infusions
Agalsidase beta (Fabrazyme) recommended dosage is 1 mg/kg body weight given every two weeks as an intravenous infusion [14]. The majority of the participants in this cohort received the infusions without incident, and some have transitioned to home infusions. To determine the effectiveness of ERT, we measured the presence of agalsidase beta IgG antibodies, plasma and urine GL-3 levels, and plasma Lyso-GL-3 levels at each six-month visit. Two individuals experienced adverse reactions to ERT infusions (patient 15 and patient 22); they both tested negative for IgE antibodies and showed no change in IgG antibodies.
2.4 Statistical analysis
We used linear mixed-effects regression analysis with varying intercepts and slopes to evaluate linear time trends of serum creatinine, eGFR, and health-related quality of life scores (SF-36 Health Survey), starting with the initiation of ERT (time = 0). The significance level for all statistical tests was set to α = 0.05. All analyses were carried out using the R language and environment for statistical computing, version 3.6.3.
3 Results
Of the 26 patients included in our study, there were 13 males and 13 females between ages 10–68 years (mean age 46.1 ± 16.3 years). This cohort consisted of five families in addition to eleven unrelated individuals. The majority of males (12/13, 92%) and the majority of females (8/13, 62%) were treated with ERT. Ages at initiation of ERT were between 6 and 65 years (mean age 39.0 ± 18.3) (Supplemental Table 1). The majority of patients tolerated ERT well, except for two individuals who had adverse reactions (patient 15 and patient 22). Both patients were IgE-negative and showed no changes in IgG antibodies; patient 22 was consistently IgG-positive throughout the course of ERT. Patient 15 had the most significant adverse event during the last infusion attempt, which resulted in intubation in the cardiac intensive care unit due to difficulty breathing, tightness in chest, rigors, and convulsions, and has not been on ERT since. Only three males were routinely IgG-positive (patients 12, 13, 24) and two males had one single IgG-positive test in the past, but had not had another one since (patient 1 and patient 7).
Clinical features of each patient are described in Supplemental Table 2. Notably, there was clinically evident renal involvement in 50% of participants; the most common feature was varying levels of proteinuria. One individual required a renal transplant. Seventy-nine percent of patients showed evidence of some form of cardiac involvement, including 46% with left ventricular hypertrophy. Two males and one female had a history of a stroke, and one male had a history of one TIA and one stroke. The most commonly observed clinical feature was peripheral neuropathic pain (20/26, 77%), followed by angiokeratomas (18/26, 69%), tinnitus (17/26, 65%), gastrointestinal symptoms, including abdominal pain, constipation, and diarrhea (16/26, 62%), and hypohidrosis/anhidrosis (16/26, 62%). Additional common findings of FD in this cohort include corneal verticillata (15/26, 58%), pulmonary involvement (6/12, 50%), lymphedema (11/26, 42%), and hearing loss (9/26, 35%). The adult males in this cohort had higher frequencies of clinical manifestations than adult females in all categories with the exception of neuropathic pain, white matter lesions, and corneal verticillata (Table 1).Table 1 Clinical manifestations of FD in this cohort.
Table 1 Adult Females
N = 13 (%) Adult Males
N = 11 (%) Pediatric Males
N = 2 (%) Total
N = 26 (%)
Mean Age at Diagnosis (Years) 36.2 34.3 3.5 32.8 ± 16.3
Currently On ERT 8 (62) 10 (91) 2 (100) 20 (77)
Mean Age ERT Initiated (Years) 51 37.5 6.5 39.0 ± 18.3
Renal Involvement 5 (38) 7 (64) 1 (50) 13 (50)
Cardiac Involvement 9/12 (75) 9/10 (90) 1 (50) 19/24 (79)
White matter Lesions 3/10 (30) 1/6 (17) N/A 4/16 (25)
Neuropathic Pain 10 (77) 8 (73) 2 (100) 20 (77)
Corneal Verticillata 10 (77) 4 (36) 1 (50) 15 (58)
Angiokeratomas 7 (54) 11 (100) 0 (0) 18 (69)
Hypohidrosis 5 (38) 10 (91) 1 (50) 16 (62)
GI Involvement 8 (62) 8 (73) 0 (0) 16 (62)
Low BMD 2/6 (33) 5/8 (63) N/A 7/14 (50)
Hearing Loss 3 (23) 6 (55) 0 (0) 9 (35)
Tinnitus 7 (54) 9 (82) 1 (50) 17 (65)
Vertigo 4/10 (40) 6/10 (60) 0/1 (0) 10/21 (48)
Pulmonary Involvement 0/5 (0) 5/6 (83) 1/1 (100) 6/12 (50)
Depression 3 (23) 3 (27) 0 (0) 6/26 (23)
Numbers provided in fractions indicate that not all patients had the corresponding assessment.
3.1 Clearance of GL-3 in plasma
Plasma-GL-3, plasma Lyso-GL-3, and urine GL-3 are biomarkers used as surrogate endpoints to monitor Fabry disease severity and progression in both treated and untreated patients. Longitudinal analysis of plasma GL-3 concentration in 17 of our patients on ERT is depicted in Fig. 1. The data showed significant reduction in GL-3 of −0.25 μg/mL per year (95% CI: −0.478 to −0.030, p = 0.026) with ERT treatment.Fig. 1 Plasma GL-3 clearance in patients on ERT. A linear mixed-effects regression analysis of plasma GL-3 with time as a predictor and varying intercepts and slopes. The figure shows observed GL-3 measurements (dots) together with model-estimated time trajectories of GL-3 for each participant (colored lines) and for the population (black line). Note that observations at time < 0 were excluded from the analysis. N = 17.
Fig. 1
While there was less data available for the deacetylated form, plasma Lyso-GL-3, the majority showed a reduction or stabilization of plasma Lyso-GL-3 concentration at last visit compared to that of the earlier time points (N = 11, data not shown).
3.2 Renal involvement
There was clinically evident renal disease in 50% (13/26) of participants as defined by albuminuria, hypertension or reduced eGFR. Renal biopsies were not done routinely. The majority demonstrated proteinuria and/or albuminuria as the sole renal manifestation. Ten patients had elevated urine albumin-to-creatine ratios, nine with microalbuminuria (5 females, 4 males) and one male with macroalbuminuria. Hypertension was seen in 32% (8/25) of patients and managed with angiotensin-converting enzyme inhibitors (ACEI) (8/26), angiotensin receptor blockers (ARB) (4/26), beta-blockers (4/26), and diuretics (6/26). Two had stage I chronic kidney disease (CKD) (patient 1 and patient 12), two had stage III CKD (patient 14 and patient 17), and one had stage IV CKD and is on hemodialysis (patient 22). Patient 23 underwent a deceased donor renal transplant due to ESRD at age 52 years and has had normal kidney function since. For those on ERT, only some patients maintained serum creatinine and estimated glomerular filtration rate (eGFR) within normal limits for age and sex. However, the data showed significant increase in serum creatinine levels of +0.02 mg/dL per year (95% CI: 0.01–0.04, p = 0.015) for both males and females on ERT in this cohort (Fig. 2A) and +0.038 mg/dL per year (95% CI: 0.01–0.06, p = 0.003, data not shown) for males alone. Further, the data showed an annual decrease in eGFR of −1.88 mL/min/1.73m2 (95% CI: −3.36 to −0.40, p = 0.013) on ERT in both males and females (Fig. 2B), and −3.16 mL/min/1.73m2 (95% CI: −4.97 to −1.35, p = 0.001, data not shown) for males alone.Fig. 2 Serum creatinine and estimated glomerular filtration rate (eGFR) of patients on ERT. The figure shows observed serum creatinine measurements (A) and eGFR measurements (B) together with model-estimated time trajectories for each participant (colored lines) and for the population (black lines). Note that observations at time < 0 were excluded from both analyses in figs. A-B. N = 18.
Fig. 2
3.3 Cardiac involvement
In our cohort, 79% (19/24) of participants had some form of cardiac involvement. Left ventricular hypertrophy (LVH) was seen in 46% (11/24) of patients, with severity ranging from borderline to severe and one confined to the septum. Severe LVH of patient 24 is shown in Fig. 3A. Of the adult males, only one was not found to have clinically evident cardiac involvement, and while his past ECGs met voltage criteria for LVH, his echocardiograms were normal showing no evidence of LVH. Left ventricular ejection fraction (LVEF) was one of the measures used to monitor cardiac disease progression of patients on ERT. LVEF was within normal range at baseline prior to ERT. Some patients fell below normal range over the course of treatment with the lowest recorded being 43% in a male patient. Of the individuals in our cohort who had cardiac imaging, 25% (6/24) had valvular abnormalities, including one patient with aortic valve sclerosis, two patients had thickened mitral valve leaflets, one patient with mild Systolic Anterior Motion (SAM) abnormality of the mitral valve, one with mitral valve prolapse, and one patient required mitral valve annuloplasty after experiencing lower extremity edema, shortness of breath and dyspnea on exertion (patient 22). Other cardiac manifestations are described in Table 2. Of those who underwent cardiac MRI, only one showed possible fibrosis of the myocardium (Fig. 3A). Those with cardiac abnormalities on echocardiogram, cardiac MRI, and/or ECG did not consistently have abnormal cardiac biomarkers, such as brain natriuretic peptide (BNP), troponin, and soluble suppression of tumourigenicity 2 (ST2), though limited data was available. Adult FD patients can have elevated lipid levels, particularly high HDL cholesterol, which may be non-responsive to long-term ERT [18]. Dyslipidemia was present in 48% (10/21) of patients in our cohort, and managed with diet, fish-oil and statins.Fig. 3 Clinical features in patients with FD. (A) Cardiac MRI showing severe concentric hypertrophy of the left ventricle with reduced chamber size. There is subendocardial linear hyperenhancement in the basal inferolateral wall suggesting myocardial scar (white arrow), consistent with FD (patient 24); (B) Axial brain MRI section following TIA event (patient 22); (C) Corneal verticillata seen by slit lamp examination. Courtesy: Dr. Pinakin Davey, Western University of Health Sciences; (D) Cluster of angiokeratomas in the groin region (patient 24).
Fig. 3Table 2 Characterization of cardiac involvement in FD patients in this cohort.
Table 2 Adult
Females Adult
Males Pediatric Males Total (%)
Cardiac Involvement 9/12 (75) 9/10 (90) 1/2 (50) 19/24 (79)
Echocardiogram Findings
Left Ventricular Hypertrophy 5/12 7/10 0/2 11/24 (46)
Borderline 1 2 0
Mild 2 3 0
Moderate 0 1 0
Severe 0 1 0
Septal 1 0 0
Valvular Abnormality 2/12 4/10 0/2 6/24 (25)
Aortic Valve 1 0 0
Mitral Valve 1 4 0
Left Atrium Dilation 2/12 2/10 0/2 4/24 (17)
Right Atrium Dilation 1/12 0/10 0/2 1/24 (4)
Electrocardiogram Findings
Conduction Abnormalities 3/12 6/10 0/2 9/24 (38)
Right Bundle Branch Block 2 5 0
Anterior Fascicular Block 1 1 0
First Degree Atrioventricular Block 1 0 0
Intraventricular Conduction Delay 0 2 0
Short PR Interval 1 2 0
Rhythm Abnormalities 5/12 8/10 1/2 14/24 (58)
Sinus Bradycardia 5 8 1
Sinus Arrhythmia 1 0 0
Premature Ventricular Complexes 0 2 0
Premature Atrial Contractions 0 1 0
Repolarization Abnormalities 5/12 3/10 0/2 8/24 (33)
T Wave Changes 2 1 0
ST/STT Changes 3 2 0
Left Axis Deviation 0/12 1/10 0/2 1/24 (4)
One male and one female in this cohort were excluded due to no cardiac imaging records available.
3.4 Cerebrovascular complications, neurologic, and neuropsychiatric symptoms
White matter lesions were present in 25% (4/16) of participants who had brain imaging. One male in this cohort (patient 22) has a history of two cerebrovascular accidents, one transient ischemic attack (TIA) at age 52 years and one stroke at age 60 years. Brain MRI of patient 22 showed multifocal and adjacent gliosis within the left cerebral hemisphere, high left frontal and parietal lobes, as well as subcortical and periventricular deep white matter T2 hyperintensity without mass effect that is most pronounced within the parietal occipital region (Fig. 3B). The stroke impacted his eyesight and word-finding ability. Patient 22 only began ERT at the age of 57 years, and passed away at the age of 63 years due to post-operative complications of abdominal surgery. Another male (patient 15) and one female (patient 14) in this cohort have a history of TIAs while on ERT. They were not taking low dose aspirin at the time.
With regard to neuropathic manifestations, 77% (20/26) of patients reported peripheral neuropathic pain, manifesting as burning, tingling, or numbness, often triggered by temperature change or strenuous activity. Of the 17 participants for whom data on the brief pain inventory (BPI) survey was available, the majority reported worsening or inconsistent pain symptoms over the duration of treatment with ERT. Decreased quality of life has been associated with FD, in part due to the debilitating episodic pain crises and burden associated with having a chronic illness. Health-related quality of life was collected using the SF-36 Health survey. Bodily pain scores obtained from the SF-36 survey also showed no improvement over the course of ERT, though this was not significant (p = 0.19; Fig. 4C). For the majority of SF-36 respondents, pain worsened over time, which was similar to what was reported in the BPI survey. Notably, there was a significant decline in physical functioning (Est. = −1.40, 95% CI: −2.73 to −0.06, p = 0.040) for patients in this cohort. The remaining health dimensions did not show significant change over time (Fig. 4A-H).Fig. 4 SF-36 health-related quality of life component scores of patients on ERT. The SF-36 Health survey measures (A) physical function, (B) physical role, (C) pain, (D) general health perception, (E) vitality, (F) social function, (G) emotional role, (H) mental health. Scores ranged from 0 (worst) to 100 (best). Observed values (dots) are shown together with model-estimated trajectories for individual patients (colored lines) and the population (black lines).
Fig. 4
Of the adult patients in this cohort, seven reported depression (7/24; 29%). One patient reported a history of depression that correlated with pain symptoms. Another patient had a history of depression with no current pain symptoms based on responses in the BPI and SF-36 Health Survey. Of note, one patient in this cohort had a history of two suicide attempts. On the other hand, one patient's testimonial described noticeable improvement in anxiety, fatigue, and executive function (termed “Fabry fog”) since initiation of ERT.
3.5 Ophthalmologic findings
Corneal verticillata, or corneal whorling, is a unique clinical feature in patients of both sexes with FD and is often used as a diagnostic tool. It is a result of GL-3 deposits and has not been linked to vision loss. Corneal verticillata was noted in 58% (15/26) of individuals, at a higher prevalence in females than males in our cohort (Fig. 3C). Vessel tortuosity is also associated with FD and is exacerbated by renal complications [19,20]. Only one patient was found to have vessel tortuosity (patient 22); this individual had stage IV CKD.
3.6 Skin manifestations
Angiokeratomas are common in Fabry patients as they occur when GL-3 accumulates in dermal endothelial cells and lead to secondary ectasia. In our cohort, 69% (18/26) had angiokeratomas (Fig. 3D).
Hypohidrosis is also a common feature of FD and may be a predisposition to acroparesthesia. In this cohort, 62% (16/26) experienced hypohidrosis or anhidrosis. Of those with hypohidrosis, 89% experienced acroparesthesia. Lymphedema of the extremities, particularly in the feet, was present in 42% (11/26) of patients in this study.
3.7 Gastrointestinal involvement
Gastrointestinal symptoms are common among FD patients and can manifest as diarrhea, constipation, nausea, vomiting, incontinence, and abdominal pain. In our cohort, gastrointestinal symptoms were present in 62% (16/26) and the most common complaint was abdominal pain. Previous studies have shown significantly improved gastrointestinal pain with ERT [21]. This was also reported by the patients in our study in the patient health survey. The majority of those on ERT reported either improved or stable abdominal pain and frequency of diarrhea based on their survey responses at last visit compared to earlier survey responses. Specifically, six patients reported improvement in abdominal pain and four reported that symptoms were stable (N = 20). Eleven reported improvement in diarrhea and one reported that symptoms were stable (N = 20). One patient's testimonial described a frequency of diarrhea multiple times daily prior to ERT that has now significantly improved since ERT initiation. He also reported noticeable return of symptoms during the time of the ERT shortage. On the other hand, worsening or recent onset of abdominal pain (3/20) and diarrhea (2/20) over the course of ERT treatment were also reported by participants in this study.
3.8 Auditory and vestibular involvement
Sensorineural hearing loss, predominantly in the high frequencies, is a common manifestation of FD and correlates with neuropathic and vascular damage [22]. In our cohort, 35% (9/26) of patients reported a history of hearing loss. The majority of those who underwent audiology assessments were found to have bilateral mild to moderate sensorineural hearing loss. Additionally, 65% (17/26) of patients reported tinnitus. Some patients do not use hearing aids due to amplification by the presence of tinnitus. Vertigo was reported in 48% of our cohort in those who recorded their response in the health survey (10/21).
3.9 Pulmonary involvement
Respiratory involvement in FD typically includes obstructive lung disease, however patients may develop interstitial restrictive lung disease. In our cohort, 50% (6/12) of those who have had spirometry testing were found to have reduced lung capacity. Of those, four had obstructive lung disease, one had possible obstructive lung disease, and one had restrictive lung disease.
3.10 Endocrine and other clinical manifestations
There is limited data in the literature that describes endocrine system involvement in FD. In our cohort, patients reported type II diabetes, hypothyroidism, hyperthyroidism, hyperparathyroidism, and vitamin D deficiency (Supplemental Table 2). Patient 8 has a history of hyperthyroidism secondary to Graves' disease, and a past history of a 1.9 mm benign thyroid nodule. Patient 21 has type II diabetes and hypothyroidism. Patient 22 has acquired hypothyroidism and hyperparathyroidism secondary to renal disease. One of the pediatric participants (patient 9) was diagnosed with growth hormone deficiency and a small pituitary gland. Not all participants in this study have had an endocrine evaluation, so this study may have missed those with subclinical endocrine dysfunction.
Low Bone Mineral Density (BMD) has been noted more recently to be a common feature of FD [23]. Impaired renal function leads to vitamin D deficiency and may be one of the contributing factors to reduced BMD in FD patients. Fifty percent of patients in this cohort who had DXA scans (7/14) were found to have low BMD. Of those, 57% (4/7) had osteopenia (T score < 1.5) and 43% (3/7) had osteoporosis (T score < 2.5). Notably, two of the seven patients (29%) had early-onset low BMD at age 32 years (patient 6) and age 36 years (patient 13). There were no prior DXA scans to measure the rate of bone loss in these two young adult males. Additionally, patient 13 spends up to 40 h per week in a sensory deprivation floatation tank, which may have contributed to bone loss (described below in section 3.13. Supplemental Treatments). Only one patient endured an ankle fracture at the age of 65 years due to a mechanical fall; none of the remaining participants reported bone fractures. Of those with low BMD, four patients had concurrent vitamin D deficiency; of those four, one had secondary hyperparathyroidism due to renal disease. All patients in this cohort were prescribed calcium and vitamin D supplementation. Those with osteoporosis were managed by an endocrinologist and prescribed bisphosphonates.
Hypertension is also a common adverse event in adult FD patients. Cardiac, renal and cerebrovascular complications increase the risk of uncontrolled hypertension in individuals with FD [24]. Hypertension was present in 32% (8/25) of patients in our cohort, the majority but not all of these individuals had some form of clinically evident renal involvement. Patients were managed on ACEIs, ARBs, beta-blockers, and diuretics.
3.11 Novel GLA variant and genotype-phenotype correlations
Our study describes 16 unique variants in the 26 total participants, including one novel variant and one atypical ‘later-onset’ cardiac variant (Table 3; Fig. 5). The novel variant c.1226_1231delCCACAG (p.P409_G411delinsR), an in-frame deletion located within a hot-spot, was identified in a 63-year-old male (patient 22) who was diagnosed later in adulthood at 57 years old following a renal biopsy that revealed stage IV chronic kidney disease. He is discussed earlier with renal, pulmonary, and cardiac involvement and a history of two cerebrovascular accidents. Additional FD-related symptoms in patient 22 include angiokeratomas, corneal verticillata, gastrointestinal involvement, lymphedema, hypohidrosis, acroparesthesia, Raynaud's syndrome, hearing loss, tinnitus, and low bone mineral density (Supplemental Table 2). Cardiac work-up for chest pain revealed a 95% blockage of the left anterior descending coronary artery requiring stent placement, which resolved except for occasional reports of arrhythmia. Endocrine dysfunction in this patient includes acquired hypothyroidism and hyperparathyroidism secondary to renal disease. Patient 22 was particularly significant among our participants because of the history of one TIA and one stroke; brain MRI revealed significant gliosis and nonspecific white matter signal changes (Fig. 3B). Patient 22 is now deceased due to post-operative complications of abdominal surgery for abdominal abscess and intra-abdominal infection.Table 3 GLA variant details.
Table 3Fam. No. Patient No. Ethnicity Nucleotide Change Protein Change Position Type Phenotype
1 1,2,3,4,5,6 Hispanic c.983G > T G328V Exon 6 Missense Classic
2 7,8 Caucasian c.1250 T > G L417R Exon 7 Missense Classic
3 9,10,11 Caucasian c.132G > T W44C Exon 1 Missense Classic
4 12 Caucasian c.568delG A190PfsX2 Exon 4 Small Deletion Classic
5 13,14 Caucasian c.1041_1042insG A348GfsX27 Exon 7 Small Insertion Classic
6 15 Hispanic c.706 T > C W236R Exon 5 Missense Classic
7 16 Caucasian c.680G > A R227Q Exon 5 Missense Classic
8 17 Caucasian c.816C > A N272K Exon 6 Missense Classic
9 18 Caucasian c.730G > A D244N Exon 5 Missense Classic/ Later Onset
10 19 Asian c.639 + 919G > A IVS4 + 919G > A Intron 4 Splicing Later Onset
11 20 Asian c.427G > C A143P Exon 3 Missense Classic
12 21 Hispanic c.639 + 4A > T IVS4 + 4A > T Intron 4 Splicing Classic
13 22 Caucasian c.1226_1231delCCACAG P409_G411delinsR Exon 7 Small Deletion Classic
14 23 Hispanic c.1088G > A R363H Exon 7 Missense Later Onset
15 24,25 Hispanic c.1072_1074delGAG E358del Exon 7 Small Deletion Classic
16 26 Caucasian c.1246C > T Q416X Exon 7 Nonsense Classic
Novel variant is bolded.
Fig. 5 Unique variants identified in the GLA gene in this cohort. A schematic representation of GLA showing the position of all variants identified, with novel variant in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Though the rest of our cohort harbors variants that have been previously reported in the literature, we present novel clinical data for certain recurrent variants. Family 3 (patients 9, 10, 11) harbor the W44C variant, a previously reported variant in a Chinese family of 12 affected members, all of whom experienced paroxysmal pain of limb extremities [25]. Interestingly, our family is of European/mixed descent, with no known Asian ancestry, and present with multi-organ involvement in addition to peripheral pain. We report one patient with the c.639 + 919G > A ‘later-onset’ cardiac variant, who, in addition to mild left ventricular hypertrophy, experiences other FD-related symptoms including angiokeratomas, gastrointestinal issues, pulmonary involvement, and hypohidrosis with heat intolerance. His mother, who also harbors the familial variant, has a history of strokes and presented at age 76 years with hypertrophic cardiomyopathy. Another example of potential genotype-phenotype correlation in our cohort is Family 1; all three females who harbor the G328V variant have audiologic involvement.
3.12 Pediatric patients
There were two male pediatric participants in this study (patient 7 and patient 9). Both patients were diagnosed before the age of five and treated with ERT at or before the onset of symptoms. Patient 9 is a seven year-old male with the W44C variant. This patient was diagnosed with FD at age 3 and began ERT at age 7 years soon after an evaluation for reactive hyperemia index of 0.94 (Reference RHI value >1.68) using an EndoPAT (Itamar Medical) device performed on a research basis, which was consistent with low peripheral vascular endothelial function. He lacked ‘classic’ FD symptoms at the time of the procedure, but ERT was recommended due to concern for deteriorating endothelial function. Initial urine GL-3 level of 569 μg/mmol creatinine resolved during the course of this study while on ERT. He is developmentally normal, however he has a past medical history remarkable for growth hormone deficiency and a small pituitary gland and has been managed on somatropin since 3 years of age.
Patient 7 from Family 2 first presented with acroparesthesia and hypohidrosis at 4 years of age and began ERT by 6 years of age. Prior to initiation of ERT, urine GL-3 level was at 370 μg/mmol and was cleared to 0 μg/mmol in less than two years on ERT. Notably, spirometry testing revealed mild obstructive airway disease, though it has not yet affected routine activity. Patient 7 and his affected mother (patient 8) present with variable symptoms of FD as shown in Supplemental Table 2.
3.13 Supplemental treatments
Several individuals in this cohort sought alternative sources for pain management. Effective pain management could impact the psychological consequences of FD, as there is a high rate of depression that is often linked to the effects of long-term pain. Patient 13 had a five-year history of frequent, severe pain crises, including in response to ERT infusion, which has led the patient to seek alternative pain management strategies in conjunction with ERT. This includes floatation in a sensory deprivation tank used for the treatment of pain and anxiety by limiting stimulation from gravity, sound and light while immersed in water baths saturated with Epsom salts to allow for buoyancy in a dark and soundless room. Patient 15 and Patient 16 also report benefit from this float tank. Important to note, extended time in the float tank may have had an adverse effect on patient 13's bone density, as he reports spending up to 40 h per week in the float tank. There was a variety of medications used in this cohort for the management of neuropathic pain. Patient 13 and 15 are prescribed cannabidiol (CBD). Patient 15 and patient 26 use non-prescription topical and oral CBD as needed during pain crises. Use of tetrahydrocannabinol (THC) was also reported. Six patients are prescribed opioid narcotic analgesics, eight patients are managed with over-the-counter analgesics, two are on antidepressants, and one on an anticonvulsant (carbamazepine).
4 Discussion
The benefit, safety, and risk of ERT have been previously investigated, though the long-term impact continues to be studied [[26], [27], [28], [29], [30]]. ERT is recommended in order to curtail disease progression or when there is already evidence of disease progression and vital organ damage. In this study, 20 of the 26 participants and all but one male (patient 15) were consistently on ERT.
All participants receiving ERT however, demonstrated a different response rate of disease progression. While ERT was shown to be effective in clearing plasma GL-3 and, in many patients maintaining serum creatinine and eGFR within normal limits in this cohort, we identified that many patients continued to have impairment of their renal function and health-related quality of life despite standard of care management. For patients with chronic kidney disease from FD, standard of care did not vary when compared to that of other etiologies. The standard management includes the evaluation for possible reversible causes of renal disease (i.e. dehydration, reduced renal perfusion, obstruction, nephrotoxicity), management of blood pressure to a target ≤130/80, maximally tolerated dose of renin-angiotensin-aldosterone system inhibitors (i.e. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone receptor antagonist), smoking cessation, dietary interventions (particularly reduced sodium and protein restricted diet), treatment of metabolic acidosis, management of anemia of CKD with erythropoiesis-stimulating agents to target Hg 10–11.5 g/dL, management of mineral and bone disorders as per 2012 KDIGO guidelines, initiation of renal replacement therapy in individuals with uremic symptoms and eGFR 5–15 mL/min/1.73 m2 or in those with eGFR <5 mL/min/1.73 m2, and referral for kidney transplant evaluation in those with eGFR 20–25 mL/min/1.73 m2.
The late initiation of ERT may have contributed to those with renal disease progression, as shown previously [31]. Furthermore, nearly all of the adult patients in this cohort had major organ involvement at baseline prior to initiation of ERT. Typically, renal function in FD gradually deteriorates to end-stage renal disease in the third to fifth decade in males [32,33]. Only one patient progressed to severe CKD requiring hemodialysis, therefore, compared to the natural history data, it is possible that ERT contributed to the prevention of renal failure by slowing disease progression. Though vital organ damage is not typically seen in pediatric patients (some cases have been documented), studies have shown evidence for the benefit of the early initiation of ERT in potentially reversing renal damage [10,29]. Patient 12 initiated ERT at age 11 years, however, developed renal involvement by age 24 years [34]. Therefore, we believe earlier initiation of ERT is required to limit deterioration of the major organs. Our two pediatric participants initiated ERT at ages 6 and 7 years, respectively, and one reported significant improvement of pain symptoms.
Genotype-phenotype association is difficult to establish in FD for many reasons. Most GLA variants are unique (private mutations) and there are few family studies. Even within families, the phenotype may vary depending on the sex, age of onset, and other genetic and epigenetic factors. There are several reports in the literature of ‘later-onset’ isolated hypertrophic cardiomyopathy in individuals with the recurrent c.639 + 919G > A variant [6,[35], [36], [37]]. While this is often considered an atypical later-onset cardiac disease variant, patient 19 presented with several other ‘classic’ FD symptoms. Studies have explored the use of Amiloride as a potential treatment for FD by modulating alternative splicing in the context of the c.639 + 919G > A variant [35]. In our cohort we identified one novel variant c.1226_1231delCCACAG (p.P409_G411delinsR) in patient 22 with a severe, ‘classic’ FD phenotype [38]. This variant is absent in The Genome Aggregation Database (gnomAD), is located in a mutational hot-spot, and thus predicted to be pathogenic [39]. Two of the 26 individuals in this cohort harbor variants amenable to migalastat, an oral chaperone therapy, but chose not to switch from ERT. Long-term outcome studies for FD patients who have switched to migalastat will be of interest for future studies.
In this cohort, nearly one third of adult patients reported depression and anxiety. Based on the patient-reported SF-36 Health Survey and Brief Pain Inventory survey, there is no significant improvement in health-related quality of life over the course of ERT. Depression, sleep apnea, and anxiety disorders are linked to both pathology and the effects of long-term pain [40]. It has been suggested that psychological counseling can have an impact on reversing or reducing effects, an important consideration to optimize patient care as needed [41]. Importantly, studies have proposed pain as an indicator for the need to initiate ERT [42]. The majority of our cohort experienced Fabry crises or acroparasthesia. There are few data on nervous system involvement of FD in women [43]. Some studies have shown hippocampal volume loss and white matter lesions in female patients with FD [44]. While we showed white matter lesions in three females and one male in this cohort, we did not include brain imaging, electrical conduction, or quantitative sensory testing for every participant in this study. Further studies assessing the outcomes of neuropathy and CNS findings with ERT are warranted.
Cardiac manifestation can be the primary and only symptom of FD in some patients and can present as early as childhood. Our study showed a higher prevalence of cardiac manifestations in both the males and females compared to previous studies [34,[45], [46], [47]]. Cardiac involvement was observed in 83% of male patients, including one pediatric male patient with sinus bradycardia, and 75% of female patients in this cohort. Left ventricular hypertrophy (LVH) was seen in 70% of males and 42% of females. This data provides supporting evidence that female patients, even those with no obvious signs or symptoms of FD, should be monitored closely for evidence of cardiac involvement and be considered for ERT. The standard of care for patients with hypertrophic cardiomyopathy due to FD did not vary when compared to the management of other etiologies and followed AHA/ACC guidelines.
We highlight the variable endocrine dysfunction and low bone mineral density in this cohort, including several with osteoporosis, which provides further evidence for the inclusion of adequate endocrine work-up in the ongoing management of all patients with FD [48].
There is a need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. Furthermore, there is a need for more and improved biomarkers to monitor disease progression. Early identification of endothelial function or other pathologies can provide insight into when ERT should be initiated in order to slow the advancement of adverse effects [49]. Additional tools and biomarkers for measuring endothelial function and other indicators of future pathologies, such as proteomics, are needed to help prevent the deterioration of vital organs [[50], [51], [52]]. Newborn screening for FD has been implemented in several states and will lead to earlier detection. Early detection is important to proactively monitor for complications of the disease, initiate ERT, and prevent co-morbidity progression [[53], [54], [55], [56]].
Since most of our participants were on ERT, we could not make comparisons between long-term outcomes of those on ERT compared to those not on ERT. Also, with the small sample size, we could not compare multiple age-matched individuals or genotype-specific individuals other than our reported families. Another limitation to this study is the long-term analysis of plasma-GL-3 over time rather than plasma Lyso-GL-3, due to the fact that we had more data points for plasma-GL-3. Plasma Lyso-GL-3 has been recently determined to be a more accurate biomarker that correlates more closely with disease phenotype in male and in female patients, with higher levels associated with increased clinical manifestation [57].
5 Conclusion
The variability of symptoms and disease progression in patients with FD, even within the same family, complicates the discernment of a genotype-phenotype correlation. ERT has been available for the treatment of FD since 2001 and has shown improved outcomes, especially when started prior to organ damage, and may reverse fatal disease progression. In this cohort, agalsidase beta infusions have been effective in clearing GL-3 levels. However, renal involvement and health-related quality of life has continued to progress in adult patients diagnosed late despite ERT infusions.
Eight females were on ERT in this study and demonstrated significant symptoms of FD, and in some cases the disease progressed to a similar degree as in the males. This study further demonstrates that females may develop significant FD symptoms and should be managed appropriately and treated as more than carriers.
Documentation of novel variants can contribute to future genotype-phenotype associations between the severity and progression of FD. Genotype-phenotype correlations and long-term outcome data on ERT and/or other FD treatments are needed, especially now that the newborn screening has led to an increase in diagnoses and earlier detection in individuals with FD.
Authors' contributions
V.K. conceived the study and supervised the overall direction and implementation of the study. All authors contributed to acquisition of data. V.K., M.D.C., D.R., D.T., Z.A.D., and G.L. analyzed the data. D.R. performed statistical analyses. M.D.C. and E.C. drafted the manuscript with input from all authors. All authors provided critical feedback and approved the final manuscript for publication.
Funding
The Fabry registry was funded by Sanofi Genzyme. This funding source had no role in data collection or analysis.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Appendix A Supplementary data
Supplementary material
Image 1
Acknowledgements
We thank the patients and their health care providers for their contribution to the study. The Fabry registry was funded by Sanofi Genzyme. This work was supported by CTSI grant UL1TR000124UCLA (Clinical and Translational Science Institute), Los Angeles, CA, USA.
Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2020.100700. | AGALSIDASE BETA, CANNABIDIOL | DrugsGivenReaction | CC BY-NC-ND | 33437642 | 18,784,055 | 2021-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chills'. | Variable clinical features of patients with Fabry disease and outcome of enzyme replacement therapy.
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme α-galactosidase A due to mutations in the GLA gene. This leads to an accumulation of globotriaosylceramide (GL-3) in many tissues, which results in progressive damage to the kidneys, heart, and nervous system. We present the molecular and clinical characteristics and long-term outcomes of FD patients from a multidisciplinary clinic at the University of California, Irvine treated with agalsidase beta enzyme replacement therapy (ERT) for 2-20 years. This cohort comprised 24 adults (11 males, 13 females) and two male children (median age 45; range 10-68 years). Of the 26 patients in this cohort, 20 were on ERT (12 males, 8 females). We describe one novel variant not previously reported in the literature in a patient with features of 'classic' FD. The vast majority of patients in this cohort presented with symptoms of 'classic' FD including peripheral neuropathic pain, some form of cardiac involvement, angiokeratomas, corneal verticillata, hypohidrosis, tinnitus, and gastrointestinal symptoms, primarily abdominal pain. The majority of males had clinically evident renal involvement. An annual eGFR reduction of -1.88 mL/min/1.73 m2/yr during the course of ERT was seen in this cohort. The most common renal presentation was proteinuria, and one individual required a renal transplant. Other common findings were pulmonary involvement, lymphedema, hearing loss, and significantly, three patients had strokes. Notably, there was a high prevalence of endocrine dysfunction and low bone mineral density, including several with osteoporosis. While enzyme replacement therapy (ERT) cleared plasma GL-3 in this cohort, there was limited improvement in renal function or health-related quality of life based on the patient-reported SF-36 Health Survey. Physical functioning significantly declined over the course of ERT treatment, which may be, in part, due to the late initiation of ERT in several patients. Further delineation of the phenotypic and genotypic spectrum in patients with FD and the long-term outcome of ERT will help improve management and treatment options for this disease.
1 Introduction
Fabry Disease (FD, OMIM#301500) is an inherited, X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficient or absent α-galactosidase A enzyme [1]. This consequently leads to the progressive deposition of globotriaosylceramide (GL-3) in several tissues throughout the body, notably in endothelial cells, smooth muscle cells, podocytes, and cardiomyocytes [2]. The absence of this enzyme may lead to multi-organ involvement including progressive renal disease, cardiac disease, cerebrovascular disease, neuropathic pain, corneal verticillata, angiokeratomas, tinnitus, hearing loss, pulmonary involvement, and gastrointestinal complaints, including abdominal pain, constipation, and diarrhea. FD is pan-ethnic and the second most prevalent lysosomal storage disorder with an estimated incidence of 1 in 40,000 to 117,000 males worldwide, though newborn screening initiatives have revealed that the incidence is much higher [[3], [4], [5], [6]]. This is likely due to the fact that early studies referred to the incidence of ‘classic’ FD, while newborn screening may identify individuals on the full phenotypic spectrum. Symptoms typically present early during childhood or adolescence. Affected males with little or absent α-galactosidase A activity (<1% of mean normal) may present with clinical features of ‘classic’ FD. Mutations that result in residual enzyme activity typically cause a ‘later-onset’ form of the disease with variable manifestations. Heterozygous females typically present with milder symptoms and later-onset and less commonly have ‘classic’ FD due to residual enzyme activity and X-chromosome inactivation pattern.
Management and guidelines of a newly diagnosed individual with FD includes documentation of symptoms, consideration of enzyme replacement therapy (ERT), and monitoring of disease progression [7,8]. ERT is provided to supplement or replace α-galactosidase A, and to slow the rate of disease progression. Thus, early identification of affected individuals is important in order to initiate ERT as early as possible [[8], [9], [10]]. Clinical evaluation during childhood is recommended if a familial mutation is known [11]. In the absence of a family history, a timely diagnosis of FD may be difficult due to the variation and unpredictability of clinical manifestations that can often be misdiagnosed as childhood growing pains. The addition of FD to newborn screening programs worldwide has allowed for the early detection of the disorder [12].
There are two types of enzyme replacement therapy: agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme); however only agalsidase beta is an approved ERT by the US Food and Drug Administration (FDA) [13,14]. There is some discussion on the most appropriate infusion as well as the efficacy and safety of the infusions, but studies have shown no adverse effects in switching therapies or an increased benefit of using one over the other [15,16]. There is one oral pharmacological chaperone therapy, migalastat (Galafold), that has been approved by the FDA [17]. This treatment is only available for adults with FD who have amenable gene variants that lead to misfolded α-galactosidase A enzyme that can be stabilized by migalastat. Once stabilized, protein folding is improved and trafficking of the α-galactosidase A enzyme to the lysosome is restored. Other treatments currently being investigated in clinical trials include plant-based ERT, gene therapy, and substrate reducing therapies.
We describe a cohort of 24 adults and two children with FD, 13 males and 13 females, who range in age from 10 to 68 years (mean age 46.1 ± 16.3). The majority of participants were on agalsidase beta ERT (20/26; 12 males, 8 females). Each participant demonstrated different response rates of symptoms and disease progression with ERT, including two individuals who developed adverse infusion reactions.
The aim of this study is to present the variable molecular and clinical features in a cohort of FD patients on ERT, treatment outcomes across organ systems, and highlight patients with previously unreported variants, to help improve treatment options and patient care.
2 Methods
2.1 Participants
This cohort comprised 13 male and 13 female patients, including two male children. All participants had a confirmed molecular diagnosis of FD and most also had recorded enzyme activity levels measured. GLA variants are listed using transcript NM_000169.2. These patients were followed over the course of a one to 12 year period in an outpatient multidisciplinary clinic at the University of California, Irvine. Earlier data were obtained from review of past medical records and the Fabry Registry, if available. Written IRB informed consent approved by UC Irvine (#2008–6631) was obtained from each participant, and all procedures were performed according to The Code of Ethics of the World Medical Association.
2.2 Clinical evaluations
Clinical surveillance visits were conducted every six months and included evaluations by a clinical geneticist, a cardiologist, and a nephrologist. The standard of care for patients with renal and cardiac disease from FD did not vary when compared to the management of other etiologies. At each visit, the following components were documented:• Quality of life surveys, including a Brief Pain Inventory (BPI) and the SF-36 Health Survey. These provided a scale for pain and represented an attempt to document how pain has influenced the quality of life of each individual. The SF-36 Health survey measures physical function, social function, physical role, emotional role, mental health, energy, pain, and general health perception. Scores ranged from 0 (worst) to 100 (best).
• The development or change in the status of clinical features specific to FD including cardiac, renal, pulmonary, and nervous system involvement, in addition to angiokeratomas, acroparesthesia, GI health, hyper or hypohidrosis, hearing loss and tinnitus, lymphedema, and corneal whorling.
• Plasma and urine GL-3 levels and plasma Lyso-GL-3 to assess overall glycolipid burden.
• Renal status and disease progression were monitored by labs: serum creatinine levels to measure the mean estimated glomerular filtration rate (eGFR), in addition to urine creatinine, urine protein, and urine albumin/micro-albumin to measure proteinuria and albuminuria. Renal biopsy was only performed if clinically indicated.
Evaluations performed at less frequent intervals:• Cardiac status was determined by echocardiography (with strain), electrocardiograms, and holter monitor screened at annual intervals in males and less frequently in females and children. Cardiac MRI was conducted every 2–3 years or as needed.
• Brain imaging was performed by MRI, MRA, and CT scan.
• Ophthalmologic exams were performed for evaluation of corneal whorls, vessel tortuosity, and lenticular opacities.
• Audiometry was performed for evaluation of sensorineural hearing loss and tinnitus, as needed.
• Dual-energy X-ray absorptiometry (DXA) & the World Health Organization (WHO) classification of bone mineral density were used to classify severity. T-scores between −1.0 and −2.5 SD were categorized as osteopenia and T-scores below −2.5 SD were categorized as osteoporosis.
2.3 ERT infusions
Agalsidase beta (Fabrazyme) recommended dosage is 1 mg/kg body weight given every two weeks as an intravenous infusion [14]. The majority of the participants in this cohort received the infusions without incident, and some have transitioned to home infusions. To determine the effectiveness of ERT, we measured the presence of agalsidase beta IgG antibodies, plasma and urine GL-3 levels, and plasma Lyso-GL-3 levels at each six-month visit. Two individuals experienced adverse reactions to ERT infusions (patient 15 and patient 22); they both tested negative for IgE antibodies and showed no change in IgG antibodies.
2.4 Statistical analysis
We used linear mixed-effects regression analysis with varying intercepts and slopes to evaluate linear time trends of serum creatinine, eGFR, and health-related quality of life scores (SF-36 Health Survey), starting with the initiation of ERT (time = 0). The significance level for all statistical tests was set to α = 0.05. All analyses were carried out using the R language and environment for statistical computing, version 3.6.3.
3 Results
Of the 26 patients included in our study, there were 13 males and 13 females between ages 10–68 years (mean age 46.1 ± 16.3 years). This cohort consisted of five families in addition to eleven unrelated individuals. The majority of males (12/13, 92%) and the majority of females (8/13, 62%) were treated with ERT. Ages at initiation of ERT were between 6 and 65 years (mean age 39.0 ± 18.3) (Supplemental Table 1). The majority of patients tolerated ERT well, except for two individuals who had adverse reactions (patient 15 and patient 22). Both patients were IgE-negative and showed no changes in IgG antibodies; patient 22 was consistently IgG-positive throughout the course of ERT. Patient 15 had the most significant adverse event during the last infusion attempt, which resulted in intubation in the cardiac intensive care unit due to difficulty breathing, tightness in chest, rigors, and convulsions, and has not been on ERT since. Only three males were routinely IgG-positive (patients 12, 13, 24) and two males had one single IgG-positive test in the past, but had not had another one since (patient 1 and patient 7).
Clinical features of each patient are described in Supplemental Table 2. Notably, there was clinically evident renal involvement in 50% of participants; the most common feature was varying levels of proteinuria. One individual required a renal transplant. Seventy-nine percent of patients showed evidence of some form of cardiac involvement, including 46% with left ventricular hypertrophy. Two males and one female had a history of a stroke, and one male had a history of one TIA and one stroke. The most commonly observed clinical feature was peripheral neuropathic pain (20/26, 77%), followed by angiokeratomas (18/26, 69%), tinnitus (17/26, 65%), gastrointestinal symptoms, including abdominal pain, constipation, and diarrhea (16/26, 62%), and hypohidrosis/anhidrosis (16/26, 62%). Additional common findings of FD in this cohort include corneal verticillata (15/26, 58%), pulmonary involvement (6/12, 50%), lymphedema (11/26, 42%), and hearing loss (9/26, 35%). The adult males in this cohort had higher frequencies of clinical manifestations than adult females in all categories with the exception of neuropathic pain, white matter lesions, and corneal verticillata (Table 1).Table 1 Clinical manifestations of FD in this cohort.
Table 1 Adult Females
N = 13 (%) Adult Males
N = 11 (%) Pediatric Males
N = 2 (%) Total
N = 26 (%)
Mean Age at Diagnosis (Years) 36.2 34.3 3.5 32.8 ± 16.3
Currently On ERT 8 (62) 10 (91) 2 (100) 20 (77)
Mean Age ERT Initiated (Years) 51 37.5 6.5 39.0 ± 18.3
Renal Involvement 5 (38) 7 (64) 1 (50) 13 (50)
Cardiac Involvement 9/12 (75) 9/10 (90) 1 (50) 19/24 (79)
White matter Lesions 3/10 (30) 1/6 (17) N/A 4/16 (25)
Neuropathic Pain 10 (77) 8 (73) 2 (100) 20 (77)
Corneal Verticillata 10 (77) 4 (36) 1 (50) 15 (58)
Angiokeratomas 7 (54) 11 (100) 0 (0) 18 (69)
Hypohidrosis 5 (38) 10 (91) 1 (50) 16 (62)
GI Involvement 8 (62) 8 (73) 0 (0) 16 (62)
Low BMD 2/6 (33) 5/8 (63) N/A 7/14 (50)
Hearing Loss 3 (23) 6 (55) 0 (0) 9 (35)
Tinnitus 7 (54) 9 (82) 1 (50) 17 (65)
Vertigo 4/10 (40) 6/10 (60) 0/1 (0) 10/21 (48)
Pulmonary Involvement 0/5 (0) 5/6 (83) 1/1 (100) 6/12 (50)
Depression 3 (23) 3 (27) 0 (0) 6/26 (23)
Numbers provided in fractions indicate that not all patients had the corresponding assessment.
3.1 Clearance of GL-3 in plasma
Plasma-GL-3, plasma Lyso-GL-3, and urine GL-3 are biomarkers used as surrogate endpoints to monitor Fabry disease severity and progression in both treated and untreated patients. Longitudinal analysis of plasma GL-3 concentration in 17 of our patients on ERT is depicted in Fig. 1. The data showed significant reduction in GL-3 of −0.25 μg/mL per year (95% CI: −0.478 to −0.030, p = 0.026) with ERT treatment.Fig. 1 Plasma GL-3 clearance in patients on ERT. A linear mixed-effects regression analysis of plasma GL-3 with time as a predictor and varying intercepts and slopes. The figure shows observed GL-3 measurements (dots) together with model-estimated time trajectories of GL-3 for each participant (colored lines) and for the population (black line). Note that observations at time < 0 were excluded from the analysis. N = 17.
Fig. 1
While there was less data available for the deacetylated form, plasma Lyso-GL-3, the majority showed a reduction or stabilization of plasma Lyso-GL-3 concentration at last visit compared to that of the earlier time points (N = 11, data not shown).
3.2 Renal involvement
There was clinically evident renal disease in 50% (13/26) of participants as defined by albuminuria, hypertension or reduced eGFR. Renal biopsies were not done routinely. The majority demonstrated proteinuria and/or albuminuria as the sole renal manifestation. Ten patients had elevated urine albumin-to-creatine ratios, nine with microalbuminuria (5 females, 4 males) and one male with macroalbuminuria. Hypertension was seen in 32% (8/25) of patients and managed with angiotensin-converting enzyme inhibitors (ACEI) (8/26), angiotensin receptor blockers (ARB) (4/26), beta-blockers (4/26), and diuretics (6/26). Two had stage I chronic kidney disease (CKD) (patient 1 and patient 12), two had stage III CKD (patient 14 and patient 17), and one had stage IV CKD and is on hemodialysis (patient 22). Patient 23 underwent a deceased donor renal transplant due to ESRD at age 52 years and has had normal kidney function since. For those on ERT, only some patients maintained serum creatinine and estimated glomerular filtration rate (eGFR) within normal limits for age and sex. However, the data showed significant increase in serum creatinine levels of +0.02 mg/dL per year (95% CI: 0.01–0.04, p = 0.015) for both males and females on ERT in this cohort (Fig. 2A) and +0.038 mg/dL per year (95% CI: 0.01–0.06, p = 0.003, data not shown) for males alone. Further, the data showed an annual decrease in eGFR of −1.88 mL/min/1.73m2 (95% CI: −3.36 to −0.40, p = 0.013) on ERT in both males and females (Fig. 2B), and −3.16 mL/min/1.73m2 (95% CI: −4.97 to −1.35, p = 0.001, data not shown) for males alone.Fig. 2 Serum creatinine and estimated glomerular filtration rate (eGFR) of patients on ERT. The figure shows observed serum creatinine measurements (A) and eGFR measurements (B) together with model-estimated time trajectories for each participant (colored lines) and for the population (black lines). Note that observations at time < 0 were excluded from both analyses in figs. A-B. N = 18.
Fig. 2
3.3 Cardiac involvement
In our cohort, 79% (19/24) of participants had some form of cardiac involvement. Left ventricular hypertrophy (LVH) was seen in 46% (11/24) of patients, with severity ranging from borderline to severe and one confined to the septum. Severe LVH of patient 24 is shown in Fig. 3A. Of the adult males, only one was not found to have clinically evident cardiac involvement, and while his past ECGs met voltage criteria for LVH, his echocardiograms were normal showing no evidence of LVH. Left ventricular ejection fraction (LVEF) was one of the measures used to monitor cardiac disease progression of patients on ERT. LVEF was within normal range at baseline prior to ERT. Some patients fell below normal range over the course of treatment with the lowest recorded being 43% in a male patient. Of the individuals in our cohort who had cardiac imaging, 25% (6/24) had valvular abnormalities, including one patient with aortic valve sclerosis, two patients had thickened mitral valve leaflets, one patient with mild Systolic Anterior Motion (SAM) abnormality of the mitral valve, one with mitral valve prolapse, and one patient required mitral valve annuloplasty after experiencing lower extremity edema, shortness of breath and dyspnea on exertion (patient 22). Other cardiac manifestations are described in Table 2. Of those who underwent cardiac MRI, only one showed possible fibrosis of the myocardium (Fig. 3A). Those with cardiac abnormalities on echocardiogram, cardiac MRI, and/or ECG did not consistently have abnormal cardiac biomarkers, such as brain natriuretic peptide (BNP), troponin, and soluble suppression of tumourigenicity 2 (ST2), though limited data was available. Adult FD patients can have elevated lipid levels, particularly high HDL cholesterol, which may be non-responsive to long-term ERT [18]. Dyslipidemia was present in 48% (10/21) of patients in our cohort, and managed with diet, fish-oil and statins.Fig. 3 Clinical features in patients with FD. (A) Cardiac MRI showing severe concentric hypertrophy of the left ventricle with reduced chamber size. There is subendocardial linear hyperenhancement in the basal inferolateral wall suggesting myocardial scar (white arrow), consistent with FD (patient 24); (B) Axial brain MRI section following TIA event (patient 22); (C) Corneal verticillata seen by slit lamp examination. Courtesy: Dr. Pinakin Davey, Western University of Health Sciences; (D) Cluster of angiokeratomas in the groin region (patient 24).
Fig. 3Table 2 Characterization of cardiac involvement in FD patients in this cohort.
Table 2 Adult
Females Adult
Males Pediatric Males Total (%)
Cardiac Involvement 9/12 (75) 9/10 (90) 1/2 (50) 19/24 (79)
Echocardiogram Findings
Left Ventricular Hypertrophy 5/12 7/10 0/2 11/24 (46)
Borderline 1 2 0
Mild 2 3 0
Moderate 0 1 0
Severe 0 1 0
Septal 1 0 0
Valvular Abnormality 2/12 4/10 0/2 6/24 (25)
Aortic Valve 1 0 0
Mitral Valve 1 4 0
Left Atrium Dilation 2/12 2/10 0/2 4/24 (17)
Right Atrium Dilation 1/12 0/10 0/2 1/24 (4)
Electrocardiogram Findings
Conduction Abnormalities 3/12 6/10 0/2 9/24 (38)
Right Bundle Branch Block 2 5 0
Anterior Fascicular Block 1 1 0
First Degree Atrioventricular Block 1 0 0
Intraventricular Conduction Delay 0 2 0
Short PR Interval 1 2 0
Rhythm Abnormalities 5/12 8/10 1/2 14/24 (58)
Sinus Bradycardia 5 8 1
Sinus Arrhythmia 1 0 0
Premature Ventricular Complexes 0 2 0
Premature Atrial Contractions 0 1 0
Repolarization Abnormalities 5/12 3/10 0/2 8/24 (33)
T Wave Changes 2 1 0
ST/STT Changes 3 2 0
Left Axis Deviation 0/12 1/10 0/2 1/24 (4)
One male and one female in this cohort were excluded due to no cardiac imaging records available.
3.4 Cerebrovascular complications, neurologic, and neuropsychiatric symptoms
White matter lesions were present in 25% (4/16) of participants who had brain imaging. One male in this cohort (patient 22) has a history of two cerebrovascular accidents, one transient ischemic attack (TIA) at age 52 years and one stroke at age 60 years. Brain MRI of patient 22 showed multifocal and adjacent gliosis within the left cerebral hemisphere, high left frontal and parietal lobes, as well as subcortical and periventricular deep white matter T2 hyperintensity without mass effect that is most pronounced within the parietal occipital region (Fig. 3B). The stroke impacted his eyesight and word-finding ability. Patient 22 only began ERT at the age of 57 years, and passed away at the age of 63 years due to post-operative complications of abdominal surgery. Another male (patient 15) and one female (patient 14) in this cohort have a history of TIAs while on ERT. They were not taking low dose aspirin at the time.
With regard to neuropathic manifestations, 77% (20/26) of patients reported peripheral neuropathic pain, manifesting as burning, tingling, or numbness, often triggered by temperature change or strenuous activity. Of the 17 participants for whom data on the brief pain inventory (BPI) survey was available, the majority reported worsening or inconsistent pain symptoms over the duration of treatment with ERT. Decreased quality of life has been associated with FD, in part due to the debilitating episodic pain crises and burden associated with having a chronic illness. Health-related quality of life was collected using the SF-36 Health survey. Bodily pain scores obtained from the SF-36 survey also showed no improvement over the course of ERT, though this was not significant (p = 0.19; Fig. 4C). For the majority of SF-36 respondents, pain worsened over time, which was similar to what was reported in the BPI survey. Notably, there was a significant decline in physical functioning (Est. = −1.40, 95% CI: −2.73 to −0.06, p = 0.040) for patients in this cohort. The remaining health dimensions did not show significant change over time (Fig. 4A-H).Fig. 4 SF-36 health-related quality of life component scores of patients on ERT. The SF-36 Health survey measures (A) physical function, (B) physical role, (C) pain, (D) general health perception, (E) vitality, (F) social function, (G) emotional role, (H) mental health. Scores ranged from 0 (worst) to 100 (best). Observed values (dots) are shown together with model-estimated trajectories for individual patients (colored lines) and the population (black lines).
Fig. 4
Of the adult patients in this cohort, seven reported depression (7/24; 29%). One patient reported a history of depression that correlated with pain symptoms. Another patient had a history of depression with no current pain symptoms based on responses in the BPI and SF-36 Health Survey. Of note, one patient in this cohort had a history of two suicide attempts. On the other hand, one patient's testimonial described noticeable improvement in anxiety, fatigue, and executive function (termed “Fabry fog”) since initiation of ERT.
3.5 Ophthalmologic findings
Corneal verticillata, or corneal whorling, is a unique clinical feature in patients of both sexes with FD and is often used as a diagnostic tool. It is a result of GL-3 deposits and has not been linked to vision loss. Corneal verticillata was noted in 58% (15/26) of individuals, at a higher prevalence in females than males in our cohort (Fig. 3C). Vessel tortuosity is also associated with FD and is exacerbated by renal complications [19,20]. Only one patient was found to have vessel tortuosity (patient 22); this individual had stage IV CKD.
3.6 Skin manifestations
Angiokeratomas are common in Fabry patients as they occur when GL-3 accumulates in dermal endothelial cells and lead to secondary ectasia. In our cohort, 69% (18/26) had angiokeratomas (Fig. 3D).
Hypohidrosis is also a common feature of FD and may be a predisposition to acroparesthesia. In this cohort, 62% (16/26) experienced hypohidrosis or anhidrosis. Of those with hypohidrosis, 89% experienced acroparesthesia. Lymphedema of the extremities, particularly in the feet, was present in 42% (11/26) of patients in this study.
3.7 Gastrointestinal involvement
Gastrointestinal symptoms are common among FD patients and can manifest as diarrhea, constipation, nausea, vomiting, incontinence, and abdominal pain. In our cohort, gastrointestinal symptoms were present in 62% (16/26) and the most common complaint was abdominal pain. Previous studies have shown significantly improved gastrointestinal pain with ERT [21]. This was also reported by the patients in our study in the patient health survey. The majority of those on ERT reported either improved or stable abdominal pain and frequency of diarrhea based on their survey responses at last visit compared to earlier survey responses. Specifically, six patients reported improvement in abdominal pain and four reported that symptoms were stable (N = 20). Eleven reported improvement in diarrhea and one reported that symptoms were stable (N = 20). One patient's testimonial described a frequency of diarrhea multiple times daily prior to ERT that has now significantly improved since ERT initiation. He also reported noticeable return of symptoms during the time of the ERT shortage. On the other hand, worsening or recent onset of abdominal pain (3/20) and diarrhea (2/20) over the course of ERT treatment were also reported by participants in this study.
3.8 Auditory and vestibular involvement
Sensorineural hearing loss, predominantly in the high frequencies, is a common manifestation of FD and correlates with neuropathic and vascular damage [22]. In our cohort, 35% (9/26) of patients reported a history of hearing loss. The majority of those who underwent audiology assessments were found to have bilateral mild to moderate sensorineural hearing loss. Additionally, 65% (17/26) of patients reported tinnitus. Some patients do not use hearing aids due to amplification by the presence of tinnitus. Vertigo was reported in 48% of our cohort in those who recorded their response in the health survey (10/21).
3.9 Pulmonary involvement
Respiratory involvement in FD typically includes obstructive lung disease, however patients may develop interstitial restrictive lung disease. In our cohort, 50% (6/12) of those who have had spirometry testing were found to have reduced lung capacity. Of those, four had obstructive lung disease, one had possible obstructive lung disease, and one had restrictive lung disease.
3.10 Endocrine and other clinical manifestations
There is limited data in the literature that describes endocrine system involvement in FD. In our cohort, patients reported type II diabetes, hypothyroidism, hyperthyroidism, hyperparathyroidism, and vitamin D deficiency (Supplemental Table 2). Patient 8 has a history of hyperthyroidism secondary to Graves' disease, and a past history of a 1.9 mm benign thyroid nodule. Patient 21 has type II diabetes and hypothyroidism. Patient 22 has acquired hypothyroidism and hyperparathyroidism secondary to renal disease. One of the pediatric participants (patient 9) was diagnosed with growth hormone deficiency and a small pituitary gland. Not all participants in this study have had an endocrine evaluation, so this study may have missed those with subclinical endocrine dysfunction.
Low Bone Mineral Density (BMD) has been noted more recently to be a common feature of FD [23]. Impaired renal function leads to vitamin D deficiency and may be one of the contributing factors to reduced BMD in FD patients. Fifty percent of patients in this cohort who had DXA scans (7/14) were found to have low BMD. Of those, 57% (4/7) had osteopenia (T score < 1.5) and 43% (3/7) had osteoporosis (T score < 2.5). Notably, two of the seven patients (29%) had early-onset low BMD at age 32 years (patient 6) and age 36 years (patient 13). There were no prior DXA scans to measure the rate of bone loss in these two young adult males. Additionally, patient 13 spends up to 40 h per week in a sensory deprivation floatation tank, which may have contributed to bone loss (described below in section 3.13. Supplemental Treatments). Only one patient endured an ankle fracture at the age of 65 years due to a mechanical fall; none of the remaining participants reported bone fractures. Of those with low BMD, four patients had concurrent vitamin D deficiency; of those four, one had secondary hyperparathyroidism due to renal disease. All patients in this cohort were prescribed calcium and vitamin D supplementation. Those with osteoporosis were managed by an endocrinologist and prescribed bisphosphonates.
Hypertension is also a common adverse event in adult FD patients. Cardiac, renal and cerebrovascular complications increase the risk of uncontrolled hypertension in individuals with FD [24]. Hypertension was present in 32% (8/25) of patients in our cohort, the majority but not all of these individuals had some form of clinically evident renal involvement. Patients were managed on ACEIs, ARBs, beta-blockers, and diuretics.
3.11 Novel GLA variant and genotype-phenotype correlations
Our study describes 16 unique variants in the 26 total participants, including one novel variant and one atypical ‘later-onset’ cardiac variant (Table 3; Fig. 5). The novel variant c.1226_1231delCCACAG (p.P409_G411delinsR), an in-frame deletion located within a hot-spot, was identified in a 63-year-old male (patient 22) who was diagnosed later in adulthood at 57 years old following a renal biopsy that revealed stage IV chronic kidney disease. He is discussed earlier with renal, pulmonary, and cardiac involvement and a history of two cerebrovascular accidents. Additional FD-related symptoms in patient 22 include angiokeratomas, corneal verticillata, gastrointestinal involvement, lymphedema, hypohidrosis, acroparesthesia, Raynaud's syndrome, hearing loss, tinnitus, and low bone mineral density (Supplemental Table 2). Cardiac work-up for chest pain revealed a 95% blockage of the left anterior descending coronary artery requiring stent placement, which resolved except for occasional reports of arrhythmia. Endocrine dysfunction in this patient includes acquired hypothyroidism and hyperparathyroidism secondary to renal disease. Patient 22 was particularly significant among our participants because of the history of one TIA and one stroke; brain MRI revealed significant gliosis and nonspecific white matter signal changes (Fig. 3B). Patient 22 is now deceased due to post-operative complications of abdominal surgery for abdominal abscess and intra-abdominal infection.Table 3 GLA variant details.
Table 3Fam. No. Patient No. Ethnicity Nucleotide Change Protein Change Position Type Phenotype
1 1,2,3,4,5,6 Hispanic c.983G > T G328V Exon 6 Missense Classic
2 7,8 Caucasian c.1250 T > G L417R Exon 7 Missense Classic
3 9,10,11 Caucasian c.132G > T W44C Exon 1 Missense Classic
4 12 Caucasian c.568delG A190PfsX2 Exon 4 Small Deletion Classic
5 13,14 Caucasian c.1041_1042insG A348GfsX27 Exon 7 Small Insertion Classic
6 15 Hispanic c.706 T > C W236R Exon 5 Missense Classic
7 16 Caucasian c.680G > A R227Q Exon 5 Missense Classic
8 17 Caucasian c.816C > A N272K Exon 6 Missense Classic
9 18 Caucasian c.730G > A D244N Exon 5 Missense Classic/ Later Onset
10 19 Asian c.639 + 919G > A IVS4 + 919G > A Intron 4 Splicing Later Onset
11 20 Asian c.427G > C A143P Exon 3 Missense Classic
12 21 Hispanic c.639 + 4A > T IVS4 + 4A > T Intron 4 Splicing Classic
13 22 Caucasian c.1226_1231delCCACAG P409_G411delinsR Exon 7 Small Deletion Classic
14 23 Hispanic c.1088G > A R363H Exon 7 Missense Later Onset
15 24,25 Hispanic c.1072_1074delGAG E358del Exon 7 Small Deletion Classic
16 26 Caucasian c.1246C > T Q416X Exon 7 Nonsense Classic
Novel variant is bolded.
Fig. 5 Unique variants identified in the GLA gene in this cohort. A schematic representation of GLA showing the position of all variants identified, with novel variant in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Though the rest of our cohort harbors variants that have been previously reported in the literature, we present novel clinical data for certain recurrent variants. Family 3 (patients 9, 10, 11) harbor the W44C variant, a previously reported variant in a Chinese family of 12 affected members, all of whom experienced paroxysmal pain of limb extremities [25]. Interestingly, our family is of European/mixed descent, with no known Asian ancestry, and present with multi-organ involvement in addition to peripheral pain. We report one patient with the c.639 + 919G > A ‘later-onset’ cardiac variant, who, in addition to mild left ventricular hypertrophy, experiences other FD-related symptoms including angiokeratomas, gastrointestinal issues, pulmonary involvement, and hypohidrosis with heat intolerance. His mother, who also harbors the familial variant, has a history of strokes and presented at age 76 years with hypertrophic cardiomyopathy. Another example of potential genotype-phenotype correlation in our cohort is Family 1; all three females who harbor the G328V variant have audiologic involvement.
3.12 Pediatric patients
There were two male pediatric participants in this study (patient 7 and patient 9). Both patients were diagnosed before the age of five and treated with ERT at or before the onset of symptoms. Patient 9 is a seven year-old male with the W44C variant. This patient was diagnosed with FD at age 3 and began ERT at age 7 years soon after an evaluation for reactive hyperemia index of 0.94 (Reference RHI value >1.68) using an EndoPAT (Itamar Medical) device performed on a research basis, which was consistent with low peripheral vascular endothelial function. He lacked ‘classic’ FD symptoms at the time of the procedure, but ERT was recommended due to concern for deteriorating endothelial function. Initial urine GL-3 level of 569 μg/mmol creatinine resolved during the course of this study while on ERT. He is developmentally normal, however he has a past medical history remarkable for growth hormone deficiency and a small pituitary gland and has been managed on somatropin since 3 years of age.
Patient 7 from Family 2 first presented with acroparesthesia and hypohidrosis at 4 years of age and began ERT by 6 years of age. Prior to initiation of ERT, urine GL-3 level was at 370 μg/mmol and was cleared to 0 μg/mmol in less than two years on ERT. Notably, spirometry testing revealed mild obstructive airway disease, though it has not yet affected routine activity. Patient 7 and his affected mother (patient 8) present with variable symptoms of FD as shown in Supplemental Table 2.
3.13 Supplemental treatments
Several individuals in this cohort sought alternative sources for pain management. Effective pain management could impact the psychological consequences of FD, as there is a high rate of depression that is often linked to the effects of long-term pain. Patient 13 had a five-year history of frequent, severe pain crises, including in response to ERT infusion, which has led the patient to seek alternative pain management strategies in conjunction with ERT. This includes floatation in a sensory deprivation tank used for the treatment of pain and anxiety by limiting stimulation from gravity, sound and light while immersed in water baths saturated with Epsom salts to allow for buoyancy in a dark and soundless room. Patient 15 and Patient 16 also report benefit from this float tank. Important to note, extended time in the float tank may have had an adverse effect on patient 13's bone density, as he reports spending up to 40 h per week in the float tank. There was a variety of medications used in this cohort for the management of neuropathic pain. Patient 13 and 15 are prescribed cannabidiol (CBD). Patient 15 and patient 26 use non-prescription topical and oral CBD as needed during pain crises. Use of tetrahydrocannabinol (THC) was also reported. Six patients are prescribed opioid narcotic analgesics, eight patients are managed with over-the-counter analgesics, two are on antidepressants, and one on an anticonvulsant (carbamazepine).
4 Discussion
The benefit, safety, and risk of ERT have been previously investigated, though the long-term impact continues to be studied [[26], [27], [28], [29], [30]]. ERT is recommended in order to curtail disease progression or when there is already evidence of disease progression and vital organ damage. In this study, 20 of the 26 participants and all but one male (patient 15) were consistently on ERT.
All participants receiving ERT however, demonstrated a different response rate of disease progression. While ERT was shown to be effective in clearing plasma GL-3 and, in many patients maintaining serum creatinine and eGFR within normal limits in this cohort, we identified that many patients continued to have impairment of their renal function and health-related quality of life despite standard of care management. For patients with chronic kidney disease from FD, standard of care did not vary when compared to that of other etiologies. The standard management includes the evaluation for possible reversible causes of renal disease (i.e. dehydration, reduced renal perfusion, obstruction, nephrotoxicity), management of blood pressure to a target ≤130/80, maximally tolerated dose of renin-angiotensin-aldosterone system inhibitors (i.e. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone receptor antagonist), smoking cessation, dietary interventions (particularly reduced sodium and protein restricted diet), treatment of metabolic acidosis, management of anemia of CKD with erythropoiesis-stimulating agents to target Hg 10–11.5 g/dL, management of mineral and bone disorders as per 2012 KDIGO guidelines, initiation of renal replacement therapy in individuals with uremic symptoms and eGFR 5–15 mL/min/1.73 m2 or in those with eGFR <5 mL/min/1.73 m2, and referral for kidney transplant evaluation in those with eGFR 20–25 mL/min/1.73 m2.
The late initiation of ERT may have contributed to those with renal disease progression, as shown previously [31]. Furthermore, nearly all of the adult patients in this cohort had major organ involvement at baseline prior to initiation of ERT. Typically, renal function in FD gradually deteriorates to end-stage renal disease in the third to fifth decade in males [32,33]. Only one patient progressed to severe CKD requiring hemodialysis, therefore, compared to the natural history data, it is possible that ERT contributed to the prevention of renal failure by slowing disease progression. Though vital organ damage is not typically seen in pediatric patients (some cases have been documented), studies have shown evidence for the benefit of the early initiation of ERT in potentially reversing renal damage [10,29]. Patient 12 initiated ERT at age 11 years, however, developed renal involvement by age 24 years [34]. Therefore, we believe earlier initiation of ERT is required to limit deterioration of the major organs. Our two pediatric participants initiated ERT at ages 6 and 7 years, respectively, and one reported significant improvement of pain symptoms.
Genotype-phenotype association is difficult to establish in FD for many reasons. Most GLA variants are unique (private mutations) and there are few family studies. Even within families, the phenotype may vary depending on the sex, age of onset, and other genetic and epigenetic factors. There are several reports in the literature of ‘later-onset’ isolated hypertrophic cardiomyopathy in individuals with the recurrent c.639 + 919G > A variant [6,[35], [36], [37]]. While this is often considered an atypical later-onset cardiac disease variant, patient 19 presented with several other ‘classic’ FD symptoms. Studies have explored the use of Amiloride as a potential treatment for FD by modulating alternative splicing in the context of the c.639 + 919G > A variant [35]. In our cohort we identified one novel variant c.1226_1231delCCACAG (p.P409_G411delinsR) in patient 22 with a severe, ‘classic’ FD phenotype [38]. This variant is absent in The Genome Aggregation Database (gnomAD), is located in a mutational hot-spot, and thus predicted to be pathogenic [39]. Two of the 26 individuals in this cohort harbor variants amenable to migalastat, an oral chaperone therapy, but chose not to switch from ERT. Long-term outcome studies for FD patients who have switched to migalastat will be of interest for future studies.
In this cohort, nearly one third of adult patients reported depression and anxiety. Based on the patient-reported SF-36 Health Survey and Brief Pain Inventory survey, there is no significant improvement in health-related quality of life over the course of ERT. Depression, sleep apnea, and anxiety disorders are linked to both pathology and the effects of long-term pain [40]. It has been suggested that psychological counseling can have an impact on reversing or reducing effects, an important consideration to optimize patient care as needed [41]. Importantly, studies have proposed pain as an indicator for the need to initiate ERT [42]. The majority of our cohort experienced Fabry crises or acroparasthesia. There are few data on nervous system involvement of FD in women [43]. Some studies have shown hippocampal volume loss and white matter lesions in female patients with FD [44]. While we showed white matter lesions in three females and one male in this cohort, we did not include brain imaging, electrical conduction, or quantitative sensory testing for every participant in this study. Further studies assessing the outcomes of neuropathy and CNS findings with ERT are warranted.
Cardiac manifestation can be the primary and only symptom of FD in some patients and can present as early as childhood. Our study showed a higher prevalence of cardiac manifestations in both the males and females compared to previous studies [34,[45], [46], [47]]. Cardiac involvement was observed in 83% of male patients, including one pediatric male patient with sinus bradycardia, and 75% of female patients in this cohort. Left ventricular hypertrophy (LVH) was seen in 70% of males and 42% of females. This data provides supporting evidence that female patients, even those with no obvious signs or symptoms of FD, should be monitored closely for evidence of cardiac involvement and be considered for ERT. The standard of care for patients with hypertrophic cardiomyopathy due to FD did not vary when compared to the management of other etiologies and followed AHA/ACC guidelines.
We highlight the variable endocrine dysfunction and low bone mineral density in this cohort, including several with osteoporosis, which provides further evidence for the inclusion of adequate endocrine work-up in the ongoing management of all patients with FD [48].
There is a need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. Furthermore, there is a need for more and improved biomarkers to monitor disease progression. Early identification of endothelial function or other pathologies can provide insight into when ERT should be initiated in order to slow the advancement of adverse effects [49]. Additional tools and biomarkers for measuring endothelial function and other indicators of future pathologies, such as proteomics, are needed to help prevent the deterioration of vital organs [[50], [51], [52]]. Newborn screening for FD has been implemented in several states and will lead to earlier detection. Early detection is important to proactively monitor for complications of the disease, initiate ERT, and prevent co-morbidity progression [[53], [54], [55], [56]].
Since most of our participants were on ERT, we could not make comparisons between long-term outcomes of those on ERT compared to those not on ERT. Also, with the small sample size, we could not compare multiple age-matched individuals or genotype-specific individuals other than our reported families. Another limitation to this study is the long-term analysis of plasma-GL-3 over time rather than plasma Lyso-GL-3, due to the fact that we had more data points for plasma-GL-3. Plasma Lyso-GL-3 has been recently determined to be a more accurate biomarker that correlates more closely with disease phenotype in male and in female patients, with higher levels associated with increased clinical manifestation [57].
5 Conclusion
The variability of symptoms and disease progression in patients with FD, even within the same family, complicates the discernment of a genotype-phenotype correlation. ERT has been available for the treatment of FD since 2001 and has shown improved outcomes, especially when started prior to organ damage, and may reverse fatal disease progression. In this cohort, agalsidase beta infusions have been effective in clearing GL-3 levels. However, renal involvement and health-related quality of life has continued to progress in adult patients diagnosed late despite ERT infusions.
Eight females were on ERT in this study and demonstrated significant symptoms of FD, and in some cases the disease progressed to a similar degree as in the males. This study further demonstrates that females may develop significant FD symptoms and should be managed appropriately and treated as more than carriers.
Documentation of novel variants can contribute to future genotype-phenotype associations between the severity and progression of FD. Genotype-phenotype correlations and long-term outcome data on ERT and/or other FD treatments are needed, especially now that the newborn screening has led to an increase in diagnoses and earlier detection in individuals with FD.
Authors' contributions
V.K. conceived the study and supervised the overall direction and implementation of the study. All authors contributed to acquisition of data. V.K., M.D.C., D.R., D.T., Z.A.D., and G.L. analyzed the data. D.R. performed statistical analyses. M.D.C. and E.C. drafted the manuscript with input from all authors. All authors provided critical feedback and approved the final manuscript for publication.
Funding
The Fabry registry was funded by Sanofi Genzyme. This funding source had no role in data collection or analysis.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Appendix A Supplementary data
Supplementary material
Image 1
Acknowledgements
We thank the patients and their health care providers for their contribution to the study. The Fabry registry was funded by Sanofi Genzyme. This work was supported by CTSI grant UL1TR000124UCLA (Clinical and Translational Science Institute), Los Angeles, CA, USA.
Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2020.100700. | AGALSIDASE BETA, CANNABIDIOL | DrugsGivenReaction | CC BY-NC-ND | 33437642 | 18,784,055 | 2021-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dyspnoea'. | Variable clinical features of patients with Fabry disease and outcome of enzyme replacement therapy.
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme α-galactosidase A due to mutations in the GLA gene. This leads to an accumulation of globotriaosylceramide (GL-3) in many tissues, which results in progressive damage to the kidneys, heart, and nervous system. We present the molecular and clinical characteristics and long-term outcomes of FD patients from a multidisciplinary clinic at the University of California, Irvine treated with agalsidase beta enzyme replacement therapy (ERT) for 2-20 years. This cohort comprised 24 adults (11 males, 13 females) and two male children (median age 45; range 10-68 years). Of the 26 patients in this cohort, 20 were on ERT (12 males, 8 females). We describe one novel variant not previously reported in the literature in a patient with features of 'classic' FD. The vast majority of patients in this cohort presented with symptoms of 'classic' FD including peripheral neuropathic pain, some form of cardiac involvement, angiokeratomas, corneal verticillata, hypohidrosis, tinnitus, and gastrointestinal symptoms, primarily abdominal pain. The majority of males had clinically evident renal involvement. An annual eGFR reduction of -1.88 mL/min/1.73 m2/yr during the course of ERT was seen in this cohort. The most common renal presentation was proteinuria, and one individual required a renal transplant. Other common findings were pulmonary involvement, lymphedema, hearing loss, and significantly, three patients had strokes. Notably, there was a high prevalence of endocrine dysfunction and low bone mineral density, including several with osteoporosis. While enzyme replacement therapy (ERT) cleared plasma GL-3 in this cohort, there was limited improvement in renal function or health-related quality of life based on the patient-reported SF-36 Health Survey. Physical functioning significantly declined over the course of ERT treatment, which may be, in part, due to the late initiation of ERT in several patients. Further delineation of the phenotypic and genotypic spectrum in patients with FD and the long-term outcome of ERT will help improve management and treatment options for this disease.
1 Introduction
Fabry Disease (FD, OMIM#301500) is an inherited, X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficient or absent α-galactosidase A enzyme [1]. This consequently leads to the progressive deposition of globotriaosylceramide (GL-3) in several tissues throughout the body, notably in endothelial cells, smooth muscle cells, podocytes, and cardiomyocytes [2]. The absence of this enzyme may lead to multi-organ involvement including progressive renal disease, cardiac disease, cerebrovascular disease, neuropathic pain, corneal verticillata, angiokeratomas, tinnitus, hearing loss, pulmonary involvement, and gastrointestinal complaints, including abdominal pain, constipation, and diarrhea. FD is pan-ethnic and the second most prevalent lysosomal storage disorder with an estimated incidence of 1 in 40,000 to 117,000 males worldwide, though newborn screening initiatives have revealed that the incidence is much higher [[3], [4], [5], [6]]. This is likely due to the fact that early studies referred to the incidence of ‘classic’ FD, while newborn screening may identify individuals on the full phenotypic spectrum. Symptoms typically present early during childhood or adolescence. Affected males with little or absent α-galactosidase A activity (<1% of mean normal) may present with clinical features of ‘classic’ FD. Mutations that result in residual enzyme activity typically cause a ‘later-onset’ form of the disease with variable manifestations. Heterozygous females typically present with milder symptoms and later-onset and less commonly have ‘classic’ FD due to residual enzyme activity and X-chromosome inactivation pattern.
Management and guidelines of a newly diagnosed individual with FD includes documentation of symptoms, consideration of enzyme replacement therapy (ERT), and monitoring of disease progression [7,8]. ERT is provided to supplement or replace α-galactosidase A, and to slow the rate of disease progression. Thus, early identification of affected individuals is important in order to initiate ERT as early as possible [[8], [9], [10]]. Clinical evaluation during childhood is recommended if a familial mutation is known [11]. In the absence of a family history, a timely diagnosis of FD may be difficult due to the variation and unpredictability of clinical manifestations that can often be misdiagnosed as childhood growing pains. The addition of FD to newborn screening programs worldwide has allowed for the early detection of the disorder [12].
There are two types of enzyme replacement therapy: agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme); however only agalsidase beta is an approved ERT by the US Food and Drug Administration (FDA) [13,14]. There is some discussion on the most appropriate infusion as well as the efficacy and safety of the infusions, but studies have shown no adverse effects in switching therapies or an increased benefit of using one over the other [15,16]. There is one oral pharmacological chaperone therapy, migalastat (Galafold), that has been approved by the FDA [17]. This treatment is only available for adults with FD who have amenable gene variants that lead to misfolded α-galactosidase A enzyme that can be stabilized by migalastat. Once stabilized, protein folding is improved and trafficking of the α-galactosidase A enzyme to the lysosome is restored. Other treatments currently being investigated in clinical trials include plant-based ERT, gene therapy, and substrate reducing therapies.
We describe a cohort of 24 adults and two children with FD, 13 males and 13 females, who range in age from 10 to 68 years (mean age 46.1 ± 16.3). The majority of participants were on agalsidase beta ERT (20/26; 12 males, 8 females). Each participant demonstrated different response rates of symptoms and disease progression with ERT, including two individuals who developed adverse infusion reactions.
The aim of this study is to present the variable molecular and clinical features in a cohort of FD patients on ERT, treatment outcomes across organ systems, and highlight patients with previously unreported variants, to help improve treatment options and patient care.
2 Methods
2.1 Participants
This cohort comprised 13 male and 13 female patients, including two male children. All participants had a confirmed molecular diagnosis of FD and most also had recorded enzyme activity levels measured. GLA variants are listed using transcript NM_000169.2. These patients were followed over the course of a one to 12 year period in an outpatient multidisciplinary clinic at the University of California, Irvine. Earlier data were obtained from review of past medical records and the Fabry Registry, if available. Written IRB informed consent approved by UC Irvine (#2008–6631) was obtained from each participant, and all procedures were performed according to The Code of Ethics of the World Medical Association.
2.2 Clinical evaluations
Clinical surveillance visits were conducted every six months and included evaluations by a clinical geneticist, a cardiologist, and a nephrologist. The standard of care for patients with renal and cardiac disease from FD did not vary when compared to the management of other etiologies. At each visit, the following components were documented:• Quality of life surveys, including a Brief Pain Inventory (BPI) and the SF-36 Health Survey. These provided a scale for pain and represented an attempt to document how pain has influenced the quality of life of each individual. The SF-36 Health survey measures physical function, social function, physical role, emotional role, mental health, energy, pain, and general health perception. Scores ranged from 0 (worst) to 100 (best).
• The development or change in the status of clinical features specific to FD including cardiac, renal, pulmonary, and nervous system involvement, in addition to angiokeratomas, acroparesthesia, GI health, hyper or hypohidrosis, hearing loss and tinnitus, lymphedema, and corneal whorling.
• Plasma and urine GL-3 levels and plasma Lyso-GL-3 to assess overall glycolipid burden.
• Renal status and disease progression were monitored by labs: serum creatinine levels to measure the mean estimated glomerular filtration rate (eGFR), in addition to urine creatinine, urine protein, and urine albumin/micro-albumin to measure proteinuria and albuminuria. Renal biopsy was only performed if clinically indicated.
Evaluations performed at less frequent intervals:• Cardiac status was determined by echocardiography (with strain), electrocardiograms, and holter monitor screened at annual intervals in males and less frequently in females and children. Cardiac MRI was conducted every 2–3 years or as needed.
• Brain imaging was performed by MRI, MRA, and CT scan.
• Ophthalmologic exams were performed for evaluation of corneal whorls, vessel tortuosity, and lenticular opacities.
• Audiometry was performed for evaluation of sensorineural hearing loss and tinnitus, as needed.
• Dual-energy X-ray absorptiometry (DXA) & the World Health Organization (WHO) classification of bone mineral density were used to classify severity. T-scores between −1.0 and −2.5 SD were categorized as osteopenia and T-scores below −2.5 SD were categorized as osteoporosis.
2.3 ERT infusions
Agalsidase beta (Fabrazyme) recommended dosage is 1 mg/kg body weight given every two weeks as an intravenous infusion [14]. The majority of the participants in this cohort received the infusions without incident, and some have transitioned to home infusions. To determine the effectiveness of ERT, we measured the presence of agalsidase beta IgG antibodies, plasma and urine GL-3 levels, and plasma Lyso-GL-3 levels at each six-month visit. Two individuals experienced adverse reactions to ERT infusions (patient 15 and patient 22); they both tested negative for IgE antibodies and showed no change in IgG antibodies.
2.4 Statistical analysis
We used linear mixed-effects regression analysis with varying intercepts and slopes to evaluate linear time trends of serum creatinine, eGFR, and health-related quality of life scores (SF-36 Health Survey), starting with the initiation of ERT (time = 0). The significance level for all statistical tests was set to α = 0.05. All analyses were carried out using the R language and environment for statistical computing, version 3.6.3.
3 Results
Of the 26 patients included in our study, there were 13 males and 13 females between ages 10–68 years (mean age 46.1 ± 16.3 years). This cohort consisted of five families in addition to eleven unrelated individuals. The majority of males (12/13, 92%) and the majority of females (8/13, 62%) were treated with ERT. Ages at initiation of ERT were between 6 and 65 years (mean age 39.0 ± 18.3) (Supplemental Table 1). The majority of patients tolerated ERT well, except for two individuals who had adverse reactions (patient 15 and patient 22). Both patients were IgE-negative and showed no changes in IgG antibodies; patient 22 was consistently IgG-positive throughout the course of ERT. Patient 15 had the most significant adverse event during the last infusion attempt, which resulted in intubation in the cardiac intensive care unit due to difficulty breathing, tightness in chest, rigors, and convulsions, and has not been on ERT since. Only three males were routinely IgG-positive (patients 12, 13, 24) and two males had one single IgG-positive test in the past, but had not had another one since (patient 1 and patient 7).
Clinical features of each patient are described in Supplemental Table 2. Notably, there was clinically evident renal involvement in 50% of participants; the most common feature was varying levels of proteinuria. One individual required a renal transplant. Seventy-nine percent of patients showed evidence of some form of cardiac involvement, including 46% with left ventricular hypertrophy. Two males and one female had a history of a stroke, and one male had a history of one TIA and one stroke. The most commonly observed clinical feature was peripheral neuropathic pain (20/26, 77%), followed by angiokeratomas (18/26, 69%), tinnitus (17/26, 65%), gastrointestinal symptoms, including abdominal pain, constipation, and diarrhea (16/26, 62%), and hypohidrosis/anhidrosis (16/26, 62%). Additional common findings of FD in this cohort include corneal verticillata (15/26, 58%), pulmonary involvement (6/12, 50%), lymphedema (11/26, 42%), and hearing loss (9/26, 35%). The adult males in this cohort had higher frequencies of clinical manifestations than adult females in all categories with the exception of neuropathic pain, white matter lesions, and corneal verticillata (Table 1).Table 1 Clinical manifestations of FD in this cohort.
Table 1 Adult Females
N = 13 (%) Adult Males
N = 11 (%) Pediatric Males
N = 2 (%) Total
N = 26 (%)
Mean Age at Diagnosis (Years) 36.2 34.3 3.5 32.8 ± 16.3
Currently On ERT 8 (62) 10 (91) 2 (100) 20 (77)
Mean Age ERT Initiated (Years) 51 37.5 6.5 39.0 ± 18.3
Renal Involvement 5 (38) 7 (64) 1 (50) 13 (50)
Cardiac Involvement 9/12 (75) 9/10 (90) 1 (50) 19/24 (79)
White matter Lesions 3/10 (30) 1/6 (17) N/A 4/16 (25)
Neuropathic Pain 10 (77) 8 (73) 2 (100) 20 (77)
Corneal Verticillata 10 (77) 4 (36) 1 (50) 15 (58)
Angiokeratomas 7 (54) 11 (100) 0 (0) 18 (69)
Hypohidrosis 5 (38) 10 (91) 1 (50) 16 (62)
GI Involvement 8 (62) 8 (73) 0 (0) 16 (62)
Low BMD 2/6 (33) 5/8 (63) N/A 7/14 (50)
Hearing Loss 3 (23) 6 (55) 0 (0) 9 (35)
Tinnitus 7 (54) 9 (82) 1 (50) 17 (65)
Vertigo 4/10 (40) 6/10 (60) 0/1 (0) 10/21 (48)
Pulmonary Involvement 0/5 (0) 5/6 (83) 1/1 (100) 6/12 (50)
Depression 3 (23) 3 (27) 0 (0) 6/26 (23)
Numbers provided in fractions indicate that not all patients had the corresponding assessment.
3.1 Clearance of GL-3 in plasma
Plasma-GL-3, plasma Lyso-GL-3, and urine GL-3 are biomarkers used as surrogate endpoints to monitor Fabry disease severity and progression in both treated and untreated patients. Longitudinal analysis of plasma GL-3 concentration in 17 of our patients on ERT is depicted in Fig. 1. The data showed significant reduction in GL-3 of −0.25 μg/mL per year (95% CI: −0.478 to −0.030, p = 0.026) with ERT treatment.Fig. 1 Plasma GL-3 clearance in patients on ERT. A linear mixed-effects regression analysis of plasma GL-3 with time as a predictor and varying intercepts and slopes. The figure shows observed GL-3 measurements (dots) together with model-estimated time trajectories of GL-3 for each participant (colored lines) and for the population (black line). Note that observations at time < 0 were excluded from the analysis. N = 17.
Fig. 1
While there was less data available for the deacetylated form, plasma Lyso-GL-3, the majority showed a reduction or stabilization of plasma Lyso-GL-3 concentration at last visit compared to that of the earlier time points (N = 11, data not shown).
3.2 Renal involvement
There was clinically evident renal disease in 50% (13/26) of participants as defined by albuminuria, hypertension or reduced eGFR. Renal biopsies were not done routinely. The majority demonstrated proteinuria and/or albuminuria as the sole renal manifestation. Ten patients had elevated urine albumin-to-creatine ratios, nine with microalbuminuria (5 females, 4 males) and one male with macroalbuminuria. Hypertension was seen in 32% (8/25) of patients and managed with angiotensin-converting enzyme inhibitors (ACEI) (8/26), angiotensin receptor blockers (ARB) (4/26), beta-blockers (4/26), and diuretics (6/26). Two had stage I chronic kidney disease (CKD) (patient 1 and patient 12), two had stage III CKD (patient 14 and patient 17), and one had stage IV CKD and is on hemodialysis (patient 22). Patient 23 underwent a deceased donor renal transplant due to ESRD at age 52 years and has had normal kidney function since. For those on ERT, only some patients maintained serum creatinine and estimated glomerular filtration rate (eGFR) within normal limits for age and sex. However, the data showed significant increase in serum creatinine levels of +0.02 mg/dL per year (95% CI: 0.01–0.04, p = 0.015) for both males and females on ERT in this cohort (Fig. 2A) and +0.038 mg/dL per year (95% CI: 0.01–0.06, p = 0.003, data not shown) for males alone. Further, the data showed an annual decrease in eGFR of −1.88 mL/min/1.73m2 (95% CI: −3.36 to −0.40, p = 0.013) on ERT in both males and females (Fig. 2B), and −3.16 mL/min/1.73m2 (95% CI: −4.97 to −1.35, p = 0.001, data not shown) for males alone.Fig. 2 Serum creatinine and estimated glomerular filtration rate (eGFR) of patients on ERT. The figure shows observed serum creatinine measurements (A) and eGFR measurements (B) together with model-estimated time trajectories for each participant (colored lines) and for the population (black lines). Note that observations at time < 0 were excluded from both analyses in figs. A-B. N = 18.
Fig. 2
3.3 Cardiac involvement
In our cohort, 79% (19/24) of participants had some form of cardiac involvement. Left ventricular hypertrophy (LVH) was seen in 46% (11/24) of patients, with severity ranging from borderline to severe and one confined to the septum. Severe LVH of patient 24 is shown in Fig. 3A. Of the adult males, only one was not found to have clinically evident cardiac involvement, and while his past ECGs met voltage criteria for LVH, his echocardiograms were normal showing no evidence of LVH. Left ventricular ejection fraction (LVEF) was one of the measures used to monitor cardiac disease progression of patients on ERT. LVEF was within normal range at baseline prior to ERT. Some patients fell below normal range over the course of treatment with the lowest recorded being 43% in a male patient. Of the individuals in our cohort who had cardiac imaging, 25% (6/24) had valvular abnormalities, including one patient with aortic valve sclerosis, two patients had thickened mitral valve leaflets, one patient with mild Systolic Anterior Motion (SAM) abnormality of the mitral valve, one with mitral valve prolapse, and one patient required mitral valve annuloplasty after experiencing lower extremity edema, shortness of breath and dyspnea on exertion (patient 22). Other cardiac manifestations are described in Table 2. Of those who underwent cardiac MRI, only one showed possible fibrosis of the myocardium (Fig. 3A). Those with cardiac abnormalities on echocardiogram, cardiac MRI, and/or ECG did not consistently have abnormal cardiac biomarkers, such as brain natriuretic peptide (BNP), troponin, and soluble suppression of tumourigenicity 2 (ST2), though limited data was available. Adult FD patients can have elevated lipid levels, particularly high HDL cholesterol, which may be non-responsive to long-term ERT [18]. Dyslipidemia was present in 48% (10/21) of patients in our cohort, and managed with diet, fish-oil and statins.Fig. 3 Clinical features in patients with FD. (A) Cardiac MRI showing severe concentric hypertrophy of the left ventricle with reduced chamber size. There is subendocardial linear hyperenhancement in the basal inferolateral wall suggesting myocardial scar (white arrow), consistent with FD (patient 24); (B) Axial brain MRI section following TIA event (patient 22); (C) Corneal verticillata seen by slit lamp examination. Courtesy: Dr. Pinakin Davey, Western University of Health Sciences; (D) Cluster of angiokeratomas in the groin region (patient 24).
Fig. 3Table 2 Characterization of cardiac involvement in FD patients in this cohort.
Table 2 Adult
Females Adult
Males Pediatric Males Total (%)
Cardiac Involvement 9/12 (75) 9/10 (90) 1/2 (50) 19/24 (79)
Echocardiogram Findings
Left Ventricular Hypertrophy 5/12 7/10 0/2 11/24 (46)
Borderline 1 2 0
Mild 2 3 0
Moderate 0 1 0
Severe 0 1 0
Septal 1 0 0
Valvular Abnormality 2/12 4/10 0/2 6/24 (25)
Aortic Valve 1 0 0
Mitral Valve 1 4 0
Left Atrium Dilation 2/12 2/10 0/2 4/24 (17)
Right Atrium Dilation 1/12 0/10 0/2 1/24 (4)
Electrocardiogram Findings
Conduction Abnormalities 3/12 6/10 0/2 9/24 (38)
Right Bundle Branch Block 2 5 0
Anterior Fascicular Block 1 1 0
First Degree Atrioventricular Block 1 0 0
Intraventricular Conduction Delay 0 2 0
Short PR Interval 1 2 0
Rhythm Abnormalities 5/12 8/10 1/2 14/24 (58)
Sinus Bradycardia 5 8 1
Sinus Arrhythmia 1 0 0
Premature Ventricular Complexes 0 2 0
Premature Atrial Contractions 0 1 0
Repolarization Abnormalities 5/12 3/10 0/2 8/24 (33)
T Wave Changes 2 1 0
ST/STT Changes 3 2 0
Left Axis Deviation 0/12 1/10 0/2 1/24 (4)
One male and one female in this cohort were excluded due to no cardiac imaging records available.
3.4 Cerebrovascular complications, neurologic, and neuropsychiatric symptoms
White matter lesions were present in 25% (4/16) of participants who had brain imaging. One male in this cohort (patient 22) has a history of two cerebrovascular accidents, one transient ischemic attack (TIA) at age 52 years and one stroke at age 60 years. Brain MRI of patient 22 showed multifocal and adjacent gliosis within the left cerebral hemisphere, high left frontal and parietal lobes, as well as subcortical and periventricular deep white matter T2 hyperintensity without mass effect that is most pronounced within the parietal occipital region (Fig. 3B). The stroke impacted his eyesight and word-finding ability. Patient 22 only began ERT at the age of 57 years, and passed away at the age of 63 years due to post-operative complications of abdominal surgery. Another male (patient 15) and one female (patient 14) in this cohort have a history of TIAs while on ERT. They were not taking low dose aspirin at the time.
With regard to neuropathic manifestations, 77% (20/26) of patients reported peripheral neuropathic pain, manifesting as burning, tingling, or numbness, often triggered by temperature change or strenuous activity. Of the 17 participants for whom data on the brief pain inventory (BPI) survey was available, the majority reported worsening or inconsistent pain symptoms over the duration of treatment with ERT. Decreased quality of life has been associated with FD, in part due to the debilitating episodic pain crises and burden associated with having a chronic illness. Health-related quality of life was collected using the SF-36 Health survey. Bodily pain scores obtained from the SF-36 survey also showed no improvement over the course of ERT, though this was not significant (p = 0.19; Fig. 4C). For the majority of SF-36 respondents, pain worsened over time, which was similar to what was reported in the BPI survey. Notably, there was a significant decline in physical functioning (Est. = −1.40, 95% CI: −2.73 to −0.06, p = 0.040) for patients in this cohort. The remaining health dimensions did not show significant change over time (Fig. 4A-H).Fig. 4 SF-36 health-related quality of life component scores of patients on ERT. The SF-36 Health survey measures (A) physical function, (B) physical role, (C) pain, (D) general health perception, (E) vitality, (F) social function, (G) emotional role, (H) mental health. Scores ranged from 0 (worst) to 100 (best). Observed values (dots) are shown together with model-estimated trajectories for individual patients (colored lines) and the population (black lines).
Fig. 4
Of the adult patients in this cohort, seven reported depression (7/24; 29%). One patient reported a history of depression that correlated with pain symptoms. Another patient had a history of depression with no current pain symptoms based on responses in the BPI and SF-36 Health Survey. Of note, one patient in this cohort had a history of two suicide attempts. On the other hand, one patient's testimonial described noticeable improvement in anxiety, fatigue, and executive function (termed “Fabry fog”) since initiation of ERT.
3.5 Ophthalmologic findings
Corneal verticillata, or corneal whorling, is a unique clinical feature in patients of both sexes with FD and is often used as a diagnostic tool. It is a result of GL-3 deposits and has not been linked to vision loss. Corneal verticillata was noted in 58% (15/26) of individuals, at a higher prevalence in females than males in our cohort (Fig. 3C). Vessel tortuosity is also associated with FD and is exacerbated by renal complications [19,20]. Only one patient was found to have vessel tortuosity (patient 22); this individual had stage IV CKD.
3.6 Skin manifestations
Angiokeratomas are common in Fabry patients as they occur when GL-3 accumulates in dermal endothelial cells and lead to secondary ectasia. In our cohort, 69% (18/26) had angiokeratomas (Fig. 3D).
Hypohidrosis is also a common feature of FD and may be a predisposition to acroparesthesia. In this cohort, 62% (16/26) experienced hypohidrosis or anhidrosis. Of those with hypohidrosis, 89% experienced acroparesthesia. Lymphedema of the extremities, particularly in the feet, was present in 42% (11/26) of patients in this study.
3.7 Gastrointestinal involvement
Gastrointestinal symptoms are common among FD patients and can manifest as diarrhea, constipation, nausea, vomiting, incontinence, and abdominal pain. In our cohort, gastrointestinal symptoms were present in 62% (16/26) and the most common complaint was abdominal pain. Previous studies have shown significantly improved gastrointestinal pain with ERT [21]. This was also reported by the patients in our study in the patient health survey. The majority of those on ERT reported either improved or stable abdominal pain and frequency of diarrhea based on their survey responses at last visit compared to earlier survey responses. Specifically, six patients reported improvement in abdominal pain and four reported that symptoms were stable (N = 20). Eleven reported improvement in diarrhea and one reported that symptoms were stable (N = 20). One patient's testimonial described a frequency of diarrhea multiple times daily prior to ERT that has now significantly improved since ERT initiation. He also reported noticeable return of symptoms during the time of the ERT shortage. On the other hand, worsening or recent onset of abdominal pain (3/20) and diarrhea (2/20) over the course of ERT treatment were also reported by participants in this study.
3.8 Auditory and vestibular involvement
Sensorineural hearing loss, predominantly in the high frequencies, is a common manifestation of FD and correlates with neuropathic and vascular damage [22]. In our cohort, 35% (9/26) of patients reported a history of hearing loss. The majority of those who underwent audiology assessments were found to have bilateral mild to moderate sensorineural hearing loss. Additionally, 65% (17/26) of patients reported tinnitus. Some patients do not use hearing aids due to amplification by the presence of tinnitus. Vertigo was reported in 48% of our cohort in those who recorded their response in the health survey (10/21).
3.9 Pulmonary involvement
Respiratory involvement in FD typically includes obstructive lung disease, however patients may develop interstitial restrictive lung disease. In our cohort, 50% (6/12) of those who have had spirometry testing were found to have reduced lung capacity. Of those, four had obstructive lung disease, one had possible obstructive lung disease, and one had restrictive lung disease.
3.10 Endocrine and other clinical manifestations
There is limited data in the literature that describes endocrine system involvement in FD. In our cohort, patients reported type II diabetes, hypothyroidism, hyperthyroidism, hyperparathyroidism, and vitamin D deficiency (Supplemental Table 2). Patient 8 has a history of hyperthyroidism secondary to Graves' disease, and a past history of a 1.9 mm benign thyroid nodule. Patient 21 has type II diabetes and hypothyroidism. Patient 22 has acquired hypothyroidism and hyperparathyroidism secondary to renal disease. One of the pediatric participants (patient 9) was diagnosed with growth hormone deficiency and a small pituitary gland. Not all participants in this study have had an endocrine evaluation, so this study may have missed those with subclinical endocrine dysfunction.
Low Bone Mineral Density (BMD) has been noted more recently to be a common feature of FD [23]. Impaired renal function leads to vitamin D deficiency and may be one of the contributing factors to reduced BMD in FD patients. Fifty percent of patients in this cohort who had DXA scans (7/14) were found to have low BMD. Of those, 57% (4/7) had osteopenia (T score < 1.5) and 43% (3/7) had osteoporosis (T score < 2.5). Notably, two of the seven patients (29%) had early-onset low BMD at age 32 years (patient 6) and age 36 years (patient 13). There were no prior DXA scans to measure the rate of bone loss in these two young adult males. Additionally, patient 13 spends up to 40 h per week in a sensory deprivation floatation tank, which may have contributed to bone loss (described below in section 3.13. Supplemental Treatments). Only one patient endured an ankle fracture at the age of 65 years due to a mechanical fall; none of the remaining participants reported bone fractures. Of those with low BMD, four patients had concurrent vitamin D deficiency; of those four, one had secondary hyperparathyroidism due to renal disease. All patients in this cohort were prescribed calcium and vitamin D supplementation. Those with osteoporosis were managed by an endocrinologist and prescribed bisphosphonates.
Hypertension is also a common adverse event in adult FD patients. Cardiac, renal and cerebrovascular complications increase the risk of uncontrolled hypertension in individuals with FD [24]. Hypertension was present in 32% (8/25) of patients in our cohort, the majority but not all of these individuals had some form of clinically evident renal involvement. Patients were managed on ACEIs, ARBs, beta-blockers, and diuretics.
3.11 Novel GLA variant and genotype-phenotype correlations
Our study describes 16 unique variants in the 26 total participants, including one novel variant and one atypical ‘later-onset’ cardiac variant (Table 3; Fig. 5). The novel variant c.1226_1231delCCACAG (p.P409_G411delinsR), an in-frame deletion located within a hot-spot, was identified in a 63-year-old male (patient 22) who was diagnosed later in adulthood at 57 years old following a renal biopsy that revealed stage IV chronic kidney disease. He is discussed earlier with renal, pulmonary, and cardiac involvement and a history of two cerebrovascular accidents. Additional FD-related symptoms in patient 22 include angiokeratomas, corneal verticillata, gastrointestinal involvement, lymphedema, hypohidrosis, acroparesthesia, Raynaud's syndrome, hearing loss, tinnitus, and low bone mineral density (Supplemental Table 2). Cardiac work-up for chest pain revealed a 95% blockage of the left anterior descending coronary artery requiring stent placement, which resolved except for occasional reports of arrhythmia. Endocrine dysfunction in this patient includes acquired hypothyroidism and hyperparathyroidism secondary to renal disease. Patient 22 was particularly significant among our participants because of the history of one TIA and one stroke; brain MRI revealed significant gliosis and nonspecific white matter signal changes (Fig. 3B). Patient 22 is now deceased due to post-operative complications of abdominal surgery for abdominal abscess and intra-abdominal infection.Table 3 GLA variant details.
Table 3Fam. No. Patient No. Ethnicity Nucleotide Change Protein Change Position Type Phenotype
1 1,2,3,4,5,6 Hispanic c.983G > T G328V Exon 6 Missense Classic
2 7,8 Caucasian c.1250 T > G L417R Exon 7 Missense Classic
3 9,10,11 Caucasian c.132G > T W44C Exon 1 Missense Classic
4 12 Caucasian c.568delG A190PfsX2 Exon 4 Small Deletion Classic
5 13,14 Caucasian c.1041_1042insG A348GfsX27 Exon 7 Small Insertion Classic
6 15 Hispanic c.706 T > C W236R Exon 5 Missense Classic
7 16 Caucasian c.680G > A R227Q Exon 5 Missense Classic
8 17 Caucasian c.816C > A N272K Exon 6 Missense Classic
9 18 Caucasian c.730G > A D244N Exon 5 Missense Classic/ Later Onset
10 19 Asian c.639 + 919G > A IVS4 + 919G > A Intron 4 Splicing Later Onset
11 20 Asian c.427G > C A143P Exon 3 Missense Classic
12 21 Hispanic c.639 + 4A > T IVS4 + 4A > T Intron 4 Splicing Classic
13 22 Caucasian c.1226_1231delCCACAG P409_G411delinsR Exon 7 Small Deletion Classic
14 23 Hispanic c.1088G > A R363H Exon 7 Missense Later Onset
15 24,25 Hispanic c.1072_1074delGAG E358del Exon 7 Small Deletion Classic
16 26 Caucasian c.1246C > T Q416X Exon 7 Nonsense Classic
Novel variant is bolded.
Fig. 5 Unique variants identified in the GLA gene in this cohort. A schematic representation of GLA showing the position of all variants identified, with novel variant in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Though the rest of our cohort harbors variants that have been previously reported in the literature, we present novel clinical data for certain recurrent variants. Family 3 (patients 9, 10, 11) harbor the W44C variant, a previously reported variant in a Chinese family of 12 affected members, all of whom experienced paroxysmal pain of limb extremities [25]. Interestingly, our family is of European/mixed descent, with no known Asian ancestry, and present with multi-organ involvement in addition to peripheral pain. We report one patient with the c.639 + 919G > A ‘later-onset’ cardiac variant, who, in addition to mild left ventricular hypertrophy, experiences other FD-related symptoms including angiokeratomas, gastrointestinal issues, pulmonary involvement, and hypohidrosis with heat intolerance. His mother, who also harbors the familial variant, has a history of strokes and presented at age 76 years with hypertrophic cardiomyopathy. Another example of potential genotype-phenotype correlation in our cohort is Family 1; all three females who harbor the G328V variant have audiologic involvement.
3.12 Pediatric patients
There were two male pediatric participants in this study (patient 7 and patient 9). Both patients were diagnosed before the age of five and treated with ERT at or before the onset of symptoms. Patient 9 is a seven year-old male with the W44C variant. This patient was diagnosed with FD at age 3 and began ERT at age 7 years soon after an evaluation for reactive hyperemia index of 0.94 (Reference RHI value >1.68) using an EndoPAT (Itamar Medical) device performed on a research basis, which was consistent with low peripheral vascular endothelial function. He lacked ‘classic’ FD symptoms at the time of the procedure, but ERT was recommended due to concern for deteriorating endothelial function. Initial urine GL-3 level of 569 μg/mmol creatinine resolved during the course of this study while on ERT. He is developmentally normal, however he has a past medical history remarkable for growth hormone deficiency and a small pituitary gland and has been managed on somatropin since 3 years of age.
Patient 7 from Family 2 first presented with acroparesthesia and hypohidrosis at 4 years of age and began ERT by 6 years of age. Prior to initiation of ERT, urine GL-3 level was at 370 μg/mmol and was cleared to 0 μg/mmol in less than two years on ERT. Notably, spirometry testing revealed mild obstructive airway disease, though it has not yet affected routine activity. Patient 7 and his affected mother (patient 8) present with variable symptoms of FD as shown in Supplemental Table 2.
3.13 Supplemental treatments
Several individuals in this cohort sought alternative sources for pain management. Effective pain management could impact the psychological consequences of FD, as there is a high rate of depression that is often linked to the effects of long-term pain. Patient 13 had a five-year history of frequent, severe pain crises, including in response to ERT infusion, which has led the patient to seek alternative pain management strategies in conjunction with ERT. This includes floatation in a sensory deprivation tank used for the treatment of pain and anxiety by limiting stimulation from gravity, sound and light while immersed in water baths saturated with Epsom salts to allow for buoyancy in a dark and soundless room. Patient 15 and Patient 16 also report benefit from this float tank. Important to note, extended time in the float tank may have had an adverse effect on patient 13's bone density, as he reports spending up to 40 h per week in the float tank. There was a variety of medications used in this cohort for the management of neuropathic pain. Patient 13 and 15 are prescribed cannabidiol (CBD). Patient 15 and patient 26 use non-prescription topical and oral CBD as needed during pain crises. Use of tetrahydrocannabinol (THC) was also reported. Six patients are prescribed opioid narcotic analgesics, eight patients are managed with over-the-counter analgesics, two are on antidepressants, and one on an anticonvulsant (carbamazepine).
4 Discussion
The benefit, safety, and risk of ERT have been previously investigated, though the long-term impact continues to be studied [[26], [27], [28], [29], [30]]. ERT is recommended in order to curtail disease progression or when there is already evidence of disease progression and vital organ damage. In this study, 20 of the 26 participants and all but one male (patient 15) were consistently on ERT.
All participants receiving ERT however, demonstrated a different response rate of disease progression. While ERT was shown to be effective in clearing plasma GL-3 and, in many patients maintaining serum creatinine and eGFR within normal limits in this cohort, we identified that many patients continued to have impairment of their renal function and health-related quality of life despite standard of care management. For patients with chronic kidney disease from FD, standard of care did not vary when compared to that of other etiologies. The standard management includes the evaluation for possible reversible causes of renal disease (i.e. dehydration, reduced renal perfusion, obstruction, nephrotoxicity), management of blood pressure to a target ≤130/80, maximally tolerated dose of renin-angiotensin-aldosterone system inhibitors (i.e. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone receptor antagonist), smoking cessation, dietary interventions (particularly reduced sodium and protein restricted diet), treatment of metabolic acidosis, management of anemia of CKD with erythropoiesis-stimulating agents to target Hg 10–11.5 g/dL, management of mineral and bone disorders as per 2012 KDIGO guidelines, initiation of renal replacement therapy in individuals with uremic symptoms and eGFR 5–15 mL/min/1.73 m2 or in those with eGFR <5 mL/min/1.73 m2, and referral for kidney transplant evaluation in those with eGFR 20–25 mL/min/1.73 m2.
The late initiation of ERT may have contributed to those with renal disease progression, as shown previously [31]. Furthermore, nearly all of the adult patients in this cohort had major organ involvement at baseline prior to initiation of ERT. Typically, renal function in FD gradually deteriorates to end-stage renal disease in the third to fifth decade in males [32,33]. Only one patient progressed to severe CKD requiring hemodialysis, therefore, compared to the natural history data, it is possible that ERT contributed to the prevention of renal failure by slowing disease progression. Though vital organ damage is not typically seen in pediatric patients (some cases have been documented), studies have shown evidence for the benefit of the early initiation of ERT in potentially reversing renal damage [10,29]. Patient 12 initiated ERT at age 11 years, however, developed renal involvement by age 24 years [34]. Therefore, we believe earlier initiation of ERT is required to limit deterioration of the major organs. Our two pediatric participants initiated ERT at ages 6 and 7 years, respectively, and one reported significant improvement of pain symptoms.
Genotype-phenotype association is difficult to establish in FD for many reasons. Most GLA variants are unique (private mutations) and there are few family studies. Even within families, the phenotype may vary depending on the sex, age of onset, and other genetic and epigenetic factors. There are several reports in the literature of ‘later-onset’ isolated hypertrophic cardiomyopathy in individuals with the recurrent c.639 + 919G > A variant [6,[35], [36], [37]]. While this is often considered an atypical later-onset cardiac disease variant, patient 19 presented with several other ‘classic’ FD symptoms. Studies have explored the use of Amiloride as a potential treatment for FD by modulating alternative splicing in the context of the c.639 + 919G > A variant [35]. In our cohort we identified one novel variant c.1226_1231delCCACAG (p.P409_G411delinsR) in patient 22 with a severe, ‘classic’ FD phenotype [38]. This variant is absent in The Genome Aggregation Database (gnomAD), is located in a mutational hot-spot, and thus predicted to be pathogenic [39]. Two of the 26 individuals in this cohort harbor variants amenable to migalastat, an oral chaperone therapy, but chose not to switch from ERT. Long-term outcome studies for FD patients who have switched to migalastat will be of interest for future studies.
In this cohort, nearly one third of adult patients reported depression and anxiety. Based on the patient-reported SF-36 Health Survey and Brief Pain Inventory survey, there is no significant improvement in health-related quality of life over the course of ERT. Depression, sleep apnea, and anxiety disorders are linked to both pathology and the effects of long-term pain [40]. It has been suggested that psychological counseling can have an impact on reversing or reducing effects, an important consideration to optimize patient care as needed [41]. Importantly, studies have proposed pain as an indicator for the need to initiate ERT [42]. The majority of our cohort experienced Fabry crises or acroparasthesia. There are few data on nervous system involvement of FD in women [43]. Some studies have shown hippocampal volume loss and white matter lesions in female patients with FD [44]. While we showed white matter lesions in three females and one male in this cohort, we did not include brain imaging, electrical conduction, or quantitative sensory testing for every participant in this study. Further studies assessing the outcomes of neuropathy and CNS findings with ERT are warranted.
Cardiac manifestation can be the primary and only symptom of FD in some patients and can present as early as childhood. Our study showed a higher prevalence of cardiac manifestations in both the males and females compared to previous studies [34,[45], [46], [47]]. Cardiac involvement was observed in 83% of male patients, including one pediatric male patient with sinus bradycardia, and 75% of female patients in this cohort. Left ventricular hypertrophy (LVH) was seen in 70% of males and 42% of females. This data provides supporting evidence that female patients, even those with no obvious signs or symptoms of FD, should be monitored closely for evidence of cardiac involvement and be considered for ERT. The standard of care for patients with hypertrophic cardiomyopathy due to FD did not vary when compared to the management of other etiologies and followed AHA/ACC guidelines.
We highlight the variable endocrine dysfunction and low bone mineral density in this cohort, including several with osteoporosis, which provides further evidence for the inclusion of adequate endocrine work-up in the ongoing management of all patients with FD [48].
There is a need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. Furthermore, there is a need for more and improved biomarkers to monitor disease progression. Early identification of endothelial function or other pathologies can provide insight into when ERT should be initiated in order to slow the advancement of adverse effects [49]. Additional tools and biomarkers for measuring endothelial function and other indicators of future pathologies, such as proteomics, are needed to help prevent the deterioration of vital organs [[50], [51], [52]]. Newborn screening for FD has been implemented in several states and will lead to earlier detection. Early detection is important to proactively monitor for complications of the disease, initiate ERT, and prevent co-morbidity progression [[53], [54], [55], [56]].
Since most of our participants were on ERT, we could not make comparisons between long-term outcomes of those on ERT compared to those not on ERT. Also, with the small sample size, we could not compare multiple age-matched individuals or genotype-specific individuals other than our reported families. Another limitation to this study is the long-term analysis of plasma-GL-3 over time rather than plasma Lyso-GL-3, due to the fact that we had more data points for plasma-GL-3. Plasma Lyso-GL-3 has been recently determined to be a more accurate biomarker that correlates more closely with disease phenotype in male and in female patients, with higher levels associated with increased clinical manifestation [57].
5 Conclusion
The variability of symptoms and disease progression in patients with FD, even within the same family, complicates the discernment of a genotype-phenotype correlation. ERT has been available for the treatment of FD since 2001 and has shown improved outcomes, especially when started prior to organ damage, and may reverse fatal disease progression. In this cohort, agalsidase beta infusions have been effective in clearing GL-3 levels. However, renal involvement and health-related quality of life has continued to progress in adult patients diagnosed late despite ERT infusions.
Eight females were on ERT in this study and demonstrated significant symptoms of FD, and in some cases the disease progressed to a similar degree as in the males. This study further demonstrates that females may develop significant FD symptoms and should be managed appropriately and treated as more than carriers.
Documentation of novel variants can contribute to future genotype-phenotype associations between the severity and progression of FD. Genotype-phenotype correlations and long-term outcome data on ERT and/or other FD treatments are needed, especially now that the newborn screening has led to an increase in diagnoses and earlier detection in individuals with FD.
Authors' contributions
V.K. conceived the study and supervised the overall direction and implementation of the study. All authors contributed to acquisition of data. V.K., M.D.C., D.R., D.T., Z.A.D., and G.L. analyzed the data. D.R. performed statistical analyses. M.D.C. and E.C. drafted the manuscript with input from all authors. All authors provided critical feedback and approved the final manuscript for publication.
Funding
The Fabry registry was funded by Sanofi Genzyme. This funding source had no role in data collection or analysis.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Appendix A Supplementary data
Supplementary material
Image 1
Acknowledgements
We thank the patients and their health care providers for their contribution to the study. The Fabry registry was funded by Sanofi Genzyme. This work was supported by CTSI grant UL1TR000124UCLA (Clinical and Translational Science Institute), Los Angeles, CA, USA.
Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2020.100700. | AGALSIDASE BETA, CANNABIDIOL | DrugsGivenReaction | CC BY-NC-ND | 33437642 | 18,784,055 | 2021-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Seizure'. | Variable clinical features of patients with Fabry disease and outcome of enzyme replacement therapy.
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme α-galactosidase A due to mutations in the GLA gene. This leads to an accumulation of globotriaosylceramide (GL-3) in many tissues, which results in progressive damage to the kidneys, heart, and nervous system. We present the molecular and clinical characteristics and long-term outcomes of FD patients from a multidisciplinary clinic at the University of California, Irvine treated with agalsidase beta enzyme replacement therapy (ERT) for 2-20 years. This cohort comprised 24 adults (11 males, 13 females) and two male children (median age 45; range 10-68 years). Of the 26 patients in this cohort, 20 were on ERT (12 males, 8 females). We describe one novel variant not previously reported in the literature in a patient with features of 'classic' FD. The vast majority of patients in this cohort presented with symptoms of 'classic' FD including peripheral neuropathic pain, some form of cardiac involvement, angiokeratomas, corneal verticillata, hypohidrosis, tinnitus, and gastrointestinal symptoms, primarily abdominal pain. The majority of males had clinically evident renal involvement. An annual eGFR reduction of -1.88 mL/min/1.73 m2/yr during the course of ERT was seen in this cohort. The most common renal presentation was proteinuria, and one individual required a renal transplant. Other common findings were pulmonary involvement, lymphedema, hearing loss, and significantly, three patients had strokes. Notably, there was a high prevalence of endocrine dysfunction and low bone mineral density, including several with osteoporosis. While enzyme replacement therapy (ERT) cleared plasma GL-3 in this cohort, there was limited improvement in renal function or health-related quality of life based on the patient-reported SF-36 Health Survey. Physical functioning significantly declined over the course of ERT treatment, which may be, in part, due to the late initiation of ERT in several patients. Further delineation of the phenotypic and genotypic spectrum in patients with FD and the long-term outcome of ERT will help improve management and treatment options for this disease.
1 Introduction
Fabry Disease (FD, OMIM#301500) is an inherited, X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficient or absent α-galactosidase A enzyme [1]. This consequently leads to the progressive deposition of globotriaosylceramide (GL-3) in several tissues throughout the body, notably in endothelial cells, smooth muscle cells, podocytes, and cardiomyocytes [2]. The absence of this enzyme may lead to multi-organ involvement including progressive renal disease, cardiac disease, cerebrovascular disease, neuropathic pain, corneal verticillata, angiokeratomas, tinnitus, hearing loss, pulmonary involvement, and gastrointestinal complaints, including abdominal pain, constipation, and diarrhea. FD is pan-ethnic and the second most prevalent lysosomal storage disorder with an estimated incidence of 1 in 40,000 to 117,000 males worldwide, though newborn screening initiatives have revealed that the incidence is much higher [[3], [4], [5], [6]]. This is likely due to the fact that early studies referred to the incidence of ‘classic’ FD, while newborn screening may identify individuals on the full phenotypic spectrum. Symptoms typically present early during childhood or adolescence. Affected males with little or absent α-galactosidase A activity (<1% of mean normal) may present with clinical features of ‘classic’ FD. Mutations that result in residual enzyme activity typically cause a ‘later-onset’ form of the disease with variable manifestations. Heterozygous females typically present with milder symptoms and later-onset and less commonly have ‘classic’ FD due to residual enzyme activity and X-chromosome inactivation pattern.
Management and guidelines of a newly diagnosed individual with FD includes documentation of symptoms, consideration of enzyme replacement therapy (ERT), and monitoring of disease progression [7,8]. ERT is provided to supplement or replace α-galactosidase A, and to slow the rate of disease progression. Thus, early identification of affected individuals is important in order to initiate ERT as early as possible [[8], [9], [10]]. Clinical evaluation during childhood is recommended if a familial mutation is known [11]. In the absence of a family history, a timely diagnosis of FD may be difficult due to the variation and unpredictability of clinical manifestations that can often be misdiagnosed as childhood growing pains. The addition of FD to newborn screening programs worldwide has allowed for the early detection of the disorder [12].
There are two types of enzyme replacement therapy: agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme); however only agalsidase beta is an approved ERT by the US Food and Drug Administration (FDA) [13,14]. There is some discussion on the most appropriate infusion as well as the efficacy and safety of the infusions, but studies have shown no adverse effects in switching therapies or an increased benefit of using one over the other [15,16]. There is one oral pharmacological chaperone therapy, migalastat (Galafold), that has been approved by the FDA [17]. This treatment is only available for adults with FD who have amenable gene variants that lead to misfolded α-galactosidase A enzyme that can be stabilized by migalastat. Once stabilized, protein folding is improved and trafficking of the α-galactosidase A enzyme to the lysosome is restored. Other treatments currently being investigated in clinical trials include plant-based ERT, gene therapy, and substrate reducing therapies.
We describe a cohort of 24 adults and two children with FD, 13 males and 13 females, who range in age from 10 to 68 years (mean age 46.1 ± 16.3). The majority of participants were on agalsidase beta ERT (20/26; 12 males, 8 females). Each participant demonstrated different response rates of symptoms and disease progression with ERT, including two individuals who developed adverse infusion reactions.
The aim of this study is to present the variable molecular and clinical features in a cohort of FD patients on ERT, treatment outcomes across organ systems, and highlight patients with previously unreported variants, to help improve treatment options and patient care.
2 Methods
2.1 Participants
This cohort comprised 13 male and 13 female patients, including two male children. All participants had a confirmed molecular diagnosis of FD and most also had recorded enzyme activity levels measured. GLA variants are listed using transcript NM_000169.2. These patients were followed over the course of a one to 12 year period in an outpatient multidisciplinary clinic at the University of California, Irvine. Earlier data were obtained from review of past medical records and the Fabry Registry, if available. Written IRB informed consent approved by UC Irvine (#2008–6631) was obtained from each participant, and all procedures were performed according to The Code of Ethics of the World Medical Association.
2.2 Clinical evaluations
Clinical surveillance visits were conducted every six months and included evaluations by a clinical geneticist, a cardiologist, and a nephrologist. The standard of care for patients with renal and cardiac disease from FD did not vary when compared to the management of other etiologies. At each visit, the following components were documented:• Quality of life surveys, including a Brief Pain Inventory (BPI) and the SF-36 Health Survey. These provided a scale for pain and represented an attempt to document how pain has influenced the quality of life of each individual. The SF-36 Health survey measures physical function, social function, physical role, emotional role, mental health, energy, pain, and general health perception. Scores ranged from 0 (worst) to 100 (best).
• The development or change in the status of clinical features specific to FD including cardiac, renal, pulmonary, and nervous system involvement, in addition to angiokeratomas, acroparesthesia, GI health, hyper or hypohidrosis, hearing loss and tinnitus, lymphedema, and corneal whorling.
• Plasma and urine GL-3 levels and plasma Lyso-GL-3 to assess overall glycolipid burden.
• Renal status and disease progression were monitored by labs: serum creatinine levels to measure the mean estimated glomerular filtration rate (eGFR), in addition to urine creatinine, urine protein, and urine albumin/micro-albumin to measure proteinuria and albuminuria. Renal biopsy was only performed if clinically indicated.
Evaluations performed at less frequent intervals:• Cardiac status was determined by echocardiography (with strain), electrocardiograms, and holter monitor screened at annual intervals in males and less frequently in females and children. Cardiac MRI was conducted every 2–3 years or as needed.
• Brain imaging was performed by MRI, MRA, and CT scan.
• Ophthalmologic exams were performed for evaluation of corneal whorls, vessel tortuosity, and lenticular opacities.
• Audiometry was performed for evaluation of sensorineural hearing loss and tinnitus, as needed.
• Dual-energy X-ray absorptiometry (DXA) & the World Health Organization (WHO) classification of bone mineral density were used to classify severity. T-scores between −1.0 and −2.5 SD were categorized as osteopenia and T-scores below −2.5 SD were categorized as osteoporosis.
2.3 ERT infusions
Agalsidase beta (Fabrazyme) recommended dosage is 1 mg/kg body weight given every two weeks as an intravenous infusion [14]. The majority of the participants in this cohort received the infusions without incident, and some have transitioned to home infusions. To determine the effectiveness of ERT, we measured the presence of agalsidase beta IgG antibodies, plasma and urine GL-3 levels, and plasma Lyso-GL-3 levels at each six-month visit. Two individuals experienced adverse reactions to ERT infusions (patient 15 and patient 22); they both tested negative for IgE antibodies and showed no change in IgG antibodies.
2.4 Statistical analysis
We used linear mixed-effects regression analysis with varying intercepts and slopes to evaluate linear time trends of serum creatinine, eGFR, and health-related quality of life scores (SF-36 Health Survey), starting with the initiation of ERT (time = 0). The significance level for all statistical tests was set to α = 0.05. All analyses were carried out using the R language and environment for statistical computing, version 3.6.3.
3 Results
Of the 26 patients included in our study, there were 13 males and 13 females between ages 10–68 years (mean age 46.1 ± 16.3 years). This cohort consisted of five families in addition to eleven unrelated individuals. The majority of males (12/13, 92%) and the majority of females (8/13, 62%) were treated with ERT. Ages at initiation of ERT were between 6 and 65 years (mean age 39.0 ± 18.3) (Supplemental Table 1). The majority of patients tolerated ERT well, except for two individuals who had adverse reactions (patient 15 and patient 22). Both patients were IgE-negative and showed no changes in IgG antibodies; patient 22 was consistently IgG-positive throughout the course of ERT. Patient 15 had the most significant adverse event during the last infusion attempt, which resulted in intubation in the cardiac intensive care unit due to difficulty breathing, tightness in chest, rigors, and convulsions, and has not been on ERT since. Only three males were routinely IgG-positive (patients 12, 13, 24) and two males had one single IgG-positive test in the past, but had not had another one since (patient 1 and patient 7).
Clinical features of each patient are described in Supplemental Table 2. Notably, there was clinically evident renal involvement in 50% of participants; the most common feature was varying levels of proteinuria. One individual required a renal transplant. Seventy-nine percent of patients showed evidence of some form of cardiac involvement, including 46% with left ventricular hypertrophy. Two males and one female had a history of a stroke, and one male had a history of one TIA and one stroke. The most commonly observed clinical feature was peripheral neuropathic pain (20/26, 77%), followed by angiokeratomas (18/26, 69%), tinnitus (17/26, 65%), gastrointestinal symptoms, including abdominal pain, constipation, and diarrhea (16/26, 62%), and hypohidrosis/anhidrosis (16/26, 62%). Additional common findings of FD in this cohort include corneal verticillata (15/26, 58%), pulmonary involvement (6/12, 50%), lymphedema (11/26, 42%), and hearing loss (9/26, 35%). The adult males in this cohort had higher frequencies of clinical manifestations than adult females in all categories with the exception of neuropathic pain, white matter lesions, and corneal verticillata (Table 1).Table 1 Clinical manifestations of FD in this cohort.
Table 1 Adult Females
N = 13 (%) Adult Males
N = 11 (%) Pediatric Males
N = 2 (%) Total
N = 26 (%)
Mean Age at Diagnosis (Years) 36.2 34.3 3.5 32.8 ± 16.3
Currently On ERT 8 (62) 10 (91) 2 (100) 20 (77)
Mean Age ERT Initiated (Years) 51 37.5 6.5 39.0 ± 18.3
Renal Involvement 5 (38) 7 (64) 1 (50) 13 (50)
Cardiac Involvement 9/12 (75) 9/10 (90) 1 (50) 19/24 (79)
White matter Lesions 3/10 (30) 1/6 (17) N/A 4/16 (25)
Neuropathic Pain 10 (77) 8 (73) 2 (100) 20 (77)
Corneal Verticillata 10 (77) 4 (36) 1 (50) 15 (58)
Angiokeratomas 7 (54) 11 (100) 0 (0) 18 (69)
Hypohidrosis 5 (38) 10 (91) 1 (50) 16 (62)
GI Involvement 8 (62) 8 (73) 0 (0) 16 (62)
Low BMD 2/6 (33) 5/8 (63) N/A 7/14 (50)
Hearing Loss 3 (23) 6 (55) 0 (0) 9 (35)
Tinnitus 7 (54) 9 (82) 1 (50) 17 (65)
Vertigo 4/10 (40) 6/10 (60) 0/1 (0) 10/21 (48)
Pulmonary Involvement 0/5 (0) 5/6 (83) 1/1 (100) 6/12 (50)
Depression 3 (23) 3 (27) 0 (0) 6/26 (23)
Numbers provided in fractions indicate that not all patients had the corresponding assessment.
3.1 Clearance of GL-3 in plasma
Plasma-GL-3, plasma Lyso-GL-3, and urine GL-3 are biomarkers used as surrogate endpoints to monitor Fabry disease severity and progression in both treated and untreated patients. Longitudinal analysis of plasma GL-3 concentration in 17 of our patients on ERT is depicted in Fig. 1. The data showed significant reduction in GL-3 of −0.25 μg/mL per year (95% CI: −0.478 to −0.030, p = 0.026) with ERT treatment.Fig. 1 Plasma GL-3 clearance in patients on ERT. A linear mixed-effects regression analysis of plasma GL-3 with time as a predictor and varying intercepts and slopes. The figure shows observed GL-3 measurements (dots) together with model-estimated time trajectories of GL-3 for each participant (colored lines) and for the population (black line). Note that observations at time < 0 were excluded from the analysis. N = 17.
Fig. 1
While there was less data available for the deacetylated form, plasma Lyso-GL-3, the majority showed a reduction or stabilization of plasma Lyso-GL-3 concentration at last visit compared to that of the earlier time points (N = 11, data not shown).
3.2 Renal involvement
There was clinically evident renal disease in 50% (13/26) of participants as defined by albuminuria, hypertension or reduced eGFR. Renal biopsies were not done routinely. The majority demonstrated proteinuria and/or albuminuria as the sole renal manifestation. Ten patients had elevated urine albumin-to-creatine ratios, nine with microalbuminuria (5 females, 4 males) and one male with macroalbuminuria. Hypertension was seen in 32% (8/25) of patients and managed with angiotensin-converting enzyme inhibitors (ACEI) (8/26), angiotensin receptor blockers (ARB) (4/26), beta-blockers (4/26), and diuretics (6/26). Two had stage I chronic kidney disease (CKD) (patient 1 and patient 12), two had stage III CKD (patient 14 and patient 17), and one had stage IV CKD and is on hemodialysis (patient 22). Patient 23 underwent a deceased donor renal transplant due to ESRD at age 52 years and has had normal kidney function since. For those on ERT, only some patients maintained serum creatinine and estimated glomerular filtration rate (eGFR) within normal limits for age and sex. However, the data showed significant increase in serum creatinine levels of +0.02 mg/dL per year (95% CI: 0.01–0.04, p = 0.015) for both males and females on ERT in this cohort (Fig. 2A) and +0.038 mg/dL per year (95% CI: 0.01–0.06, p = 0.003, data not shown) for males alone. Further, the data showed an annual decrease in eGFR of −1.88 mL/min/1.73m2 (95% CI: −3.36 to −0.40, p = 0.013) on ERT in both males and females (Fig. 2B), and −3.16 mL/min/1.73m2 (95% CI: −4.97 to −1.35, p = 0.001, data not shown) for males alone.Fig. 2 Serum creatinine and estimated glomerular filtration rate (eGFR) of patients on ERT. The figure shows observed serum creatinine measurements (A) and eGFR measurements (B) together with model-estimated time trajectories for each participant (colored lines) and for the population (black lines). Note that observations at time < 0 were excluded from both analyses in figs. A-B. N = 18.
Fig. 2
3.3 Cardiac involvement
In our cohort, 79% (19/24) of participants had some form of cardiac involvement. Left ventricular hypertrophy (LVH) was seen in 46% (11/24) of patients, with severity ranging from borderline to severe and one confined to the septum. Severe LVH of patient 24 is shown in Fig. 3A. Of the adult males, only one was not found to have clinically evident cardiac involvement, and while his past ECGs met voltage criteria for LVH, his echocardiograms were normal showing no evidence of LVH. Left ventricular ejection fraction (LVEF) was one of the measures used to monitor cardiac disease progression of patients on ERT. LVEF was within normal range at baseline prior to ERT. Some patients fell below normal range over the course of treatment with the lowest recorded being 43% in a male patient. Of the individuals in our cohort who had cardiac imaging, 25% (6/24) had valvular abnormalities, including one patient with aortic valve sclerosis, two patients had thickened mitral valve leaflets, one patient with mild Systolic Anterior Motion (SAM) abnormality of the mitral valve, one with mitral valve prolapse, and one patient required mitral valve annuloplasty after experiencing lower extremity edema, shortness of breath and dyspnea on exertion (patient 22). Other cardiac manifestations are described in Table 2. Of those who underwent cardiac MRI, only one showed possible fibrosis of the myocardium (Fig. 3A). Those with cardiac abnormalities on echocardiogram, cardiac MRI, and/or ECG did not consistently have abnormal cardiac biomarkers, such as brain natriuretic peptide (BNP), troponin, and soluble suppression of tumourigenicity 2 (ST2), though limited data was available. Adult FD patients can have elevated lipid levels, particularly high HDL cholesterol, which may be non-responsive to long-term ERT [18]. Dyslipidemia was present in 48% (10/21) of patients in our cohort, and managed with diet, fish-oil and statins.Fig. 3 Clinical features in patients with FD. (A) Cardiac MRI showing severe concentric hypertrophy of the left ventricle with reduced chamber size. There is subendocardial linear hyperenhancement in the basal inferolateral wall suggesting myocardial scar (white arrow), consistent with FD (patient 24); (B) Axial brain MRI section following TIA event (patient 22); (C) Corneal verticillata seen by slit lamp examination. Courtesy: Dr. Pinakin Davey, Western University of Health Sciences; (D) Cluster of angiokeratomas in the groin region (patient 24).
Fig. 3Table 2 Characterization of cardiac involvement in FD patients in this cohort.
Table 2 Adult
Females Adult
Males Pediatric Males Total (%)
Cardiac Involvement 9/12 (75) 9/10 (90) 1/2 (50) 19/24 (79)
Echocardiogram Findings
Left Ventricular Hypertrophy 5/12 7/10 0/2 11/24 (46)
Borderline 1 2 0
Mild 2 3 0
Moderate 0 1 0
Severe 0 1 0
Septal 1 0 0
Valvular Abnormality 2/12 4/10 0/2 6/24 (25)
Aortic Valve 1 0 0
Mitral Valve 1 4 0
Left Atrium Dilation 2/12 2/10 0/2 4/24 (17)
Right Atrium Dilation 1/12 0/10 0/2 1/24 (4)
Electrocardiogram Findings
Conduction Abnormalities 3/12 6/10 0/2 9/24 (38)
Right Bundle Branch Block 2 5 0
Anterior Fascicular Block 1 1 0
First Degree Atrioventricular Block 1 0 0
Intraventricular Conduction Delay 0 2 0
Short PR Interval 1 2 0
Rhythm Abnormalities 5/12 8/10 1/2 14/24 (58)
Sinus Bradycardia 5 8 1
Sinus Arrhythmia 1 0 0
Premature Ventricular Complexes 0 2 0
Premature Atrial Contractions 0 1 0
Repolarization Abnormalities 5/12 3/10 0/2 8/24 (33)
T Wave Changes 2 1 0
ST/STT Changes 3 2 0
Left Axis Deviation 0/12 1/10 0/2 1/24 (4)
One male and one female in this cohort were excluded due to no cardiac imaging records available.
3.4 Cerebrovascular complications, neurologic, and neuropsychiatric symptoms
White matter lesions were present in 25% (4/16) of participants who had brain imaging. One male in this cohort (patient 22) has a history of two cerebrovascular accidents, one transient ischemic attack (TIA) at age 52 years and one stroke at age 60 years. Brain MRI of patient 22 showed multifocal and adjacent gliosis within the left cerebral hemisphere, high left frontal and parietal lobes, as well as subcortical and periventricular deep white matter T2 hyperintensity without mass effect that is most pronounced within the parietal occipital region (Fig. 3B). The stroke impacted his eyesight and word-finding ability. Patient 22 only began ERT at the age of 57 years, and passed away at the age of 63 years due to post-operative complications of abdominal surgery. Another male (patient 15) and one female (patient 14) in this cohort have a history of TIAs while on ERT. They were not taking low dose aspirin at the time.
With regard to neuropathic manifestations, 77% (20/26) of patients reported peripheral neuropathic pain, manifesting as burning, tingling, or numbness, often triggered by temperature change or strenuous activity. Of the 17 participants for whom data on the brief pain inventory (BPI) survey was available, the majority reported worsening or inconsistent pain symptoms over the duration of treatment with ERT. Decreased quality of life has been associated with FD, in part due to the debilitating episodic pain crises and burden associated with having a chronic illness. Health-related quality of life was collected using the SF-36 Health survey. Bodily pain scores obtained from the SF-36 survey also showed no improvement over the course of ERT, though this was not significant (p = 0.19; Fig. 4C). For the majority of SF-36 respondents, pain worsened over time, which was similar to what was reported in the BPI survey. Notably, there was a significant decline in physical functioning (Est. = −1.40, 95% CI: −2.73 to −0.06, p = 0.040) for patients in this cohort. The remaining health dimensions did not show significant change over time (Fig. 4A-H).Fig. 4 SF-36 health-related quality of life component scores of patients on ERT. The SF-36 Health survey measures (A) physical function, (B) physical role, (C) pain, (D) general health perception, (E) vitality, (F) social function, (G) emotional role, (H) mental health. Scores ranged from 0 (worst) to 100 (best). Observed values (dots) are shown together with model-estimated trajectories for individual patients (colored lines) and the population (black lines).
Fig. 4
Of the adult patients in this cohort, seven reported depression (7/24; 29%). One patient reported a history of depression that correlated with pain symptoms. Another patient had a history of depression with no current pain symptoms based on responses in the BPI and SF-36 Health Survey. Of note, one patient in this cohort had a history of two suicide attempts. On the other hand, one patient's testimonial described noticeable improvement in anxiety, fatigue, and executive function (termed “Fabry fog”) since initiation of ERT.
3.5 Ophthalmologic findings
Corneal verticillata, or corneal whorling, is a unique clinical feature in patients of both sexes with FD and is often used as a diagnostic tool. It is a result of GL-3 deposits and has not been linked to vision loss. Corneal verticillata was noted in 58% (15/26) of individuals, at a higher prevalence in females than males in our cohort (Fig. 3C). Vessel tortuosity is also associated with FD and is exacerbated by renal complications [19,20]. Only one patient was found to have vessel tortuosity (patient 22); this individual had stage IV CKD.
3.6 Skin manifestations
Angiokeratomas are common in Fabry patients as they occur when GL-3 accumulates in dermal endothelial cells and lead to secondary ectasia. In our cohort, 69% (18/26) had angiokeratomas (Fig. 3D).
Hypohidrosis is also a common feature of FD and may be a predisposition to acroparesthesia. In this cohort, 62% (16/26) experienced hypohidrosis or anhidrosis. Of those with hypohidrosis, 89% experienced acroparesthesia. Lymphedema of the extremities, particularly in the feet, was present in 42% (11/26) of patients in this study.
3.7 Gastrointestinal involvement
Gastrointestinal symptoms are common among FD patients and can manifest as diarrhea, constipation, nausea, vomiting, incontinence, and abdominal pain. In our cohort, gastrointestinal symptoms were present in 62% (16/26) and the most common complaint was abdominal pain. Previous studies have shown significantly improved gastrointestinal pain with ERT [21]. This was also reported by the patients in our study in the patient health survey. The majority of those on ERT reported either improved or stable abdominal pain and frequency of diarrhea based on their survey responses at last visit compared to earlier survey responses. Specifically, six patients reported improvement in abdominal pain and four reported that symptoms were stable (N = 20). Eleven reported improvement in diarrhea and one reported that symptoms were stable (N = 20). One patient's testimonial described a frequency of diarrhea multiple times daily prior to ERT that has now significantly improved since ERT initiation. He also reported noticeable return of symptoms during the time of the ERT shortage. On the other hand, worsening or recent onset of abdominal pain (3/20) and diarrhea (2/20) over the course of ERT treatment were also reported by participants in this study.
3.8 Auditory and vestibular involvement
Sensorineural hearing loss, predominantly in the high frequencies, is a common manifestation of FD and correlates with neuropathic and vascular damage [22]. In our cohort, 35% (9/26) of patients reported a history of hearing loss. The majority of those who underwent audiology assessments were found to have bilateral mild to moderate sensorineural hearing loss. Additionally, 65% (17/26) of patients reported tinnitus. Some patients do not use hearing aids due to amplification by the presence of tinnitus. Vertigo was reported in 48% of our cohort in those who recorded their response in the health survey (10/21).
3.9 Pulmonary involvement
Respiratory involvement in FD typically includes obstructive lung disease, however patients may develop interstitial restrictive lung disease. In our cohort, 50% (6/12) of those who have had spirometry testing were found to have reduced lung capacity. Of those, four had obstructive lung disease, one had possible obstructive lung disease, and one had restrictive lung disease.
3.10 Endocrine and other clinical manifestations
There is limited data in the literature that describes endocrine system involvement in FD. In our cohort, patients reported type II diabetes, hypothyroidism, hyperthyroidism, hyperparathyroidism, and vitamin D deficiency (Supplemental Table 2). Patient 8 has a history of hyperthyroidism secondary to Graves' disease, and a past history of a 1.9 mm benign thyroid nodule. Patient 21 has type II diabetes and hypothyroidism. Patient 22 has acquired hypothyroidism and hyperparathyroidism secondary to renal disease. One of the pediatric participants (patient 9) was diagnosed with growth hormone deficiency and a small pituitary gland. Not all participants in this study have had an endocrine evaluation, so this study may have missed those with subclinical endocrine dysfunction.
Low Bone Mineral Density (BMD) has been noted more recently to be a common feature of FD [23]. Impaired renal function leads to vitamin D deficiency and may be one of the contributing factors to reduced BMD in FD patients. Fifty percent of patients in this cohort who had DXA scans (7/14) were found to have low BMD. Of those, 57% (4/7) had osteopenia (T score < 1.5) and 43% (3/7) had osteoporosis (T score < 2.5). Notably, two of the seven patients (29%) had early-onset low BMD at age 32 years (patient 6) and age 36 years (patient 13). There were no prior DXA scans to measure the rate of bone loss in these two young adult males. Additionally, patient 13 spends up to 40 h per week in a sensory deprivation floatation tank, which may have contributed to bone loss (described below in section 3.13. Supplemental Treatments). Only one patient endured an ankle fracture at the age of 65 years due to a mechanical fall; none of the remaining participants reported bone fractures. Of those with low BMD, four patients had concurrent vitamin D deficiency; of those four, one had secondary hyperparathyroidism due to renal disease. All patients in this cohort were prescribed calcium and vitamin D supplementation. Those with osteoporosis were managed by an endocrinologist and prescribed bisphosphonates.
Hypertension is also a common adverse event in adult FD patients. Cardiac, renal and cerebrovascular complications increase the risk of uncontrolled hypertension in individuals with FD [24]. Hypertension was present in 32% (8/25) of patients in our cohort, the majority but not all of these individuals had some form of clinically evident renal involvement. Patients were managed on ACEIs, ARBs, beta-blockers, and diuretics.
3.11 Novel GLA variant and genotype-phenotype correlations
Our study describes 16 unique variants in the 26 total participants, including one novel variant and one atypical ‘later-onset’ cardiac variant (Table 3; Fig. 5). The novel variant c.1226_1231delCCACAG (p.P409_G411delinsR), an in-frame deletion located within a hot-spot, was identified in a 63-year-old male (patient 22) who was diagnosed later in adulthood at 57 years old following a renal biopsy that revealed stage IV chronic kidney disease. He is discussed earlier with renal, pulmonary, and cardiac involvement and a history of two cerebrovascular accidents. Additional FD-related symptoms in patient 22 include angiokeratomas, corneal verticillata, gastrointestinal involvement, lymphedema, hypohidrosis, acroparesthesia, Raynaud's syndrome, hearing loss, tinnitus, and low bone mineral density (Supplemental Table 2). Cardiac work-up for chest pain revealed a 95% blockage of the left anterior descending coronary artery requiring stent placement, which resolved except for occasional reports of arrhythmia. Endocrine dysfunction in this patient includes acquired hypothyroidism and hyperparathyroidism secondary to renal disease. Patient 22 was particularly significant among our participants because of the history of one TIA and one stroke; brain MRI revealed significant gliosis and nonspecific white matter signal changes (Fig. 3B). Patient 22 is now deceased due to post-operative complications of abdominal surgery for abdominal abscess and intra-abdominal infection.Table 3 GLA variant details.
Table 3Fam. No. Patient No. Ethnicity Nucleotide Change Protein Change Position Type Phenotype
1 1,2,3,4,5,6 Hispanic c.983G > T G328V Exon 6 Missense Classic
2 7,8 Caucasian c.1250 T > G L417R Exon 7 Missense Classic
3 9,10,11 Caucasian c.132G > T W44C Exon 1 Missense Classic
4 12 Caucasian c.568delG A190PfsX2 Exon 4 Small Deletion Classic
5 13,14 Caucasian c.1041_1042insG A348GfsX27 Exon 7 Small Insertion Classic
6 15 Hispanic c.706 T > C W236R Exon 5 Missense Classic
7 16 Caucasian c.680G > A R227Q Exon 5 Missense Classic
8 17 Caucasian c.816C > A N272K Exon 6 Missense Classic
9 18 Caucasian c.730G > A D244N Exon 5 Missense Classic/ Later Onset
10 19 Asian c.639 + 919G > A IVS4 + 919G > A Intron 4 Splicing Later Onset
11 20 Asian c.427G > C A143P Exon 3 Missense Classic
12 21 Hispanic c.639 + 4A > T IVS4 + 4A > T Intron 4 Splicing Classic
13 22 Caucasian c.1226_1231delCCACAG P409_G411delinsR Exon 7 Small Deletion Classic
14 23 Hispanic c.1088G > A R363H Exon 7 Missense Later Onset
15 24,25 Hispanic c.1072_1074delGAG E358del Exon 7 Small Deletion Classic
16 26 Caucasian c.1246C > T Q416X Exon 7 Nonsense Classic
Novel variant is bolded.
Fig. 5 Unique variants identified in the GLA gene in this cohort. A schematic representation of GLA showing the position of all variants identified, with novel variant in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Though the rest of our cohort harbors variants that have been previously reported in the literature, we present novel clinical data for certain recurrent variants. Family 3 (patients 9, 10, 11) harbor the W44C variant, a previously reported variant in a Chinese family of 12 affected members, all of whom experienced paroxysmal pain of limb extremities [25]. Interestingly, our family is of European/mixed descent, with no known Asian ancestry, and present with multi-organ involvement in addition to peripheral pain. We report one patient with the c.639 + 919G > A ‘later-onset’ cardiac variant, who, in addition to mild left ventricular hypertrophy, experiences other FD-related symptoms including angiokeratomas, gastrointestinal issues, pulmonary involvement, and hypohidrosis with heat intolerance. His mother, who also harbors the familial variant, has a history of strokes and presented at age 76 years with hypertrophic cardiomyopathy. Another example of potential genotype-phenotype correlation in our cohort is Family 1; all three females who harbor the G328V variant have audiologic involvement.
3.12 Pediatric patients
There were two male pediatric participants in this study (patient 7 and patient 9). Both patients were diagnosed before the age of five and treated with ERT at or before the onset of symptoms. Patient 9 is a seven year-old male with the W44C variant. This patient was diagnosed with FD at age 3 and began ERT at age 7 years soon after an evaluation for reactive hyperemia index of 0.94 (Reference RHI value >1.68) using an EndoPAT (Itamar Medical) device performed on a research basis, which was consistent with low peripheral vascular endothelial function. He lacked ‘classic’ FD symptoms at the time of the procedure, but ERT was recommended due to concern for deteriorating endothelial function. Initial urine GL-3 level of 569 μg/mmol creatinine resolved during the course of this study while on ERT. He is developmentally normal, however he has a past medical history remarkable for growth hormone deficiency and a small pituitary gland and has been managed on somatropin since 3 years of age.
Patient 7 from Family 2 first presented with acroparesthesia and hypohidrosis at 4 years of age and began ERT by 6 years of age. Prior to initiation of ERT, urine GL-3 level was at 370 μg/mmol and was cleared to 0 μg/mmol in less than two years on ERT. Notably, spirometry testing revealed mild obstructive airway disease, though it has not yet affected routine activity. Patient 7 and his affected mother (patient 8) present with variable symptoms of FD as shown in Supplemental Table 2.
3.13 Supplemental treatments
Several individuals in this cohort sought alternative sources for pain management. Effective pain management could impact the psychological consequences of FD, as there is a high rate of depression that is often linked to the effects of long-term pain. Patient 13 had a five-year history of frequent, severe pain crises, including in response to ERT infusion, which has led the patient to seek alternative pain management strategies in conjunction with ERT. This includes floatation in a sensory deprivation tank used for the treatment of pain and anxiety by limiting stimulation from gravity, sound and light while immersed in water baths saturated with Epsom salts to allow for buoyancy in a dark and soundless room. Patient 15 and Patient 16 also report benefit from this float tank. Important to note, extended time in the float tank may have had an adverse effect on patient 13's bone density, as he reports spending up to 40 h per week in the float tank. There was a variety of medications used in this cohort for the management of neuropathic pain. Patient 13 and 15 are prescribed cannabidiol (CBD). Patient 15 and patient 26 use non-prescription topical and oral CBD as needed during pain crises. Use of tetrahydrocannabinol (THC) was also reported. Six patients are prescribed opioid narcotic analgesics, eight patients are managed with over-the-counter analgesics, two are on antidepressants, and one on an anticonvulsant (carbamazepine).
4 Discussion
The benefit, safety, and risk of ERT have been previously investigated, though the long-term impact continues to be studied [[26], [27], [28], [29], [30]]. ERT is recommended in order to curtail disease progression or when there is already evidence of disease progression and vital organ damage. In this study, 20 of the 26 participants and all but one male (patient 15) were consistently on ERT.
All participants receiving ERT however, demonstrated a different response rate of disease progression. While ERT was shown to be effective in clearing plasma GL-3 and, in many patients maintaining serum creatinine and eGFR within normal limits in this cohort, we identified that many patients continued to have impairment of their renal function and health-related quality of life despite standard of care management. For patients with chronic kidney disease from FD, standard of care did not vary when compared to that of other etiologies. The standard management includes the evaluation for possible reversible causes of renal disease (i.e. dehydration, reduced renal perfusion, obstruction, nephrotoxicity), management of blood pressure to a target ≤130/80, maximally tolerated dose of renin-angiotensin-aldosterone system inhibitors (i.e. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone receptor antagonist), smoking cessation, dietary interventions (particularly reduced sodium and protein restricted diet), treatment of metabolic acidosis, management of anemia of CKD with erythropoiesis-stimulating agents to target Hg 10–11.5 g/dL, management of mineral and bone disorders as per 2012 KDIGO guidelines, initiation of renal replacement therapy in individuals with uremic symptoms and eGFR 5–15 mL/min/1.73 m2 or in those with eGFR <5 mL/min/1.73 m2, and referral for kidney transplant evaluation in those with eGFR 20–25 mL/min/1.73 m2.
The late initiation of ERT may have contributed to those with renal disease progression, as shown previously [31]. Furthermore, nearly all of the adult patients in this cohort had major organ involvement at baseline prior to initiation of ERT. Typically, renal function in FD gradually deteriorates to end-stage renal disease in the third to fifth decade in males [32,33]. Only one patient progressed to severe CKD requiring hemodialysis, therefore, compared to the natural history data, it is possible that ERT contributed to the prevention of renal failure by slowing disease progression. Though vital organ damage is not typically seen in pediatric patients (some cases have been documented), studies have shown evidence for the benefit of the early initiation of ERT in potentially reversing renal damage [10,29]. Patient 12 initiated ERT at age 11 years, however, developed renal involvement by age 24 years [34]. Therefore, we believe earlier initiation of ERT is required to limit deterioration of the major organs. Our two pediatric participants initiated ERT at ages 6 and 7 years, respectively, and one reported significant improvement of pain symptoms.
Genotype-phenotype association is difficult to establish in FD for many reasons. Most GLA variants are unique (private mutations) and there are few family studies. Even within families, the phenotype may vary depending on the sex, age of onset, and other genetic and epigenetic factors. There are several reports in the literature of ‘later-onset’ isolated hypertrophic cardiomyopathy in individuals with the recurrent c.639 + 919G > A variant [6,[35], [36], [37]]. While this is often considered an atypical later-onset cardiac disease variant, patient 19 presented with several other ‘classic’ FD symptoms. Studies have explored the use of Amiloride as a potential treatment for FD by modulating alternative splicing in the context of the c.639 + 919G > A variant [35]. In our cohort we identified one novel variant c.1226_1231delCCACAG (p.P409_G411delinsR) in patient 22 with a severe, ‘classic’ FD phenotype [38]. This variant is absent in The Genome Aggregation Database (gnomAD), is located in a mutational hot-spot, and thus predicted to be pathogenic [39]. Two of the 26 individuals in this cohort harbor variants amenable to migalastat, an oral chaperone therapy, but chose not to switch from ERT. Long-term outcome studies for FD patients who have switched to migalastat will be of interest for future studies.
In this cohort, nearly one third of adult patients reported depression and anxiety. Based on the patient-reported SF-36 Health Survey and Brief Pain Inventory survey, there is no significant improvement in health-related quality of life over the course of ERT. Depression, sleep apnea, and anxiety disorders are linked to both pathology and the effects of long-term pain [40]. It has been suggested that psychological counseling can have an impact on reversing or reducing effects, an important consideration to optimize patient care as needed [41]. Importantly, studies have proposed pain as an indicator for the need to initiate ERT [42]. The majority of our cohort experienced Fabry crises or acroparasthesia. There are few data on nervous system involvement of FD in women [43]. Some studies have shown hippocampal volume loss and white matter lesions in female patients with FD [44]. While we showed white matter lesions in three females and one male in this cohort, we did not include brain imaging, electrical conduction, or quantitative sensory testing for every participant in this study. Further studies assessing the outcomes of neuropathy and CNS findings with ERT are warranted.
Cardiac manifestation can be the primary and only symptom of FD in some patients and can present as early as childhood. Our study showed a higher prevalence of cardiac manifestations in both the males and females compared to previous studies [34,[45], [46], [47]]. Cardiac involvement was observed in 83% of male patients, including one pediatric male patient with sinus bradycardia, and 75% of female patients in this cohort. Left ventricular hypertrophy (LVH) was seen in 70% of males and 42% of females. This data provides supporting evidence that female patients, even those with no obvious signs or symptoms of FD, should be monitored closely for evidence of cardiac involvement and be considered for ERT. The standard of care for patients with hypertrophic cardiomyopathy due to FD did not vary when compared to the management of other etiologies and followed AHA/ACC guidelines.
We highlight the variable endocrine dysfunction and low bone mineral density in this cohort, including several with osteoporosis, which provides further evidence for the inclusion of adequate endocrine work-up in the ongoing management of all patients with FD [48].
There is a need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. Furthermore, there is a need for more and improved biomarkers to monitor disease progression. Early identification of endothelial function or other pathologies can provide insight into when ERT should be initiated in order to slow the advancement of adverse effects [49]. Additional tools and biomarkers for measuring endothelial function and other indicators of future pathologies, such as proteomics, are needed to help prevent the deterioration of vital organs [[50], [51], [52]]. Newborn screening for FD has been implemented in several states and will lead to earlier detection. Early detection is important to proactively monitor for complications of the disease, initiate ERT, and prevent co-morbidity progression [[53], [54], [55], [56]].
Since most of our participants were on ERT, we could not make comparisons between long-term outcomes of those on ERT compared to those not on ERT. Also, with the small sample size, we could not compare multiple age-matched individuals or genotype-specific individuals other than our reported families. Another limitation to this study is the long-term analysis of plasma-GL-3 over time rather than plasma Lyso-GL-3, due to the fact that we had more data points for plasma-GL-3. Plasma Lyso-GL-3 has been recently determined to be a more accurate biomarker that correlates more closely with disease phenotype in male and in female patients, with higher levels associated with increased clinical manifestation [57].
5 Conclusion
The variability of symptoms and disease progression in patients with FD, even within the same family, complicates the discernment of a genotype-phenotype correlation. ERT has been available for the treatment of FD since 2001 and has shown improved outcomes, especially when started prior to organ damage, and may reverse fatal disease progression. In this cohort, agalsidase beta infusions have been effective in clearing GL-3 levels. However, renal involvement and health-related quality of life has continued to progress in adult patients diagnosed late despite ERT infusions.
Eight females were on ERT in this study and demonstrated significant symptoms of FD, and in some cases the disease progressed to a similar degree as in the males. This study further demonstrates that females may develop significant FD symptoms and should be managed appropriately and treated as more than carriers.
Documentation of novel variants can contribute to future genotype-phenotype associations between the severity and progression of FD. Genotype-phenotype correlations and long-term outcome data on ERT and/or other FD treatments are needed, especially now that the newborn screening has led to an increase in diagnoses and earlier detection in individuals with FD.
Authors' contributions
V.K. conceived the study and supervised the overall direction and implementation of the study. All authors contributed to acquisition of data. V.K., M.D.C., D.R., D.T., Z.A.D., and G.L. analyzed the data. D.R. performed statistical analyses. M.D.C. and E.C. drafted the manuscript with input from all authors. All authors provided critical feedback and approved the final manuscript for publication.
Funding
The Fabry registry was funded by Sanofi Genzyme. This funding source had no role in data collection or analysis.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Appendix A Supplementary data
Supplementary material
Image 1
Acknowledgements
We thank the patients and their health care providers for their contribution to the study. The Fabry registry was funded by Sanofi Genzyme. This work was supported by CTSI grant UL1TR000124UCLA (Clinical and Translational Science Institute), Los Angeles, CA, USA.
Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2020.100700. | AGALSIDASE BETA, CANNABIDIOL | DrugsGivenReaction | CC BY-NC-ND | 33437642 | 18,784,055 | 2021-03 |
What was the dosage of drug 'AGALSIDASE BETA'? | Variable clinical features of patients with Fabry disease and outcome of enzyme replacement therapy.
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme α-galactosidase A due to mutations in the GLA gene. This leads to an accumulation of globotriaosylceramide (GL-3) in many tissues, which results in progressive damage to the kidneys, heart, and nervous system. We present the molecular and clinical characteristics and long-term outcomes of FD patients from a multidisciplinary clinic at the University of California, Irvine treated with agalsidase beta enzyme replacement therapy (ERT) for 2-20 years. This cohort comprised 24 adults (11 males, 13 females) and two male children (median age 45; range 10-68 years). Of the 26 patients in this cohort, 20 were on ERT (12 males, 8 females). We describe one novel variant not previously reported in the literature in a patient with features of 'classic' FD. The vast majority of patients in this cohort presented with symptoms of 'classic' FD including peripheral neuropathic pain, some form of cardiac involvement, angiokeratomas, corneal verticillata, hypohidrosis, tinnitus, and gastrointestinal symptoms, primarily abdominal pain. The majority of males had clinically evident renal involvement. An annual eGFR reduction of -1.88 mL/min/1.73 m2/yr during the course of ERT was seen in this cohort. The most common renal presentation was proteinuria, and one individual required a renal transplant. Other common findings were pulmonary involvement, lymphedema, hearing loss, and significantly, three patients had strokes. Notably, there was a high prevalence of endocrine dysfunction and low bone mineral density, including several with osteoporosis. While enzyme replacement therapy (ERT) cleared plasma GL-3 in this cohort, there was limited improvement in renal function or health-related quality of life based on the patient-reported SF-36 Health Survey. Physical functioning significantly declined over the course of ERT treatment, which may be, in part, due to the late initiation of ERT in several patients. Further delineation of the phenotypic and genotypic spectrum in patients with FD and the long-term outcome of ERT will help improve management and treatment options for this disease.
1 Introduction
Fabry Disease (FD, OMIM#301500) is an inherited, X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficient or absent α-galactosidase A enzyme [1]. This consequently leads to the progressive deposition of globotriaosylceramide (GL-3) in several tissues throughout the body, notably in endothelial cells, smooth muscle cells, podocytes, and cardiomyocytes [2]. The absence of this enzyme may lead to multi-organ involvement including progressive renal disease, cardiac disease, cerebrovascular disease, neuropathic pain, corneal verticillata, angiokeratomas, tinnitus, hearing loss, pulmonary involvement, and gastrointestinal complaints, including abdominal pain, constipation, and diarrhea. FD is pan-ethnic and the second most prevalent lysosomal storage disorder with an estimated incidence of 1 in 40,000 to 117,000 males worldwide, though newborn screening initiatives have revealed that the incidence is much higher [[3], [4], [5], [6]]. This is likely due to the fact that early studies referred to the incidence of ‘classic’ FD, while newborn screening may identify individuals on the full phenotypic spectrum. Symptoms typically present early during childhood or adolescence. Affected males with little or absent α-galactosidase A activity (<1% of mean normal) may present with clinical features of ‘classic’ FD. Mutations that result in residual enzyme activity typically cause a ‘later-onset’ form of the disease with variable manifestations. Heterozygous females typically present with milder symptoms and later-onset and less commonly have ‘classic’ FD due to residual enzyme activity and X-chromosome inactivation pattern.
Management and guidelines of a newly diagnosed individual with FD includes documentation of symptoms, consideration of enzyme replacement therapy (ERT), and monitoring of disease progression [7,8]. ERT is provided to supplement or replace α-galactosidase A, and to slow the rate of disease progression. Thus, early identification of affected individuals is important in order to initiate ERT as early as possible [[8], [9], [10]]. Clinical evaluation during childhood is recommended if a familial mutation is known [11]. In the absence of a family history, a timely diagnosis of FD may be difficult due to the variation and unpredictability of clinical manifestations that can often be misdiagnosed as childhood growing pains. The addition of FD to newborn screening programs worldwide has allowed for the early detection of the disorder [12].
There are two types of enzyme replacement therapy: agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme); however only agalsidase beta is an approved ERT by the US Food and Drug Administration (FDA) [13,14]. There is some discussion on the most appropriate infusion as well as the efficacy and safety of the infusions, but studies have shown no adverse effects in switching therapies or an increased benefit of using one over the other [15,16]. There is one oral pharmacological chaperone therapy, migalastat (Galafold), that has been approved by the FDA [17]. This treatment is only available for adults with FD who have amenable gene variants that lead to misfolded α-galactosidase A enzyme that can be stabilized by migalastat. Once stabilized, protein folding is improved and trafficking of the α-galactosidase A enzyme to the lysosome is restored. Other treatments currently being investigated in clinical trials include plant-based ERT, gene therapy, and substrate reducing therapies.
We describe a cohort of 24 adults and two children with FD, 13 males and 13 females, who range in age from 10 to 68 years (mean age 46.1 ± 16.3). The majority of participants were on agalsidase beta ERT (20/26; 12 males, 8 females). Each participant demonstrated different response rates of symptoms and disease progression with ERT, including two individuals who developed adverse infusion reactions.
The aim of this study is to present the variable molecular and clinical features in a cohort of FD patients on ERT, treatment outcomes across organ systems, and highlight patients with previously unreported variants, to help improve treatment options and patient care.
2 Methods
2.1 Participants
This cohort comprised 13 male and 13 female patients, including two male children. All participants had a confirmed molecular diagnosis of FD and most also had recorded enzyme activity levels measured. GLA variants are listed using transcript NM_000169.2. These patients were followed over the course of a one to 12 year period in an outpatient multidisciplinary clinic at the University of California, Irvine. Earlier data were obtained from review of past medical records and the Fabry Registry, if available. Written IRB informed consent approved by UC Irvine (#2008–6631) was obtained from each participant, and all procedures were performed according to The Code of Ethics of the World Medical Association.
2.2 Clinical evaluations
Clinical surveillance visits were conducted every six months and included evaluations by a clinical geneticist, a cardiologist, and a nephrologist. The standard of care for patients with renal and cardiac disease from FD did not vary when compared to the management of other etiologies. At each visit, the following components were documented:• Quality of life surveys, including a Brief Pain Inventory (BPI) and the SF-36 Health Survey. These provided a scale for pain and represented an attempt to document how pain has influenced the quality of life of each individual. The SF-36 Health survey measures physical function, social function, physical role, emotional role, mental health, energy, pain, and general health perception. Scores ranged from 0 (worst) to 100 (best).
• The development or change in the status of clinical features specific to FD including cardiac, renal, pulmonary, and nervous system involvement, in addition to angiokeratomas, acroparesthesia, GI health, hyper or hypohidrosis, hearing loss and tinnitus, lymphedema, and corneal whorling.
• Plasma and urine GL-3 levels and plasma Lyso-GL-3 to assess overall glycolipid burden.
• Renal status and disease progression were monitored by labs: serum creatinine levels to measure the mean estimated glomerular filtration rate (eGFR), in addition to urine creatinine, urine protein, and urine albumin/micro-albumin to measure proteinuria and albuminuria. Renal biopsy was only performed if clinically indicated.
Evaluations performed at less frequent intervals:• Cardiac status was determined by echocardiography (with strain), electrocardiograms, and holter monitor screened at annual intervals in males and less frequently in females and children. Cardiac MRI was conducted every 2–3 years or as needed.
• Brain imaging was performed by MRI, MRA, and CT scan.
• Ophthalmologic exams were performed for evaluation of corneal whorls, vessel tortuosity, and lenticular opacities.
• Audiometry was performed for evaluation of sensorineural hearing loss and tinnitus, as needed.
• Dual-energy X-ray absorptiometry (DXA) & the World Health Organization (WHO) classification of bone mineral density were used to classify severity. T-scores between −1.0 and −2.5 SD were categorized as osteopenia and T-scores below −2.5 SD were categorized as osteoporosis.
2.3 ERT infusions
Agalsidase beta (Fabrazyme) recommended dosage is 1 mg/kg body weight given every two weeks as an intravenous infusion [14]. The majority of the participants in this cohort received the infusions without incident, and some have transitioned to home infusions. To determine the effectiveness of ERT, we measured the presence of agalsidase beta IgG antibodies, plasma and urine GL-3 levels, and plasma Lyso-GL-3 levels at each six-month visit. Two individuals experienced adverse reactions to ERT infusions (patient 15 and patient 22); they both tested negative for IgE antibodies and showed no change in IgG antibodies.
2.4 Statistical analysis
We used linear mixed-effects regression analysis with varying intercepts and slopes to evaluate linear time trends of serum creatinine, eGFR, and health-related quality of life scores (SF-36 Health Survey), starting with the initiation of ERT (time = 0). The significance level for all statistical tests was set to α = 0.05. All analyses were carried out using the R language and environment for statistical computing, version 3.6.3.
3 Results
Of the 26 patients included in our study, there were 13 males and 13 females between ages 10–68 years (mean age 46.1 ± 16.3 years). This cohort consisted of five families in addition to eleven unrelated individuals. The majority of males (12/13, 92%) and the majority of females (8/13, 62%) were treated with ERT. Ages at initiation of ERT were between 6 and 65 years (mean age 39.0 ± 18.3) (Supplemental Table 1). The majority of patients tolerated ERT well, except for two individuals who had adverse reactions (patient 15 and patient 22). Both patients were IgE-negative and showed no changes in IgG antibodies; patient 22 was consistently IgG-positive throughout the course of ERT. Patient 15 had the most significant adverse event during the last infusion attempt, which resulted in intubation in the cardiac intensive care unit due to difficulty breathing, tightness in chest, rigors, and convulsions, and has not been on ERT since. Only three males were routinely IgG-positive (patients 12, 13, 24) and two males had one single IgG-positive test in the past, but had not had another one since (patient 1 and patient 7).
Clinical features of each patient are described in Supplemental Table 2. Notably, there was clinically evident renal involvement in 50% of participants; the most common feature was varying levels of proteinuria. One individual required a renal transplant. Seventy-nine percent of patients showed evidence of some form of cardiac involvement, including 46% with left ventricular hypertrophy. Two males and one female had a history of a stroke, and one male had a history of one TIA and one stroke. The most commonly observed clinical feature was peripheral neuropathic pain (20/26, 77%), followed by angiokeratomas (18/26, 69%), tinnitus (17/26, 65%), gastrointestinal symptoms, including abdominal pain, constipation, and diarrhea (16/26, 62%), and hypohidrosis/anhidrosis (16/26, 62%). Additional common findings of FD in this cohort include corneal verticillata (15/26, 58%), pulmonary involvement (6/12, 50%), lymphedema (11/26, 42%), and hearing loss (9/26, 35%). The adult males in this cohort had higher frequencies of clinical manifestations than adult females in all categories with the exception of neuropathic pain, white matter lesions, and corneal verticillata (Table 1).Table 1 Clinical manifestations of FD in this cohort.
Table 1 Adult Females
N = 13 (%) Adult Males
N = 11 (%) Pediatric Males
N = 2 (%) Total
N = 26 (%)
Mean Age at Diagnosis (Years) 36.2 34.3 3.5 32.8 ± 16.3
Currently On ERT 8 (62) 10 (91) 2 (100) 20 (77)
Mean Age ERT Initiated (Years) 51 37.5 6.5 39.0 ± 18.3
Renal Involvement 5 (38) 7 (64) 1 (50) 13 (50)
Cardiac Involvement 9/12 (75) 9/10 (90) 1 (50) 19/24 (79)
White matter Lesions 3/10 (30) 1/6 (17) N/A 4/16 (25)
Neuropathic Pain 10 (77) 8 (73) 2 (100) 20 (77)
Corneal Verticillata 10 (77) 4 (36) 1 (50) 15 (58)
Angiokeratomas 7 (54) 11 (100) 0 (0) 18 (69)
Hypohidrosis 5 (38) 10 (91) 1 (50) 16 (62)
GI Involvement 8 (62) 8 (73) 0 (0) 16 (62)
Low BMD 2/6 (33) 5/8 (63) N/A 7/14 (50)
Hearing Loss 3 (23) 6 (55) 0 (0) 9 (35)
Tinnitus 7 (54) 9 (82) 1 (50) 17 (65)
Vertigo 4/10 (40) 6/10 (60) 0/1 (0) 10/21 (48)
Pulmonary Involvement 0/5 (0) 5/6 (83) 1/1 (100) 6/12 (50)
Depression 3 (23) 3 (27) 0 (0) 6/26 (23)
Numbers provided in fractions indicate that not all patients had the corresponding assessment.
3.1 Clearance of GL-3 in plasma
Plasma-GL-3, plasma Lyso-GL-3, and urine GL-3 are biomarkers used as surrogate endpoints to monitor Fabry disease severity and progression in both treated and untreated patients. Longitudinal analysis of plasma GL-3 concentration in 17 of our patients on ERT is depicted in Fig. 1. The data showed significant reduction in GL-3 of −0.25 μg/mL per year (95% CI: −0.478 to −0.030, p = 0.026) with ERT treatment.Fig. 1 Plasma GL-3 clearance in patients on ERT. A linear mixed-effects regression analysis of plasma GL-3 with time as a predictor and varying intercepts and slopes. The figure shows observed GL-3 measurements (dots) together with model-estimated time trajectories of GL-3 for each participant (colored lines) and for the population (black line). Note that observations at time < 0 were excluded from the analysis. N = 17.
Fig. 1
While there was less data available for the deacetylated form, plasma Lyso-GL-3, the majority showed a reduction or stabilization of plasma Lyso-GL-3 concentration at last visit compared to that of the earlier time points (N = 11, data not shown).
3.2 Renal involvement
There was clinically evident renal disease in 50% (13/26) of participants as defined by albuminuria, hypertension or reduced eGFR. Renal biopsies were not done routinely. The majority demonstrated proteinuria and/or albuminuria as the sole renal manifestation. Ten patients had elevated urine albumin-to-creatine ratios, nine with microalbuminuria (5 females, 4 males) and one male with macroalbuminuria. Hypertension was seen in 32% (8/25) of patients and managed with angiotensin-converting enzyme inhibitors (ACEI) (8/26), angiotensin receptor blockers (ARB) (4/26), beta-blockers (4/26), and diuretics (6/26). Two had stage I chronic kidney disease (CKD) (patient 1 and patient 12), two had stage III CKD (patient 14 and patient 17), and one had stage IV CKD and is on hemodialysis (patient 22). Patient 23 underwent a deceased donor renal transplant due to ESRD at age 52 years and has had normal kidney function since. For those on ERT, only some patients maintained serum creatinine and estimated glomerular filtration rate (eGFR) within normal limits for age and sex. However, the data showed significant increase in serum creatinine levels of +0.02 mg/dL per year (95% CI: 0.01–0.04, p = 0.015) for both males and females on ERT in this cohort (Fig. 2A) and +0.038 mg/dL per year (95% CI: 0.01–0.06, p = 0.003, data not shown) for males alone. Further, the data showed an annual decrease in eGFR of −1.88 mL/min/1.73m2 (95% CI: −3.36 to −0.40, p = 0.013) on ERT in both males and females (Fig. 2B), and −3.16 mL/min/1.73m2 (95% CI: −4.97 to −1.35, p = 0.001, data not shown) for males alone.Fig. 2 Serum creatinine and estimated glomerular filtration rate (eGFR) of patients on ERT. The figure shows observed serum creatinine measurements (A) and eGFR measurements (B) together with model-estimated time trajectories for each participant (colored lines) and for the population (black lines). Note that observations at time < 0 were excluded from both analyses in figs. A-B. N = 18.
Fig. 2
3.3 Cardiac involvement
In our cohort, 79% (19/24) of participants had some form of cardiac involvement. Left ventricular hypertrophy (LVH) was seen in 46% (11/24) of patients, with severity ranging from borderline to severe and one confined to the septum. Severe LVH of patient 24 is shown in Fig. 3A. Of the adult males, only one was not found to have clinically evident cardiac involvement, and while his past ECGs met voltage criteria for LVH, his echocardiograms were normal showing no evidence of LVH. Left ventricular ejection fraction (LVEF) was one of the measures used to monitor cardiac disease progression of patients on ERT. LVEF was within normal range at baseline prior to ERT. Some patients fell below normal range over the course of treatment with the lowest recorded being 43% in a male patient. Of the individuals in our cohort who had cardiac imaging, 25% (6/24) had valvular abnormalities, including one patient with aortic valve sclerosis, two patients had thickened mitral valve leaflets, one patient with mild Systolic Anterior Motion (SAM) abnormality of the mitral valve, one with mitral valve prolapse, and one patient required mitral valve annuloplasty after experiencing lower extremity edema, shortness of breath and dyspnea on exertion (patient 22). Other cardiac manifestations are described in Table 2. Of those who underwent cardiac MRI, only one showed possible fibrosis of the myocardium (Fig. 3A). Those with cardiac abnormalities on echocardiogram, cardiac MRI, and/or ECG did not consistently have abnormal cardiac biomarkers, such as brain natriuretic peptide (BNP), troponin, and soluble suppression of tumourigenicity 2 (ST2), though limited data was available. Adult FD patients can have elevated lipid levels, particularly high HDL cholesterol, which may be non-responsive to long-term ERT [18]. Dyslipidemia was present in 48% (10/21) of patients in our cohort, and managed with diet, fish-oil and statins.Fig. 3 Clinical features in patients with FD. (A) Cardiac MRI showing severe concentric hypertrophy of the left ventricle with reduced chamber size. There is subendocardial linear hyperenhancement in the basal inferolateral wall suggesting myocardial scar (white arrow), consistent with FD (patient 24); (B) Axial brain MRI section following TIA event (patient 22); (C) Corneal verticillata seen by slit lamp examination. Courtesy: Dr. Pinakin Davey, Western University of Health Sciences; (D) Cluster of angiokeratomas in the groin region (patient 24).
Fig. 3Table 2 Characterization of cardiac involvement in FD patients in this cohort.
Table 2 Adult
Females Adult
Males Pediatric Males Total (%)
Cardiac Involvement 9/12 (75) 9/10 (90) 1/2 (50) 19/24 (79)
Echocardiogram Findings
Left Ventricular Hypertrophy 5/12 7/10 0/2 11/24 (46)
Borderline 1 2 0
Mild 2 3 0
Moderate 0 1 0
Severe 0 1 0
Septal 1 0 0
Valvular Abnormality 2/12 4/10 0/2 6/24 (25)
Aortic Valve 1 0 0
Mitral Valve 1 4 0
Left Atrium Dilation 2/12 2/10 0/2 4/24 (17)
Right Atrium Dilation 1/12 0/10 0/2 1/24 (4)
Electrocardiogram Findings
Conduction Abnormalities 3/12 6/10 0/2 9/24 (38)
Right Bundle Branch Block 2 5 0
Anterior Fascicular Block 1 1 0
First Degree Atrioventricular Block 1 0 0
Intraventricular Conduction Delay 0 2 0
Short PR Interval 1 2 0
Rhythm Abnormalities 5/12 8/10 1/2 14/24 (58)
Sinus Bradycardia 5 8 1
Sinus Arrhythmia 1 0 0
Premature Ventricular Complexes 0 2 0
Premature Atrial Contractions 0 1 0
Repolarization Abnormalities 5/12 3/10 0/2 8/24 (33)
T Wave Changes 2 1 0
ST/STT Changes 3 2 0
Left Axis Deviation 0/12 1/10 0/2 1/24 (4)
One male and one female in this cohort were excluded due to no cardiac imaging records available.
3.4 Cerebrovascular complications, neurologic, and neuropsychiatric symptoms
White matter lesions were present in 25% (4/16) of participants who had brain imaging. One male in this cohort (patient 22) has a history of two cerebrovascular accidents, one transient ischemic attack (TIA) at age 52 years and one stroke at age 60 years. Brain MRI of patient 22 showed multifocal and adjacent gliosis within the left cerebral hemisphere, high left frontal and parietal lobes, as well as subcortical and periventricular deep white matter T2 hyperintensity without mass effect that is most pronounced within the parietal occipital region (Fig. 3B). The stroke impacted his eyesight and word-finding ability. Patient 22 only began ERT at the age of 57 years, and passed away at the age of 63 years due to post-operative complications of abdominal surgery. Another male (patient 15) and one female (patient 14) in this cohort have a history of TIAs while on ERT. They were not taking low dose aspirin at the time.
With regard to neuropathic manifestations, 77% (20/26) of patients reported peripheral neuropathic pain, manifesting as burning, tingling, or numbness, often triggered by temperature change or strenuous activity. Of the 17 participants for whom data on the brief pain inventory (BPI) survey was available, the majority reported worsening or inconsistent pain symptoms over the duration of treatment with ERT. Decreased quality of life has been associated with FD, in part due to the debilitating episodic pain crises and burden associated with having a chronic illness. Health-related quality of life was collected using the SF-36 Health survey. Bodily pain scores obtained from the SF-36 survey also showed no improvement over the course of ERT, though this was not significant (p = 0.19; Fig. 4C). For the majority of SF-36 respondents, pain worsened over time, which was similar to what was reported in the BPI survey. Notably, there was a significant decline in physical functioning (Est. = −1.40, 95% CI: −2.73 to −0.06, p = 0.040) for patients in this cohort. The remaining health dimensions did not show significant change over time (Fig. 4A-H).Fig. 4 SF-36 health-related quality of life component scores of patients on ERT. The SF-36 Health survey measures (A) physical function, (B) physical role, (C) pain, (D) general health perception, (E) vitality, (F) social function, (G) emotional role, (H) mental health. Scores ranged from 0 (worst) to 100 (best). Observed values (dots) are shown together with model-estimated trajectories for individual patients (colored lines) and the population (black lines).
Fig. 4
Of the adult patients in this cohort, seven reported depression (7/24; 29%). One patient reported a history of depression that correlated with pain symptoms. Another patient had a history of depression with no current pain symptoms based on responses in the BPI and SF-36 Health Survey. Of note, one patient in this cohort had a history of two suicide attempts. On the other hand, one patient's testimonial described noticeable improvement in anxiety, fatigue, and executive function (termed “Fabry fog”) since initiation of ERT.
3.5 Ophthalmologic findings
Corneal verticillata, or corneal whorling, is a unique clinical feature in patients of both sexes with FD and is often used as a diagnostic tool. It is a result of GL-3 deposits and has not been linked to vision loss. Corneal verticillata was noted in 58% (15/26) of individuals, at a higher prevalence in females than males in our cohort (Fig. 3C). Vessel tortuosity is also associated with FD and is exacerbated by renal complications [19,20]. Only one patient was found to have vessel tortuosity (patient 22); this individual had stage IV CKD.
3.6 Skin manifestations
Angiokeratomas are common in Fabry patients as they occur when GL-3 accumulates in dermal endothelial cells and lead to secondary ectasia. In our cohort, 69% (18/26) had angiokeratomas (Fig. 3D).
Hypohidrosis is also a common feature of FD and may be a predisposition to acroparesthesia. In this cohort, 62% (16/26) experienced hypohidrosis or anhidrosis. Of those with hypohidrosis, 89% experienced acroparesthesia. Lymphedema of the extremities, particularly in the feet, was present in 42% (11/26) of patients in this study.
3.7 Gastrointestinal involvement
Gastrointestinal symptoms are common among FD patients and can manifest as diarrhea, constipation, nausea, vomiting, incontinence, and abdominal pain. In our cohort, gastrointestinal symptoms were present in 62% (16/26) and the most common complaint was abdominal pain. Previous studies have shown significantly improved gastrointestinal pain with ERT [21]. This was also reported by the patients in our study in the patient health survey. The majority of those on ERT reported either improved or stable abdominal pain and frequency of diarrhea based on their survey responses at last visit compared to earlier survey responses. Specifically, six patients reported improvement in abdominal pain and four reported that symptoms were stable (N = 20). Eleven reported improvement in diarrhea and one reported that symptoms were stable (N = 20). One patient's testimonial described a frequency of diarrhea multiple times daily prior to ERT that has now significantly improved since ERT initiation. He also reported noticeable return of symptoms during the time of the ERT shortage. On the other hand, worsening or recent onset of abdominal pain (3/20) and diarrhea (2/20) over the course of ERT treatment were also reported by participants in this study.
3.8 Auditory and vestibular involvement
Sensorineural hearing loss, predominantly in the high frequencies, is a common manifestation of FD and correlates with neuropathic and vascular damage [22]. In our cohort, 35% (9/26) of patients reported a history of hearing loss. The majority of those who underwent audiology assessments were found to have bilateral mild to moderate sensorineural hearing loss. Additionally, 65% (17/26) of patients reported tinnitus. Some patients do not use hearing aids due to amplification by the presence of tinnitus. Vertigo was reported in 48% of our cohort in those who recorded their response in the health survey (10/21).
3.9 Pulmonary involvement
Respiratory involvement in FD typically includes obstructive lung disease, however patients may develop interstitial restrictive lung disease. In our cohort, 50% (6/12) of those who have had spirometry testing were found to have reduced lung capacity. Of those, four had obstructive lung disease, one had possible obstructive lung disease, and one had restrictive lung disease.
3.10 Endocrine and other clinical manifestations
There is limited data in the literature that describes endocrine system involvement in FD. In our cohort, patients reported type II diabetes, hypothyroidism, hyperthyroidism, hyperparathyroidism, and vitamin D deficiency (Supplemental Table 2). Patient 8 has a history of hyperthyroidism secondary to Graves' disease, and a past history of a 1.9 mm benign thyroid nodule. Patient 21 has type II diabetes and hypothyroidism. Patient 22 has acquired hypothyroidism and hyperparathyroidism secondary to renal disease. One of the pediatric participants (patient 9) was diagnosed with growth hormone deficiency and a small pituitary gland. Not all participants in this study have had an endocrine evaluation, so this study may have missed those with subclinical endocrine dysfunction.
Low Bone Mineral Density (BMD) has been noted more recently to be a common feature of FD [23]. Impaired renal function leads to vitamin D deficiency and may be one of the contributing factors to reduced BMD in FD patients. Fifty percent of patients in this cohort who had DXA scans (7/14) were found to have low BMD. Of those, 57% (4/7) had osteopenia (T score < 1.5) and 43% (3/7) had osteoporosis (T score < 2.5). Notably, two of the seven patients (29%) had early-onset low BMD at age 32 years (patient 6) and age 36 years (patient 13). There were no prior DXA scans to measure the rate of bone loss in these two young adult males. Additionally, patient 13 spends up to 40 h per week in a sensory deprivation floatation tank, which may have contributed to bone loss (described below in section 3.13. Supplemental Treatments). Only one patient endured an ankle fracture at the age of 65 years due to a mechanical fall; none of the remaining participants reported bone fractures. Of those with low BMD, four patients had concurrent vitamin D deficiency; of those four, one had secondary hyperparathyroidism due to renal disease. All patients in this cohort were prescribed calcium and vitamin D supplementation. Those with osteoporosis were managed by an endocrinologist and prescribed bisphosphonates.
Hypertension is also a common adverse event in adult FD patients. Cardiac, renal and cerebrovascular complications increase the risk of uncontrolled hypertension in individuals with FD [24]. Hypertension was present in 32% (8/25) of patients in our cohort, the majority but not all of these individuals had some form of clinically evident renal involvement. Patients were managed on ACEIs, ARBs, beta-blockers, and diuretics.
3.11 Novel GLA variant and genotype-phenotype correlations
Our study describes 16 unique variants in the 26 total participants, including one novel variant and one atypical ‘later-onset’ cardiac variant (Table 3; Fig. 5). The novel variant c.1226_1231delCCACAG (p.P409_G411delinsR), an in-frame deletion located within a hot-spot, was identified in a 63-year-old male (patient 22) who was diagnosed later in adulthood at 57 years old following a renal biopsy that revealed stage IV chronic kidney disease. He is discussed earlier with renal, pulmonary, and cardiac involvement and a history of two cerebrovascular accidents. Additional FD-related symptoms in patient 22 include angiokeratomas, corneal verticillata, gastrointestinal involvement, lymphedema, hypohidrosis, acroparesthesia, Raynaud's syndrome, hearing loss, tinnitus, and low bone mineral density (Supplemental Table 2). Cardiac work-up for chest pain revealed a 95% blockage of the left anterior descending coronary artery requiring stent placement, which resolved except for occasional reports of arrhythmia. Endocrine dysfunction in this patient includes acquired hypothyroidism and hyperparathyroidism secondary to renal disease. Patient 22 was particularly significant among our participants because of the history of one TIA and one stroke; brain MRI revealed significant gliosis and nonspecific white matter signal changes (Fig. 3B). Patient 22 is now deceased due to post-operative complications of abdominal surgery for abdominal abscess and intra-abdominal infection.Table 3 GLA variant details.
Table 3Fam. No. Patient No. Ethnicity Nucleotide Change Protein Change Position Type Phenotype
1 1,2,3,4,5,6 Hispanic c.983G > T G328V Exon 6 Missense Classic
2 7,8 Caucasian c.1250 T > G L417R Exon 7 Missense Classic
3 9,10,11 Caucasian c.132G > T W44C Exon 1 Missense Classic
4 12 Caucasian c.568delG A190PfsX2 Exon 4 Small Deletion Classic
5 13,14 Caucasian c.1041_1042insG A348GfsX27 Exon 7 Small Insertion Classic
6 15 Hispanic c.706 T > C W236R Exon 5 Missense Classic
7 16 Caucasian c.680G > A R227Q Exon 5 Missense Classic
8 17 Caucasian c.816C > A N272K Exon 6 Missense Classic
9 18 Caucasian c.730G > A D244N Exon 5 Missense Classic/ Later Onset
10 19 Asian c.639 + 919G > A IVS4 + 919G > A Intron 4 Splicing Later Onset
11 20 Asian c.427G > C A143P Exon 3 Missense Classic
12 21 Hispanic c.639 + 4A > T IVS4 + 4A > T Intron 4 Splicing Classic
13 22 Caucasian c.1226_1231delCCACAG P409_G411delinsR Exon 7 Small Deletion Classic
14 23 Hispanic c.1088G > A R363H Exon 7 Missense Later Onset
15 24,25 Hispanic c.1072_1074delGAG E358del Exon 7 Small Deletion Classic
16 26 Caucasian c.1246C > T Q416X Exon 7 Nonsense Classic
Novel variant is bolded.
Fig. 5 Unique variants identified in the GLA gene in this cohort. A schematic representation of GLA showing the position of all variants identified, with novel variant in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Though the rest of our cohort harbors variants that have been previously reported in the literature, we present novel clinical data for certain recurrent variants. Family 3 (patients 9, 10, 11) harbor the W44C variant, a previously reported variant in a Chinese family of 12 affected members, all of whom experienced paroxysmal pain of limb extremities [25]. Interestingly, our family is of European/mixed descent, with no known Asian ancestry, and present with multi-organ involvement in addition to peripheral pain. We report one patient with the c.639 + 919G > A ‘later-onset’ cardiac variant, who, in addition to mild left ventricular hypertrophy, experiences other FD-related symptoms including angiokeratomas, gastrointestinal issues, pulmonary involvement, and hypohidrosis with heat intolerance. His mother, who also harbors the familial variant, has a history of strokes and presented at age 76 years with hypertrophic cardiomyopathy. Another example of potential genotype-phenotype correlation in our cohort is Family 1; all three females who harbor the G328V variant have audiologic involvement.
3.12 Pediatric patients
There were two male pediatric participants in this study (patient 7 and patient 9). Both patients were diagnosed before the age of five and treated with ERT at or before the onset of symptoms. Patient 9 is a seven year-old male with the W44C variant. This patient was diagnosed with FD at age 3 and began ERT at age 7 years soon after an evaluation for reactive hyperemia index of 0.94 (Reference RHI value >1.68) using an EndoPAT (Itamar Medical) device performed on a research basis, which was consistent with low peripheral vascular endothelial function. He lacked ‘classic’ FD symptoms at the time of the procedure, but ERT was recommended due to concern for deteriorating endothelial function. Initial urine GL-3 level of 569 μg/mmol creatinine resolved during the course of this study while on ERT. He is developmentally normal, however he has a past medical history remarkable for growth hormone deficiency and a small pituitary gland and has been managed on somatropin since 3 years of age.
Patient 7 from Family 2 first presented with acroparesthesia and hypohidrosis at 4 years of age and began ERT by 6 years of age. Prior to initiation of ERT, urine GL-3 level was at 370 μg/mmol and was cleared to 0 μg/mmol in less than two years on ERT. Notably, spirometry testing revealed mild obstructive airway disease, though it has not yet affected routine activity. Patient 7 and his affected mother (patient 8) present with variable symptoms of FD as shown in Supplemental Table 2.
3.13 Supplemental treatments
Several individuals in this cohort sought alternative sources for pain management. Effective pain management could impact the psychological consequences of FD, as there is a high rate of depression that is often linked to the effects of long-term pain. Patient 13 had a five-year history of frequent, severe pain crises, including in response to ERT infusion, which has led the patient to seek alternative pain management strategies in conjunction with ERT. This includes floatation in a sensory deprivation tank used for the treatment of pain and anxiety by limiting stimulation from gravity, sound and light while immersed in water baths saturated with Epsom salts to allow for buoyancy in a dark and soundless room. Patient 15 and Patient 16 also report benefit from this float tank. Important to note, extended time in the float tank may have had an adverse effect on patient 13's bone density, as he reports spending up to 40 h per week in the float tank. There was a variety of medications used in this cohort for the management of neuropathic pain. Patient 13 and 15 are prescribed cannabidiol (CBD). Patient 15 and patient 26 use non-prescription topical and oral CBD as needed during pain crises. Use of tetrahydrocannabinol (THC) was also reported. Six patients are prescribed opioid narcotic analgesics, eight patients are managed with over-the-counter analgesics, two are on antidepressants, and one on an anticonvulsant (carbamazepine).
4 Discussion
The benefit, safety, and risk of ERT have been previously investigated, though the long-term impact continues to be studied [[26], [27], [28], [29], [30]]. ERT is recommended in order to curtail disease progression or when there is already evidence of disease progression and vital organ damage. In this study, 20 of the 26 participants and all but one male (patient 15) were consistently on ERT.
All participants receiving ERT however, demonstrated a different response rate of disease progression. While ERT was shown to be effective in clearing plasma GL-3 and, in many patients maintaining serum creatinine and eGFR within normal limits in this cohort, we identified that many patients continued to have impairment of their renal function and health-related quality of life despite standard of care management. For patients with chronic kidney disease from FD, standard of care did not vary when compared to that of other etiologies. The standard management includes the evaluation for possible reversible causes of renal disease (i.e. dehydration, reduced renal perfusion, obstruction, nephrotoxicity), management of blood pressure to a target ≤130/80, maximally tolerated dose of renin-angiotensin-aldosterone system inhibitors (i.e. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone receptor antagonist), smoking cessation, dietary interventions (particularly reduced sodium and protein restricted diet), treatment of metabolic acidosis, management of anemia of CKD with erythropoiesis-stimulating agents to target Hg 10–11.5 g/dL, management of mineral and bone disorders as per 2012 KDIGO guidelines, initiation of renal replacement therapy in individuals with uremic symptoms and eGFR 5–15 mL/min/1.73 m2 or in those with eGFR <5 mL/min/1.73 m2, and referral for kidney transplant evaluation in those with eGFR 20–25 mL/min/1.73 m2.
The late initiation of ERT may have contributed to those with renal disease progression, as shown previously [31]. Furthermore, nearly all of the adult patients in this cohort had major organ involvement at baseline prior to initiation of ERT. Typically, renal function in FD gradually deteriorates to end-stage renal disease in the third to fifth decade in males [32,33]. Only one patient progressed to severe CKD requiring hemodialysis, therefore, compared to the natural history data, it is possible that ERT contributed to the prevention of renal failure by slowing disease progression. Though vital organ damage is not typically seen in pediatric patients (some cases have been documented), studies have shown evidence for the benefit of the early initiation of ERT in potentially reversing renal damage [10,29]. Patient 12 initiated ERT at age 11 years, however, developed renal involvement by age 24 years [34]. Therefore, we believe earlier initiation of ERT is required to limit deterioration of the major organs. Our two pediatric participants initiated ERT at ages 6 and 7 years, respectively, and one reported significant improvement of pain symptoms.
Genotype-phenotype association is difficult to establish in FD for many reasons. Most GLA variants are unique (private mutations) and there are few family studies. Even within families, the phenotype may vary depending on the sex, age of onset, and other genetic and epigenetic factors. There are several reports in the literature of ‘later-onset’ isolated hypertrophic cardiomyopathy in individuals with the recurrent c.639 + 919G > A variant [6,[35], [36], [37]]. While this is often considered an atypical later-onset cardiac disease variant, patient 19 presented with several other ‘classic’ FD symptoms. Studies have explored the use of Amiloride as a potential treatment for FD by modulating alternative splicing in the context of the c.639 + 919G > A variant [35]. In our cohort we identified one novel variant c.1226_1231delCCACAG (p.P409_G411delinsR) in patient 22 with a severe, ‘classic’ FD phenotype [38]. This variant is absent in The Genome Aggregation Database (gnomAD), is located in a mutational hot-spot, and thus predicted to be pathogenic [39]. Two of the 26 individuals in this cohort harbor variants amenable to migalastat, an oral chaperone therapy, but chose not to switch from ERT. Long-term outcome studies for FD patients who have switched to migalastat will be of interest for future studies.
In this cohort, nearly one third of adult patients reported depression and anxiety. Based on the patient-reported SF-36 Health Survey and Brief Pain Inventory survey, there is no significant improvement in health-related quality of life over the course of ERT. Depression, sleep apnea, and anxiety disorders are linked to both pathology and the effects of long-term pain [40]. It has been suggested that psychological counseling can have an impact on reversing or reducing effects, an important consideration to optimize patient care as needed [41]. Importantly, studies have proposed pain as an indicator for the need to initiate ERT [42]. The majority of our cohort experienced Fabry crises or acroparasthesia. There are few data on nervous system involvement of FD in women [43]. Some studies have shown hippocampal volume loss and white matter lesions in female patients with FD [44]. While we showed white matter lesions in three females and one male in this cohort, we did not include brain imaging, electrical conduction, or quantitative sensory testing for every participant in this study. Further studies assessing the outcomes of neuropathy and CNS findings with ERT are warranted.
Cardiac manifestation can be the primary and only symptom of FD in some patients and can present as early as childhood. Our study showed a higher prevalence of cardiac manifestations in both the males and females compared to previous studies [34,[45], [46], [47]]. Cardiac involvement was observed in 83% of male patients, including one pediatric male patient with sinus bradycardia, and 75% of female patients in this cohort. Left ventricular hypertrophy (LVH) was seen in 70% of males and 42% of females. This data provides supporting evidence that female patients, even those with no obvious signs or symptoms of FD, should be monitored closely for evidence of cardiac involvement and be considered for ERT. The standard of care for patients with hypertrophic cardiomyopathy due to FD did not vary when compared to the management of other etiologies and followed AHA/ACC guidelines.
We highlight the variable endocrine dysfunction and low bone mineral density in this cohort, including several with osteoporosis, which provides further evidence for the inclusion of adequate endocrine work-up in the ongoing management of all patients with FD [48].
There is a need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. Furthermore, there is a need for more and improved biomarkers to monitor disease progression. Early identification of endothelial function or other pathologies can provide insight into when ERT should be initiated in order to slow the advancement of adverse effects [49]. Additional tools and biomarkers for measuring endothelial function and other indicators of future pathologies, such as proteomics, are needed to help prevent the deterioration of vital organs [[50], [51], [52]]. Newborn screening for FD has been implemented in several states and will lead to earlier detection. Early detection is important to proactively monitor for complications of the disease, initiate ERT, and prevent co-morbidity progression [[53], [54], [55], [56]].
Since most of our participants were on ERT, we could not make comparisons between long-term outcomes of those on ERT compared to those not on ERT. Also, with the small sample size, we could not compare multiple age-matched individuals or genotype-specific individuals other than our reported families. Another limitation to this study is the long-term analysis of plasma-GL-3 over time rather than plasma Lyso-GL-3, due to the fact that we had more data points for plasma-GL-3. Plasma Lyso-GL-3 has been recently determined to be a more accurate biomarker that correlates more closely with disease phenotype in male and in female patients, with higher levels associated with increased clinical manifestation [57].
5 Conclusion
The variability of symptoms and disease progression in patients with FD, even within the same family, complicates the discernment of a genotype-phenotype correlation. ERT has been available for the treatment of FD since 2001 and has shown improved outcomes, especially when started prior to organ damage, and may reverse fatal disease progression. In this cohort, agalsidase beta infusions have been effective in clearing GL-3 levels. However, renal involvement and health-related quality of life has continued to progress in adult patients diagnosed late despite ERT infusions.
Eight females were on ERT in this study and demonstrated significant symptoms of FD, and in some cases the disease progressed to a similar degree as in the males. This study further demonstrates that females may develop significant FD symptoms and should be managed appropriately and treated as more than carriers.
Documentation of novel variants can contribute to future genotype-phenotype associations between the severity and progression of FD. Genotype-phenotype correlations and long-term outcome data on ERT and/or other FD treatments are needed, especially now that the newborn screening has led to an increase in diagnoses and earlier detection in individuals with FD.
Authors' contributions
V.K. conceived the study and supervised the overall direction and implementation of the study. All authors contributed to acquisition of data. V.K., M.D.C., D.R., D.T., Z.A.D., and G.L. analyzed the data. D.R. performed statistical analyses. M.D.C. and E.C. drafted the manuscript with input from all authors. All authors provided critical feedback and approved the final manuscript for publication.
Funding
The Fabry registry was funded by Sanofi Genzyme. This funding source had no role in data collection or analysis.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Appendix A Supplementary data
Supplementary material
Image 1
Acknowledgements
We thank the patients and their health care providers for their contribution to the study. The Fabry registry was funded by Sanofi Genzyme. This work was supported by CTSI grant UL1TR000124UCLA (Clinical and Translational Science Institute), Los Angeles, CA, USA.
Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymgmr.2020.100700. | 1 MG/KG, QOW | DrugDosageText | CC BY-NC-ND | 33437642 | 18,784,055 | 2021-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Atrial flutter'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID, CARBOPROST TROMETHAMINE, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Foetal exposure during pregnancy'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID, CARBOPROST TROMETHAMINE, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Low birth weight baby'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID, CARBOPROST TROMETHAMINE, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Maternal exposure during pregnancy'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CARBOPROST TROMETHAMINE, CYANOCOBALAMIN\FOLIC ACID\IRON, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,807,528 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pre-eclampsia'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CARBOPROST TROMETHAMINE, CYANOCOBALAMIN\FOLIC ACID\IRON, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,807,528 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Premature baby'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID, CARBOPROST TROMETHAMINE, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Premature delivery'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CARBOPROST TROMETHAMINE, CYANOCOBALAMIN\FOLIC ACID\IRON, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,807,528 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Tachycardia foetal'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID, CARBOPROST TROMETHAMINE, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Therapeutic response decreased'. | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | ACETAMINOPHEN\BUTALBITAL\CAFFEINE, CARBOPROST TROMETHAMINE, CYANOCOBALAMIN\FOLIC ACID\IRON, DEXAMETHASONE, DIGOXIN, ELTROMBOPAG OLAMINE, HYDROMORPHONE, MAGNESIUM, MAGNESIUM OXIDE, METHYLERGONOVINE, PREDNISONE, RITUXIMAB, SODIUM CHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY-NC-ND | 33437657 | 18,807,528 | 2021-01 |
What was the administration route of drug 'CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the administration route of drug 'CARBOPROST TROMETHAMINE'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the administration route of drug 'CYANOCOBALAMIN\FOLIC ACID\IRON'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33437657 | 18,807,528 | 2021-01 |
What was the administration route of drug 'METHYLERGONOVINE'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the administration route of drug 'TRANEXAMIC ACID'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | Transplacental | DrugAdministrationRoute | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the dosage of drug 'CALCIUM ASCORBATE\CALCIUM THREONATE\CYANOCOBALAMIN\FOLIC ACID\IRON\SUCCINIC ACID'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | MOTHER DOSE: UNK (Q10AM) | DrugDosageText | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the dosage of drug 'CARBOPROST TROMETHAMINE'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | MATERNAL DOSE:UNK | DrugDosageText | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the dosage of drug 'CYANOCOBALAMIN\FOLIC ACID\IRON'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | UNK UNK, QD | DrugDosageText | CC BY-NC-ND | 33437657 | 18,807,528 | 2021-01 |
What was the dosage of drug 'DEXAMETHASONE'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | MATERNAL DOSE:UNK | DrugDosageText | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the dosage of drug 'METHYLERGONOVINE'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | MATERNAL DOSE:UNK | DrugDosageText | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the dosage of drug 'TRANEXAMIC ACID'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | MATERNAL DOSE:UNK | DrugDosageText | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
What was the outcome of reaction 'Tachycardia foetal'? | Eltrombopag use for refractory immune thrombocytopenia in pregnancy: A case report.
Immune thrombocytopenic purpura (ITP) is a rare autoimmune disorder that involves platelet destruction in the spleen. Eltrombopag (Promacta®), a thrombopoietin agonist, has been used in non-pregnant patients to manage ITP, but few cases of its use in pregnancy have been reported.
We present a case of a pregnant patient at 26 weeks of gestation with severe refractory ITP. After first-line therapies failed, the patient was treated with the drug eltrombopag. The patient had no response to initial therapy, and the fetus developed supraventricular tachycardia (SVT). This resolved with maternal digoxin but the patient elected to stop the eltrombopag. The patient refused further experimental and second-line treatments, and after a multidisciplinary meeting a decision was made to deliver by cesarean section at 30 weeks of gestation due to severe refractory ITP and allow other therapies to be tried postpartum. Preeclampsia and neonatal atrial flutter were encountered in the postpartum period but both mother and baby had good outcomes.
Refractory ITP in pregnancy is not well studied. Eltrombobag could have maternal and fetal side-effects but a multidisciplinary approach to management leads to favorable maternal and fetal outcomes.
1 Introduction
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder that involves anti-platelet glycoprotein antibodies that stimulate platelet destruction in the spleen. It is a rare complication in pregnancy that accounts for about 3–4% of cases of thrombocytopenia in pregnancy [1]. Maternal complications of ITP in pregnancy include mild to severe bleeding [2,3].
Current consensus supports the use of corticosteroids and intravenous immune globulin (IVIG) for ITP in pregnancy [4]. Anti-D immune globulin and platelet transfusions are also therapeutic options for patients with severe ITP. However, platelet transfusions have limited utility in ITP due to rapid platelet destruction. Splenectomies are about 90% effective in refractory ITP [5]; however, in pregnancy they may have associated maternal and fetal risks. The two most commonly used medications in the non-pregnant population are rituximab and thrombopoietin receptor agonists (eltrombopag and romiplostim), but neither is well studied in this population.
Eltrombopag (Promacta®) is a thrombopoietin receptor agonist that interacts with the transmembrane domain of the thrombopoietin receptor, leading to increased platelet production [6]. Reported side-effects include headaches, upper respiratory tract infections, nasopharyngitis, fatigue, back and bone pain, anemia, cataracts, pneumonia, liver enzyme and bilirubin elevation, and thrombotic events [[7], [8], [9]]. Based on limited data in pregnant women, eltrombopag is designated pregnancy category C [10]. We present a report of maternal eltrombopag use with possible side-effects of preeclampsia and fetal supraventricular tachycardia (SVT). We also offer a perspective on the multidisciplinary approach to ITP in pregnancy.
2 Case Presentation
A 35-year-old patient, G2P1001, with a history of ITP, presented at 26 weeks of gestation to a local emergency room with unremitting epistaxis and was found to have platelets of 12 × 109/L and hemoglobin of 6.2 g/dL. She received blood products and intravenous dexamethasone, and was transferred to a tertiary care center. Her history was significant for unprovoked nosebleeds throughout childhood and postpartum hemorrhage in a prior pregnancy requiring blood transfusion. She reported no significant family history of bleeding disorders. A hematologist had seen her two weeks earlier, when her platelets were 77 × 109/L and hemoglobin 6.3 g/dL, and recommended B12 injections and prednisone but the patient did not take them due to concern about potential fetal side-effects.
On hospital day 2, the patient's platelet count was 9 × 109/L and epistaxis stopped with a nasal tamponade device. A low-lying placenta was noted on ultrasound with an estimated fetal weight in the 39th percentile. A multidisciplinary approach was taken, with the involvement of maternal fetal medicine (MFM), obstetrics, anesthesia, neonatology and hematology, in order to work up the case and plan management. Testing included a bone marrow biopsy, preeclampsia, anemia and infectious workups. The workup still led to the diagnosis of ITP by exclusion.
The patient received multiple rounds of IVIG and high-dose steroids, along with platelet transfusions, but with minimal response. Platelets remained less than 10 × 109/L, and epistaxis continued. The patient was counseled by MFM and hematology on the options of splenectomy and eltrombopag. The patient elected to proceed with eltrombopag; she received 25 mg daily for 5 days with minimal response, with platelets less than 10 × 109/L. The eltrombopag dose was increased to 50 mg daily. On the second day of increased dose therapy, at 28 weeks of gestation, fetal SVT was noted, with a fetal heart rate of 210–220 beats per minute. Pediatric cardiology was consulted and a fetal echocardiogram showed no structural malformations or hydrops fetalis. Maternal oral digoxin was prescribed and the fetal heart rate returned to normal (as shown in Fig. 1).Fig. 1 Fetal heart tracing with the transition point from fetal SVT to normal fetal heart rate with a baseline of 160 beats per minute.
Fig. 1
The patient was counseled by MFM and hematology but she elected to discontinue the eltrombopag. She was concerned about the potential effects on the fetus, even with no further fetal SVT while taking the digoxin and with no confirmation that the SVT was from the eltrombopag. She also did not wish to undergo splenectomy or consider rituximab during pregnancy due to possible risks to the fetus.
A multidisciplinary meeting was held and options were reviewed. Due to the patient having declined other treatment options and concerns for serious adverse events, such as intracranial or vaginal hemorrhage, she was offered elective preterm delivery or expectant management. The patient decided on elective preterm delivery to avoid further adverse outcomes and to allow for further treatment postpartum. She understood the risks of preterm delivery and elected to proceed. The decision was thus made for delivery in a planned setting due to severe refractory ITP.
Cesarean delivery, due to low-lying placenta, was performed at 30 weeks of gestation. The patient's platelets were 11 × 109/L. Surgery was performed under general anesthesia and she received 4 superpacks of platelets, 3 units packed red blood cells, 2 units fresh frozen plasma, 1 L albumin and 120 mL cell saver. She received carboprost tromethamine, methylergonovine and tranexamic acid intraoperatively due to uterine atony. The quantitative blood loss was 1500 mL. A live vertex female neonate was born with Apgar scores of 4 and 7 at 1 and 5 min respectively, with weight 1370 g (44th percentile). The cord blood gases did not show acidosis. The neonate was transferred to the neonatal intensive care unit (NICU) on a continuous positive airway pressure device. The patient was extubated after surgery and transferred to the recovery room.
On the first postoperative day, maternal platelets were 10 × 109/L. The patient was started on rituximab, eltrombopag and intravenous steroids. She developed mild-range blood pressures (defined as systolic 140–159 and diastolic 90–109) and severe, unrelenting headache,and was diagnosed with preeclampsia with severe features. She received magnesium sulfate for 24 h for seizure prophylaxis. The patient was discharged home on post-operative day 10 with a platelet count 18 × 109/L after 2 cycles of rituximab. The patient was advised not to breastfeed while using eltrombopag (rat pup exposure via lactation was noted in animal studies) [11]. One month postoperatively her platelets were 45 × 109/L and at two months 78 × 109/L.
On day of life 1, the neonate's platelets were 274 × 109/L. At 2.5 h of life, atrial flutter was noted with 2:1 conduction, as shown in Fig. 2. Adenosine was given with continued atrial flutter and decreased atrioventricular conduction, thus the neonate was cardioverted to normal sinus rhythm. Echocardiography was performed and no structural abnormalities were noted. The neonate remained in sinus rhythm until day of life 10, when atrial flutter returned and cardioversion was performed again. The neonate was started on sotalol and switched to propranolol prior to discharge. The neonate was discharged on day of life 71, when platelets were 511 × 109/L.Fig. 2 Neonatal electrocardiogram from telemetry strip demonstrating atrial flutter on day of life 1. The second line demonstrates atrial flutter with 2:1 atrioventricular conduction and the third line shows a higher degree of block.
Fig. 2
3 Discussion
Refractory ITP is diagnosed when first-line therapies fail. Only splenectomy, romiplostim, rituximab and eltrombopag have been considered and studied in pregnancy, with limited data available. In animal studies of eltrombopag, there was evidence of embryo lethality and fetal growth restriction at maternally toxic doses [12]. Case reports of maternal use during pregnancy have demonstrated development of antepartum preeclampsia, growth restriction and preterm birth [[13], [14], [15]]. There are no reports of fetal or maternal SVT in patients treated with eltrombopag or romiplostim.
In our case, eltrombopag was ineffective during pregnancy. Postpartum combination therapy helped achieve response, thus we cannot make conclusions that the eltrombopag alone was beneficial. In our case, the patient declined treatment options and thus patient counseling and planning by multiple providers helped to achieve good maternal and fetal outcomes.
Fetal SVT and postpartum preeclampsia were noted during treatment with eltrombopag. The timing of the events and lack of cardiac structural abnormalities originally led us to believe the fetal SVT might be a side-effect of eltrombopag. Fetal SVT, including atrial flutter, is an uncommon arrhythmia, sometimes associated with fetal accessory pathways, structural anomalies, or hereditary channelopathies [16]. After birth, the neonate had sustained atrial flutter even without further exposure to eltrombopag. In retrospect and upon discussion with the neonatologists, we believe the drug could have triggered the arrhythmia in a fetus with an underlying predisposition, but the sustained atrial flutter was likely unrelated to the medication. Decision for delivery in patients with refractory ITP is crucial and usually needs a multidisciplinary approach, especially when preterm delivery is being considered.
4 Conclusion
This is the fourth known report of eltrombopag use in pregnancy. In the other cases, it had a positive impact with minimal side-effects on pregnant women with refractory ITP [10,[13], [14], [15]]. Our case is the first report known to us of minimal response and fetal SVT. Although we cannot prove causation, this is the first report in the literature and we believe it is important for others to be aware of when considering eltrombopag use in pregnancy. We again noted the finding of preeclampsia in a patient treated with eltrombopag. More research needs to be done to determine association and causation with these side-effects, and efficacy of the drug. We still believe this medication has potential for benefit in pregnant women. We agree that we would trial the medication in the future with close observation for side-effects in both mother and fetus, and updated counseling on risks and benefits. In addition, further research would be extremely valuable in assessing the use and risks of this drug in pregnancy. Our case highlights the importance of a multidisciplinary approach in the management of refractory ITP to achieve optimal outcomes.
Contributors
All authors contributed equally to the preparation of this case report.
Conflict of Interest
Dr. Friedman reports personal fees from AstraZeneca, which is outside the scope of the submitted work. The other authors declare that they have no conflict of interest.
Funding
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Patient Consent
The patient gave signed consent and authorized a case report to be written on behalf of advancing medical knowledge. She understands that the case report is fully de-identified. She was given the opportunity to write a section for the paper from her perspective but declined.
Provenance and Peer Review
This case report was peer reviewed. | Recovered | ReactionOutcome | CC BY-NC-ND | 33437657 | 18,795,659 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Interstitial lung disease'. | Early-onset interstitial pneumonitis in a patient with advanced non-small cell lung cancer treated with crizotinib and osimertinib.
Both crizotinib and osimertinib have been reported to have an adverse effect of interstitial pneumonitis in the treatment of non-small cell lung cancer (NSCLC). Here, we report the case of a 60-year-old male patient with advanced NSCLC resistant to osimertinib. Crizotinib was administered in combination with osimertinib due to elevated mesenchymal epithelial transition (MET) copy number amplification. However, early-onset interstitial pneumonitis occurred within two days.
Introduction
Mesenchymal epithelial transition (MET) amplification is one of the mechanisms of epidermal growth‐factor receptor tyrosine kinase inhibitor (EGFR‐TKI) acquired resistance. As a MET inhibitor, the efficiency of crizotinib in the treatment of MET amplification has been confirmed. Most patients tolerate crizontinib and osimertinib well. Interstitial pneumonitis is a rare and serious adverse effect with tyrosine kinase inhibitor (TKI) treatment, but the mortality rate is high once it occurs. The currently reported risk factors for EGFR‐TKI‐related interstitial lung disease (ILD) include age, being male, a previous history of interstitial lung disease, and complicated heart disease. A small sample of retrospective research data reported that the combined use of the two drugs did not increase the incidence of side effects.
1
Here, we report a case of early‐onset interstitial pneumonia following the combined use of crizontinib and osimertinib.
Case report
In May 2017, a 60‐year‐old male patient, who presented with a cough, was admitted to our hospital. His chest computed tomography (CT) scan in May 2017 had revealed an opacity in the left lung. CT‐guided lung biopsy and PET/CT results confirmed a diagnosis of adenocarcinoma with multiple bone metastases. This patient had no history of smoking or pulmonary disease.
He subsequently received three cycles of chemotherapy with pemetrexed plus carboplatin, with a partial response. Next‐generation sequencing (NGS) of tumor tissue showed an EGFR 19del mutation (mutant allele frequency [MAF], 47.2%), MET amplification (copy number 2.1), and TP53 (MAF, 36.5%) mutation. Gefitinib (250 mg/day) treatment was initiated in August 2017. Disease progression was documented until March 2019. NGS of plasma ctDNA revealed an EGFR T790M mutation, and osimertinib (80 mg/day) was administered. The patient did not receive any radiation therapy to the chest. He subsequently visited our hospital with a complaint of dyspnea in April 2020. Chest CT showed lung cancer which had invaded the carina, causing left lung atelectasis (Fig 1a). Bronchoscopic biopsy revealed adenocarcinoma. NGS of tumor tissue showed EGFR 19del mutation (MAF, 79.3%), met amplification (copy number 6.6), and TP53 mutation (MAF, 53.5%) without EGFR T790M mutation. Interventional rigid bronchoscopy was performed to install covered self‐expandable metallic stents and relieve dyspnea in the patient. Postoperative chest radiography showed left lung re‐expansion. Crizotinib (250 mg/twice per day) was initiated. Two days later, he presented with mild dyspnea. Chest CT showed mild bilateral pleural effusion and mild ground‐glass opacities (GGOs) in the right lung (Fig 1b). Seven days later, his dyspnea progressed with respiratory failure. Acute pulmonary embolism was excluded by CT pulmonary angiogram but moderate bilateral pleural effusion, diffuse severe bilateral GGO infiltration and mild consolidation in both lungs were found (Fig 1c). The patient had no peripheral edema, no crackles in the lungs, brain natriuretic peptide was mildly elevated, and echocardiography indicated a normal left ventricular ejection fraction. The bronchoalveolar lavage fluid did not reveal pathogenic microorganisms (bacterial and fungal culture negative, acid‐fast bacilli negative, pneumocystis jiroveci negative), and transbronchial biopsy of the right lung showed organizing pneumonia (Fig 2). The tumor shrank significantly after seven days of treatment (Fig 3).
Figure 1 (a) CT scan shows left lung atelectasis. (b). CT scan shows multiple mild diffuse ground‐glass opacity (GGO) in the right lung after two days of crizotinib and osimertinib administration. (c) CT scan shows multiple severe diffuse GGO in both lungs after seven days of crizotinib and osimertinib treatment. (d) CT scan taken six weeks later shows diffuse GGO absorbed after glucocorticoid therapy.
Figure 2 Organizing pneumonia in transbronchial lung biopsy of the right lung (HE, HPF).
Figure 3 The tumor shrank significantly after seven days combination therapy of crizotinib and osimertinib (red arrows a, before; b, after).
Life‐threatening TKI‐induced interstitial pneumonitis was considered. Crizotinib and osimertinib were stopped immediately. Glucocorticoids were initiated at 80 mg, and the dose was tapered over two months. After the symptoms had been relieved, pemetrexed and carboplatin chemotherapy was administered. Six weeks later, CT scan showed a remarkably absorbed diffuse GGO (Fig 1d). The patient in this study is currently under follow‐up.
Discussion
Elevated MET copy number amplification in this patient may be a resistance mechanism to EGFR‐TKI. Combination therapy has been reported to have a better overall response rate (ORR), progression‐free survival (PFS) and overall survival (OS) in patients than monotherapy,
1
,
2
although there have been some successful reports of crizotinib monotherapy in EGFR‐mutant NSCLC that acquired MET amplification.
In this study, we report a case of early‐onset life‐threatening drug related interstitial pneumonitis caused by the addition of crizotinib to osimertinib. Interstitial pneumonitis occurred within two days which was confirmed by CT scan. The incidence of interstitial pneumonia caused by EGFR‐TKI drugs has been previously reported to be 1.1%–1.6%, and the incidence of cases above grade 3 was 0.49%–0.9%.
3
,
4
However, this is significantly higher in the Japanese population. The mechanism behind the pulmonary toxicity of these drugs is unclear. EGFR is expressed in type II alveolar epithelial cells and is involved in alveolar wall repair. EGFR‐TKIs may interfere with the alveolar repair mechanism. In previous reports, the onset time of interstitial lung disease (ILD) with erlotinib ranged from 4–47 days, while that of osimertinib ranged from 17–230 days. The median onset of ILD from the initiation of crizotinib therapy was 23 days (range: 3–763 days).
5
The EGFR and ALK dual inhibitor brigatinib reportedly induced similar early‐onset drug related interstitial pneumonitis.
6
Supplemental oxygen and systemic glucocorticoids are usually needed. Rechallenge of TKIs after acute lung injury is controversial. Clinicians should therefore be cautious of the potential side effects, particularly early onset interstitial pneumonitis, when using two TKI drugs.
Disclosure
The authors report no conflicts of interest in this work. | CRIZOTINIB, OSIMERTINIB | DrugsGivenReaction | CC BY | 33438350 | 18,819,261 | 2021-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Therapy partial responder'. | Cutaneous angiosarcoma clinically presenting as Quincke's edema.
We report a case of a 75-year-old man with facial edema that also affected the periorbital area who was admitted to the hospital with the suspected diagnosis of Quincke's edema. The diagnosis of cutaneous angiosarcoma was made by microscopic examination and immunohistochemical staining. Chemotherapy was initially initiated because the angiosarcoma was unresectable and the radiation situation was difficult. Therapy has to be switched to second and third line therapy due to disease progression. The case illustrates the complexity of diagnosis and therapy in patients with cutaneous angiosarcoma.
pmcFalldarstellung
Anamnese
Ein 75-jähriger Patient stellte sich 2017 erstmalig mit einer seit ca. 4 Wochen aufgefallenen Gesichtsschwellung in unserer Klinik vor. Unter der Verdachtsdiagnose eines Quincke-Ödems war bereits eine ambulante Therapie mit Steroiden und Antihistaminika per os eingeleitet worden – zum Zeitpunkt der Vorstellung ohne nachhaltigen Therapieerfolg.
Klinischer Befund
Der Patient präsentierte sich in gutem Allgemeinzustand mit einem ausgeprägten Ödem und Erythem der gesamten Gesichtshaut unter Betonung der oberen Gesichtshälfte, insbesondere der Ober- und Unterlider, links etwas ausgeprägter als rechts (Abb. 1a). Die Hautveränderungen setzten sich bis auf das frontale und hoch parietale Kapillitium fort.
Verdachtsdiagnose
Unter der Verdachtsdiagnose eines ambulant therapierefraktären Quincke-Ödems wurde der Patient stationär aufgenommen, und es wurde eine intensivierte intravenöse Therapie mit Antihistaminika (2 mg Clemastin 2‑mal täglich) und Prednisolon (100 mg 1‑mal täglich) eingeleitet. Nach 2 Tagen eines unveränderten Hautbefundes wurde eine Probebiopsie aus dem prominentesten Schwellungsareal an der Stirn entnommen.
Histologie
Der histologische Befund zeigte über das gesamte Korium verteilt zahlreiche schlitzförmige Gefäßspalten mit prominentem Endothel und Zellatypien (Abb. 1d). In der immunhistologischen Untersuchung waren die atypischen Zellen positiv für CD31 und D2–40 sowie negativ für HHV‑8 (Abb. 1e). Weiterhin wiesen sie eine sehr hohe proliferative Aktivität auf (Ki-67 >60 %). In der Zusammenschau der Befunde wurde somit ein kutanes Angiosarkom diagnostiziert.
Therapie und Verlauf
Um die Tumorausdehnung zu beurteilen, wurden zahlreiche Mappingbiopsien an den sichtbaren Randbereichen durchgeführt. Deren histologische Beurteilung erbrachte eine großflächige Ausdehnung des Tumors vom Kapillitium hochparietal über die Stirn und das obere Mittelgesicht bis retroaurikulär beidseits. Ein daraufhin durchgeführtes Tumorstaging mittels PET(Positronenemissionstomographie)-CT(Computertomographie) zeigte die Ausdehnung des Angiosarkoms am Kapillitium von beidseits okzipital (links > rechts) über temporal nach parietal reichend (Abb. 1f), ergab aber erfreulicherweise keinen Hinweis auf eine Metastasierung. Der Fall wurde in unserem interdisziplinären Hauttumorboard vorgestellt und diskutiert. Es wurde empfohlen, bei nicht resektablem Primärbefund und schwieriger Bestrahlungssituation aufgrund der Beteiligung beider Periorbitalareale eine primäre Chemotherapie mit Paclitaxel 80 mg/m2 Q1W einzuleiten. Hierunter kam es rasch zu einer klinischen Besserung mit teilweise wieder möglicher Öffnung des linken Auges (Abb. 1b). Eine Reevaluation mittels PET-CT 8 Wochen nach Therapiebeginn bestätigte die deutliche Abnahme des Glukosemetabolismus im ursprünglichen Tumorareal (Abb. 1g), sodass die Therapie mit Paclitaxel bei partieller Remission fortgesetzt wurde. Fünf Monate nach Therapiebeginn kam es zu einer erneuten Zunahme der periorbitalen Ödeme. Es erfolgte eine Therapieumstellung auf liposomales Doxorubicin (Caelyx 20 mg/m2 Q2W). Bei massiver Schwellung der Augenlider mit vollständigem Verschluss des linken Auges nach 4 Zyklen Caelyx (Abb. 1c) wurde aufgrund der raschen Krankheitsprogression die Therapie auf den Multikinaseinhibitor Pazopanib 800 mg/Tag umgestellt. Hierunter war bereits nach wenigen Wochen eine deutliche klinische Besserung bei zunächst guter Verträglichkeit zu verzeichnen. Allerdings entwickelte der Patient 3 Monate nach Therapiebeginn eine ausgedehnte Lungenarterienembolie, aufgrund derer die Therapie mit Pazopanib pausiert werden musste. In der Folge ergab sich rasch ein deutlicher Progress des Angiosarkoms mit erneut fast vollständigem Lidverschluss links, sodass eine weitere Therapie trotz vorliegender Lungenarterienembolie notwendig wurde. Von unserem interdisziplinären Hauttumorboard wurden eine palliative Strahlentherapie sowie die Umstellung der Systemtherapie auf den PD-1-Inhibitor Pembrolizumab empfohlen. Noch vor der geplanten Einleitung dieser Therapie entwickelte der Patient eine Staphylococcus-aureus-Sepsis und verstarb daran, ungefähr 1 Jahr nach der Erstdiagnose des kutanen Angiosarkoms.
Diskussion
Das kutane Angiosarkom stellt mit einem Anteil von 1–2 % der Weichteilsarkome und 5 % der kutanen Sarkome eine sehr seltene Tumorentität dar [1]. Die Tumoren treten bei ungefähr 50 % der Patienten im Kopf-Hals-Bereich auf [1]. Das idiopathische kutane Angiosarkom betrifft v. a. ältere Menschen mit deutlicher Bevorzugung des männlichen Geschlechts (Verhältnis 3 zu 1) [1]. Die Klinik ist oft unspezifisch und die Prognose aufgrund hoher Lokalrezidivraten und früher hämatogener Metastasierung schlecht [1]. Eine aktuelle Metaanalyse kam zu dem Ergebnis, dass die mittlere Fünfjahresüberlebensrate von Patienten mit Angiosarkom nur 33,5 % beträgt [2]. Weiterhin wurden anhand dieser Analyse ein Lebensalter über 70 Jahre, eine primäre Tumorausdehnung größer 5 cm sowie die Lokalisation im Kopfbereich als Prädiktoren für eine schlechte Prognose identifiziert [2].
Unser Fall führt die Schwierigkeit der Diagnosestellung sowie die Komplexität des Behandlungsverlaufes bei Patienten mit kutanem Angiosarkom vor Augen. Wie bei unserem Fall kommt es beim kutanen Angiosarkom häufig zu einer Verschleppung der Diagnosestellung durch die klinische Ähnlichkeit zu inflammatorischen Dermatosen. So können die Frühstadien mit teigigen Ödemen und Erythemen nicht nur an ein Quincke-Ödem denken lassen, sondern auch an eine Rosazea oder ein Erysipel erinnern. Erschwerend kommt hinzu, dass die Mehrzahl der Patienten asymptomatisch ist und sich erst spät aufgrund von Blutung, Ödemen und/oder Ulzerationen ärztlich vorstellt [3]. Ein weiteres Problem besteht darin, dass es klinisch nur schwer möglich ist, die tatsächliche Tumorausdehnung zu bestimmen. Mappingbiopsien können hier mehr Sicherheit bringen, oftmals wachsen die Tumoren jedoch primär multifokal, was eine operative Sanierung erschwert [1]. Bei eingeschränkter Operabilität stellt die primäre Chemo- und/oder Strahlentherapie die Therapie der Wahl dar. Auch Kinaseinhibitoren, wie in unserem Fall Pazopanib, die die Angiogenese des Tumors hemmen, können vielversprechende Therapieoptionen darstellen. Die Wirksamkeit des Multikinaseinhibitors Pazopanib beim Angiosarkom wurde in mehreren klinischen Studien untersucht. So wurden in der PALETTE-Studie 369 Patienten mit Weichgewebssarkomen nach dem Zufallsprinzip mit 800 mg Pazopanib (n = 246) oder Placebo (n = 123) behandelt [4]. Das mediane progressionsfreie Überleben in dieser Phase-III-Studie betrug 4,6 Monate für Pazopanib im Vergleich zu 1,6 Monate für Placebo (Hazard Ratio [HR] 0,31; 95 %-KI [Konfidenzintervall] 0,24–0,40; p < 0,0001). Das Gesamtüberleben der Patienten betrug 12,5 Monate mit Pazopanib gegenüber 10,7 Monaten unter Placebo (HR 0,86; 95 %-KI 0,67–1,11; p = 0,25). Die PALETTE-Studie konnte somit eine Überlegenheit von Pazopanib gegenüber Placebo nachweisen [4], woraufhin die Substanz von der FDA (Food and Drug Administration) für die Indikation Weichteilsarkom zugelassen wurde. Aktuell werden Patienten im Rahmen einer Phase-III-Studie zur Bewertung der Wirksamkeit von Pazopanib zur Behandlung von Patienten mit fortgeschrittenem kutanem Angiosarkom in Japan rekrutiert (JCOG1605, JCOG-PACS).
In unserem vorliegenden Fall war nach Therapieversagen der Standardschemata eine Therapieumstellung auf Pembrolizumab geplant, da eine Therapie mit PD-1-Inhibitoren in Vergleich zu Chemotherapeutika besser verträglich ist. Die Erfahrungen mit Immuncheckpointinhibitoren beim kutanen Angiosarkom und die entsprechende Datenlage sind noch limitiert, was unter anderem daran liegt, dass zwar in zahlreichen Studien die Rolle der Immuntherapie bei Weichteilsarkomen untersucht wird, allerdings seltene Subtypen wie das Angiosarkom oft von einer Teilnahme ausgeschlossen werden. Die Ergebnisse größerer Studien stehen noch aus (NCT02815995), allerdings weisen Fallberichte und Zwischenberichte aus klinischen Studien darauf hin, dass Angiosarkome auf die Immuntherapie ansprechen könnten [5–7]. Die 2016 veröffentlichte multizentrische Phase-II-Studie SARC028 untersuchte die Wirksamkeit und Verträglichkeit des PD-1-Inhibitors Pembrolizumab bei je 40 Patienten mit fortgeschrittenen Weichteil- und Knochensarkomen [8]. Die Ergebnisse zeigten ein Ansprechen auf Pembrolizumab bei 18 % der Patienten mit Weichteilsarkom [8]. Dies deutet auf eine Wirksamkeit von PD-1-Inhibitoren bei der Behandlung von Sarkomen hin. Aktuell werden Sarkompatienten in der SAINT-Studie rekrutiert, in der unter anderem das Zytostatikum Trabectedin und die CTLA-4-Hemmung mit Immuntherapie kombiniert werden (Ipilimumab und Nivolumab, NCT03138161) [9]. Das Angiosarkomprojekt zur genetischen Sequenzierung von Angiosarkomproben hat gezeigt, dass manche kutane Angiosarkome UV-Mutationssignaturen aufweisen, wie sie beim Melanom gefunden wurden [10]. Angesichts der hohen Mutationslast beim Melanom und der relativ hohen Ansprechraten auf die Immuntherapie bietet dies eine mögliche Hypothese, um die Hinweise auf ein Ansprechen von PD-1-Inhibitoren bei kutanen Angiosarkomen zu erklären.
Fazit für die Praxis
Der geschilderte Fall verdeutlicht, dass die Planung der Diagnostik und Therapiestrategie bei Patienten mit kutanen Angiosarkomen komplex ist.
Es empfiehlt sich, diese interdisziplinär abzustimmen sowie die Patienten an ein erfahrenes Sarkomzentrum anzubinden.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Einhaltung ethischer Richtlinien
Interessenkonflikt
M. Albrecht erhielt eine Reisekostenunterstützung von Novartis und Merck Serono. L. Zimmer war als Beraterin tätig und/oder erhielt Honorare von Roche, Bristol-Myers Squibb, Merck Sharp und Dohme, Novartis, Pierre Fabre und Sanofi sowie Reisekostenunterstützung von Bristol-Myers Squibb, Merck Sharp und Dohme, Amgen und Pierre Fabre und Novartis. E. Livingstone empfing Beraterhonorare und/oder Referentenhonorare von Amgen, Actelion, Roche, BMS, MSD, Novartis, Janssen, Medac sowie Reisekostenzuschüsse oder -erstattungen von Amgen, MSD, BMS, Pierre Fabre, Sun Pharma und Novartis. R. Hamacher gab an, Reisekostenzuschüsse oder -erstattungen von Lilly, Novartis und PharmaMar sowie ein Honorar von Lilly erhalten zu haben. D. Schadendorf erhielt Honorare für eine Beratertätigkeit und/oder Vorträge von Roche, Novartis, Bristol-Myers Squibb, Merck, Amgen, Boehringer Ingelheim und Leo sowie Reisekostenunterstützung von Roche, Novartis, Bristol-Myers Squibb, Merck, Amgen, Boehringer Ingelheim und Leo. S. Ugurel erklärte, Forschungsunterstützung von Bristol-Myers Squibb und Merck Serono, Honorare für eine Beratertätigkeit und/oder Vorträge von Bristol-Myers Squibb, Merck Sharp und Dohme, Merck Serono, Novartis und Roche sowie Reisekostenunterstützung von Bristol-Myers Squibb, Merck Sharp und Dohme erhalten zu haben. E. Hadaschik und S. Bauer 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. | PACLITAXEL | DrugsGivenReaction | CC BY | 33439269 | 20,205,395 | 2021-09 |
What was the dosage of drug 'PACLITAXEL'? | Cutaneous angiosarcoma clinically presenting as Quincke's edema.
We report a case of a 75-year-old man with facial edema that also affected the periorbital area who was admitted to the hospital with the suspected diagnosis of Quincke's edema. The diagnosis of cutaneous angiosarcoma was made by microscopic examination and immunohistochemical staining. Chemotherapy was initially initiated because the angiosarcoma was unresectable and the radiation situation was difficult. Therapy has to be switched to second and third line therapy due to disease progression. The case illustrates the complexity of diagnosis and therapy in patients with cutaneous angiosarcoma.
pmcFalldarstellung
Anamnese
Ein 75-jähriger Patient stellte sich 2017 erstmalig mit einer seit ca. 4 Wochen aufgefallenen Gesichtsschwellung in unserer Klinik vor. Unter der Verdachtsdiagnose eines Quincke-Ödems war bereits eine ambulante Therapie mit Steroiden und Antihistaminika per os eingeleitet worden – zum Zeitpunkt der Vorstellung ohne nachhaltigen Therapieerfolg.
Klinischer Befund
Der Patient präsentierte sich in gutem Allgemeinzustand mit einem ausgeprägten Ödem und Erythem der gesamten Gesichtshaut unter Betonung der oberen Gesichtshälfte, insbesondere der Ober- und Unterlider, links etwas ausgeprägter als rechts (Abb. 1a). Die Hautveränderungen setzten sich bis auf das frontale und hoch parietale Kapillitium fort.
Verdachtsdiagnose
Unter der Verdachtsdiagnose eines ambulant therapierefraktären Quincke-Ödems wurde der Patient stationär aufgenommen, und es wurde eine intensivierte intravenöse Therapie mit Antihistaminika (2 mg Clemastin 2‑mal täglich) und Prednisolon (100 mg 1‑mal täglich) eingeleitet. Nach 2 Tagen eines unveränderten Hautbefundes wurde eine Probebiopsie aus dem prominentesten Schwellungsareal an der Stirn entnommen.
Histologie
Der histologische Befund zeigte über das gesamte Korium verteilt zahlreiche schlitzförmige Gefäßspalten mit prominentem Endothel und Zellatypien (Abb. 1d). In der immunhistologischen Untersuchung waren die atypischen Zellen positiv für CD31 und D2–40 sowie negativ für HHV‑8 (Abb. 1e). Weiterhin wiesen sie eine sehr hohe proliferative Aktivität auf (Ki-67 >60 %). In der Zusammenschau der Befunde wurde somit ein kutanes Angiosarkom diagnostiziert.
Therapie und Verlauf
Um die Tumorausdehnung zu beurteilen, wurden zahlreiche Mappingbiopsien an den sichtbaren Randbereichen durchgeführt. Deren histologische Beurteilung erbrachte eine großflächige Ausdehnung des Tumors vom Kapillitium hochparietal über die Stirn und das obere Mittelgesicht bis retroaurikulär beidseits. Ein daraufhin durchgeführtes Tumorstaging mittels PET(Positronenemissionstomographie)-CT(Computertomographie) zeigte die Ausdehnung des Angiosarkoms am Kapillitium von beidseits okzipital (links > rechts) über temporal nach parietal reichend (Abb. 1f), ergab aber erfreulicherweise keinen Hinweis auf eine Metastasierung. Der Fall wurde in unserem interdisziplinären Hauttumorboard vorgestellt und diskutiert. Es wurde empfohlen, bei nicht resektablem Primärbefund und schwieriger Bestrahlungssituation aufgrund der Beteiligung beider Periorbitalareale eine primäre Chemotherapie mit Paclitaxel 80 mg/m2 Q1W einzuleiten. Hierunter kam es rasch zu einer klinischen Besserung mit teilweise wieder möglicher Öffnung des linken Auges (Abb. 1b). Eine Reevaluation mittels PET-CT 8 Wochen nach Therapiebeginn bestätigte die deutliche Abnahme des Glukosemetabolismus im ursprünglichen Tumorareal (Abb. 1g), sodass die Therapie mit Paclitaxel bei partieller Remission fortgesetzt wurde. Fünf Monate nach Therapiebeginn kam es zu einer erneuten Zunahme der periorbitalen Ödeme. Es erfolgte eine Therapieumstellung auf liposomales Doxorubicin (Caelyx 20 mg/m2 Q2W). Bei massiver Schwellung der Augenlider mit vollständigem Verschluss des linken Auges nach 4 Zyklen Caelyx (Abb. 1c) wurde aufgrund der raschen Krankheitsprogression die Therapie auf den Multikinaseinhibitor Pazopanib 800 mg/Tag umgestellt. Hierunter war bereits nach wenigen Wochen eine deutliche klinische Besserung bei zunächst guter Verträglichkeit zu verzeichnen. Allerdings entwickelte der Patient 3 Monate nach Therapiebeginn eine ausgedehnte Lungenarterienembolie, aufgrund derer die Therapie mit Pazopanib pausiert werden musste. In der Folge ergab sich rasch ein deutlicher Progress des Angiosarkoms mit erneut fast vollständigem Lidverschluss links, sodass eine weitere Therapie trotz vorliegender Lungenarterienembolie notwendig wurde. Von unserem interdisziplinären Hauttumorboard wurden eine palliative Strahlentherapie sowie die Umstellung der Systemtherapie auf den PD-1-Inhibitor Pembrolizumab empfohlen. Noch vor der geplanten Einleitung dieser Therapie entwickelte der Patient eine Staphylococcus-aureus-Sepsis und verstarb daran, ungefähr 1 Jahr nach der Erstdiagnose des kutanen Angiosarkoms.
Diskussion
Das kutane Angiosarkom stellt mit einem Anteil von 1–2 % der Weichteilsarkome und 5 % der kutanen Sarkome eine sehr seltene Tumorentität dar [1]. Die Tumoren treten bei ungefähr 50 % der Patienten im Kopf-Hals-Bereich auf [1]. Das idiopathische kutane Angiosarkom betrifft v. a. ältere Menschen mit deutlicher Bevorzugung des männlichen Geschlechts (Verhältnis 3 zu 1) [1]. Die Klinik ist oft unspezifisch und die Prognose aufgrund hoher Lokalrezidivraten und früher hämatogener Metastasierung schlecht [1]. Eine aktuelle Metaanalyse kam zu dem Ergebnis, dass die mittlere Fünfjahresüberlebensrate von Patienten mit Angiosarkom nur 33,5 % beträgt [2]. Weiterhin wurden anhand dieser Analyse ein Lebensalter über 70 Jahre, eine primäre Tumorausdehnung größer 5 cm sowie die Lokalisation im Kopfbereich als Prädiktoren für eine schlechte Prognose identifiziert [2].
Unser Fall führt die Schwierigkeit der Diagnosestellung sowie die Komplexität des Behandlungsverlaufes bei Patienten mit kutanem Angiosarkom vor Augen. Wie bei unserem Fall kommt es beim kutanen Angiosarkom häufig zu einer Verschleppung der Diagnosestellung durch die klinische Ähnlichkeit zu inflammatorischen Dermatosen. So können die Frühstadien mit teigigen Ödemen und Erythemen nicht nur an ein Quincke-Ödem denken lassen, sondern auch an eine Rosazea oder ein Erysipel erinnern. Erschwerend kommt hinzu, dass die Mehrzahl der Patienten asymptomatisch ist und sich erst spät aufgrund von Blutung, Ödemen und/oder Ulzerationen ärztlich vorstellt [3]. Ein weiteres Problem besteht darin, dass es klinisch nur schwer möglich ist, die tatsächliche Tumorausdehnung zu bestimmen. Mappingbiopsien können hier mehr Sicherheit bringen, oftmals wachsen die Tumoren jedoch primär multifokal, was eine operative Sanierung erschwert [1]. Bei eingeschränkter Operabilität stellt die primäre Chemo- und/oder Strahlentherapie die Therapie der Wahl dar. Auch Kinaseinhibitoren, wie in unserem Fall Pazopanib, die die Angiogenese des Tumors hemmen, können vielversprechende Therapieoptionen darstellen. Die Wirksamkeit des Multikinaseinhibitors Pazopanib beim Angiosarkom wurde in mehreren klinischen Studien untersucht. So wurden in der PALETTE-Studie 369 Patienten mit Weichgewebssarkomen nach dem Zufallsprinzip mit 800 mg Pazopanib (n = 246) oder Placebo (n = 123) behandelt [4]. Das mediane progressionsfreie Überleben in dieser Phase-III-Studie betrug 4,6 Monate für Pazopanib im Vergleich zu 1,6 Monate für Placebo (Hazard Ratio [HR] 0,31; 95 %-KI [Konfidenzintervall] 0,24–0,40; p < 0,0001). Das Gesamtüberleben der Patienten betrug 12,5 Monate mit Pazopanib gegenüber 10,7 Monaten unter Placebo (HR 0,86; 95 %-KI 0,67–1,11; p = 0,25). Die PALETTE-Studie konnte somit eine Überlegenheit von Pazopanib gegenüber Placebo nachweisen [4], woraufhin die Substanz von der FDA (Food and Drug Administration) für die Indikation Weichteilsarkom zugelassen wurde. Aktuell werden Patienten im Rahmen einer Phase-III-Studie zur Bewertung der Wirksamkeit von Pazopanib zur Behandlung von Patienten mit fortgeschrittenem kutanem Angiosarkom in Japan rekrutiert (JCOG1605, JCOG-PACS).
In unserem vorliegenden Fall war nach Therapieversagen der Standardschemata eine Therapieumstellung auf Pembrolizumab geplant, da eine Therapie mit PD-1-Inhibitoren in Vergleich zu Chemotherapeutika besser verträglich ist. Die Erfahrungen mit Immuncheckpointinhibitoren beim kutanen Angiosarkom und die entsprechende Datenlage sind noch limitiert, was unter anderem daran liegt, dass zwar in zahlreichen Studien die Rolle der Immuntherapie bei Weichteilsarkomen untersucht wird, allerdings seltene Subtypen wie das Angiosarkom oft von einer Teilnahme ausgeschlossen werden. Die Ergebnisse größerer Studien stehen noch aus (NCT02815995), allerdings weisen Fallberichte und Zwischenberichte aus klinischen Studien darauf hin, dass Angiosarkome auf die Immuntherapie ansprechen könnten [5–7]. Die 2016 veröffentlichte multizentrische Phase-II-Studie SARC028 untersuchte die Wirksamkeit und Verträglichkeit des PD-1-Inhibitors Pembrolizumab bei je 40 Patienten mit fortgeschrittenen Weichteil- und Knochensarkomen [8]. Die Ergebnisse zeigten ein Ansprechen auf Pembrolizumab bei 18 % der Patienten mit Weichteilsarkom [8]. Dies deutet auf eine Wirksamkeit von PD-1-Inhibitoren bei der Behandlung von Sarkomen hin. Aktuell werden Sarkompatienten in der SAINT-Studie rekrutiert, in der unter anderem das Zytostatikum Trabectedin und die CTLA-4-Hemmung mit Immuntherapie kombiniert werden (Ipilimumab und Nivolumab, NCT03138161) [9]. Das Angiosarkomprojekt zur genetischen Sequenzierung von Angiosarkomproben hat gezeigt, dass manche kutane Angiosarkome UV-Mutationssignaturen aufweisen, wie sie beim Melanom gefunden wurden [10]. Angesichts der hohen Mutationslast beim Melanom und der relativ hohen Ansprechraten auf die Immuntherapie bietet dies eine mögliche Hypothese, um die Hinweise auf ein Ansprechen von PD-1-Inhibitoren bei kutanen Angiosarkomen zu erklären.
Fazit für die Praxis
Der geschilderte Fall verdeutlicht, dass die Planung der Diagnostik und Therapiestrategie bei Patienten mit kutanen Angiosarkomen komplex ist.
Es empfiehlt sich, diese interdisziplinär abzustimmen sowie die Patienten an ein erfahrenes Sarkomzentrum anzubinden.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Einhaltung ethischer Richtlinien
Interessenkonflikt
M. Albrecht erhielt eine Reisekostenunterstützung von Novartis und Merck Serono. L. Zimmer war als Beraterin tätig und/oder erhielt Honorare von Roche, Bristol-Myers Squibb, Merck Sharp und Dohme, Novartis, Pierre Fabre und Sanofi sowie Reisekostenunterstützung von Bristol-Myers Squibb, Merck Sharp und Dohme, Amgen und Pierre Fabre und Novartis. E. Livingstone empfing Beraterhonorare und/oder Referentenhonorare von Amgen, Actelion, Roche, BMS, MSD, Novartis, Janssen, Medac sowie Reisekostenzuschüsse oder -erstattungen von Amgen, MSD, BMS, Pierre Fabre, Sun Pharma und Novartis. R. Hamacher gab an, Reisekostenzuschüsse oder -erstattungen von Lilly, Novartis und PharmaMar sowie ein Honorar von Lilly erhalten zu haben. D. Schadendorf erhielt Honorare für eine Beratertätigkeit und/oder Vorträge von Roche, Novartis, Bristol-Myers Squibb, Merck, Amgen, Boehringer Ingelheim und Leo sowie Reisekostenunterstützung von Roche, Novartis, Bristol-Myers Squibb, Merck, Amgen, Boehringer Ingelheim und Leo. S. Ugurel erklärte, Forschungsunterstützung von Bristol-Myers Squibb und Merck Serono, Honorare für eine Beratertätigkeit und/oder Vorträge von Bristol-Myers Squibb, Merck Sharp und Dohme, Merck Serono, Novartis und Roche sowie Reisekostenunterstützung von Bristol-Myers Squibb, Merck Sharp und Dohme erhalten zu haben. E. Hadaschik und S. Bauer 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. | 80 MILLIGRAM/SQ. METER, Q1W | DrugDosageText | CC BY | 33439269 | 20,205,395 | 2021-09 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Sepsis'. | Long term follow-up of pediatric-onset Evans syndrome: broad immunopathological manifestations and high treatment burden.
Pediatric-onset Evans syndrome (pES) is defined by both immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) before the age of 18 years. There have been no comprehensive long-term studies of this rare disease, which can be associated to various immunopathological manifestations (IMs). We report outcomes of the 151 patients with pES and more than 5 years of follow-up from the nationwide French prospective OBS'CEREVANCE cohort. Median age at final follow-up was 18.5 (6.8-50.0) years and the median follow-up period was 11.3 (5.1-38.0) years. At 10 years, ITP and AIHA were in sustained complete remission in 54.5% and 78.4% of patients, respectively. The frequency and number of clinical and biological IMs increased with age: at 20 years old, 74% had at least one clinical cIM. A wide range of cIMs occurred, mainly lymphoproliferation, dermatological, gastrointestinal/hepatic and pneumological IMs. The number of cIMs was associated with a subsequent increase in the number of second-line treatments received (other than steroids and immunoglobulins; hazard ratio, 1.4; 95% confidence interval, 1.15-1.60; p = 0.0002, Cox proportional hazards method). Survival at 15 years after diagnosis was 84%. Death occurred at a median age of 18 (1.7-31.5) years, and the most frequent cause was infection. The number of second-line treatments and severe/recurrent infections were independently associated with mortality. In conclusion, longterm outcomes of pES showed remission of cytopenias but frequent IMs linked to high secondline treatment burden. Mortality was associated to drugs and/or underlying immunodeficiencies, and adolescents-young adults are a high-risk subgroup.
pmcIntroduction
The presence of both immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) defines Evans syndrome (ES). Pediatric-onset ES (pES) is a rare disease, and approximately ten new cases are diagnosed every year in France, which has a population of 66 million.1 Since its first description in 1951 by Robert Evans,2 our understanding of pES has been based on small retrospective cohorts with limited follow-up.3-7 In 2004, the French Rare Disease Center CEREVANCE set up the prospective national cohort OBS’CEREVANCE, which includes children with AIHA, chronic ITP persisting for more than 12 months (cITP), and pES.8
Preliminary reports from this cohort and previously published studies showed that pES is a chronic disease with a high rate of relapse for both types of cytopenias. 1,3,4,7,9 Mortality rates across studies have ranged from 7–36%.1,3-7 In addition to cytopenias, immunopathological manifestations (IM) such as autoimmune/autoinflammatory organ diseases, lymphoproliferation, and hypogammaglobulinemia have been reported in 70–80% of patients with pES.4,5,8,10 In an undetermined number of cases, pES is thought to be “secondary” and caused by an underlying disease, classically systemic lupus erythematosus (SLE) or autoimmune lymphoproliferative syndrome (ALPS).1,11,12 Recently, genetic analyses found a heterogeneous genetic background in up to 65% of a subset of 80 patients from the OBS’CEREVANCE cohort. These patients carried variants in genes that are linked to primary immunodeficiencies (PID) or involved in immune responses.13
Overall, outcomes and the long-term course of pES are poorly understood. There have been no comprehensive longitudinal studies including both cytopenia and IM. In addition, the transition to adulthood is often particularly challenging for patients with chronic pediatric diseases.14 Adolescents–young adults (AYA) outcomes have not been investigated in patients with pES, and whether the disease improves with age is unknown. In a clinical setting, the possibility to identify high-risk patients would be extremely helpful in the management of this complex disease. Here, we describe the long-term course of hematological IM and treatments received throughout childhood into adulthood in patients with pES from the OBS’CEREVANCE cohort. We aimed to identify clinically relevant factors associated to the occurrence of IM, the number of second-line treatments received and mortality. Particularly, we investigated the impact of the number second-line treatment received and splenectomy on mortality.
Methods
OBS’CEREVANCE prospective national cohort
Inclusion and exclusion criteria are shown in the Online Supplementary Table S1.1,8,15 Data collected have been previously detailed.1,8 Patients were included if <18 years old at first cytopenia diagnosis. The coordinating center gathered and analyzed all data from the medical team in charge in real time, enabling prospective follow-up even after the pediatric-to-adult transition. The CEREVANCE group recommends scheduling clinical and biological follow-up at least every 6-12 months. Some patients underwent genetic analyses, as previously described.13 Written informed consent was obtained from parents and eligible patients. The cohort study was approved by the Institutional Ethics Committee (CPPRB-A; Bordeaux, France) and the database was registered with the national data protection authority (CNIL, 1396823V0).
Patient selection
Patients with pES, defined as the simultaneous (within 1 month) or sequential association of ITP and AIHA, were included if at least 5 years of follow-up data were available after the first cytopenia diagnosis. In order to provide a complete mortality report, all patients, including those with less than 5 years of follow-up data, were included in the survival analyses. The data were extracted on 21 June 2019.
Definitions
Initial cytopenia refers to the onset of ITP or AIHA (whichever occurred first) and does not take autoimmune neutropenia (AIN) into account. The IM categories were separated in clinical (cIM) and biological (bIM). pES was defined as secondary if a diagnosis of SLE or PID was made during the follow-up period. SLE diagnosis was made according to the Systemic Lupus International Collaborating Clinics Classification criteria (SLICC).16 ALPS diagnosis was based on international criteria.17 Second-line treatments were all immunomodulatory or immunosuppressive treatments, including splenectomy but excluding steroids and therapeutic intravenous immunoglobulins (IVIG). Sustained complete remission (CR) was defined as remission persisting until final follow-up, regardless of ongoing treatments.
For analyses by age, patients were assessed annually from birth (for IM and treatments) or from cytopenia onset (for AIHA and ITP), until final follow-up. Occasional treatments (e.g., splenectomy and rituximab) were considered as ongoing if these occurred during the previous year. Further details are stated in the Online Supplementary Table S1.
Statistical analyses
Continuous and categorical variables were compared using Wilcoxon–Mann–Whitney non-parametric test and Fisher’s exact test, respectively. Correlations were tested using the Pearson correlation coefficient. Survival and cumulative incidence estimates were based on the Kaplan–Meier method and compared using log-rank test. Patients of OBS’CEREVANCE cohort with isolated cITP or AIHA were used for comparison in survival analyses (unpublished data). The Cox proportional hazards method was used to analyze factors associated with time-dependent variables (i.e., time to CR, AIN, cIM, and second-line treatment, as well as survival). The potential cumulative and/or time-dependent nature of variables was taken in account. Proportionality of hazard was assessed for each variable. Logistic regression was used to analyze factors associated with severe or recurrent infections. Variables that were statistically significant in the univariate analyses were included in the multivariate analyses. We investigated associations with the following characteristics and events: sex, consanguinity, cIM/cancer in a first-degree relative, age at first and second cytopenia, sequence of cytopenia, AIN, hypogammaglobulinemia, time to ITP/AIHA CR, severe/recurrent infections, number of cIM, number of second-line treatments. The 95% Confidence Intervals (CI) for hazard ratios (HR) and odds ratios (OR) were not adjusted for multiple testing and should not be used to infer definitive effects. All tests were two-sided and a P-value <0.05 was considered statistically significant. Statistical analyses were performed using R (ver. 4.0; R Development Core Team) and GraphPad Prism (ver. 8; GraphPad Software, Inc., San Diego, CA, USA) software.
Results
Population
Of the 216 patients with pES, 151 were included in this study (Online Supplementary Figure S1). They were followed from 25 different centers. Patient characteristics are shown in Table 1. The median (min–max) follow-up time from the first cytopenia diagnosis was 11.3 years (range, 5.1–38.0 years). Median age at final follow-up was 18.5 years (range, 6.8–50.0 years). In 20 cases (15%), follow-up was discontinued because the patient was considered cured (n=11) or lost to follow-up (n=9). Median age at loss to follow-up was 18.4 years (range, 6.8-25.1 years).
Hematological outcomes
AIN developed in 43 patients (28.5%). It was diagnosed within 1 month before or after first cytopenia onset in 23 of 43 cases (53.5%), more than 1 month before in two cases (4.7%), and more than 1 month after in 18 cases (41.9%; maximal delay, 12.4 years). In all cases, the diagnosis was made before the age of 18 years (median age, 6.8 years; range, 0.6–16.2 years).
ITP and AIHA flare rates at 5 years of follow-up were calculated for the 61 alive patients who did not receive a second-line treatment during this period. Forty-eight patients (79%) had experienced an ITP flare and seven (11%) an AIHA flare.
The proportion of patients achieving sustained CR for ITP and AIHA steadily increased after cytopenia onset (Figure 1A). At 5 and 10 years, ITP was in sustained CR in 40.5% and 62.3% of patients (P=0.02) and AIHA was in sustained CR in 54.5% and 74.1% of patients (P=0.001), respectively. Sustained CR was achieved earlier for AIHA than ITP (median time to CR, 4.0 years vs. 7.0 years; P=0.01). At the final follow-up of the 135 surviving patients, the numbers of patients in CR, partial remission, and no remission were 126 (83%), five (3%), and one (1%) for AIHA and 119 (79%), eight (5%), and five (3%) for ITP, respectively (missing data in three cases). Forty-six patients (34%) had no treatment ongoing at last followup. No particular characteristic was associated with AIHA or ITP CR, including cIM and bIM.
Table 1. Patient characteristics.
Over the first three decades, the proportions of patients achieving sustained CR increased with age (Figure 1B). ITP and AIHA were in CR in 26% and 30% of cases at 10 years compared to 50% and 72% at 20 years, respectively (P<0.001 for both comparisons).
Immunopathological manifestations
A total of 122 of 151 patients (81%) had at least one IM. The data for each category and specific diagnosis are shown in the Online Supplementary Table S2.
cIM developed in 100 of 151 patients (66%). A total of 47 patients (31%) had two or more IM and 22 (15%) patients had three or more IM (Online Supplementary Figure S2A). Patients with no cIM had shorter median follow-up times (9.7 years vs. 13 years; P=0.0002) and were younger when data were collected (15 years vs. 20 years; P<0.0001). A cIM was diagnosed before the first cytopenia in 21 of 100, simultaneously in 13, and after in 66 cases (median delay, 3.7 years [range, 0.2–20.5 years]; Figure 2A). Among the 185 cIM, 29 (16%) were diagnosed before any second-line treatment. No cIM category had a statistically significant difference in frequency before and after first second-line treatment. The number of cIM increased with age. At 10 compared to 20 years old, 37% and 74% of patients had at least one cIM and 9% and 34% of patients had at least two cIM, respectively (P<0.001 for both comparisons; Figure 2B).
The most common cIM categories were lymphoproliferation (n=71), dermatological (n= 26), gastrointestinal/hepatic (n=23) and pneumological manifestations (n=16, Figure 3; Online Supplementary Figure S2B). The most frequent cIM diagnosis are shown in Table 2. Thirteen patients developed a neurological manifestation as previously described.10 Four patients had a hematological malignancy (age at diagnosis): Hodgkin lymphoma (16 years), juvenile myelomonocytic leukemia (20 years), large granular lymphocytic leukemia (21 years) and angioimmunoblastic Tcell lymphoma (29 years). Older age at ES diagnosis (HR 1.09; 95% CI: 1.01–1.17; P=0.02), cIM/cancer in a firstdegree relative (HR 1.64; 95% CI: 1.1–2.4; P=0.006), and the presence of AIN were independently associated with the number of cIM (HR 2.41; 95% CI: 1.5–3.8; P=0.0002).
Biological IM (bIM) were diagnosed in 101 of 151 patients (67%), and the frequency of bIM also increased with the age (Figure 2C). Hypogammaglobulinemia was the most frequently diagnosed bIM (n=54), including 44 cases diagnosed prior to any anti-CD20 treatment. Among those 54 patients, 25 (46%) received immunoglobulin replacement therapy. SLE and ALPS biomarkers were present (regardless of whether patients met the diagnostic criteria) in 42 and 24 patients, respectively. At 10 and 20 years of age, 39% and 75% of patients had at least one bIM, respectively (P<0.001).
Patients with bIM were more likely to have cIM (79% vs. 40%; P<0.001), and patients with cIM were more likely to have bIM (80% vs. 41%; P<0.001) but the correlation between the number of bIM and cIM was low (r=0.27; P<0.001).
Secondary pediatric-onset Evans syndrome
In 37 patients (24.5%), pES eventually revealed a SLE or a PID unknown at cytopenia onset.
Eleven patients (7.3%) eventually met the SLE SLICC diagnostic criteria.16 These patients were older at first cytopenia (median age 13 years vs. 5 years; P=0.007) and almost exclusively female (one of 88 males [1%] and ten of 63 females [16%]); P<0.001).
Figure 1. Hematological outcomes. (A) Cumulative incidence of patients achieving a sustained complete remission (CR). Among the 23 patients without sustained CR for autoimmune hemolytic anemia (AIHA), four (17%) had achieved sustained CR for immune thrombocytopenic purpura (ITP). Conversely, among the 32 patients without sustained CR for ITP, 13 (40%) had achieved sustained CR for AIHA. (B) Percentage of patients with a sustained complete remission according to age.
Seven patients (4.6%) met the diagnostic criteria for ALPS after pES onset which prompted targeted genetic analysis.17 Overall, 66 of 151 patients (44%) underwent genetic analyses as previously described.13 Among them, 26 (39%) patients were considered to have a PID (including the seven with ALPS). They carried a heterozygous pathogenic variant in CTLA4 (n=7), TNFRSF6 (germline n=6, somatic n=1), STAT3 (n=5), PIK3CD (n=1), CBL (n=1), and KRAS (somatic n=1) or a homozygous/compound heterozygous pathogenic variants in LRBA (n=3) and RAG1 (n=1). Compared to the 40 other patients, the 26 with a PID had more cIM (2 [range, 1-5] vs. 1 [range, 0-4], P=0.008) and a trend toward more bIM as shown by Wilcoxon–Mann–Whitney sum ranks comparison but same medians (1 [range, 0-3] vs. 1 [range, 0-2], P=0.029). There was no statistically significant difference in the median time to ITP CR (4.7 years vs. 8.0 years, P=0.26) and to AIHA CR (5.5 years vs. 5.5 years, P>0.9), the number of second-line treatment received (3 [range, 0-9] vs. 2 [range, 0-6]; P=0.057) and mortality (two of 26 [7.7%] vs. three of 40 [7.5%]; P>0.9).
Figure 2. Immunopathological manifestations. (A) Age at first clinical immunopathological manifestation (cIM) diagnosis and at first cytopenia. Pearson correlation coefficient r=0.42, P<0.0001. There was no difference in the median age at first cIM and at first cytopenia in terms of the number of cIM (data not shown). (B) Cumulative incidence of cIM according to age. Half of the patients had developed a cIM by the age of 13.5 years and a second IM by the age of 27 years. (C) Cumulative incidence of any biological IM (bIM), as well as each category. Half of the patients had at least one bIM diagnosed by 13.2 years of age. The biological workup was not standardized and was made at the clinician’s discretion. SLE: systemic lupus erythematosus; ALPS: autoimmune lymphoproliferative syndrome.
Figure 3. Immunopathological manifestations and other associated manifestations. Individual occurrence of autoimmune neutropenia, clinical immunopathological manifestations (cIM), biological IM (bIM), atopy, severe or recurrent infections, and malignancies. Each column represents a patient. The patients are ordered according to their cIM, from the most (lymphoproliferation) to the least (hematological, other) frequent. Hypoγ: hypogammaglobulinemia; SLE: systemic lupus erythematosus; ALPS: autoimmune lymphoproliferative syndrome.
Table 2. Most frequent clinical immunopathological manifestations diagnosis.
Treatments
All except two patients (98.6%) had received at least one first-line treatment course. Second-line treatments (regardless of the hematological and/or extra-hematological indication) were required in 117 of 151 (77%) patients (Online Supplementary Figure 3A). Patients who did not receive any second-line treatment had shorter median follow- up times (10.5 years vs. 12.3 years; P=0.017). The median number of second-line treatments received was two (range, 0–9).
The number of second-line treatments received increased with the time elapsed since first cytopenia without reaching a plateau (Online Supplementary Figure 3B). After a sustained CR for both ITP and AIHA achieved, the number of treatments received had continued to increase: at 5 years after CR of both cytopenias, 67% of patients who achieved CR for both ITP and AIHA had received a new first and/or second-line treatments and 31% had received a new second-line treatment (Online Supplementary Figure S3C).
The number of second-line treatments received increased with age, particularly after the first decade (Figure 4A). At 10 and 20 years, 47% and 88% of patients had received a second-line treatment, respectively (P<0.001). The number of patients receiving ongoing treatments also increased with age (Figure 4B). At 10 and 20 years, 27% and 69% of patients had received an active second-line treatment, respectively (P<0.001). At the final follow-up, patients with a cIM had received more secondline treatments (median, 3 vs. 1; P<0.0001).
The most frequently used second-line treatments were rituximab (n=79; 52%), azathioprine (n=55; 36%), splenectomy (n=36; 24%), and mycophenolate (n=29; 19%; Online Supplementary Table S3).
The number of cIM was associated with a subsequent increase in the number of second-line treatments received (HR 1.4; 95% CI: 1.15–1.60; P=0.0002). On the contrary, the number of second-line treatment was not associated to a subsequent increase in the number of cIM in univariate analysis (HR 1.09; 95% CI: 0.98–1.22; P=0.11).
Infections
In total, 53 (35%) patients had severe or recurrent infections (Online Supplementary Table S4). The most frequent were herpes zoster (n=17), sinusitis/otitis media (n=15), pneumopathy (n=12), and bronchiectasis (n=11). Patients with infections had more cIM (median, 2 vs. 1; P<0.0001), a higher incidence of hypogammaglobulinemia (53% vs. 28%; P=0.003), and received more second-line treatments (median 3 vs. 1; P<0.0001). Among the 16 patients with severe infection, nine (63%) were receiving an active treatment at infection time.
Figure 4. Second-line treatments. (A) Total number of second-line treatments received according to the age. (B) Number of second-line treatments ongoing according to age.
Severe/recurrent infections were independently associated with hypogammaglobulinemia (OR 2.4; 95% CI: 1.10–5.33; P=0.03) and the number of second-line treatments (OR 1.34; 95% CI: 1.13–1.71; P=0.002).
Mortality
Sixteen of the 151 patients followed for more than 5 years (10.6%) died, and seven other patients died before the fifth year of follow-up (23 deaths in total, 22 with available data). Patient survival at 5, 10, and 15 years after the first cytopenia was 97%, 92%, and 84%, respectively (Figure 5A). Mortality rates in patients with pES were higher than those in patients with cITP or AIHA alone (P<0.0001 for both comparisons).
Deaths occurred regularly throughout the follow-up period (median delay after first cytopenia diagnosis, 8.9 years [range, 0.1–24.3 years]) and at a median age of 18.0 years (range, 1.7–31.5 years) (Figure 5B). In the majority of these patients, cytopenia was under control at the time of death: 15 (65%) and 19 (83%) patients had CR or partial remission from ITP and AIHA, respectively (Figure 5C). Mortality was linked to the disease, the treatment, or both in eight (36%), two (9%), and twelve (55%) cases, respectively. The most frequent cause of death was infections (n=12 [52%]; Online Supplementary Table S5). Four patients (18%) died of a hemorrhage, and all were less than 13 years of age. The patients who died from a hemorrhage were younger than those who died from an infection (median 10 years vs. 18 years; P=0.03). All of these patients, except for one who died in the first month of a cerebral hemorrhage, had at least one cIM. Eight of the patients (36%) had hypogammaglobulinemia.
Figure 5. Long-term survival. (A) Survival estimate of patients in terms of time from first cytopenia. At 10-year follow- up, survival rates among patients with chronic immune thrombocytopenic purpura (ITP) alone, autoimmune hemolytic anemia (AIHA) alone and pediatric-onset Evans syndrome (pES) were 100%, 99% and 92%, respectively. (B) Mortality is shown in terms of time from first cytopenia, as well as age. Individual values are shown as dots with medians and interquartile ranges shown as lines. (C) Hematological status at death. CR: complete remission; PR: partial remission; NR: no remission.
The patients who died had received more second-line treatments than the others in the cohort (median 3 vs. 2; P=0.02), including splenectomy, which was more common in this subgroup (56% vs. 20%; P=0.003). Patients who had received more than two second-line treatments had a three-fold increase in the risk of death compared to those who had received two or less (11 of 65 [16.9%] vs. five of 86 [5.8%], P=0.03). At death, 81% of patients were receiving ongoing second-line treatment. The number of second-line treatments (HR 1.3; 95% CI: 1.1–1.6; P=0.004) and severe/recurrent infections (HR 3.4; 95% CI: 1.2–9.7; P=0.02) were independently associated with a higher risk of mortality after 5 years of follow-up.
Discussion
This large follow-up study of pES patients included more than 1,900 patient-years. Over the long term, AIHA and ITP were sustainably controlled in the majority of patients. Conversely, clinical and biological IM increased in frequency and number with increasing patient age, finally affecting almost all adult patients. The number of cIM was associated with a subsequent increase in the number of second-line treatments received. Mortality was high, frequently occurred while cytopenias were in remission, and most deaths concerned AYA. Two characteristics were associated to mortality: severe or recurrent infections and the number of second-line treatments received. Overall, the age-related clinical picture showed similar trends for all patients, shifting from cytopenia to increased IM, a greater treatment burden, and an increased risk of mortality.
In setting up a nationwide cohort, the CEREVANCE group tried to ensure unbiased patient inclusion in this study. Omitting patients with less than 5 years of followup data limited any bias due to short-term follow-up, which probably accounts for many of the discrepancies between previous studies. Indeed, our median follow-up period was more than twice as long as in previous studies (median 4.8 years [range, 3–7 years]).1,3–7 However, although the trends reported here are clear, some factors may also influence the estimates. The loss to follow-up mainly concerned AYA and few patients were followed after the age of 20 years. As well, the CEREVANCE group recommends clinical and biological follow-up at least every 6-12 months but local practice or patients’ phenotype (such as the presence of cIM) may have influenced biological testing.
Sustained CR was eventually achieved for both types of cytopenia in the vast majority of patients, although this often took many years, especially for ITP (>10 years for one-third of our patients). Because active treatments are used to treat most AYA (notably because of cIM), hematological CR may be drug induced and it is impossible to determine whether an underlying hematological autoimmunity is still present. The higher rate of sustained CR in ITP among patients with pES compared to patients with cITP alone may be due to more patients with pES receiving treatment.18
One of the most striking findings in this study was the progressive increase in the frequency and number of IM. A range of cIM, affecting almost every organ, were identified and developed independently of cytopenias. These findings clearly show that pES is a marker for a more general tendency toward immunodeficiencies while we cannot exclude a contribution of the second-line treatments received to some IM. The underlying etiology is not completely understood and may vary among patients, with both genetic and environmental factors being important. Consequently, pES may be considered a composite syndrome with several overlapping subgroups of secondary pES. One of these subgroups includes patients with PID. Classically, ALPS has been associated with pES.12 In this study, only 4% of patients were diagnosed with ALPS based on well-defined criteria, despite evocative biological “ALPS-like” abnormalities in a larger proportion of patients.17 This observation is consistent with our previous study,13 which showed that more immune-response genes are potentially involved in pES than initially suspected. 13,19–21 However, pES rarely comports as a Mendelian disease,1 and some of these variants may be predisposing rather than disease-causing alleles. Even in patients carrying a variant in a monogenic PID gene (e.g., TNFRFS6 or CTLA4),13,22 the altered genes show incomplete penetrance.23,24 We were unable to evaluate the proportion of patients who met common variable immunodeficiency disorders diagnostic criteria,25 as vaccine responses were not evaluable in all cases due to secondline treatments received. A second subgroup includes patients with SLE, although the prevalence of this subgroup is controversial.11,26,27 Our cohort suggests that SLE eventually occurs almost exclusively within the known atrisk population of female adolescents and is frequent in this subgroup, as it developed in seven of 15 (47%) of the females >12 years old.26 Despite its heterogeneity, the course of pES, in terms of age-related changes and trends, was similar for the majority of patients. The spectrum of IM described here is probably influenced by the underlying etiology, and further analyses are needed to understand the determinant of IM. The long-term follow-up of the present study confirms that the subgroup of patients with identified PID had more cIM.13
Most patients required second-line treatments. These treatments reflect local practices and we cannot draw conclusions regarding their efficacy. We were unable to investigate the risk associated to specific treatments given the high heterogeneity in second-line treatment combinations and duration as well as the changes in management practices since the cohort onset in 2004. The rapid initial increase in second-line treatments is partly due to the high rate of early relapse and the current practice of administering steroid-sparing agents to treat pES.28 However, the presence of cytopenia is not the only reason for using these drugs and first- and second-line treatments were also used after CR of both cytopenias. cIM are important in determining the number of second-line treatments used, but bIM may also play a role, particularly in patients with SLE biomarkers, who are frequently given hydroxychloroquine. Nevertheless, second-line treatments are rarely selected based on a single factor. Patients with pES often have bIM and cIM, and the whole clinical picture needs to be assessed before selecting a treatment strategy. As previously reported,13 approximately one-third of patients may carry alterations in genes that are potentially accessible to targeted therapy.29–31 Given the high burden of second-line treatments and their association with infections and mortality, the CEREVANCE network has proposed implementing genetic analyses for all patients with pES to limit the use of immunosuppressive and toxic drugs.
Comprehensively, the pES clinical picture changes as patients age. From 10 to 20 years of age, cytopenia tends to be controlled but IM are more prevalent, and active second- line treatments are used in more than two-thirds of patients during the pediatric-to-adult transition. Overall, as patients age, the illness becomes more severe and the risk of mortality increases. Both IM and treatment burden contribute to the infection-related mortality peak observed at the end of the second decade. The patients who died had received more second-line treatments, including splenectomy. Because these two parameters are correlated (r=0.60; P<0.0001), the number of deaths was too low to determine whether splenectomy alone was a risk factor of mortality per se or a marker of severity.
In conclusion, pES must now be considered a complex multi-systemic disease in which cytopenias frequently present fewer challenges than IM and infections in longterm follow-up. Adult patients with pES form a specific subgroup, distinct from older adults with ES.32 Multidisciplinary follow-up of patients with pES is needed and must focus on IM screening, genetic diagnosis, infections prevention, patient-tailored drugs development, and AYA management. Specifically, the infection burden may be reduced by ensuring up-to-date vaccinations, eradicating chronic infections, and using adequate antimicrobial prophylaxis or immunoglobulin replacements. As in several chronic pediatric diseases,33 dedicated child-to-adult transition programs are warranted to improve outcomes in patients with pES.
Supplementary Material
Supplementary Appendix
Acknowledgments
The list of collaborators is given in the Online Supplementary Appendix. The authors would like to thank all of the patients, families, medical and para-medical teams involved in the CEREVANCE prospective cohort study from 2004 onwards. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, RITUXIMAB, VINCRISTINE | DrugsGivenReaction | CC BY-NC | 33440924 | 20,702,383 | 2021-01-14 |
What was the outcome of reaction 'Sepsis'? | Long term follow-up of pediatric-onset Evans syndrome: broad immunopathological manifestations and high treatment burden.
Pediatric-onset Evans syndrome (pES) is defined by both immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) before the age of 18 years. There have been no comprehensive long-term studies of this rare disease, which can be associated to various immunopathological manifestations (IMs). We report outcomes of the 151 patients with pES and more than 5 years of follow-up from the nationwide French prospective OBS'CEREVANCE cohort. Median age at final follow-up was 18.5 (6.8-50.0) years and the median follow-up period was 11.3 (5.1-38.0) years. At 10 years, ITP and AIHA were in sustained complete remission in 54.5% and 78.4% of patients, respectively. The frequency and number of clinical and biological IMs increased with age: at 20 years old, 74% had at least one clinical cIM. A wide range of cIMs occurred, mainly lymphoproliferation, dermatological, gastrointestinal/hepatic and pneumological IMs. The number of cIMs was associated with a subsequent increase in the number of second-line treatments received (other than steroids and immunoglobulins; hazard ratio, 1.4; 95% confidence interval, 1.15-1.60; p = 0.0002, Cox proportional hazards method). Survival at 15 years after diagnosis was 84%. Death occurred at a median age of 18 (1.7-31.5) years, and the most frequent cause was infection. The number of second-line treatments and severe/recurrent infections were independently associated with mortality. In conclusion, longterm outcomes of pES showed remission of cytopenias but frequent IMs linked to high secondline treatment burden. Mortality was associated to drugs and/or underlying immunodeficiencies, and adolescents-young adults are a high-risk subgroup.
pmcIntroduction
The presence of both immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) defines Evans syndrome (ES). Pediatric-onset ES (pES) is a rare disease, and approximately ten new cases are diagnosed every year in France, which has a population of 66 million.1 Since its first description in 1951 by Robert Evans,2 our understanding of pES has been based on small retrospective cohorts with limited follow-up.3-7 In 2004, the French Rare Disease Center CEREVANCE set up the prospective national cohort OBS’CEREVANCE, which includes children with AIHA, chronic ITP persisting for more than 12 months (cITP), and pES.8
Preliminary reports from this cohort and previously published studies showed that pES is a chronic disease with a high rate of relapse for both types of cytopenias. 1,3,4,7,9 Mortality rates across studies have ranged from 7–36%.1,3-7 In addition to cytopenias, immunopathological manifestations (IM) such as autoimmune/autoinflammatory organ diseases, lymphoproliferation, and hypogammaglobulinemia have been reported in 70–80% of patients with pES.4,5,8,10 In an undetermined number of cases, pES is thought to be “secondary” and caused by an underlying disease, classically systemic lupus erythematosus (SLE) or autoimmune lymphoproliferative syndrome (ALPS).1,11,12 Recently, genetic analyses found a heterogeneous genetic background in up to 65% of a subset of 80 patients from the OBS’CEREVANCE cohort. These patients carried variants in genes that are linked to primary immunodeficiencies (PID) or involved in immune responses.13
Overall, outcomes and the long-term course of pES are poorly understood. There have been no comprehensive longitudinal studies including both cytopenia and IM. In addition, the transition to adulthood is often particularly challenging for patients with chronic pediatric diseases.14 Adolescents–young adults (AYA) outcomes have not been investigated in patients with pES, and whether the disease improves with age is unknown. In a clinical setting, the possibility to identify high-risk patients would be extremely helpful in the management of this complex disease. Here, we describe the long-term course of hematological IM and treatments received throughout childhood into adulthood in patients with pES from the OBS’CEREVANCE cohort. We aimed to identify clinically relevant factors associated to the occurrence of IM, the number of second-line treatments received and mortality. Particularly, we investigated the impact of the number second-line treatment received and splenectomy on mortality.
Methods
OBS’CEREVANCE prospective national cohort
Inclusion and exclusion criteria are shown in the Online Supplementary Table S1.1,8,15 Data collected have been previously detailed.1,8 Patients were included if <18 years old at first cytopenia diagnosis. The coordinating center gathered and analyzed all data from the medical team in charge in real time, enabling prospective follow-up even after the pediatric-to-adult transition. The CEREVANCE group recommends scheduling clinical and biological follow-up at least every 6-12 months. Some patients underwent genetic analyses, as previously described.13 Written informed consent was obtained from parents and eligible patients. The cohort study was approved by the Institutional Ethics Committee (CPPRB-A; Bordeaux, France) and the database was registered with the national data protection authority (CNIL, 1396823V0).
Patient selection
Patients with pES, defined as the simultaneous (within 1 month) or sequential association of ITP and AIHA, were included if at least 5 years of follow-up data were available after the first cytopenia diagnosis. In order to provide a complete mortality report, all patients, including those with less than 5 years of follow-up data, were included in the survival analyses. The data were extracted on 21 June 2019.
Definitions
Initial cytopenia refers to the onset of ITP or AIHA (whichever occurred first) and does not take autoimmune neutropenia (AIN) into account. The IM categories were separated in clinical (cIM) and biological (bIM). pES was defined as secondary if a diagnosis of SLE or PID was made during the follow-up period. SLE diagnosis was made according to the Systemic Lupus International Collaborating Clinics Classification criteria (SLICC).16 ALPS diagnosis was based on international criteria.17 Second-line treatments were all immunomodulatory or immunosuppressive treatments, including splenectomy but excluding steroids and therapeutic intravenous immunoglobulins (IVIG). Sustained complete remission (CR) was defined as remission persisting until final follow-up, regardless of ongoing treatments.
For analyses by age, patients were assessed annually from birth (for IM and treatments) or from cytopenia onset (for AIHA and ITP), until final follow-up. Occasional treatments (e.g., splenectomy and rituximab) were considered as ongoing if these occurred during the previous year. Further details are stated in the Online Supplementary Table S1.
Statistical analyses
Continuous and categorical variables were compared using Wilcoxon–Mann–Whitney non-parametric test and Fisher’s exact test, respectively. Correlations were tested using the Pearson correlation coefficient. Survival and cumulative incidence estimates were based on the Kaplan–Meier method and compared using log-rank test. Patients of OBS’CEREVANCE cohort with isolated cITP or AIHA were used for comparison in survival analyses (unpublished data). The Cox proportional hazards method was used to analyze factors associated with time-dependent variables (i.e., time to CR, AIN, cIM, and second-line treatment, as well as survival). The potential cumulative and/or time-dependent nature of variables was taken in account. Proportionality of hazard was assessed for each variable. Logistic regression was used to analyze factors associated with severe or recurrent infections. Variables that were statistically significant in the univariate analyses were included in the multivariate analyses. We investigated associations with the following characteristics and events: sex, consanguinity, cIM/cancer in a first-degree relative, age at first and second cytopenia, sequence of cytopenia, AIN, hypogammaglobulinemia, time to ITP/AIHA CR, severe/recurrent infections, number of cIM, number of second-line treatments. The 95% Confidence Intervals (CI) for hazard ratios (HR) and odds ratios (OR) were not adjusted for multiple testing and should not be used to infer definitive effects. All tests were two-sided and a P-value <0.05 was considered statistically significant. Statistical analyses were performed using R (ver. 4.0; R Development Core Team) and GraphPad Prism (ver. 8; GraphPad Software, Inc., San Diego, CA, USA) software.
Results
Population
Of the 216 patients with pES, 151 were included in this study (Online Supplementary Figure S1). They were followed from 25 different centers. Patient characteristics are shown in Table 1. The median (min–max) follow-up time from the first cytopenia diagnosis was 11.3 years (range, 5.1–38.0 years). Median age at final follow-up was 18.5 years (range, 6.8–50.0 years). In 20 cases (15%), follow-up was discontinued because the patient was considered cured (n=11) or lost to follow-up (n=9). Median age at loss to follow-up was 18.4 years (range, 6.8-25.1 years).
Hematological outcomes
AIN developed in 43 patients (28.5%). It was diagnosed within 1 month before or after first cytopenia onset in 23 of 43 cases (53.5%), more than 1 month before in two cases (4.7%), and more than 1 month after in 18 cases (41.9%; maximal delay, 12.4 years). In all cases, the diagnosis was made before the age of 18 years (median age, 6.8 years; range, 0.6–16.2 years).
ITP and AIHA flare rates at 5 years of follow-up were calculated for the 61 alive patients who did not receive a second-line treatment during this period. Forty-eight patients (79%) had experienced an ITP flare and seven (11%) an AIHA flare.
The proportion of patients achieving sustained CR for ITP and AIHA steadily increased after cytopenia onset (Figure 1A). At 5 and 10 years, ITP was in sustained CR in 40.5% and 62.3% of patients (P=0.02) and AIHA was in sustained CR in 54.5% and 74.1% of patients (P=0.001), respectively. Sustained CR was achieved earlier for AIHA than ITP (median time to CR, 4.0 years vs. 7.0 years; P=0.01). At the final follow-up of the 135 surviving patients, the numbers of patients in CR, partial remission, and no remission were 126 (83%), five (3%), and one (1%) for AIHA and 119 (79%), eight (5%), and five (3%) for ITP, respectively (missing data in three cases). Forty-six patients (34%) had no treatment ongoing at last followup. No particular characteristic was associated with AIHA or ITP CR, including cIM and bIM.
Table 1. Patient characteristics.
Over the first three decades, the proportions of patients achieving sustained CR increased with age (Figure 1B). ITP and AIHA were in CR in 26% and 30% of cases at 10 years compared to 50% and 72% at 20 years, respectively (P<0.001 for both comparisons).
Immunopathological manifestations
A total of 122 of 151 patients (81%) had at least one IM. The data for each category and specific diagnosis are shown in the Online Supplementary Table S2.
cIM developed in 100 of 151 patients (66%). A total of 47 patients (31%) had two or more IM and 22 (15%) patients had three or more IM (Online Supplementary Figure S2A). Patients with no cIM had shorter median follow-up times (9.7 years vs. 13 years; P=0.0002) and were younger when data were collected (15 years vs. 20 years; P<0.0001). A cIM was diagnosed before the first cytopenia in 21 of 100, simultaneously in 13, and after in 66 cases (median delay, 3.7 years [range, 0.2–20.5 years]; Figure 2A). Among the 185 cIM, 29 (16%) were diagnosed before any second-line treatment. No cIM category had a statistically significant difference in frequency before and after first second-line treatment. The number of cIM increased with age. At 10 compared to 20 years old, 37% and 74% of patients had at least one cIM and 9% and 34% of patients had at least two cIM, respectively (P<0.001 for both comparisons; Figure 2B).
The most common cIM categories were lymphoproliferation (n=71), dermatological (n= 26), gastrointestinal/hepatic (n=23) and pneumological manifestations (n=16, Figure 3; Online Supplementary Figure S2B). The most frequent cIM diagnosis are shown in Table 2. Thirteen patients developed a neurological manifestation as previously described.10 Four patients had a hematological malignancy (age at diagnosis): Hodgkin lymphoma (16 years), juvenile myelomonocytic leukemia (20 years), large granular lymphocytic leukemia (21 years) and angioimmunoblastic Tcell lymphoma (29 years). Older age at ES diagnosis (HR 1.09; 95% CI: 1.01–1.17; P=0.02), cIM/cancer in a firstdegree relative (HR 1.64; 95% CI: 1.1–2.4; P=0.006), and the presence of AIN were independently associated with the number of cIM (HR 2.41; 95% CI: 1.5–3.8; P=0.0002).
Biological IM (bIM) were diagnosed in 101 of 151 patients (67%), and the frequency of bIM also increased with the age (Figure 2C). Hypogammaglobulinemia was the most frequently diagnosed bIM (n=54), including 44 cases diagnosed prior to any anti-CD20 treatment. Among those 54 patients, 25 (46%) received immunoglobulin replacement therapy. SLE and ALPS biomarkers were present (regardless of whether patients met the diagnostic criteria) in 42 and 24 patients, respectively. At 10 and 20 years of age, 39% and 75% of patients had at least one bIM, respectively (P<0.001).
Patients with bIM were more likely to have cIM (79% vs. 40%; P<0.001), and patients with cIM were more likely to have bIM (80% vs. 41%; P<0.001) but the correlation between the number of bIM and cIM was low (r=0.27; P<0.001).
Secondary pediatric-onset Evans syndrome
In 37 patients (24.5%), pES eventually revealed a SLE or a PID unknown at cytopenia onset.
Eleven patients (7.3%) eventually met the SLE SLICC diagnostic criteria.16 These patients were older at first cytopenia (median age 13 years vs. 5 years; P=0.007) and almost exclusively female (one of 88 males [1%] and ten of 63 females [16%]); P<0.001).
Figure 1. Hematological outcomes. (A) Cumulative incidence of patients achieving a sustained complete remission (CR). Among the 23 patients without sustained CR for autoimmune hemolytic anemia (AIHA), four (17%) had achieved sustained CR for immune thrombocytopenic purpura (ITP). Conversely, among the 32 patients without sustained CR for ITP, 13 (40%) had achieved sustained CR for AIHA. (B) Percentage of patients with a sustained complete remission according to age.
Seven patients (4.6%) met the diagnostic criteria for ALPS after pES onset which prompted targeted genetic analysis.17 Overall, 66 of 151 patients (44%) underwent genetic analyses as previously described.13 Among them, 26 (39%) patients were considered to have a PID (including the seven with ALPS). They carried a heterozygous pathogenic variant in CTLA4 (n=7), TNFRSF6 (germline n=6, somatic n=1), STAT3 (n=5), PIK3CD (n=1), CBL (n=1), and KRAS (somatic n=1) or a homozygous/compound heterozygous pathogenic variants in LRBA (n=3) and RAG1 (n=1). Compared to the 40 other patients, the 26 with a PID had more cIM (2 [range, 1-5] vs. 1 [range, 0-4], P=0.008) and a trend toward more bIM as shown by Wilcoxon–Mann–Whitney sum ranks comparison but same medians (1 [range, 0-3] vs. 1 [range, 0-2], P=0.029). There was no statistically significant difference in the median time to ITP CR (4.7 years vs. 8.0 years, P=0.26) and to AIHA CR (5.5 years vs. 5.5 years, P>0.9), the number of second-line treatment received (3 [range, 0-9] vs. 2 [range, 0-6]; P=0.057) and mortality (two of 26 [7.7%] vs. three of 40 [7.5%]; P>0.9).
Figure 2. Immunopathological manifestations. (A) Age at first clinical immunopathological manifestation (cIM) diagnosis and at first cytopenia. Pearson correlation coefficient r=0.42, P<0.0001. There was no difference in the median age at first cIM and at first cytopenia in terms of the number of cIM (data not shown). (B) Cumulative incidence of cIM according to age. Half of the patients had developed a cIM by the age of 13.5 years and a second IM by the age of 27 years. (C) Cumulative incidence of any biological IM (bIM), as well as each category. Half of the patients had at least one bIM diagnosed by 13.2 years of age. The biological workup was not standardized and was made at the clinician’s discretion. SLE: systemic lupus erythematosus; ALPS: autoimmune lymphoproliferative syndrome.
Figure 3. Immunopathological manifestations and other associated manifestations. Individual occurrence of autoimmune neutropenia, clinical immunopathological manifestations (cIM), biological IM (bIM), atopy, severe or recurrent infections, and malignancies. Each column represents a patient. The patients are ordered according to their cIM, from the most (lymphoproliferation) to the least (hematological, other) frequent. Hypoγ: hypogammaglobulinemia; SLE: systemic lupus erythematosus; ALPS: autoimmune lymphoproliferative syndrome.
Table 2. Most frequent clinical immunopathological manifestations diagnosis.
Treatments
All except two patients (98.6%) had received at least one first-line treatment course. Second-line treatments (regardless of the hematological and/or extra-hematological indication) were required in 117 of 151 (77%) patients (Online Supplementary Figure 3A). Patients who did not receive any second-line treatment had shorter median follow- up times (10.5 years vs. 12.3 years; P=0.017). The median number of second-line treatments received was two (range, 0–9).
The number of second-line treatments received increased with the time elapsed since first cytopenia without reaching a plateau (Online Supplementary Figure 3B). After a sustained CR for both ITP and AIHA achieved, the number of treatments received had continued to increase: at 5 years after CR of both cytopenias, 67% of patients who achieved CR for both ITP and AIHA had received a new first and/or second-line treatments and 31% had received a new second-line treatment (Online Supplementary Figure S3C).
The number of second-line treatments received increased with age, particularly after the first decade (Figure 4A). At 10 and 20 years, 47% and 88% of patients had received a second-line treatment, respectively (P<0.001). The number of patients receiving ongoing treatments also increased with age (Figure 4B). At 10 and 20 years, 27% and 69% of patients had received an active second-line treatment, respectively (P<0.001). At the final follow-up, patients with a cIM had received more secondline treatments (median, 3 vs. 1; P<0.0001).
The most frequently used second-line treatments were rituximab (n=79; 52%), azathioprine (n=55; 36%), splenectomy (n=36; 24%), and mycophenolate (n=29; 19%; Online Supplementary Table S3).
The number of cIM was associated with a subsequent increase in the number of second-line treatments received (HR 1.4; 95% CI: 1.15–1.60; P=0.0002). On the contrary, the number of second-line treatment was not associated to a subsequent increase in the number of cIM in univariate analysis (HR 1.09; 95% CI: 0.98–1.22; P=0.11).
Infections
In total, 53 (35%) patients had severe or recurrent infections (Online Supplementary Table S4). The most frequent were herpes zoster (n=17), sinusitis/otitis media (n=15), pneumopathy (n=12), and bronchiectasis (n=11). Patients with infections had more cIM (median, 2 vs. 1; P<0.0001), a higher incidence of hypogammaglobulinemia (53% vs. 28%; P=0.003), and received more second-line treatments (median 3 vs. 1; P<0.0001). Among the 16 patients with severe infection, nine (63%) were receiving an active treatment at infection time.
Figure 4. Second-line treatments. (A) Total number of second-line treatments received according to the age. (B) Number of second-line treatments ongoing according to age.
Severe/recurrent infections were independently associated with hypogammaglobulinemia (OR 2.4; 95% CI: 1.10–5.33; P=0.03) and the number of second-line treatments (OR 1.34; 95% CI: 1.13–1.71; P=0.002).
Mortality
Sixteen of the 151 patients followed for more than 5 years (10.6%) died, and seven other patients died before the fifth year of follow-up (23 deaths in total, 22 with available data). Patient survival at 5, 10, and 15 years after the first cytopenia was 97%, 92%, and 84%, respectively (Figure 5A). Mortality rates in patients with pES were higher than those in patients with cITP or AIHA alone (P<0.0001 for both comparisons).
Deaths occurred regularly throughout the follow-up period (median delay after first cytopenia diagnosis, 8.9 years [range, 0.1–24.3 years]) and at a median age of 18.0 years (range, 1.7–31.5 years) (Figure 5B). In the majority of these patients, cytopenia was under control at the time of death: 15 (65%) and 19 (83%) patients had CR or partial remission from ITP and AIHA, respectively (Figure 5C). Mortality was linked to the disease, the treatment, or both in eight (36%), two (9%), and twelve (55%) cases, respectively. The most frequent cause of death was infections (n=12 [52%]; Online Supplementary Table S5). Four patients (18%) died of a hemorrhage, and all were less than 13 years of age. The patients who died from a hemorrhage were younger than those who died from an infection (median 10 years vs. 18 years; P=0.03). All of these patients, except for one who died in the first month of a cerebral hemorrhage, had at least one cIM. Eight of the patients (36%) had hypogammaglobulinemia.
Figure 5. Long-term survival. (A) Survival estimate of patients in terms of time from first cytopenia. At 10-year follow- up, survival rates among patients with chronic immune thrombocytopenic purpura (ITP) alone, autoimmune hemolytic anemia (AIHA) alone and pediatric-onset Evans syndrome (pES) were 100%, 99% and 92%, respectively. (B) Mortality is shown in terms of time from first cytopenia, as well as age. Individual values are shown as dots with medians and interquartile ranges shown as lines. (C) Hematological status at death. CR: complete remission; PR: partial remission; NR: no remission.
The patients who died had received more second-line treatments than the others in the cohort (median 3 vs. 2; P=0.02), including splenectomy, which was more common in this subgroup (56% vs. 20%; P=0.003). Patients who had received more than two second-line treatments had a three-fold increase in the risk of death compared to those who had received two or less (11 of 65 [16.9%] vs. five of 86 [5.8%], P=0.03). At death, 81% of patients were receiving ongoing second-line treatment. The number of second-line treatments (HR 1.3; 95% CI: 1.1–1.6; P=0.004) and severe/recurrent infections (HR 3.4; 95% CI: 1.2–9.7; P=0.02) were independently associated with a higher risk of mortality after 5 years of follow-up.
Discussion
This large follow-up study of pES patients included more than 1,900 patient-years. Over the long term, AIHA and ITP were sustainably controlled in the majority of patients. Conversely, clinical and biological IM increased in frequency and number with increasing patient age, finally affecting almost all adult patients. The number of cIM was associated with a subsequent increase in the number of second-line treatments received. Mortality was high, frequently occurred while cytopenias were in remission, and most deaths concerned AYA. Two characteristics were associated to mortality: severe or recurrent infections and the number of second-line treatments received. Overall, the age-related clinical picture showed similar trends for all patients, shifting from cytopenia to increased IM, a greater treatment burden, and an increased risk of mortality.
In setting up a nationwide cohort, the CEREVANCE group tried to ensure unbiased patient inclusion in this study. Omitting patients with less than 5 years of followup data limited any bias due to short-term follow-up, which probably accounts for many of the discrepancies between previous studies. Indeed, our median follow-up period was more than twice as long as in previous studies (median 4.8 years [range, 3–7 years]).1,3–7 However, although the trends reported here are clear, some factors may also influence the estimates. The loss to follow-up mainly concerned AYA and few patients were followed after the age of 20 years. As well, the CEREVANCE group recommends clinical and biological follow-up at least every 6-12 months but local practice or patients’ phenotype (such as the presence of cIM) may have influenced biological testing.
Sustained CR was eventually achieved for both types of cytopenia in the vast majority of patients, although this often took many years, especially for ITP (>10 years for one-third of our patients). Because active treatments are used to treat most AYA (notably because of cIM), hematological CR may be drug induced and it is impossible to determine whether an underlying hematological autoimmunity is still present. The higher rate of sustained CR in ITP among patients with pES compared to patients with cITP alone may be due to more patients with pES receiving treatment.18
One of the most striking findings in this study was the progressive increase in the frequency and number of IM. A range of cIM, affecting almost every organ, were identified and developed independently of cytopenias. These findings clearly show that pES is a marker for a more general tendency toward immunodeficiencies while we cannot exclude a contribution of the second-line treatments received to some IM. The underlying etiology is not completely understood and may vary among patients, with both genetic and environmental factors being important. Consequently, pES may be considered a composite syndrome with several overlapping subgroups of secondary pES. One of these subgroups includes patients with PID. Classically, ALPS has been associated with pES.12 In this study, only 4% of patients were diagnosed with ALPS based on well-defined criteria, despite evocative biological “ALPS-like” abnormalities in a larger proportion of patients.17 This observation is consistent with our previous study,13 which showed that more immune-response genes are potentially involved in pES than initially suspected. 13,19–21 However, pES rarely comports as a Mendelian disease,1 and some of these variants may be predisposing rather than disease-causing alleles. Even in patients carrying a variant in a monogenic PID gene (e.g., TNFRFS6 or CTLA4),13,22 the altered genes show incomplete penetrance.23,24 We were unable to evaluate the proportion of patients who met common variable immunodeficiency disorders diagnostic criteria,25 as vaccine responses were not evaluable in all cases due to secondline treatments received. A second subgroup includes patients with SLE, although the prevalence of this subgroup is controversial.11,26,27 Our cohort suggests that SLE eventually occurs almost exclusively within the known atrisk population of female adolescents and is frequent in this subgroup, as it developed in seven of 15 (47%) of the females >12 years old.26 Despite its heterogeneity, the course of pES, in terms of age-related changes and trends, was similar for the majority of patients. The spectrum of IM described here is probably influenced by the underlying etiology, and further analyses are needed to understand the determinant of IM. The long-term follow-up of the present study confirms that the subgroup of patients with identified PID had more cIM.13
Most patients required second-line treatments. These treatments reflect local practices and we cannot draw conclusions regarding their efficacy. We were unable to investigate the risk associated to specific treatments given the high heterogeneity in second-line treatment combinations and duration as well as the changes in management practices since the cohort onset in 2004. The rapid initial increase in second-line treatments is partly due to the high rate of early relapse and the current practice of administering steroid-sparing agents to treat pES.28 However, the presence of cytopenia is not the only reason for using these drugs and first- and second-line treatments were also used after CR of both cytopenias. cIM are important in determining the number of second-line treatments used, but bIM may also play a role, particularly in patients with SLE biomarkers, who are frequently given hydroxychloroquine. Nevertheless, second-line treatments are rarely selected based on a single factor. Patients with pES often have bIM and cIM, and the whole clinical picture needs to be assessed before selecting a treatment strategy. As previously reported,13 approximately one-third of patients may carry alterations in genes that are potentially accessible to targeted therapy.29–31 Given the high burden of second-line treatments and their association with infections and mortality, the CEREVANCE network has proposed implementing genetic analyses for all patients with pES to limit the use of immunosuppressive and toxic drugs.
Comprehensively, the pES clinical picture changes as patients age. From 10 to 20 years of age, cytopenia tends to be controlled but IM are more prevalent, and active second- line treatments are used in more than two-thirds of patients during the pediatric-to-adult transition. Overall, as patients age, the illness becomes more severe and the risk of mortality increases. Both IM and treatment burden contribute to the infection-related mortality peak observed at the end of the second decade. The patients who died had received more second-line treatments, including splenectomy. Because these two parameters are correlated (r=0.60; P<0.0001), the number of deaths was too low to determine whether splenectomy alone was a risk factor of mortality per se or a marker of severity.
In conclusion, pES must now be considered a complex multi-systemic disease in which cytopenias frequently present fewer challenges than IM and infections in longterm follow-up. Adult patients with pES form a specific subgroup, distinct from older adults with ES.32 Multidisciplinary follow-up of patients with pES is needed and must focus on IM screening, genetic diagnosis, infections prevention, patient-tailored drugs development, and AYA management. Specifically, the infection burden may be reduced by ensuring up-to-date vaccinations, eradicating chronic infections, and using adequate antimicrobial prophylaxis or immunoglobulin replacements. As in several chronic pediatric diseases,33 dedicated child-to-adult transition programs are warranted to improve outcomes in patients with pES.
Supplementary Material
Supplementary Appendix
Acknowledgments
The list of collaborators is given in the Online Supplementary Appendix. The authors would like to thank all of the patients, families, medical and para-medical teams involved in the CEREVANCE prospective cohort study from 2004 onwards. | Fatal | ReactionOutcome | CC BY-NC | 33440924 | 20,702,383 | 2021-01-14 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cardiac disorder'. | A virtual-hybrid approach to launching a cardio-oncology clinic during a pandemic.
BACKGROUND
As cardiovascular disease is a leading cause of death in cancer survivors, the new subspecialty of Cardio-Oncology has emerged to address prevention, monitoring, and management of cardiovascular toxicities to cancer therapies. During the coronavirus disease of 2019 (COVID-19) pandemic, we developed a Virtual-Hybrid Approach to build a de novo Cardio-Oncology Clinic.
METHODS
We conceptualized a Virtual-Hybrid Approach including three arms: information seeking in locations with existing Cardio-Oncology clinics, information gathering at the location for a new clinic, and information sharing to report clinic-building outcomes. A retrospective review of outcomes included collection and synthesis of data from our first 3 months (at pandemic peak) on types of appointments, cancers, drugs, and cardiotoxicities. Data were presented using descriptive statistics.
RESULTS
A de-novo Cardio-Oncology clinic was developed structured from the ground up to integrate virtual and in-person care in a hybrid and innovative model, using the three arms of the Virtual-Hybrid Approach. First, we garnered in-person and virtual preparation through hands-on experiences, training, and discussions in existing Cardio-Oncology Clinics and conferences. Next, we gleaned information through virtual inquiry and niche-building. With partners throughout the institution, a virtual referral process was established for outpatient referrals and inpatient e-consult referrals to actualize a hybrid care spectrum for our patients administered by a multidisciplinary hybrid care team of clinicians, ancillary support staff, and clinical pharmacists. Among the multi-subspecialty clinic sessions, approximately 50% were in Cardio-Oncology, 20% in Preventive Cardiology, and 30% in General Cardiology. In the hybrid model, the Heart & Vascular Center had started to re-open, allowing for 65% of our visits to be in person. In additional analyses, the most frequent cardiovascular diagnosis was cardiomyopathy (34%), the most common cancer drug leading to referral was trastuzumab (29%), and the most prevalent cancer type was breast cancer (42%).
CONCLUSIONS
This Virtual-Hybrid Approach and retrospective review provides guidance and information regarding initiating a brand-new Cardio-Oncology Clinic during the pandemic for cancer patients/survivors. This report also furnishes virtual resources for patients, virtual tools for oncologists, cardiologists, and administrators tasked with starting new clinics during the pandemic, and innovative future directions for this digital pandemic to post-pandemic era.
Introduction
Cardio-Oncology care has been adjusted in the COVID-19 pandemic with limited in-person clinic or hospital visits, increased use of teleconsultation, less frequent imaging, increased reliance on biomarkers, and considerations of differential diagnoses involving COVID-19 when evaluating cancer patients or survivors for possible cardiovascular toxicity [1]. Monitoring and management algorithms have been developed to help guide virtual care [2–4]. In the pandemic, we have changed the way in which we provide healthcare services at our clinics and institutions. This has challenged us to restructure current systems for the safety of our patients.
Various forms of innovation have come to bear in the pandemic, including telemedicine, digital health, artificial intelligence, social media, informatics, big data, and precision medicine [5, 6]. Telemedicine is the primary form of innovation that has been most developed in the pandemic [2, 5, 7]. Social media has been very helpful for dissemination of information, as well as education, and has been integral for creating online groups for support and determining the best ways for proceeding in the pandemic and advocating for our patients and colleagues in this period [5, 6]. In addition, the Doximity social media application has been valuable to practices across the nation, due to its telehealth platform (Doximity Video and Phone; https://www.doximity.com/dialer-video).
Despite the growing need, and allowances made during the pandemic, many centers do not have formal Cardio-Oncology clinics. Starting a new clinic can be challenging. The COVID-19 pandemic has made the process significantly more difficult, with the need to minimize exposure and maximize patient safety.
Currently, limited information is available on how to start a Cardio-Oncology Clinic during a pandemic, albeit given the high risk of morbidity or mortality in COVID-19-positive patients who also have cancer or CVD [8–13]. Several institutions have published on their experiences with starting in-person Cardio-Oncology clinics prior to the pandemic [8, 14–17]. One group has reported on their conversion from existing in-person Cardio-Oncology visits to telemedicine consultations, seeing 11 patients virtually within a few weeks [7]. Many have considered implications of the pandemic on the practice and study of cardio-oncology [2, 4, 5, 18–20], and two groups have suggested models for clinics converting from existing in-person care to televisits [4, 5]. Yet, no groups have directly addressed steps for de novo virtual-hybrid clinic formation within the limitations of the pandemic and without conversion of a pre-existing Cardio-Oncology clinic.
Our report offers a template for other centers to develop their own new Cardio-Oncology clinics during the pandemic. We determined a Virtual-Hybrid Approach to clinic launch, with both virtual and in-person elements of three key arms: information seeking where there are existing Cardio-Oncology Clinics in place, information gathering where the clinic will be built, and information sharing to report on initial patient data demonstrating the success of the launch (Fig. 1). We then performed retrospective chart review to collect and synthesize data on the types of appointments (new versus established, virtual versus in-person), cancers (e.g., breast, prostate, leukemia, lung), cancer drugs, and cardiovascular toxicities (e.g., cardiomyopathy, hypertension) for patients seen virtually or in person in our new Cardio-Oncology clinic at Froedtert Hospital and Medical College of Wisconsin (F&MCW). Here, we will discuss our findings in the context of previous publications on launching Cardio-Oncology Clinics prior to the pandemic. Our results will present distributions of cancer drugs and types, and cardiovascular diagnoses, similar to previous publications on Cardio-Oncology clinic-building. However, we will differentiate and illuminate the techniques that leverage the virtual underpinnings of pandemic clinic-building. We submit that it is feasible to establish a new Cardio-Oncology Clinic for cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy during a pandemic, providing optimal care for new patients in the midst of the need for safety and minimizing exposure. We also propose virtual resources for patients and clinicians and describe innovative future directions in the pandemic and post-pandemic period.
Fig. 1 The Virtual-Hybrid Approach to Cardio-Oncology Clinic-building in the pandemic
Methods
Virtual-hybrid approach
We pursued a Virtual-Hybrid Approach of information seeking, information gathering, and information sharing (Fig. 1). For information seeking, we focused on institutions that already had a Cardio-Oncology clinic in place. Prior to the COVID-19 pandemic, substantial experience was gained at an established in-person Cardio-Oncology clinic at a world-renowned leading hospital. Published manuscripts on pre-pandemic building and operations of Cardio-Oncology clinics at other institutions were reviewed. Extensive networking with leaders of Cardio-Oncology clinics around the United States and in the United Kingdom was accomplished at regional, national, and international meetings in person and on social media in a hybrid approach. These meetings were attended in person pre-COVID-19 and virtually during the pandemic. For information gathering, we addressed the location in which the new Cardio-Oncology clinic would be built. We learned about existing resources in the destination Heart & Vascular Center and existing needs and patients in the destination partner cancer center. We networked with physicians, advanced practice providers (APPs), nurses, service line leaders, and administrators in the Heart & Vascular Center and the cancer center, as well as in primary care and other supporting specialties. The subsequent Cardio-Oncology clinic sessions were intermingled with other subspecialty areas, to optimize availability for patient visits while filling clinic slots and tailoring spectrum of care to emerging patient needs. Cardio-Oncology patient visits were included in multi-subspecialty clinics.
Retrospective study design
For quantitative data, we pursued a retrospective observational study to determine the distribution of outpatient visits in the first 3 months of our virtual-hybrid Cardio-Oncology Clinic. We reviewed data from charts of patients (all were 18 years of age or older) who received outpatient care from the new Director of Cardio-Oncology at F&MCW between April 15, 2020 and July 17, 2020 to determine which of these patients were considered to be in Cardio-Oncology, Preventive Cardiology, or General Cardiology. We collated all three to determine the percentage of patients seen in Cardio-Oncology, compared to the other two specialties. Preventive Cardiology was collated as a partner clinic to help build the Preventive Cardio-Oncology component of the Cardio-Oncology Clinic, to help apply established principles for prevention. From among the multi-subspecialty clinic sessions, we determined the proportion of patients who were specifically cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy and thereby seen in the Cardio-Oncology Clinic. Next, we identified the distribution of cardiovascular toxicities in cancer patients or survivors seen in the Cardio-Oncology Clinic. We also evaluated the spectrum of cancer drugs received by cancer patients or survivors seen in the Cardio-Oncology Clinic. In addition, we summarized the types of cancers in patients seen in the Cardio-Oncology Clinic. Finally, we assessed the frequency of virtual visits during the course of the pandemic over our first 3 months for patients seen in the Cardio-Oncology Clinic. This retrospective review was approved by the F&MCW IRB; HIPAA informed consent was waived for this minimal risk study, which did not involve any form of intervention and was conducted in compliance with good clinical and research practice. The team designed and carried out the study with reliance on virtual communication tools.
Data collection and analysis
Data gathering, management, and analysis were conducted at F&MCW. We collected patient-related, disease-related, treatment-related, and outcome-related data, particularly patient sex, type of appointment (new versus established, virtual versus in-person), type of cancer (e.g., breast, prostate, leukemia, lung), type of cancer drug, and type of cardiovascular toxicity (e.g., cardiomyopathy, hypertension). In order to minimize any risk of breaching patient confidentiality, all data collection occurred on institutional-based computing environments with de-identified data used for analyses. There were no alternative procedures for the subjects as this is a retrospective review of data that are not amenable to prospective collection and review. Descriptive graphs or tables of patient-, disease-, treatment-, and outcome-related variables distributions were prepared, with no comparisons made needing statistical tests.
Results
Virtual preparation
Preparation for starting the Cardio-Oncology clinic followed a Virtual-Hybrid Approach (Fig. 1, left). Five overarching factors employing virtual communication methods emerged to ensure the successful launching of the clinic. Team and individual experience and exposure to various areas of interest in Cardio-Oncology were achieved and assessed before and during the pandemic. Far-reaching connections to experts and potential collaborators in the field were developed and exercised. Close contact with the institution launching this clinic was important to determine the resources available; these resources dictated the strategy and potential outcomes of the clinic. Importantly, the expectations of others for the Cardio-Oncology clinic were determined and incorporated. Finally, recognition of the limitations that exist at the destination institution guided care and goal setting.
Virtual inquiry
Before initiating the Cardio-Oncology clinic in the destination institution, existing structures, patient base, and needs in the Heart & Vascular Center as well as the Cancer Center were evaluated, adhering to pandemic protocols (Fig. 1, middle). Pre-existing building blocks for the planned Cardio-Oncology clinic were assessed, and the partner Preventive Cardiology clinic was investigated. We also evaluated characteristics of the cancer center patient population to best position the clinic for success.
Virtual niche-building
Five main aspects of niche-building were pursued. Partnerships with Vascular and Cancer Center physicians, advanced practice providers, and service line leaders were developed to initiate and grow the clinic (Fig. 1, middle). The Cardio-Oncology team and clinic flexibility were demonstrated through openness to taking quicksteps. Presentations were made at Grand Rounds and rounds across the institution in Cardiology, Hematology/Oncology, Radiation Oncology, Surgical Oncology, Internal Medicine, and Family Medicine to promote the clinic capabilities. Collaborative solutions for problems facing fields complementing Cardio-Oncology developed trust and collaboration. Teamwork was developed by leveraging diversity of perspectives and virtual communication technologies, to establish effective patient care despite COVID-19 limitations.
Hybrid care Spectrum
The hybrid F&MCW Cardio-Oncology Clinic was initiated and established in the outpatient setting, in close partnership with the Preventive Cardiology Clinic, Cancer Center, and inpatient Cardiology Consult and Hematology/Oncology teams (Fig. 1, right).
Initial and subsequent visits have been completed in person or by video, with phone visits also available for virtual return visits if patients without adept and available smartphone use have limited ability to appear in person. Virtual patient visits over our first 3 months occurred with the use of telemedicine platforms integrated with Epic (via MyChart for patients and Haiku/Canto for clinicians), or using the Doximity video call function. Patients with in-person appointments are screened appropriately on arrival for signs or symptoms of COVID-19 or exposure, following institutional protocols. Wearing masks is required of all patients, and each patient can be accompanied by a family member; some choose to also wear gloves or face shields. There is sufficient room for maintaining social distancing in the clinic waiting room and hallways.
Innovation
Current innovation in the clinic also includes Virtual Clinician Tools and Virtual Patient Resources (Fig. 2). For clinicians, the links for an AHA CME course on Novel Concepts, Current Debates and Treatment Considerations in Cardio-Oncology, an online Cardio-Oncology Compendium hosting risk assessment clinical decision aids, Cardio-Oncology Drug Regimen and Acronym Databases, and UPTODATE access for reviewing Cardio-Oncology drug information are supplied. For patients, the video from the International Cardio-Oncology Society explaining the Cardio-Oncology subspecialty, American College of Cardiology (ACC) mobile health (mHealth) CardioSmart education app and website, Cancer Heart Talk mini-podcast series accessed via SoundCloud app and website, Cardio-Oncology Frequently Asked Questions, and ChemoCare website are provided for patient-facing Cardio-Oncology and heart anatomy and physiology education, engagement, and awareness. Virtual Resources for Preventive Cardio-Oncology are also made available to our patients. These include the American Heart Association (AHA) Physical Activity Recommendations, AHA Life’s Simple 7 Webpages, American Society For Preventive Cardiology Online Coaching Webpages in partnership with Intervent, and the Become An Ex Smoking Cessation Support Webpages in partnership with Mayo Clinic. The resources are provided in the Epic patient portal MyChart, and more ways to make the resources accessible to a broad and diverse patient population are in development. Future innovation in the clinic will explore contemporary initiatives connecting patients and their safely guarded data with their permission with wearable devices, health information technology, informatics, artificial intelligence, personalized medicine, and additional mobile health (mHealth) applications.
Fig. 2 Virtual patient and clinician education and resources. Available online as PDF with hot links in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual visit infrastructure and timeline
There was no pre-existing Cardio-Oncology program at the time of launching our de novo Virtual/Hybrid Cardio-Oncology Clinic. The newly recruited Director of Cardio-Oncology was tasked with the responsibility of launching the new clinic, with support from the Heart & Vascular Center clinic administrators, medical director, and Cardiology Division and Department of Medicine leadership. Prior to opening the Cardio-Oncology Clinic, the Heart & Vascular Center initiated virtual conversion then additionally collaborated with Inception Health (MCW’s innovation lab company) over the course of 4 weeks to iteratively develop the clinical informatics infrastructure for virtual visits. The video visits were designed to function using clinician’s personal smartphones, iPads, and tablets, with direct web browser video links from the electronic health record mobile application. Direct video calls through the Doximity mobile application were also approved. Existing Inception Health personnel re-allocated their time in order to adopt and maintain responsibility for the virtual component of all ambulatory clinics across the health system, in partnership with medical and administrative directors of each clinic area, such as the Heart & Vascular Center. No additional costs or hires were pursued to facilitate the development of the virtual visit infrastructure and timeline. Existing resources and personnel were re-allocated to virtual visit design to enable building the virtual clinics in the Heart & Vascular Center. To assist clinicians and billing compliance colleagues, note templates were created for video and phone visits to indicate patient informed consent for virtual visits due to the pandemic, as well as to capture limited appropriate physical examinations, in addition to the amount of time spent on records review and real-time medical counseling.
Initial consults were electronically triaged by either a cardiologist or a cardiology fellow supervised by a cardiologist. Each triage team determined which consults would be appropriate as virtual video visits, versus in-person to occur once the Heart and Vascular Center started re-opening routine physical visits, or whether patients needed to be evaluated urgently in person. During the first week of operation, the brand-new Cardio-Oncology Clinic started entirely virtually with only video and phone visits. As the Heart and Vascular Center re-opened for physical patient visits the following week, from week 2 through the remainder of the first 3 months the Cardio-Oncology Clinic had both virtual and in-person visits integrated throughout each clinic session weekly, based on whether patients were new and whether they had smart device or computer functionality available.
Virtual referral network and process
Cardio-Oncology patient assessment begins within a referral network before the patient arrives at a Cardio-Oncology clinic. Consequently, forming a virtual referral network and enacting a user-friendly virtual referral process was a key component of building the Cardio-Oncology clinic during the pandemic. All referral patterns and networks for our de novo Cardio-Oncology Clinic were built from the ground up. Initial referrals were from within our health system; this quickly expanded to consults from outside of our health system encompassing the entire state. Patients were referred to our Clinic by clinicians or by self-referrals. Some of our local patients connected to us after being introduced to us by their clinicians in other states or through family members in other states who learned about us from their own clinicians or community-based Cardiology society outreach events.
Referrals across the institution and outside of our health system have come to us from the Divisions of Hematology and Oncology, Internal Medicine, Family Medicine, Surgical Oncology/Breast Clinic, and Survivorship Clinics. From the cancer center’s perspective, there may be many “triggers” that would warrant a Cardio-Oncology referral. For example, an abnormal ECG, an abnormal echocardiogram, cardiovascular symptoms, previous cardiovascular history (e.g., coronary artery disease, hypertension, cardiomyopathy) particularly in a patient who previously underwent treatment or is beginning new treatment with cardiotoxic neoplastic medications or radiation therapy and is at high risk of cardiovascular toxicity, or those in preparation for stem cell transplant, or oncologic surgery. Referral protocols were determined based on standard practice, discussions with colleagues in Medical and Radiation Oncology, Hematology, Bone Marrow Transplant, Surgical Oncology/Breast Clinic, Children’s Hospital, Radiation Oncology, Primary Care, and updated literature reviews.
Cancer survivors are at a higher risk than the general population for cardiovascular morbidity and mortality. If a cancer survivor needing to be evaluated is already under the care of a cardiologist, the referring provider can reach out to their cardiologist for guidance on the appropriate CV surveillance. If they do not already have a cardiologist, a Cardio-Oncology consult should be requested. The Cardio-Oncology consult can be placed using a direct Cardio-Oncology button within the universally available Cardiovascular Consult order panel. Referrers can also place a General Cardiology consult and mention the Cardio-Oncology physician by name as requested by the clinician or patient. An E-Consult functionality is also being implemented for those patients who need to be assessed sooner than the next available appointment, or for those patients who may not need a full Cardio-Oncology evaluation, or if referring providers are uncertain. The e-consult can also be placed as a second opinion requested by the inpatient Cardiology Consult team.
The inpatient Cardiology Consult service will continue to directly address inpatient consults from the inpatient hematology/oncology services. The inpatient Cardiology Consult service can collaborate with the Cardio-Oncology Clinic via formal Cardio-Oncology E-consults in the electronic health record Epic if a specific focused question arises regarding Cardio-Oncology relevant to the care of individual currently hospitalized patients that have already been formally evaluated by the inpatient Cardiology Consult service. After a patient has been formally evaluated by the inpatient Cardiology Consult service, if the patient is appropriate for outpatient follow up in the Cardiology clinic with Cardio-Oncology, this should be communicated to the primary Hematology/Oncology service. If appropriate at the time of consultation, the inpatient cardiology consult service can make the follow-up appointment. Oftentimes, this patient population remains in the inpatient setting for several weeks. If this is the case, the Cardiology clinic phone number and clinician information should be provided to the primary service to do so prior to the patient being discharged from the hospital.
Virtual-hybrid multidisciplinary team
It is important to develop a multidisciplinary team and initially focus on allocation of pre-existing resources. Accordingly, some roles among our Cardio-Oncology clinic personnel are shared with other subspecialties. Our virtual-hybrid multidisciplinary Cardio-Oncology Clinic personnel include physicians, a nurse practitioner (NP), a nurse, a research support specialist, medical assistants, pharmacists, administrative assistants, and administrators. All personnel with pre-existing in-person roles and practices re-allocated a portion of their time to the development and practice of virtual visits.
Our clinic and partners consist of board-certified Cardiologists with special training in various cardiac subspecialties (e.g., cardio-oncology, preventive cardiology, heart failure and transplant, electrophysiology, interventional cardiology), who collaborate closely with our cancer experts. Our physicians together specialize in the prevention, diagnosis, and treatment of heart and vascular disorders resulting from side effects of cancer therapy. Our comprehensive team of advanced practice providers, nurses, and pharmacists work alongside our physicians to care for patients from the moment of cancer diagnosis through life’s survivorship journey. The NP typically sees established patients when needed to follow up on imaging, intervention, or diagnostic and management plans, and may also see select new patients. In complex cases, the NP discusses the care of established patients with both the cardio-oncologist and the referring clinician. The nurse assists with patient triage and communications (including addressing patient requests and queries), liaises closely with the nurse practitioner and pharmacists, and educates patients on Cardio-Oncology using virtual materials. Our clinical pharmacists function at the highest level of their advanced training, similar to all clinic personnel, and assist with medication education, review, titration, discussion, and prescription, particularly for heart failure, hypertension, hyperlipidemia, and smoking cessation, as well as commenting on potential drug interactions.
For Preventive Cardio-Oncology, we additionally partner with our dietitians and exercise physiologists to help advise our patients on nutrition and exercise plans, as well as our colleagues in cardiopulmonary stress testing where applicable. Further, in the pandemic, we provide patients with free online coaching options for lifestyle modification (Fig. 2). We also direct patients to AHA webpages with guidance on pursuing ideal cardiovascular health.
Virtual-hybrid patient flow
Once a referral is placed by the designated order buttons in the electronic health record, central schedulers or the Cardio-Oncology Clinic administrative assistant schedule the new patient for a video or in-person visit (Fig. 3). The clinic administrative assistant works closely with our health professionals in our interdisciplinary advanced subspecialty clinic to gather relevant clinical reports and history pertinent to patient appointments. Virtual medical assistants contact patients a few days before their appointments to confirm and troubleshoot virtual connectivity. On the appointment day, medical assistants then ‘room’ patients for virtual or in-person visits by preparing patients for their medical visits (including reviewing medications and in-person or at-home virtual vital signs), and also rechecking virtual connectivity for video visits. The clinician then completes the visit virtually or in-person and introduces the patient to the range of electronic resources available. Following the visit, the clinical administrative assistant arranges follow-up testing and appointments.
Fig. 3 Virtual-Hybrid Patient Flow Chart. Admin = Administrative; MA = Medical Assistant; MD = Medical Doctor; NP = Nurse Practitioner
Virtual risk assessment
Baseline risk assessment and follow-up start with oncology and primary care [21]. Asymptomatic low risk patients with low-risk treatment plans can have continued assessment and follow-up by oncology and primary care in partnership. Patients who have symptoms, are at high risk based on their history, or are planned for high-risk treatment plans should be referred to Cardio-Oncology for prevention, monitoring, and management recommendations. Recommendations should adhere to society expert consensus, scientific statements, and guidelines for prevention, surveillance, and survivorship, and optimize CVD risk and medications [21]. A putative risk score based on medication-related and patient-related risk factors can be used to guide monitoring and management recommendations for most Cardio-Oncology patients [22], and can be used in a virtual clinical decision aid (https://tinyurl.com/CardioOncCDA) (Fig. 4). Specific risk scores are also available for adults treated with anthracyclines, trastuzumab, or other drugs, or for adult survivors of childhood cancers [9–13].
Fig. 4 Virtual Cardio-Oncology clinical decision aid (CDA). Risk assessment (https://tinyurl.com/CardioOncCDA) to guide monitoring and management recommendations regarding development of cardiomyopathy for most Cardio-Oncology patients; a CDA specifically for women with early breast cancer is also available in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual management algorithms
Evidence-based management algorithms have been selected or developed as adjunctive resources for inpatient teams. They are available online in a virtual collection for use in the inpatient setting by the inpatient Cardiology Consult service or hematology/oncology teams to assist with diagnosis and treatment of cardiovascular toxicities from cancer therapies or cancer itself. The algorithms cover cardiomyopathy from anthracyclines or trastuzumab, planned chemotherapy with pre-existing cardiomyopathy, neurohormonal therapy or dexrazoxane for cardioprotection, myocarditis, persistent malignant pericardial effusion, hypertension, surveillance after radiation therapy or drugs that cause ischemia, malignant pericardial effusion, and other salient topics frequently encountered.
Virtual community engagement
The local, regional, national, and international community was virtually engaged via social media posts on Twitter (using #MCWCardioOnc on @DrBrownCares or @PrevCardioOnc), podcasts hosted by the MCW CTSI (available on iTunes, Google, and Apple podcast platforms), Heart Success podcast series, and Cancer Heart Talk brief 15-min mini-podcast series (available on SoundCloud). Perspectives were also published for international community engagement in the Women Heart Alliance newsletter, as well as on the AHA Early Career Blog, ACC Women in Cardiology Blog, CardioOncTrain.Com Blog, and PrevCardioOnc.Com Blog. Virtual continuing medical education (CME) presentations were also given at the Wisconsin state ACC annual conference meeting, Midwest ACC annual conference meeting, Southeast ACC annual conference meeting, Brazilian Cardio-Oncology Symposium, and the Ohio State Cardio-Oncology CME conference, then subsequently at the AHA and ACC annual national scientific sessions.
Distribution of patient data
In our multi-subspecialty clinic visits (virtual and in-person integrated and combined; n = 182; 136 new and 47 returns), approximately 50% of patient visits were in Cardio-Oncology, 20% were in Preventive Cardio-Oncology, and 30% were in General Cardiology (Fig. 5a). Overall among Cardio-Oncology visits, 65% were in person, consistent with early and safe clinic re-opening in a hybrid model, with 19% by video and 16% by phone, with the fraction by phone decreasing over time as patients and clinic personnel became more adept with troubleshooting video. Of new patients, 77% were in person, and the remainder by video. No Cardio-Oncology patients presenting in person developed any signs or symptoms concerning for COVID-19.
Fig. 5 Initial Clinic-Building Outcomes Using the Virtual-Hybrid Approach. a Distribution of Cardio-Oncology, Preventive Cardio-Oncology, and General Cardiology patients seen in our multi-subspecialty clinic visits. b Distribution of cardiovascular diagnosis or indication for referral. c Distribution of cancer drugs. d Distribution of cancer types
The most frequent cardiovascular diagnosis or indication for referral was cardiomyopathy (34%) (Fig. 5b). Other diagnoses included decrease in global longitudinal strain, diastolic congestive heart failure, hypertension, myocarditis, dyspnea, chest pain, palpitations, survivorship, risk assessment, and pre-bone marrow transplant, among other cardiovascular diagnoses or visit indications. The most frequent cancer drug was trastuzumab (29%) (Fig. 5c), managed according to a novel algorithm developed in our de novo Virtual-Hybrid Cardio-Oncology Clinic based on the recent publication indicating the safety of continuation of trastuzumab for left ventricular ejection fraction of 40% or greater [23] (Fig. 6). The second most frequent cancer drug was anthracycline (24%). Other drugs included, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), endocrine therapies, and investigational therapeutics, among others. The most frequent cancer type in our clinic was breast cancer (42%) (Fig. 5d). These trends in cardiovascular diagnosis or indication and cancer drugs or types were similar in assessments of virtual visits alone, with the most frequent being cardiomyopathy (43%), trastuzumab (41%), and breast cancer (44%), respectively. The findings of similar cardiovascular and cancer distributions in virtual versus in-person visits indicated an optimal qualitative return on resource and personnel investment.
Fig. 6 Algorithm for Continuation of Trastuzumab Therapy with Mild LV Dysfunction
Imaging and medication titration
In our clinic, a distribution of cardiovascular diagnoses determines the imaging needed for each patient (Fig. 5b). Therefore, a number of imaging modalities are useful to our patients (e.g., echocardiography, computed tomography with or without angiography, magnetic resonance imaging, coronary angiography, myocardial perfusion imaging). Our most frequently used imaging modality is echocardiography. The frequency of obtaining echocardiograms has depended on each patient’s condition and cancer treatment. A substantial portion of patients coming to us on trastuzumab have needed an echocardiogram every 1–3 months, depending on the extent of adverse effects on left ventricular ejection fraction (LVEF) or strain In these patients, medication titration has occurred approximately every 2 weeks, and for very symptomatic patients with volume overload, they have often been seen weekly..
In our management algorithms, early referral prior to the onset of symptoms has been emphasized, especially in cancer patients or survivors with a history of cardiovascular disease, cardiotoxic neoplastic agents, or a high risk of cardiovascular toxicity. This has provided an opportunity for us to assess and discuss ways to optimize the benefit to risk ratio of continuing with the current cancer treatment plan, and more importantly how and when to put cardioprotective measures in place to facilitate safe cancer therapy. Such discussions have also resulted in closer monitoring. Some conditions have warranted proceeding to other modalities of non-invasive imaging, such as cardiac MRI if myocarditis is suspected. For cases in which coronary artery disease is suspected, our patients undergo functional assessment of their coronaries with a stress test or anatomical assessment with a coronary CT scan or invasive coronary angiography. In our practice, only exercise stress tests were halted due to the pandemic. Every other form of imaging including rest and stress echo, as well as MRI and nuclear medicine have remained readily available for those with cardiovascular toxicities or individuals considered to be at moderate or high risk. This allowed us to adhere to pre-pandemic imaging recommendations tailored during the pandemic to limit imaging if possible to those who are at higher risk for cardiovascular toxicities or who have already been diagnosed with these adverse effects [1–4] (e.g., Fig. 6).
Discussion
The COVID-19 pandemic has inevitably compelled leaders of healthcare clinics to rethink and restructure approaches to deliver optimal care for patients. Our brand-new Cardio-Oncology clinic has been built to thrive in this new pandemic landscape by utilizing virtual technology as one of the key components of our clinic-building and care model since its inception. While existing clinics have reinvented their operations through the uptake of technology, our clinic has been able to capitalize on this resource to deliver virtual-hybrid care from the start. Virtual communication has proved useful to coordinate referral networks and care among providers within a multidisciplinary team across different clinics and departments. We see a variety of cancer patients, types, and drugs (Fig. 5), and our distribution results are generally congruent with reports from other leading cardio-oncology clinics [8, 14, 15, 17, 22].
Various methods have been developed for risk assessment to help guide providers and patients in determining the appropriate guidelines for care. We offer the use of virtual risk assessment tools such as the computed risk scores based on medication- and patient-related risk factors [22] (Fig. 4) (https://tinyurl.com/CardioOncCompendium), as well as recommendations for establishing cross-provider partnerships to continuously evaluate risk [21]. Other online databases containing useful information and guidelines are readily accessible and can help guide clinical practices. We encourage use of these virtual tools, which can further facilitate collaborative Cardio-Oncology care in the pandemic. Our conversations with international colleagues have suggested additional utility of these virtual tools beyond the pandemic. The online resources can be very helpful in settings where clinical practitioners work alone without support from nurses, pharmacists, nutrition specialists, or exercise physiologists.
Virtual-hybrid care has extended the care team’s capabilities for delivering and maintaining patient education and follow-up. The internet continues to be a robust resource, containing a wealth of health information that is easily accessible to the general population. Various mobile applications and electronic devices have also been developed in recent years to educate, track, and manage patients’ health and lifestyles. While these tools provide patients with greater accessibility and independence, they also create a valuable opportunity for healthcare providers to further engage patients. In a virtual-hybrid model, this becomes increasingly important, as patients may frequently transition between virtual and in-person visits. Forming care partnerships with patients through these virtual information and health-tracking resources becomes crucial in the continuity of care and proper health maintenance as we move through the pandemic.
Our most frequent cardiovascular diagnosis was cardiomyopathy (34%), which is reflective of the management need that first helped start the emerging field of Cardio-Oncology, and is similar to the most frequent cardiovascular diagnosis noted by clinicians from several other leading centers (20–35%) [24, 25]. However, Cardio-Oncology has grown remarkably over the last 10–20 years, with a wide spectrum of cardiovascular diagnoses and indications for referral (Fig. 5b) [8, 14]. Accordingly, at some other leading centers, the most frequent cardiovascular diagnosis or indication for referral has been reported as hypertension [14], arrythmia [17], or comprehensive risk assessment prior to beginning of therapy to optimize cardioprotection [8] in the practice of Preventive Cardio-Oncology [21]. This illustrates an opportunity for growth in our Clinic, to increase the fraction of high-risk patients who undergo comprehensive cardiovascular risk evaluation and management of risk factors prior to administration of cardiotoxic therapy.
Our clinic cares for patients with a range of cancer types (Fig. 5d). Individuals with breast, lung, and hematologic cancers represent a substantial proportion of our patient population, similar to other Cardio-Oncology clinics [8, 14, 22, 26]. The most frequent cancer diagnosis, breast cancer (43%), is consistent with reports from other leading cardio-oncology clinics such as the Mayo Clinic (39.2%) [15] and the Cleveland Clinic in Florida (44.3%) [14]. While hematologic malignancies such as leukemia and lymphoma represented 29% of our patients and was the second most prevalent cancer within our cohort, they comprised the most frequent forms of cancer at other cardio-oncology clinics such as at the Moffitt Cancer Center (31%) [8] and at UCLA (32.70%) [25]. However, the absolute difference was relatively insignificant. Overall, similar to these established cardio-oncology clinics, we receive patients from across a variety of cancers.
A wide breadth of cancer therapeutics is associated with cardiotoxicity [21]. Anthracyclines associate with cardiomyopathy, especially when used with trastuzumab. Targeted therapies (e.g., TKIs) can cause new or worsening of pre-existing hypertension. ICIs are associated with an increased incidence of myocarditis. Some cytotoxic chemotherapeutics, such as cisplatin, increase the risk of venous thromboembolism, and antimetabolites such as fluoropyrimidines have long been associated with a broad range of cardiotoxicities. Radiation therapy is associated with ischemic heart disease, valve dysfunction, conduction abnormalities, pericardial disease, and cardiomyopathy. Patients with cancer who have developed cardiovascular toxicity or who may be at high risk for cardiovascular toxicity should be referred to the Cardio-Oncology clinic for close follow-up.
The most frequently used cancer medication used among our patients was trastuzumab (29%), with the second most frequent being anthracyclines (24%). This was similar to other institutions, with anthracyclines and trastuzumab among the most common cancer drugs in their Cardio-Oncology clinics. Yet, anthracyclines were typically noted more commonly than trastuzumab. The Cleveland Clinic in Florida saw patients most commonly treated with radiation (40%), followed by anthracyclines (26.8%) [14]. The Moffitt Cancer Center most frequently had patients who were treated with anthracyclines (52%), with HER2 targeted therapies representing 27% of the cancer drugs [8]. This difference may reflect a high frequency of patients with HER+ breast cancer in our population (diagnosed by a ratio of HER2 to chromosome 17 signals on dual probe fluorescent in situ hybridization ≥2 or ≥ 6 HER2 signals/cell [27]), as well as the keen attention to a substantial fall in left ventricular ejection fraction or global longitudinal strain as a potential prognostic factor in our patients, per American Society of Echocardiography (ASE) guidelines [28].
All of these patient data distributions were obtained in the context of the Virtual-Hybrid patient flow in our de novo Cardio-Oncology Clinic (Fig. 3), that can be modeled by other future Virtual-Hybrid Cardio-Oncology clinics initiated during the pandemic. Table 1 compares the first few weeks of our de novo C-O clinic setup model with two published manuscripts describing conversion of pre-existing in-person C-O clinics to providing telehealth visits as an option for patients. The table shows similar numbers of patients seen in the initial periods of the clinics, although the numbers in our new clinic went from 0 to 10 in the first 3 weeks, compared to going from up to 40 patients weekly to 11 patients in 2.5 weeks for a group that converted their in-person clinic to a virtual option. Overall, CV diagnoses and cancer types were comparable; distributions of cancer drugs were not reported by the other group. Important differences were noted. Most of our patients in the pandemic were new (90%), given the de novo status of the Cardio-Oncology Clinic, while the converted virtual clinic of another group initially focused on established patients for > 50% of their patient visits. While key personnel were also the same (e.g., physician, advanced practice provider, nurse or nurse coordinator), we also report virtual versions of supportive staffing patterns, including the virtual scheduling and rooming process and pharmacy and lifestyle modification visits. Additionally, trainees have been integrally involved in the establishment of our Cardio-Oncology Clinic, with residents training in program-building, and medical students and fellows training in ambulatory cardio-oncology clinical practice and cardio-oncology critical thinking, respectively. Finally, besides the patient flow (Fig. 3) and de novo nature of our Virtual-Hybrid clinic initiated in the pandemic, our unique contribution may be the virtual resources, compared to the essential “webside manner” [7] or an alternative algorithm [4] for triaging virtual or in-person visits to the physician or advanced practice provider (Table 1).
Table 1 Comparisons Among Clinic Models Described In The Pandemic
Virtual-Hybrid Clinic Telehealth Clinic (14) Triage Clinic (4)
Model Type De Novo Conversion Conversion
Time Frame Compared 3 weeks 2.5 weeks Not reported
Number of Patients 10 11 Not reported
New Visits (%) 90 45 Not reported
Variety of CV Diagnoses Yes Yes Not reported
Variety of CA Types Yes Yes Not reported
Variety of CA Drugs Yes Unknown Not reported
Referrals Yes Unknown Yes
Scheduling Yes Unknown Not reported
Rooming Process Yes Unknown Not Reported
Virtual AA Yes Unknown Not Reported
Virtual MA Yes Unknown Not Reported
Virtual Physician Yes Yes Yes
Virtual APP Yes Unknown Yes
Virtual Pharmacist Yes Unknown Not Reported
Virtual Nurse (Coordinator) Yes Unknown Yes
Unique Contribution Virtual Resources Webside Manner Triage Algorithm
Similar to the formation or conversion of Cardio-Oncology clinics, many protocols for treatment regimens and cancer patients are yet to be standardized. While no standard protocols have been widely adopted at Cardio-Oncology practices, various institutions and writing groups have proposed some approaches (e.g., ASE or ASCO guidelines). We have collaboratively developed institutional algorithms for various cardiovascular toxicities and medications based on existing scientific statements, society guidelines, expert consensus statements, and manuscripts from leading cardio-oncology research institutions. The goal is to adopt, adapt, develop, and continuously update these algorithms, as new literature arises in order to establish best practices and an institutional standard of care.
Conclusion
Starting a new Cardio-Oncology Clinic in the pandemic has its challenges, and yet for our patients can be invaluable. Appropriately competing priorities in the pandemic can limit the scheduling of meetings and gathering of people together in one virtual room to discuss a mutual vision. Gathering resources for patient and clinician education can also be formidable, as can social distancing and obtaining important imaging. However, multiple virtual one-on-one or small group meetings can be beneficial for building institutional relationships. Similarly, virtual visits have risen to the challenge to ensure maintenance of patient care throughout the pandemic. Modifications have also been made to enable safety and distancing during imaging. With the benefit of these adjustments to address the challenge, this report provides a foundation for initiating a cardio-oncology clinic in the pandemic, with virtual resources and tools to equip patients and clinicians.
In the future, we will also lay out a roadmap for initiation of comprehensive cardio-oncology programs with the five pillars of patient care, education, research, community engagement, and innovation in the era of digital transformation accelerated by the pandemic. Novel risk modifiers and risk attenuation methods, such as breast arterial calcification, clonal hematopoiesis of indeterminate potential, and Cardio-Oncology prehabilitation, habilitation, and rehabilitation will also be addressed. Future innovation to implement recommendations from clinical trials across the nation currently underway that utilize mobile health or web-based diet and physical activity interventions and/or seek to determine the impact of cardioprotective pharmacotherapy in Preventive Cardio-Oncology will also be assessed (ClinicalTrials.Gov: NCT01988571, NCT02943590, NCT02562716, NCT01968200, NCT03265574, NCT03760588, NCT03386383, NCT02244411, NCT03223753). Many of these studies incorporate virtual technologies that will be very helpful during and after the pandemic as we continue pursuit of digital transformation.
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Acknowledgements
We are grateful to the following individuals, as well as all of the departments and divisions at Froedtert & MCW that have rallied together to help us establish the hybrid Cardio-Oncology clinic or to see our patients needing subspecialty care, or to partner with us in referrals from the inpatient service, especially the following clinicians: Stacey Gardiner MD, Jason Rubenstein MD, David Ishizawar MD, Jalaj Garg MD, Andrew Rosenblum MD. We are also grateful to Nicole Lohr MD PhD and the clinical sections at the Zablocki Veterans Administration Medical Center that have also helped us establish an affiliate Cardio-Oncology Clinic in tandem at the VAMC in Milwaukee, WI.
Authors’ contributions
SAB conceived of the study, obtained, analyzed and interpreted the data, and made a substantial contribution to the writing of the manuscript; SP made a substantial contribution to the writing of the manuscript; DR made a substantial contribution to the writing of the manuscript; SZ obtained the data and made a substantial contribution to the writing of the manuscript; ML interpreted the data and made a substantial contribution to the writing of the manuscript; TN made a substantial contribution to the writing of the manuscript; BS analyzed the data and made a substantial contribution to the writing of the manuscript; RM made a substantial contribution to the writing of the manuscript; JMac made a substantial contribution to the writing of the manuscript; KD made a substantial contribution to the writing of the manuscript; JMes made a substantial contribution to the writing of the manuscript; DM made a substantial contribution to the writing of the manuscript; JS made a substantial contribution to the writing of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was associated with this work.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Froedtert & MCW Institutional Review Board (ID PRO00038807); HIPAA informed consent was waived for this minimal risk study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests. | TRASTUZUMAB | DrugsGivenReaction | CC BY | 33441188 | 18,793,759 | 2021-01-13 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cardiac function test abnormal'. | A virtual-hybrid approach to launching a cardio-oncology clinic during a pandemic.
BACKGROUND
As cardiovascular disease is a leading cause of death in cancer survivors, the new subspecialty of Cardio-Oncology has emerged to address prevention, monitoring, and management of cardiovascular toxicities to cancer therapies. During the coronavirus disease of 2019 (COVID-19) pandemic, we developed a Virtual-Hybrid Approach to build a de novo Cardio-Oncology Clinic.
METHODS
We conceptualized a Virtual-Hybrid Approach including three arms: information seeking in locations with existing Cardio-Oncology clinics, information gathering at the location for a new clinic, and information sharing to report clinic-building outcomes. A retrospective review of outcomes included collection and synthesis of data from our first 3 months (at pandemic peak) on types of appointments, cancers, drugs, and cardiotoxicities. Data were presented using descriptive statistics.
RESULTS
A de-novo Cardio-Oncology clinic was developed structured from the ground up to integrate virtual and in-person care in a hybrid and innovative model, using the three arms of the Virtual-Hybrid Approach. First, we garnered in-person and virtual preparation through hands-on experiences, training, and discussions in existing Cardio-Oncology Clinics and conferences. Next, we gleaned information through virtual inquiry and niche-building. With partners throughout the institution, a virtual referral process was established for outpatient referrals and inpatient e-consult referrals to actualize a hybrid care spectrum for our patients administered by a multidisciplinary hybrid care team of clinicians, ancillary support staff, and clinical pharmacists. Among the multi-subspecialty clinic sessions, approximately 50% were in Cardio-Oncology, 20% in Preventive Cardiology, and 30% in General Cardiology. In the hybrid model, the Heart & Vascular Center had started to re-open, allowing for 65% of our visits to be in person. In additional analyses, the most frequent cardiovascular diagnosis was cardiomyopathy (34%), the most common cancer drug leading to referral was trastuzumab (29%), and the most prevalent cancer type was breast cancer (42%).
CONCLUSIONS
This Virtual-Hybrid Approach and retrospective review provides guidance and information regarding initiating a brand-new Cardio-Oncology Clinic during the pandemic for cancer patients/survivors. This report also furnishes virtual resources for patients, virtual tools for oncologists, cardiologists, and administrators tasked with starting new clinics during the pandemic, and innovative future directions for this digital pandemic to post-pandemic era.
Introduction
Cardio-Oncology care has been adjusted in the COVID-19 pandemic with limited in-person clinic or hospital visits, increased use of teleconsultation, less frequent imaging, increased reliance on biomarkers, and considerations of differential diagnoses involving COVID-19 when evaluating cancer patients or survivors for possible cardiovascular toxicity [1]. Monitoring and management algorithms have been developed to help guide virtual care [2–4]. In the pandemic, we have changed the way in which we provide healthcare services at our clinics and institutions. This has challenged us to restructure current systems for the safety of our patients.
Various forms of innovation have come to bear in the pandemic, including telemedicine, digital health, artificial intelligence, social media, informatics, big data, and precision medicine [5, 6]. Telemedicine is the primary form of innovation that has been most developed in the pandemic [2, 5, 7]. Social media has been very helpful for dissemination of information, as well as education, and has been integral for creating online groups for support and determining the best ways for proceeding in the pandemic and advocating for our patients and colleagues in this period [5, 6]. In addition, the Doximity social media application has been valuable to practices across the nation, due to its telehealth platform (Doximity Video and Phone; https://www.doximity.com/dialer-video).
Despite the growing need, and allowances made during the pandemic, many centers do not have formal Cardio-Oncology clinics. Starting a new clinic can be challenging. The COVID-19 pandemic has made the process significantly more difficult, with the need to minimize exposure and maximize patient safety.
Currently, limited information is available on how to start a Cardio-Oncology Clinic during a pandemic, albeit given the high risk of morbidity or mortality in COVID-19-positive patients who also have cancer or CVD [8–13]. Several institutions have published on their experiences with starting in-person Cardio-Oncology clinics prior to the pandemic [8, 14–17]. One group has reported on their conversion from existing in-person Cardio-Oncology visits to telemedicine consultations, seeing 11 patients virtually within a few weeks [7]. Many have considered implications of the pandemic on the practice and study of cardio-oncology [2, 4, 5, 18–20], and two groups have suggested models for clinics converting from existing in-person care to televisits [4, 5]. Yet, no groups have directly addressed steps for de novo virtual-hybrid clinic formation within the limitations of the pandemic and without conversion of a pre-existing Cardio-Oncology clinic.
Our report offers a template for other centers to develop their own new Cardio-Oncology clinics during the pandemic. We determined a Virtual-Hybrid Approach to clinic launch, with both virtual and in-person elements of three key arms: information seeking where there are existing Cardio-Oncology Clinics in place, information gathering where the clinic will be built, and information sharing to report on initial patient data demonstrating the success of the launch (Fig. 1). We then performed retrospective chart review to collect and synthesize data on the types of appointments (new versus established, virtual versus in-person), cancers (e.g., breast, prostate, leukemia, lung), cancer drugs, and cardiovascular toxicities (e.g., cardiomyopathy, hypertension) for patients seen virtually or in person in our new Cardio-Oncology clinic at Froedtert Hospital and Medical College of Wisconsin (F&MCW). Here, we will discuss our findings in the context of previous publications on launching Cardio-Oncology Clinics prior to the pandemic. Our results will present distributions of cancer drugs and types, and cardiovascular diagnoses, similar to previous publications on Cardio-Oncology clinic-building. However, we will differentiate and illuminate the techniques that leverage the virtual underpinnings of pandemic clinic-building. We submit that it is feasible to establish a new Cardio-Oncology Clinic for cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy during a pandemic, providing optimal care for new patients in the midst of the need for safety and minimizing exposure. We also propose virtual resources for patients and clinicians and describe innovative future directions in the pandemic and post-pandemic period.
Fig. 1 The Virtual-Hybrid Approach to Cardio-Oncology Clinic-building in the pandemic
Methods
Virtual-hybrid approach
We pursued a Virtual-Hybrid Approach of information seeking, information gathering, and information sharing (Fig. 1). For information seeking, we focused on institutions that already had a Cardio-Oncology clinic in place. Prior to the COVID-19 pandemic, substantial experience was gained at an established in-person Cardio-Oncology clinic at a world-renowned leading hospital. Published manuscripts on pre-pandemic building and operations of Cardio-Oncology clinics at other institutions were reviewed. Extensive networking with leaders of Cardio-Oncology clinics around the United States and in the United Kingdom was accomplished at regional, national, and international meetings in person and on social media in a hybrid approach. These meetings were attended in person pre-COVID-19 and virtually during the pandemic. For information gathering, we addressed the location in which the new Cardio-Oncology clinic would be built. We learned about existing resources in the destination Heart & Vascular Center and existing needs and patients in the destination partner cancer center. We networked with physicians, advanced practice providers (APPs), nurses, service line leaders, and administrators in the Heart & Vascular Center and the cancer center, as well as in primary care and other supporting specialties. The subsequent Cardio-Oncology clinic sessions were intermingled with other subspecialty areas, to optimize availability for patient visits while filling clinic slots and tailoring spectrum of care to emerging patient needs. Cardio-Oncology patient visits were included in multi-subspecialty clinics.
Retrospective study design
For quantitative data, we pursued a retrospective observational study to determine the distribution of outpatient visits in the first 3 months of our virtual-hybrid Cardio-Oncology Clinic. We reviewed data from charts of patients (all were 18 years of age or older) who received outpatient care from the new Director of Cardio-Oncology at F&MCW between April 15, 2020 and July 17, 2020 to determine which of these patients were considered to be in Cardio-Oncology, Preventive Cardiology, or General Cardiology. We collated all three to determine the percentage of patients seen in Cardio-Oncology, compared to the other two specialties. Preventive Cardiology was collated as a partner clinic to help build the Preventive Cardio-Oncology component of the Cardio-Oncology Clinic, to help apply established principles for prevention. From among the multi-subspecialty clinic sessions, we determined the proportion of patients who were specifically cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy and thereby seen in the Cardio-Oncology Clinic. Next, we identified the distribution of cardiovascular toxicities in cancer patients or survivors seen in the Cardio-Oncology Clinic. We also evaluated the spectrum of cancer drugs received by cancer patients or survivors seen in the Cardio-Oncology Clinic. In addition, we summarized the types of cancers in patients seen in the Cardio-Oncology Clinic. Finally, we assessed the frequency of virtual visits during the course of the pandemic over our first 3 months for patients seen in the Cardio-Oncology Clinic. This retrospective review was approved by the F&MCW IRB; HIPAA informed consent was waived for this minimal risk study, which did not involve any form of intervention and was conducted in compliance with good clinical and research practice. The team designed and carried out the study with reliance on virtual communication tools.
Data collection and analysis
Data gathering, management, and analysis were conducted at F&MCW. We collected patient-related, disease-related, treatment-related, and outcome-related data, particularly patient sex, type of appointment (new versus established, virtual versus in-person), type of cancer (e.g., breast, prostate, leukemia, lung), type of cancer drug, and type of cardiovascular toxicity (e.g., cardiomyopathy, hypertension). In order to minimize any risk of breaching patient confidentiality, all data collection occurred on institutional-based computing environments with de-identified data used for analyses. There were no alternative procedures for the subjects as this is a retrospective review of data that are not amenable to prospective collection and review. Descriptive graphs or tables of patient-, disease-, treatment-, and outcome-related variables distributions were prepared, with no comparisons made needing statistical tests.
Results
Virtual preparation
Preparation for starting the Cardio-Oncology clinic followed a Virtual-Hybrid Approach (Fig. 1, left). Five overarching factors employing virtual communication methods emerged to ensure the successful launching of the clinic. Team and individual experience and exposure to various areas of interest in Cardio-Oncology were achieved and assessed before and during the pandemic. Far-reaching connections to experts and potential collaborators in the field were developed and exercised. Close contact with the institution launching this clinic was important to determine the resources available; these resources dictated the strategy and potential outcomes of the clinic. Importantly, the expectations of others for the Cardio-Oncology clinic were determined and incorporated. Finally, recognition of the limitations that exist at the destination institution guided care and goal setting.
Virtual inquiry
Before initiating the Cardio-Oncology clinic in the destination institution, existing structures, patient base, and needs in the Heart & Vascular Center as well as the Cancer Center were evaluated, adhering to pandemic protocols (Fig. 1, middle). Pre-existing building blocks for the planned Cardio-Oncology clinic were assessed, and the partner Preventive Cardiology clinic was investigated. We also evaluated characteristics of the cancer center patient population to best position the clinic for success.
Virtual niche-building
Five main aspects of niche-building were pursued. Partnerships with Vascular and Cancer Center physicians, advanced practice providers, and service line leaders were developed to initiate and grow the clinic (Fig. 1, middle). The Cardio-Oncology team and clinic flexibility were demonstrated through openness to taking quicksteps. Presentations were made at Grand Rounds and rounds across the institution in Cardiology, Hematology/Oncology, Radiation Oncology, Surgical Oncology, Internal Medicine, and Family Medicine to promote the clinic capabilities. Collaborative solutions for problems facing fields complementing Cardio-Oncology developed trust and collaboration. Teamwork was developed by leveraging diversity of perspectives and virtual communication technologies, to establish effective patient care despite COVID-19 limitations.
Hybrid care Spectrum
The hybrid F&MCW Cardio-Oncology Clinic was initiated and established in the outpatient setting, in close partnership with the Preventive Cardiology Clinic, Cancer Center, and inpatient Cardiology Consult and Hematology/Oncology teams (Fig. 1, right).
Initial and subsequent visits have been completed in person or by video, with phone visits also available for virtual return visits if patients without adept and available smartphone use have limited ability to appear in person. Virtual patient visits over our first 3 months occurred with the use of telemedicine platforms integrated with Epic (via MyChart for patients and Haiku/Canto for clinicians), or using the Doximity video call function. Patients with in-person appointments are screened appropriately on arrival for signs or symptoms of COVID-19 or exposure, following institutional protocols. Wearing masks is required of all patients, and each patient can be accompanied by a family member; some choose to also wear gloves or face shields. There is sufficient room for maintaining social distancing in the clinic waiting room and hallways.
Innovation
Current innovation in the clinic also includes Virtual Clinician Tools and Virtual Patient Resources (Fig. 2). For clinicians, the links for an AHA CME course on Novel Concepts, Current Debates and Treatment Considerations in Cardio-Oncology, an online Cardio-Oncology Compendium hosting risk assessment clinical decision aids, Cardio-Oncology Drug Regimen and Acronym Databases, and UPTODATE access for reviewing Cardio-Oncology drug information are supplied. For patients, the video from the International Cardio-Oncology Society explaining the Cardio-Oncology subspecialty, American College of Cardiology (ACC) mobile health (mHealth) CardioSmart education app and website, Cancer Heart Talk mini-podcast series accessed via SoundCloud app and website, Cardio-Oncology Frequently Asked Questions, and ChemoCare website are provided for patient-facing Cardio-Oncology and heart anatomy and physiology education, engagement, and awareness. Virtual Resources for Preventive Cardio-Oncology are also made available to our patients. These include the American Heart Association (AHA) Physical Activity Recommendations, AHA Life’s Simple 7 Webpages, American Society For Preventive Cardiology Online Coaching Webpages in partnership with Intervent, and the Become An Ex Smoking Cessation Support Webpages in partnership with Mayo Clinic. The resources are provided in the Epic patient portal MyChart, and more ways to make the resources accessible to a broad and diverse patient population are in development. Future innovation in the clinic will explore contemporary initiatives connecting patients and their safely guarded data with their permission with wearable devices, health information technology, informatics, artificial intelligence, personalized medicine, and additional mobile health (mHealth) applications.
Fig. 2 Virtual patient and clinician education and resources. Available online as PDF with hot links in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual visit infrastructure and timeline
There was no pre-existing Cardio-Oncology program at the time of launching our de novo Virtual/Hybrid Cardio-Oncology Clinic. The newly recruited Director of Cardio-Oncology was tasked with the responsibility of launching the new clinic, with support from the Heart & Vascular Center clinic administrators, medical director, and Cardiology Division and Department of Medicine leadership. Prior to opening the Cardio-Oncology Clinic, the Heart & Vascular Center initiated virtual conversion then additionally collaborated with Inception Health (MCW’s innovation lab company) over the course of 4 weeks to iteratively develop the clinical informatics infrastructure for virtual visits. The video visits were designed to function using clinician’s personal smartphones, iPads, and tablets, with direct web browser video links from the electronic health record mobile application. Direct video calls through the Doximity mobile application were also approved. Existing Inception Health personnel re-allocated their time in order to adopt and maintain responsibility for the virtual component of all ambulatory clinics across the health system, in partnership with medical and administrative directors of each clinic area, such as the Heart & Vascular Center. No additional costs or hires were pursued to facilitate the development of the virtual visit infrastructure and timeline. Existing resources and personnel were re-allocated to virtual visit design to enable building the virtual clinics in the Heart & Vascular Center. To assist clinicians and billing compliance colleagues, note templates were created for video and phone visits to indicate patient informed consent for virtual visits due to the pandemic, as well as to capture limited appropriate physical examinations, in addition to the amount of time spent on records review and real-time medical counseling.
Initial consults were electronically triaged by either a cardiologist or a cardiology fellow supervised by a cardiologist. Each triage team determined which consults would be appropriate as virtual video visits, versus in-person to occur once the Heart and Vascular Center started re-opening routine physical visits, or whether patients needed to be evaluated urgently in person. During the first week of operation, the brand-new Cardio-Oncology Clinic started entirely virtually with only video and phone visits. As the Heart and Vascular Center re-opened for physical patient visits the following week, from week 2 through the remainder of the first 3 months the Cardio-Oncology Clinic had both virtual and in-person visits integrated throughout each clinic session weekly, based on whether patients were new and whether they had smart device or computer functionality available.
Virtual referral network and process
Cardio-Oncology patient assessment begins within a referral network before the patient arrives at a Cardio-Oncology clinic. Consequently, forming a virtual referral network and enacting a user-friendly virtual referral process was a key component of building the Cardio-Oncology clinic during the pandemic. All referral patterns and networks for our de novo Cardio-Oncology Clinic were built from the ground up. Initial referrals were from within our health system; this quickly expanded to consults from outside of our health system encompassing the entire state. Patients were referred to our Clinic by clinicians or by self-referrals. Some of our local patients connected to us after being introduced to us by their clinicians in other states or through family members in other states who learned about us from their own clinicians or community-based Cardiology society outreach events.
Referrals across the institution and outside of our health system have come to us from the Divisions of Hematology and Oncology, Internal Medicine, Family Medicine, Surgical Oncology/Breast Clinic, and Survivorship Clinics. From the cancer center’s perspective, there may be many “triggers” that would warrant a Cardio-Oncology referral. For example, an abnormal ECG, an abnormal echocardiogram, cardiovascular symptoms, previous cardiovascular history (e.g., coronary artery disease, hypertension, cardiomyopathy) particularly in a patient who previously underwent treatment or is beginning new treatment with cardiotoxic neoplastic medications or radiation therapy and is at high risk of cardiovascular toxicity, or those in preparation for stem cell transplant, or oncologic surgery. Referral protocols were determined based on standard practice, discussions with colleagues in Medical and Radiation Oncology, Hematology, Bone Marrow Transplant, Surgical Oncology/Breast Clinic, Children’s Hospital, Radiation Oncology, Primary Care, and updated literature reviews.
Cancer survivors are at a higher risk than the general population for cardiovascular morbidity and mortality. If a cancer survivor needing to be evaluated is already under the care of a cardiologist, the referring provider can reach out to their cardiologist for guidance on the appropriate CV surveillance. If they do not already have a cardiologist, a Cardio-Oncology consult should be requested. The Cardio-Oncology consult can be placed using a direct Cardio-Oncology button within the universally available Cardiovascular Consult order panel. Referrers can also place a General Cardiology consult and mention the Cardio-Oncology physician by name as requested by the clinician or patient. An E-Consult functionality is also being implemented for those patients who need to be assessed sooner than the next available appointment, or for those patients who may not need a full Cardio-Oncology evaluation, or if referring providers are uncertain. The e-consult can also be placed as a second opinion requested by the inpatient Cardiology Consult team.
The inpatient Cardiology Consult service will continue to directly address inpatient consults from the inpatient hematology/oncology services. The inpatient Cardiology Consult service can collaborate with the Cardio-Oncology Clinic via formal Cardio-Oncology E-consults in the electronic health record Epic if a specific focused question arises regarding Cardio-Oncology relevant to the care of individual currently hospitalized patients that have already been formally evaluated by the inpatient Cardiology Consult service. After a patient has been formally evaluated by the inpatient Cardiology Consult service, if the patient is appropriate for outpatient follow up in the Cardiology clinic with Cardio-Oncology, this should be communicated to the primary Hematology/Oncology service. If appropriate at the time of consultation, the inpatient cardiology consult service can make the follow-up appointment. Oftentimes, this patient population remains in the inpatient setting for several weeks. If this is the case, the Cardiology clinic phone number and clinician information should be provided to the primary service to do so prior to the patient being discharged from the hospital.
Virtual-hybrid multidisciplinary team
It is important to develop a multidisciplinary team and initially focus on allocation of pre-existing resources. Accordingly, some roles among our Cardio-Oncology clinic personnel are shared with other subspecialties. Our virtual-hybrid multidisciplinary Cardio-Oncology Clinic personnel include physicians, a nurse practitioner (NP), a nurse, a research support specialist, medical assistants, pharmacists, administrative assistants, and administrators. All personnel with pre-existing in-person roles and practices re-allocated a portion of their time to the development and practice of virtual visits.
Our clinic and partners consist of board-certified Cardiologists with special training in various cardiac subspecialties (e.g., cardio-oncology, preventive cardiology, heart failure and transplant, electrophysiology, interventional cardiology), who collaborate closely with our cancer experts. Our physicians together specialize in the prevention, diagnosis, and treatment of heart and vascular disorders resulting from side effects of cancer therapy. Our comprehensive team of advanced practice providers, nurses, and pharmacists work alongside our physicians to care for patients from the moment of cancer diagnosis through life’s survivorship journey. The NP typically sees established patients when needed to follow up on imaging, intervention, or diagnostic and management plans, and may also see select new patients. In complex cases, the NP discusses the care of established patients with both the cardio-oncologist and the referring clinician. The nurse assists with patient triage and communications (including addressing patient requests and queries), liaises closely with the nurse practitioner and pharmacists, and educates patients on Cardio-Oncology using virtual materials. Our clinical pharmacists function at the highest level of their advanced training, similar to all clinic personnel, and assist with medication education, review, titration, discussion, and prescription, particularly for heart failure, hypertension, hyperlipidemia, and smoking cessation, as well as commenting on potential drug interactions.
For Preventive Cardio-Oncology, we additionally partner with our dietitians and exercise physiologists to help advise our patients on nutrition and exercise plans, as well as our colleagues in cardiopulmonary stress testing where applicable. Further, in the pandemic, we provide patients with free online coaching options for lifestyle modification (Fig. 2). We also direct patients to AHA webpages with guidance on pursuing ideal cardiovascular health.
Virtual-hybrid patient flow
Once a referral is placed by the designated order buttons in the electronic health record, central schedulers or the Cardio-Oncology Clinic administrative assistant schedule the new patient for a video or in-person visit (Fig. 3). The clinic administrative assistant works closely with our health professionals in our interdisciplinary advanced subspecialty clinic to gather relevant clinical reports and history pertinent to patient appointments. Virtual medical assistants contact patients a few days before their appointments to confirm and troubleshoot virtual connectivity. On the appointment day, medical assistants then ‘room’ patients for virtual or in-person visits by preparing patients for their medical visits (including reviewing medications and in-person or at-home virtual vital signs), and also rechecking virtual connectivity for video visits. The clinician then completes the visit virtually or in-person and introduces the patient to the range of electronic resources available. Following the visit, the clinical administrative assistant arranges follow-up testing and appointments.
Fig. 3 Virtual-Hybrid Patient Flow Chart. Admin = Administrative; MA = Medical Assistant; MD = Medical Doctor; NP = Nurse Practitioner
Virtual risk assessment
Baseline risk assessment and follow-up start with oncology and primary care [21]. Asymptomatic low risk patients with low-risk treatment plans can have continued assessment and follow-up by oncology and primary care in partnership. Patients who have symptoms, are at high risk based on their history, or are planned for high-risk treatment plans should be referred to Cardio-Oncology for prevention, monitoring, and management recommendations. Recommendations should adhere to society expert consensus, scientific statements, and guidelines for prevention, surveillance, and survivorship, and optimize CVD risk and medications [21]. A putative risk score based on medication-related and patient-related risk factors can be used to guide monitoring and management recommendations for most Cardio-Oncology patients [22], and can be used in a virtual clinical decision aid (https://tinyurl.com/CardioOncCDA) (Fig. 4). Specific risk scores are also available for adults treated with anthracyclines, trastuzumab, or other drugs, or for adult survivors of childhood cancers [9–13].
Fig. 4 Virtual Cardio-Oncology clinical decision aid (CDA). Risk assessment (https://tinyurl.com/CardioOncCDA) to guide monitoring and management recommendations regarding development of cardiomyopathy for most Cardio-Oncology patients; a CDA specifically for women with early breast cancer is also available in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual management algorithms
Evidence-based management algorithms have been selected or developed as adjunctive resources for inpatient teams. They are available online in a virtual collection for use in the inpatient setting by the inpatient Cardiology Consult service or hematology/oncology teams to assist with diagnosis and treatment of cardiovascular toxicities from cancer therapies or cancer itself. The algorithms cover cardiomyopathy from anthracyclines or trastuzumab, planned chemotherapy with pre-existing cardiomyopathy, neurohormonal therapy or dexrazoxane for cardioprotection, myocarditis, persistent malignant pericardial effusion, hypertension, surveillance after radiation therapy or drugs that cause ischemia, malignant pericardial effusion, and other salient topics frequently encountered.
Virtual community engagement
The local, regional, national, and international community was virtually engaged via social media posts on Twitter (using #MCWCardioOnc on @DrBrownCares or @PrevCardioOnc), podcasts hosted by the MCW CTSI (available on iTunes, Google, and Apple podcast platforms), Heart Success podcast series, and Cancer Heart Talk brief 15-min mini-podcast series (available on SoundCloud). Perspectives were also published for international community engagement in the Women Heart Alliance newsletter, as well as on the AHA Early Career Blog, ACC Women in Cardiology Blog, CardioOncTrain.Com Blog, and PrevCardioOnc.Com Blog. Virtual continuing medical education (CME) presentations were also given at the Wisconsin state ACC annual conference meeting, Midwest ACC annual conference meeting, Southeast ACC annual conference meeting, Brazilian Cardio-Oncology Symposium, and the Ohio State Cardio-Oncology CME conference, then subsequently at the AHA and ACC annual national scientific sessions.
Distribution of patient data
In our multi-subspecialty clinic visits (virtual and in-person integrated and combined; n = 182; 136 new and 47 returns), approximately 50% of patient visits were in Cardio-Oncology, 20% were in Preventive Cardio-Oncology, and 30% were in General Cardiology (Fig. 5a). Overall among Cardio-Oncology visits, 65% were in person, consistent with early and safe clinic re-opening in a hybrid model, with 19% by video and 16% by phone, with the fraction by phone decreasing over time as patients and clinic personnel became more adept with troubleshooting video. Of new patients, 77% were in person, and the remainder by video. No Cardio-Oncology patients presenting in person developed any signs or symptoms concerning for COVID-19.
Fig. 5 Initial Clinic-Building Outcomes Using the Virtual-Hybrid Approach. a Distribution of Cardio-Oncology, Preventive Cardio-Oncology, and General Cardiology patients seen in our multi-subspecialty clinic visits. b Distribution of cardiovascular diagnosis or indication for referral. c Distribution of cancer drugs. d Distribution of cancer types
The most frequent cardiovascular diagnosis or indication for referral was cardiomyopathy (34%) (Fig. 5b). Other diagnoses included decrease in global longitudinal strain, diastolic congestive heart failure, hypertension, myocarditis, dyspnea, chest pain, palpitations, survivorship, risk assessment, and pre-bone marrow transplant, among other cardiovascular diagnoses or visit indications. The most frequent cancer drug was trastuzumab (29%) (Fig. 5c), managed according to a novel algorithm developed in our de novo Virtual-Hybrid Cardio-Oncology Clinic based on the recent publication indicating the safety of continuation of trastuzumab for left ventricular ejection fraction of 40% or greater [23] (Fig. 6). The second most frequent cancer drug was anthracycline (24%). Other drugs included, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), endocrine therapies, and investigational therapeutics, among others. The most frequent cancer type in our clinic was breast cancer (42%) (Fig. 5d). These trends in cardiovascular diagnosis or indication and cancer drugs or types were similar in assessments of virtual visits alone, with the most frequent being cardiomyopathy (43%), trastuzumab (41%), and breast cancer (44%), respectively. The findings of similar cardiovascular and cancer distributions in virtual versus in-person visits indicated an optimal qualitative return on resource and personnel investment.
Fig. 6 Algorithm for Continuation of Trastuzumab Therapy with Mild LV Dysfunction
Imaging and medication titration
In our clinic, a distribution of cardiovascular diagnoses determines the imaging needed for each patient (Fig. 5b). Therefore, a number of imaging modalities are useful to our patients (e.g., echocardiography, computed tomography with or without angiography, magnetic resonance imaging, coronary angiography, myocardial perfusion imaging). Our most frequently used imaging modality is echocardiography. The frequency of obtaining echocardiograms has depended on each patient’s condition and cancer treatment. A substantial portion of patients coming to us on trastuzumab have needed an echocardiogram every 1–3 months, depending on the extent of adverse effects on left ventricular ejection fraction (LVEF) or strain In these patients, medication titration has occurred approximately every 2 weeks, and for very symptomatic patients with volume overload, they have often been seen weekly..
In our management algorithms, early referral prior to the onset of symptoms has been emphasized, especially in cancer patients or survivors with a history of cardiovascular disease, cardiotoxic neoplastic agents, or a high risk of cardiovascular toxicity. This has provided an opportunity for us to assess and discuss ways to optimize the benefit to risk ratio of continuing with the current cancer treatment plan, and more importantly how and when to put cardioprotective measures in place to facilitate safe cancer therapy. Such discussions have also resulted in closer monitoring. Some conditions have warranted proceeding to other modalities of non-invasive imaging, such as cardiac MRI if myocarditis is suspected. For cases in which coronary artery disease is suspected, our patients undergo functional assessment of their coronaries with a stress test or anatomical assessment with a coronary CT scan or invasive coronary angiography. In our practice, only exercise stress tests were halted due to the pandemic. Every other form of imaging including rest and stress echo, as well as MRI and nuclear medicine have remained readily available for those with cardiovascular toxicities or individuals considered to be at moderate or high risk. This allowed us to adhere to pre-pandemic imaging recommendations tailored during the pandemic to limit imaging if possible to those who are at higher risk for cardiovascular toxicities or who have already been diagnosed with these adverse effects [1–4] (e.g., Fig. 6).
Discussion
The COVID-19 pandemic has inevitably compelled leaders of healthcare clinics to rethink and restructure approaches to deliver optimal care for patients. Our brand-new Cardio-Oncology clinic has been built to thrive in this new pandemic landscape by utilizing virtual technology as one of the key components of our clinic-building and care model since its inception. While existing clinics have reinvented their operations through the uptake of technology, our clinic has been able to capitalize on this resource to deliver virtual-hybrid care from the start. Virtual communication has proved useful to coordinate referral networks and care among providers within a multidisciplinary team across different clinics and departments. We see a variety of cancer patients, types, and drugs (Fig. 5), and our distribution results are generally congruent with reports from other leading cardio-oncology clinics [8, 14, 15, 17, 22].
Various methods have been developed for risk assessment to help guide providers and patients in determining the appropriate guidelines for care. We offer the use of virtual risk assessment tools such as the computed risk scores based on medication- and patient-related risk factors [22] (Fig. 4) (https://tinyurl.com/CardioOncCompendium), as well as recommendations for establishing cross-provider partnerships to continuously evaluate risk [21]. Other online databases containing useful information and guidelines are readily accessible and can help guide clinical practices. We encourage use of these virtual tools, which can further facilitate collaborative Cardio-Oncology care in the pandemic. Our conversations with international colleagues have suggested additional utility of these virtual tools beyond the pandemic. The online resources can be very helpful in settings where clinical practitioners work alone without support from nurses, pharmacists, nutrition specialists, or exercise physiologists.
Virtual-hybrid care has extended the care team’s capabilities for delivering and maintaining patient education and follow-up. The internet continues to be a robust resource, containing a wealth of health information that is easily accessible to the general population. Various mobile applications and electronic devices have also been developed in recent years to educate, track, and manage patients’ health and lifestyles. While these tools provide patients with greater accessibility and independence, they also create a valuable opportunity for healthcare providers to further engage patients. In a virtual-hybrid model, this becomes increasingly important, as patients may frequently transition between virtual and in-person visits. Forming care partnerships with patients through these virtual information and health-tracking resources becomes crucial in the continuity of care and proper health maintenance as we move through the pandemic.
Our most frequent cardiovascular diagnosis was cardiomyopathy (34%), which is reflective of the management need that first helped start the emerging field of Cardio-Oncology, and is similar to the most frequent cardiovascular diagnosis noted by clinicians from several other leading centers (20–35%) [24, 25]. However, Cardio-Oncology has grown remarkably over the last 10–20 years, with a wide spectrum of cardiovascular diagnoses and indications for referral (Fig. 5b) [8, 14]. Accordingly, at some other leading centers, the most frequent cardiovascular diagnosis or indication for referral has been reported as hypertension [14], arrythmia [17], or comprehensive risk assessment prior to beginning of therapy to optimize cardioprotection [8] in the practice of Preventive Cardio-Oncology [21]. This illustrates an opportunity for growth in our Clinic, to increase the fraction of high-risk patients who undergo comprehensive cardiovascular risk evaluation and management of risk factors prior to administration of cardiotoxic therapy.
Our clinic cares for patients with a range of cancer types (Fig. 5d). Individuals with breast, lung, and hematologic cancers represent a substantial proportion of our patient population, similar to other Cardio-Oncology clinics [8, 14, 22, 26]. The most frequent cancer diagnosis, breast cancer (43%), is consistent with reports from other leading cardio-oncology clinics such as the Mayo Clinic (39.2%) [15] and the Cleveland Clinic in Florida (44.3%) [14]. While hematologic malignancies such as leukemia and lymphoma represented 29% of our patients and was the second most prevalent cancer within our cohort, they comprised the most frequent forms of cancer at other cardio-oncology clinics such as at the Moffitt Cancer Center (31%) [8] and at UCLA (32.70%) [25]. However, the absolute difference was relatively insignificant. Overall, similar to these established cardio-oncology clinics, we receive patients from across a variety of cancers.
A wide breadth of cancer therapeutics is associated with cardiotoxicity [21]. Anthracyclines associate with cardiomyopathy, especially when used with trastuzumab. Targeted therapies (e.g., TKIs) can cause new or worsening of pre-existing hypertension. ICIs are associated with an increased incidence of myocarditis. Some cytotoxic chemotherapeutics, such as cisplatin, increase the risk of venous thromboembolism, and antimetabolites such as fluoropyrimidines have long been associated with a broad range of cardiotoxicities. Radiation therapy is associated with ischemic heart disease, valve dysfunction, conduction abnormalities, pericardial disease, and cardiomyopathy. Patients with cancer who have developed cardiovascular toxicity or who may be at high risk for cardiovascular toxicity should be referred to the Cardio-Oncology clinic for close follow-up.
The most frequently used cancer medication used among our patients was trastuzumab (29%), with the second most frequent being anthracyclines (24%). This was similar to other institutions, with anthracyclines and trastuzumab among the most common cancer drugs in their Cardio-Oncology clinics. Yet, anthracyclines were typically noted more commonly than trastuzumab. The Cleveland Clinic in Florida saw patients most commonly treated with radiation (40%), followed by anthracyclines (26.8%) [14]. The Moffitt Cancer Center most frequently had patients who were treated with anthracyclines (52%), with HER2 targeted therapies representing 27% of the cancer drugs [8]. This difference may reflect a high frequency of patients with HER+ breast cancer in our population (diagnosed by a ratio of HER2 to chromosome 17 signals on dual probe fluorescent in situ hybridization ≥2 or ≥ 6 HER2 signals/cell [27]), as well as the keen attention to a substantial fall in left ventricular ejection fraction or global longitudinal strain as a potential prognostic factor in our patients, per American Society of Echocardiography (ASE) guidelines [28].
All of these patient data distributions were obtained in the context of the Virtual-Hybrid patient flow in our de novo Cardio-Oncology Clinic (Fig. 3), that can be modeled by other future Virtual-Hybrid Cardio-Oncology clinics initiated during the pandemic. Table 1 compares the first few weeks of our de novo C-O clinic setup model with two published manuscripts describing conversion of pre-existing in-person C-O clinics to providing telehealth visits as an option for patients. The table shows similar numbers of patients seen in the initial periods of the clinics, although the numbers in our new clinic went from 0 to 10 in the first 3 weeks, compared to going from up to 40 patients weekly to 11 patients in 2.5 weeks for a group that converted their in-person clinic to a virtual option. Overall, CV diagnoses and cancer types were comparable; distributions of cancer drugs were not reported by the other group. Important differences were noted. Most of our patients in the pandemic were new (90%), given the de novo status of the Cardio-Oncology Clinic, while the converted virtual clinic of another group initially focused on established patients for > 50% of their patient visits. While key personnel were also the same (e.g., physician, advanced practice provider, nurse or nurse coordinator), we also report virtual versions of supportive staffing patterns, including the virtual scheduling and rooming process and pharmacy and lifestyle modification visits. Additionally, trainees have been integrally involved in the establishment of our Cardio-Oncology Clinic, with residents training in program-building, and medical students and fellows training in ambulatory cardio-oncology clinical practice and cardio-oncology critical thinking, respectively. Finally, besides the patient flow (Fig. 3) and de novo nature of our Virtual-Hybrid clinic initiated in the pandemic, our unique contribution may be the virtual resources, compared to the essential “webside manner” [7] or an alternative algorithm [4] for triaging virtual or in-person visits to the physician or advanced practice provider (Table 1).
Table 1 Comparisons Among Clinic Models Described In The Pandemic
Virtual-Hybrid Clinic Telehealth Clinic (14) Triage Clinic (4)
Model Type De Novo Conversion Conversion
Time Frame Compared 3 weeks 2.5 weeks Not reported
Number of Patients 10 11 Not reported
New Visits (%) 90 45 Not reported
Variety of CV Diagnoses Yes Yes Not reported
Variety of CA Types Yes Yes Not reported
Variety of CA Drugs Yes Unknown Not reported
Referrals Yes Unknown Yes
Scheduling Yes Unknown Not reported
Rooming Process Yes Unknown Not Reported
Virtual AA Yes Unknown Not Reported
Virtual MA Yes Unknown Not Reported
Virtual Physician Yes Yes Yes
Virtual APP Yes Unknown Yes
Virtual Pharmacist Yes Unknown Not Reported
Virtual Nurse (Coordinator) Yes Unknown Yes
Unique Contribution Virtual Resources Webside Manner Triage Algorithm
Similar to the formation or conversion of Cardio-Oncology clinics, many protocols for treatment regimens and cancer patients are yet to be standardized. While no standard protocols have been widely adopted at Cardio-Oncology practices, various institutions and writing groups have proposed some approaches (e.g., ASE or ASCO guidelines). We have collaboratively developed institutional algorithms for various cardiovascular toxicities and medications based on existing scientific statements, society guidelines, expert consensus statements, and manuscripts from leading cardio-oncology research institutions. The goal is to adopt, adapt, develop, and continuously update these algorithms, as new literature arises in order to establish best practices and an institutional standard of care.
Conclusion
Starting a new Cardio-Oncology Clinic in the pandemic has its challenges, and yet for our patients can be invaluable. Appropriately competing priorities in the pandemic can limit the scheduling of meetings and gathering of people together in one virtual room to discuss a mutual vision. Gathering resources for patient and clinician education can also be formidable, as can social distancing and obtaining important imaging. However, multiple virtual one-on-one or small group meetings can be beneficial for building institutional relationships. Similarly, virtual visits have risen to the challenge to ensure maintenance of patient care throughout the pandemic. Modifications have also been made to enable safety and distancing during imaging. With the benefit of these adjustments to address the challenge, this report provides a foundation for initiating a cardio-oncology clinic in the pandemic, with virtual resources and tools to equip patients and clinicians.
In the future, we will also lay out a roadmap for initiation of comprehensive cardio-oncology programs with the five pillars of patient care, education, research, community engagement, and innovation in the era of digital transformation accelerated by the pandemic. Novel risk modifiers and risk attenuation methods, such as breast arterial calcification, clonal hematopoiesis of indeterminate potential, and Cardio-Oncology prehabilitation, habilitation, and rehabilitation will also be addressed. Future innovation to implement recommendations from clinical trials across the nation currently underway that utilize mobile health or web-based diet and physical activity interventions and/or seek to determine the impact of cardioprotective pharmacotherapy in Preventive Cardio-Oncology will also be assessed (ClinicalTrials.Gov: NCT01988571, NCT02943590, NCT02562716, NCT01968200, NCT03265574, NCT03760588, NCT03386383, NCT02244411, NCT03223753). Many of these studies incorporate virtual technologies that will be very helpful during and after the pandemic as we continue pursuit of digital transformation.
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Acknowledgements
We are grateful to the following individuals, as well as all of the departments and divisions at Froedtert & MCW that have rallied together to help us establish the hybrid Cardio-Oncology clinic or to see our patients needing subspecialty care, or to partner with us in referrals from the inpatient service, especially the following clinicians: Stacey Gardiner MD, Jason Rubenstein MD, David Ishizawar MD, Jalaj Garg MD, Andrew Rosenblum MD. We are also grateful to Nicole Lohr MD PhD and the clinical sections at the Zablocki Veterans Administration Medical Center that have also helped us establish an affiliate Cardio-Oncology Clinic in tandem at the VAMC in Milwaukee, WI.
Authors’ contributions
SAB conceived of the study, obtained, analyzed and interpreted the data, and made a substantial contribution to the writing of the manuscript; SP made a substantial contribution to the writing of the manuscript; DR made a substantial contribution to the writing of the manuscript; SZ obtained the data and made a substantial contribution to the writing of the manuscript; ML interpreted the data and made a substantial contribution to the writing of the manuscript; TN made a substantial contribution to the writing of the manuscript; BS analyzed the data and made a substantial contribution to the writing of the manuscript; RM made a substantial contribution to the writing of the manuscript; JMac made a substantial contribution to the writing of the manuscript; KD made a substantial contribution to the writing of the manuscript; JMes made a substantial contribution to the writing of the manuscript; DM made a substantial contribution to the writing of the manuscript; JS made a substantial contribution to the writing of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was associated with this work.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Froedtert & MCW Institutional Review Board (ID PRO00038807); HIPAA informed consent was waived for this minimal risk study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests. | TRASTUZUMAB | DrugsGivenReaction | CC BY | 33441188 | 18,793,759 | 2021-01-13 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cardiomyopathy'. | A virtual-hybrid approach to launching a cardio-oncology clinic during a pandemic.
BACKGROUND
As cardiovascular disease is a leading cause of death in cancer survivors, the new subspecialty of Cardio-Oncology has emerged to address prevention, monitoring, and management of cardiovascular toxicities to cancer therapies. During the coronavirus disease of 2019 (COVID-19) pandemic, we developed a Virtual-Hybrid Approach to build a de novo Cardio-Oncology Clinic.
METHODS
We conceptualized a Virtual-Hybrid Approach including three arms: information seeking in locations with existing Cardio-Oncology clinics, information gathering at the location for a new clinic, and information sharing to report clinic-building outcomes. A retrospective review of outcomes included collection and synthesis of data from our first 3 months (at pandemic peak) on types of appointments, cancers, drugs, and cardiotoxicities. Data were presented using descriptive statistics.
RESULTS
A de-novo Cardio-Oncology clinic was developed structured from the ground up to integrate virtual and in-person care in a hybrid and innovative model, using the three arms of the Virtual-Hybrid Approach. First, we garnered in-person and virtual preparation through hands-on experiences, training, and discussions in existing Cardio-Oncology Clinics and conferences. Next, we gleaned information through virtual inquiry and niche-building. With partners throughout the institution, a virtual referral process was established for outpatient referrals and inpatient e-consult referrals to actualize a hybrid care spectrum for our patients administered by a multidisciplinary hybrid care team of clinicians, ancillary support staff, and clinical pharmacists. Among the multi-subspecialty clinic sessions, approximately 50% were in Cardio-Oncology, 20% in Preventive Cardiology, and 30% in General Cardiology. In the hybrid model, the Heart & Vascular Center had started to re-open, allowing for 65% of our visits to be in person. In additional analyses, the most frequent cardiovascular diagnosis was cardiomyopathy (34%), the most common cancer drug leading to referral was trastuzumab (29%), and the most prevalent cancer type was breast cancer (42%).
CONCLUSIONS
This Virtual-Hybrid Approach and retrospective review provides guidance and information regarding initiating a brand-new Cardio-Oncology Clinic during the pandemic for cancer patients/survivors. This report also furnishes virtual resources for patients, virtual tools for oncologists, cardiologists, and administrators tasked with starting new clinics during the pandemic, and innovative future directions for this digital pandemic to post-pandemic era.
Introduction
Cardio-Oncology care has been adjusted in the COVID-19 pandemic with limited in-person clinic or hospital visits, increased use of teleconsultation, less frequent imaging, increased reliance on biomarkers, and considerations of differential diagnoses involving COVID-19 when evaluating cancer patients or survivors for possible cardiovascular toxicity [1]. Monitoring and management algorithms have been developed to help guide virtual care [2–4]. In the pandemic, we have changed the way in which we provide healthcare services at our clinics and institutions. This has challenged us to restructure current systems for the safety of our patients.
Various forms of innovation have come to bear in the pandemic, including telemedicine, digital health, artificial intelligence, social media, informatics, big data, and precision medicine [5, 6]. Telemedicine is the primary form of innovation that has been most developed in the pandemic [2, 5, 7]. Social media has been very helpful for dissemination of information, as well as education, and has been integral for creating online groups for support and determining the best ways for proceeding in the pandemic and advocating for our patients and colleagues in this period [5, 6]. In addition, the Doximity social media application has been valuable to practices across the nation, due to its telehealth platform (Doximity Video and Phone; https://www.doximity.com/dialer-video).
Despite the growing need, and allowances made during the pandemic, many centers do not have formal Cardio-Oncology clinics. Starting a new clinic can be challenging. The COVID-19 pandemic has made the process significantly more difficult, with the need to minimize exposure and maximize patient safety.
Currently, limited information is available on how to start a Cardio-Oncology Clinic during a pandemic, albeit given the high risk of morbidity or mortality in COVID-19-positive patients who also have cancer or CVD [8–13]. Several institutions have published on their experiences with starting in-person Cardio-Oncology clinics prior to the pandemic [8, 14–17]. One group has reported on their conversion from existing in-person Cardio-Oncology visits to telemedicine consultations, seeing 11 patients virtually within a few weeks [7]. Many have considered implications of the pandemic on the practice and study of cardio-oncology [2, 4, 5, 18–20], and two groups have suggested models for clinics converting from existing in-person care to televisits [4, 5]. Yet, no groups have directly addressed steps for de novo virtual-hybrid clinic formation within the limitations of the pandemic and without conversion of a pre-existing Cardio-Oncology clinic.
Our report offers a template for other centers to develop their own new Cardio-Oncology clinics during the pandemic. We determined a Virtual-Hybrid Approach to clinic launch, with both virtual and in-person elements of three key arms: information seeking where there are existing Cardio-Oncology Clinics in place, information gathering where the clinic will be built, and information sharing to report on initial patient data demonstrating the success of the launch (Fig. 1). We then performed retrospective chart review to collect and synthesize data on the types of appointments (new versus established, virtual versus in-person), cancers (e.g., breast, prostate, leukemia, lung), cancer drugs, and cardiovascular toxicities (e.g., cardiomyopathy, hypertension) for patients seen virtually or in person in our new Cardio-Oncology clinic at Froedtert Hospital and Medical College of Wisconsin (F&MCW). Here, we will discuss our findings in the context of previous publications on launching Cardio-Oncology Clinics prior to the pandemic. Our results will present distributions of cancer drugs and types, and cardiovascular diagnoses, similar to previous publications on Cardio-Oncology clinic-building. However, we will differentiate and illuminate the techniques that leverage the virtual underpinnings of pandemic clinic-building. We submit that it is feasible to establish a new Cardio-Oncology Clinic for cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy during a pandemic, providing optimal care for new patients in the midst of the need for safety and minimizing exposure. We also propose virtual resources for patients and clinicians and describe innovative future directions in the pandemic and post-pandemic period.
Fig. 1 The Virtual-Hybrid Approach to Cardio-Oncology Clinic-building in the pandemic
Methods
Virtual-hybrid approach
We pursued a Virtual-Hybrid Approach of information seeking, information gathering, and information sharing (Fig. 1). For information seeking, we focused on institutions that already had a Cardio-Oncology clinic in place. Prior to the COVID-19 pandemic, substantial experience was gained at an established in-person Cardio-Oncology clinic at a world-renowned leading hospital. Published manuscripts on pre-pandemic building and operations of Cardio-Oncology clinics at other institutions were reviewed. Extensive networking with leaders of Cardio-Oncology clinics around the United States and in the United Kingdom was accomplished at regional, national, and international meetings in person and on social media in a hybrid approach. These meetings were attended in person pre-COVID-19 and virtually during the pandemic. For information gathering, we addressed the location in which the new Cardio-Oncology clinic would be built. We learned about existing resources in the destination Heart & Vascular Center and existing needs and patients in the destination partner cancer center. We networked with physicians, advanced practice providers (APPs), nurses, service line leaders, and administrators in the Heart & Vascular Center and the cancer center, as well as in primary care and other supporting specialties. The subsequent Cardio-Oncology clinic sessions were intermingled with other subspecialty areas, to optimize availability for patient visits while filling clinic slots and tailoring spectrum of care to emerging patient needs. Cardio-Oncology patient visits were included in multi-subspecialty clinics.
Retrospective study design
For quantitative data, we pursued a retrospective observational study to determine the distribution of outpatient visits in the first 3 months of our virtual-hybrid Cardio-Oncology Clinic. We reviewed data from charts of patients (all were 18 years of age or older) who received outpatient care from the new Director of Cardio-Oncology at F&MCW between April 15, 2020 and July 17, 2020 to determine which of these patients were considered to be in Cardio-Oncology, Preventive Cardiology, or General Cardiology. We collated all three to determine the percentage of patients seen in Cardio-Oncology, compared to the other two specialties. Preventive Cardiology was collated as a partner clinic to help build the Preventive Cardio-Oncology component of the Cardio-Oncology Clinic, to help apply established principles for prevention. From among the multi-subspecialty clinic sessions, we determined the proportion of patients who were specifically cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy and thereby seen in the Cardio-Oncology Clinic. Next, we identified the distribution of cardiovascular toxicities in cancer patients or survivors seen in the Cardio-Oncology Clinic. We also evaluated the spectrum of cancer drugs received by cancer patients or survivors seen in the Cardio-Oncology Clinic. In addition, we summarized the types of cancers in patients seen in the Cardio-Oncology Clinic. Finally, we assessed the frequency of virtual visits during the course of the pandemic over our first 3 months for patients seen in the Cardio-Oncology Clinic. This retrospective review was approved by the F&MCW IRB; HIPAA informed consent was waived for this minimal risk study, which did not involve any form of intervention and was conducted in compliance with good clinical and research practice. The team designed and carried out the study with reliance on virtual communication tools.
Data collection and analysis
Data gathering, management, and analysis were conducted at F&MCW. We collected patient-related, disease-related, treatment-related, and outcome-related data, particularly patient sex, type of appointment (new versus established, virtual versus in-person), type of cancer (e.g., breast, prostate, leukemia, lung), type of cancer drug, and type of cardiovascular toxicity (e.g., cardiomyopathy, hypertension). In order to minimize any risk of breaching patient confidentiality, all data collection occurred on institutional-based computing environments with de-identified data used for analyses. There were no alternative procedures for the subjects as this is a retrospective review of data that are not amenable to prospective collection and review. Descriptive graphs or tables of patient-, disease-, treatment-, and outcome-related variables distributions were prepared, with no comparisons made needing statistical tests.
Results
Virtual preparation
Preparation for starting the Cardio-Oncology clinic followed a Virtual-Hybrid Approach (Fig. 1, left). Five overarching factors employing virtual communication methods emerged to ensure the successful launching of the clinic. Team and individual experience and exposure to various areas of interest in Cardio-Oncology were achieved and assessed before and during the pandemic. Far-reaching connections to experts and potential collaborators in the field were developed and exercised. Close contact with the institution launching this clinic was important to determine the resources available; these resources dictated the strategy and potential outcomes of the clinic. Importantly, the expectations of others for the Cardio-Oncology clinic were determined and incorporated. Finally, recognition of the limitations that exist at the destination institution guided care and goal setting.
Virtual inquiry
Before initiating the Cardio-Oncology clinic in the destination institution, existing structures, patient base, and needs in the Heart & Vascular Center as well as the Cancer Center were evaluated, adhering to pandemic protocols (Fig. 1, middle). Pre-existing building blocks for the planned Cardio-Oncology clinic were assessed, and the partner Preventive Cardiology clinic was investigated. We also evaluated characteristics of the cancer center patient population to best position the clinic for success.
Virtual niche-building
Five main aspects of niche-building were pursued. Partnerships with Vascular and Cancer Center physicians, advanced practice providers, and service line leaders were developed to initiate and grow the clinic (Fig. 1, middle). The Cardio-Oncology team and clinic flexibility were demonstrated through openness to taking quicksteps. Presentations were made at Grand Rounds and rounds across the institution in Cardiology, Hematology/Oncology, Radiation Oncology, Surgical Oncology, Internal Medicine, and Family Medicine to promote the clinic capabilities. Collaborative solutions for problems facing fields complementing Cardio-Oncology developed trust and collaboration. Teamwork was developed by leveraging diversity of perspectives and virtual communication technologies, to establish effective patient care despite COVID-19 limitations.
Hybrid care Spectrum
The hybrid F&MCW Cardio-Oncology Clinic was initiated and established in the outpatient setting, in close partnership with the Preventive Cardiology Clinic, Cancer Center, and inpatient Cardiology Consult and Hematology/Oncology teams (Fig. 1, right).
Initial and subsequent visits have been completed in person or by video, with phone visits also available for virtual return visits if patients without adept and available smartphone use have limited ability to appear in person. Virtual patient visits over our first 3 months occurred with the use of telemedicine platforms integrated with Epic (via MyChart for patients and Haiku/Canto for clinicians), or using the Doximity video call function. Patients with in-person appointments are screened appropriately on arrival for signs or symptoms of COVID-19 or exposure, following institutional protocols. Wearing masks is required of all patients, and each patient can be accompanied by a family member; some choose to also wear gloves or face shields. There is sufficient room for maintaining social distancing in the clinic waiting room and hallways.
Innovation
Current innovation in the clinic also includes Virtual Clinician Tools and Virtual Patient Resources (Fig. 2). For clinicians, the links for an AHA CME course on Novel Concepts, Current Debates and Treatment Considerations in Cardio-Oncology, an online Cardio-Oncology Compendium hosting risk assessment clinical decision aids, Cardio-Oncology Drug Regimen and Acronym Databases, and UPTODATE access for reviewing Cardio-Oncology drug information are supplied. For patients, the video from the International Cardio-Oncology Society explaining the Cardio-Oncology subspecialty, American College of Cardiology (ACC) mobile health (mHealth) CardioSmart education app and website, Cancer Heart Talk mini-podcast series accessed via SoundCloud app and website, Cardio-Oncology Frequently Asked Questions, and ChemoCare website are provided for patient-facing Cardio-Oncology and heart anatomy and physiology education, engagement, and awareness. Virtual Resources for Preventive Cardio-Oncology are also made available to our patients. These include the American Heart Association (AHA) Physical Activity Recommendations, AHA Life’s Simple 7 Webpages, American Society For Preventive Cardiology Online Coaching Webpages in partnership with Intervent, and the Become An Ex Smoking Cessation Support Webpages in partnership with Mayo Clinic. The resources are provided in the Epic patient portal MyChart, and more ways to make the resources accessible to a broad and diverse patient population are in development. Future innovation in the clinic will explore contemporary initiatives connecting patients and their safely guarded data with their permission with wearable devices, health information technology, informatics, artificial intelligence, personalized medicine, and additional mobile health (mHealth) applications.
Fig. 2 Virtual patient and clinician education and resources. Available online as PDF with hot links in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual visit infrastructure and timeline
There was no pre-existing Cardio-Oncology program at the time of launching our de novo Virtual/Hybrid Cardio-Oncology Clinic. The newly recruited Director of Cardio-Oncology was tasked with the responsibility of launching the new clinic, with support from the Heart & Vascular Center clinic administrators, medical director, and Cardiology Division and Department of Medicine leadership. Prior to opening the Cardio-Oncology Clinic, the Heart & Vascular Center initiated virtual conversion then additionally collaborated with Inception Health (MCW’s innovation lab company) over the course of 4 weeks to iteratively develop the clinical informatics infrastructure for virtual visits. The video visits were designed to function using clinician’s personal smartphones, iPads, and tablets, with direct web browser video links from the electronic health record mobile application. Direct video calls through the Doximity mobile application were also approved. Existing Inception Health personnel re-allocated their time in order to adopt and maintain responsibility for the virtual component of all ambulatory clinics across the health system, in partnership with medical and administrative directors of each clinic area, such as the Heart & Vascular Center. No additional costs or hires were pursued to facilitate the development of the virtual visit infrastructure and timeline. Existing resources and personnel were re-allocated to virtual visit design to enable building the virtual clinics in the Heart & Vascular Center. To assist clinicians and billing compliance colleagues, note templates were created for video and phone visits to indicate patient informed consent for virtual visits due to the pandemic, as well as to capture limited appropriate physical examinations, in addition to the amount of time spent on records review and real-time medical counseling.
Initial consults were electronically triaged by either a cardiologist or a cardiology fellow supervised by a cardiologist. Each triage team determined which consults would be appropriate as virtual video visits, versus in-person to occur once the Heart and Vascular Center started re-opening routine physical visits, or whether patients needed to be evaluated urgently in person. During the first week of operation, the brand-new Cardio-Oncology Clinic started entirely virtually with only video and phone visits. As the Heart and Vascular Center re-opened for physical patient visits the following week, from week 2 through the remainder of the first 3 months the Cardio-Oncology Clinic had both virtual and in-person visits integrated throughout each clinic session weekly, based on whether patients were new and whether they had smart device or computer functionality available.
Virtual referral network and process
Cardio-Oncology patient assessment begins within a referral network before the patient arrives at a Cardio-Oncology clinic. Consequently, forming a virtual referral network and enacting a user-friendly virtual referral process was a key component of building the Cardio-Oncology clinic during the pandemic. All referral patterns and networks for our de novo Cardio-Oncology Clinic were built from the ground up. Initial referrals were from within our health system; this quickly expanded to consults from outside of our health system encompassing the entire state. Patients were referred to our Clinic by clinicians or by self-referrals. Some of our local patients connected to us after being introduced to us by their clinicians in other states or through family members in other states who learned about us from their own clinicians or community-based Cardiology society outreach events.
Referrals across the institution and outside of our health system have come to us from the Divisions of Hematology and Oncology, Internal Medicine, Family Medicine, Surgical Oncology/Breast Clinic, and Survivorship Clinics. From the cancer center’s perspective, there may be many “triggers” that would warrant a Cardio-Oncology referral. For example, an abnormal ECG, an abnormal echocardiogram, cardiovascular symptoms, previous cardiovascular history (e.g., coronary artery disease, hypertension, cardiomyopathy) particularly in a patient who previously underwent treatment or is beginning new treatment with cardiotoxic neoplastic medications or radiation therapy and is at high risk of cardiovascular toxicity, or those in preparation for stem cell transplant, or oncologic surgery. Referral protocols were determined based on standard practice, discussions with colleagues in Medical and Radiation Oncology, Hematology, Bone Marrow Transplant, Surgical Oncology/Breast Clinic, Children’s Hospital, Radiation Oncology, Primary Care, and updated literature reviews.
Cancer survivors are at a higher risk than the general population for cardiovascular morbidity and mortality. If a cancer survivor needing to be evaluated is already under the care of a cardiologist, the referring provider can reach out to their cardiologist for guidance on the appropriate CV surveillance. If they do not already have a cardiologist, a Cardio-Oncology consult should be requested. The Cardio-Oncology consult can be placed using a direct Cardio-Oncology button within the universally available Cardiovascular Consult order panel. Referrers can also place a General Cardiology consult and mention the Cardio-Oncology physician by name as requested by the clinician or patient. An E-Consult functionality is also being implemented for those patients who need to be assessed sooner than the next available appointment, or for those patients who may not need a full Cardio-Oncology evaluation, or if referring providers are uncertain. The e-consult can also be placed as a second opinion requested by the inpatient Cardiology Consult team.
The inpatient Cardiology Consult service will continue to directly address inpatient consults from the inpatient hematology/oncology services. The inpatient Cardiology Consult service can collaborate with the Cardio-Oncology Clinic via formal Cardio-Oncology E-consults in the electronic health record Epic if a specific focused question arises regarding Cardio-Oncology relevant to the care of individual currently hospitalized patients that have already been formally evaluated by the inpatient Cardiology Consult service. After a patient has been formally evaluated by the inpatient Cardiology Consult service, if the patient is appropriate for outpatient follow up in the Cardiology clinic with Cardio-Oncology, this should be communicated to the primary Hematology/Oncology service. If appropriate at the time of consultation, the inpatient cardiology consult service can make the follow-up appointment. Oftentimes, this patient population remains in the inpatient setting for several weeks. If this is the case, the Cardiology clinic phone number and clinician information should be provided to the primary service to do so prior to the patient being discharged from the hospital.
Virtual-hybrid multidisciplinary team
It is important to develop a multidisciplinary team and initially focus on allocation of pre-existing resources. Accordingly, some roles among our Cardio-Oncology clinic personnel are shared with other subspecialties. Our virtual-hybrid multidisciplinary Cardio-Oncology Clinic personnel include physicians, a nurse practitioner (NP), a nurse, a research support specialist, medical assistants, pharmacists, administrative assistants, and administrators. All personnel with pre-existing in-person roles and practices re-allocated a portion of their time to the development and practice of virtual visits.
Our clinic and partners consist of board-certified Cardiologists with special training in various cardiac subspecialties (e.g., cardio-oncology, preventive cardiology, heart failure and transplant, electrophysiology, interventional cardiology), who collaborate closely with our cancer experts. Our physicians together specialize in the prevention, diagnosis, and treatment of heart and vascular disorders resulting from side effects of cancer therapy. Our comprehensive team of advanced practice providers, nurses, and pharmacists work alongside our physicians to care for patients from the moment of cancer diagnosis through life’s survivorship journey. The NP typically sees established patients when needed to follow up on imaging, intervention, or diagnostic and management plans, and may also see select new patients. In complex cases, the NP discusses the care of established patients with both the cardio-oncologist and the referring clinician. The nurse assists with patient triage and communications (including addressing patient requests and queries), liaises closely with the nurse practitioner and pharmacists, and educates patients on Cardio-Oncology using virtual materials. Our clinical pharmacists function at the highest level of their advanced training, similar to all clinic personnel, and assist with medication education, review, titration, discussion, and prescription, particularly for heart failure, hypertension, hyperlipidemia, and smoking cessation, as well as commenting on potential drug interactions.
For Preventive Cardio-Oncology, we additionally partner with our dietitians and exercise physiologists to help advise our patients on nutrition and exercise plans, as well as our colleagues in cardiopulmonary stress testing where applicable. Further, in the pandemic, we provide patients with free online coaching options for lifestyle modification (Fig. 2). We also direct patients to AHA webpages with guidance on pursuing ideal cardiovascular health.
Virtual-hybrid patient flow
Once a referral is placed by the designated order buttons in the electronic health record, central schedulers or the Cardio-Oncology Clinic administrative assistant schedule the new patient for a video or in-person visit (Fig. 3). The clinic administrative assistant works closely with our health professionals in our interdisciplinary advanced subspecialty clinic to gather relevant clinical reports and history pertinent to patient appointments. Virtual medical assistants contact patients a few days before their appointments to confirm and troubleshoot virtual connectivity. On the appointment day, medical assistants then ‘room’ patients for virtual or in-person visits by preparing patients for their medical visits (including reviewing medications and in-person or at-home virtual vital signs), and also rechecking virtual connectivity for video visits. The clinician then completes the visit virtually or in-person and introduces the patient to the range of electronic resources available. Following the visit, the clinical administrative assistant arranges follow-up testing and appointments.
Fig. 3 Virtual-Hybrid Patient Flow Chart. Admin = Administrative; MA = Medical Assistant; MD = Medical Doctor; NP = Nurse Practitioner
Virtual risk assessment
Baseline risk assessment and follow-up start with oncology and primary care [21]. Asymptomatic low risk patients with low-risk treatment plans can have continued assessment and follow-up by oncology and primary care in partnership. Patients who have symptoms, are at high risk based on their history, or are planned for high-risk treatment plans should be referred to Cardio-Oncology for prevention, monitoring, and management recommendations. Recommendations should adhere to society expert consensus, scientific statements, and guidelines for prevention, surveillance, and survivorship, and optimize CVD risk and medications [21]. A putative risk score based on medication-related and patient-related risk factors can be used to guide monitoring and management recommendations for most Cardio-Oncology patients [22], and can be used in a virtual clinical decision aid (https://tinyurl.com/CardioOncCDA) (Fig. 4). Specific risk scores are also available for adults treated with anthracyclines, trastuzumab, or other drugs, or for adult survivors of childhood cancers [9–13].
Fig. 4 Virtual Cardio-Oncology clinical decision aid (CDA). Risk assessment (https://tinyurl.com/CardioOncCDA) to guide monitoring and management recommendations regarding development of cardiomyopathy for most Cardio-Oncology patients; a CDA specifically for women with early breast cancer is also available in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual management algorithms
Evidence-based management algorithms have been selected or developed as adjunctive resources for inpatient teams. They are available online in a virtual collection for use in the inpatient setting by the inpatient Cardiology Consult service or hematology/oncology teams to assist with diagnosis and treatment of cardiovascular toxicities from cancer therapies or cancer itself. The algorithms cover cardiomyopathy from anthracyclines or trastuzumab, planned chemotherapy with pre-existing cardiomyopathy, neurohormonal therapy or dexrazoxane for cardioprotection, myocarditis, persistent malignant pericardial effusion, hypertension, surveillance after radiation therapy or drugs that cause ischemia, malignant pericardial effusion, and other salient topics frequently encountered.
Virtual community engagement
The local, regional, national, and international community was virtually engaged via social media posts on Twitter (using #MCWCardioOnc on @DrBrownCares or @PrevCardioOnc), podcasts hosted by the MCW CTSI (available on iTunes, Google, and Apple podcast platforms), Heart Success podcast series, and Cancer Heart Talk brief 15-min mini-podcast series (available on SoundCloud). Perspectives were also published for international community engagement in the Women Heart Alliance newsletter, as well as on the AHA Early Career Blog, ACC Women in Cardiology Blog, CardioOncTrain.Com Blog, and PrevCardioOnc.Com Blog. Virtual continuing medical education (CME) presentations were also given at the Wisconsin state ACC annual conference meeting, Midwest ACC annual conference meeting, Southeast ACC annual conference meeting, Brazilian Cardio-Oncology Symposium, and the Ohio State Cardio-Oncology CME conference, then subsequently at the AHA and ACC annual national scientific sessions.
Distribution of patient data
In our multi-subspecialty clinic visits (virtual and in-person integrated and combined; n = 182; 136 new and 47 returns), approximately 50% of patient visits were in Cardio-Oncology, 20% were in Preventive Cardio-Oncology, and 30% were in General Cardiology (Fig. 5a). Overall among Cardio-Oncology visits, 65% were in person, consistent with early and safe clinic re-opening in a hybrid model, with 19% by video and 16% by phone, with the fraction by phone decreasing over time as patients and clinic personnel became more adept with troubleshooting video. Of new patients, 77% were in person, and the remainder by video. No Cardio-Oncology patients presenting in person developed any signs or symptoms concerning for COVID-19.
Fig. 5 Initial Clinic-Building Outcomes Using the Virtual-Hybrid Approach. a Distribution of Cardio-Oncology, Preventive Cardio-Oncology, and General Cardiology patients seen in our multi-subspecialty clinic visits. b Distribution of cardiovascular diagnosis or indication for referral. c Distribution of cancer drugs. d Distribution of cancer types
The most frequent cardiovascular diagnosis or indication for referral was cardiomyopathy (34%) (Fig. 5b). Other diagnoses included decrease in global longitudinal strain, diastolic congestive heart failure, hypertension, myocarditis, dyspnea, chest pain, palpitations, survivorship, risk assessment, and pre-bone marrow transplant, among other cardiovascular diagnoses or visit indications. The most frequent cancer drug was trastuzumab (29%) (Fig. 5c), managed according to a novel algorithm developed in our de novo Virtual-Hybrid Cardio-Oncology Clinic based on the recent publication indicating the safety of continuation of trastuzumab for left ventricular ejection fraction of 40% or greater [23] (Fig. 6). The second most frequent cancer drug was anthracycline (24%). Other drugs included, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), endocrine therapies, and investigational therapeutics, among others. The most frequent cancer type in our clinic was breast cancer (42%) (Fig. 5d). These trends in cardiovascular diagnosis or indication and cancer drugs or types were similar in assessments of virtual visits alone, with the most frequent being cardiomyopathy (43%), trastuzumab (41%), and breast cancer (44%), respectively. The findings of similar cardiovascular and cancer distributions in virtual versus in-person visits indicated an optimal qualitative return on resource and personnel investment.
Fig. 6 Algorithm for Continuation of Trastuzumab Therapy with Mild LV Dysfunction
Imaging and medication titration
In our clinic, a distribution of cardiovascular diagnoses determines the imaging needed for each patient (Fig. 5b). Therefore, a number of imaging modalities are useful to our patients (e.g., echocardiography, computed tomography with or without angiography, magnetic resonance imaging, coronary angiography, myocardial perfusion imaging). Our most frequently used imaging modality is echocardiography. The frequency of obtaining echocardiograms has depended on each patient’s condition and cancer treatment. A substantial portion of patients coming to us on trastuzumab have needed an echocardiogram every 1–3 months, depending on the extent of adverse effects on left ventricular ejection fraction (LVEF) or strain In these patients, medication titration has occurred approximately every 2 weeks, and for very symptomatic patients with volume overload, they have often been seen weekly..
In our management algorithms, early referral prior to the onset of symptoms has been emphasized, especially in cancer patients or survivors with a history of cardiovascular disease, cardiotoxic neoplastic agents, or a high risk of cardiovascular toxicity. This has provided an opportunity for us to assess and discuss ways to optimize the benefit to risk ratio of continuing with the current cancer treatment plan, and more importantly how and when to put cardioprotective measures in place to facilitate safe cancer therapy. Such discussions have also resulted in closer monitoring. Some conditions have warranted proceeding to other modalities of non-invasive imaging, such as cardiac MRI if myocarditis is suspected. For cases in which coronary artery disease is suspected, our patients undergo functional assessment of their coronaries with a stress test or anatomical assessment with a coronary CT scan or invasive coronary angiography. In our practice, only exercise stress tests were halted due to the pandemic. Every other form of imaging including rest and stress echo, as well as MRI and nuclear medicine have remained readily available for those with cardiovascular toxicities or individuals considered to be at moderate or high risk. This allowed us to adhere to pre-pandemic imaging recommendations tailored during the pandemic to limit imaging if possible to those who are at higher risk for cardiovascular toxicities or who have already been diagnosed with these adverse effects [1–4] (e.g., Fig. 6).
Discussion
The COVID-19 pandemic has inevitably compelled leaders of healthcare clinics to rethink and restructure approaches to deliver optimal care for patients. Our brand-new Cardio-Oncology clinic has been built to thrive in this new pandemic landscape by utilizing virtual technology as one of the key components of our clinic-building and care model since its inception. While existing clinics have reinvented their operations through the uptake of technology, our clinic has been able to capitalize on this resource to deliver virtual-hybrid care from the start. Virtual communication has proved useful to coordinate referral networks and care among providers within a multidisciplinary team across different clinics and departments. We see a variety of cancer patients, types, and drugs (Fig. 5), and our distribution results are generally congruent with reports from other leading cardio-oncology clinics [8, 14, 15, 17, 22].
Various methods have been developed for risk assessment to help guide providers and patients in determining the appropriate guidelines for care. We offer the use of virtual risk assessment tools such as the computed risk scores based on medication- and patient-related risk factors [22] (Fig. 4) (https://tinyurl.com/CardioOncCompendium), as well as recommendations for establishing cross-provider partnerships to continuously evaluate risk [21]. Other online databases containing useful information and guidelines are readily accessible and can help guide clinical practices. We encourage use of these virtual tools, which can further facilitate collaborative Cardio-Oncology care in the pandemic. Our conversations with international colleagues have suggested additional utility of these virtual tools beyond the pandemic. The online resources can be very helpful in settings where clinical practitioners work alone without support from nurses, pharmacists, nutrition specialists, or exercise physiologists.
Virtual-hybrid care has extended the care team’s capabilities for delivering and maintaining patient education and follow-up. The internet continues to be a robust resource, containing a wealth of health information that is easily accessible to the general population. Various mobile applications and electronic devices have also been developed in recent years to educate, track, and manage patients’ health and lifestyles. While these tools provide patients with greater accessibility and independence, they also create a valuable opportunity for healthcare providers to further engage patients. In a virtual-hybrid model, this becomes increasingly important, as patients may frequently transition between virtual and in-person visits. Forming care partnerships with patients through these virtual information and health-tracking resources becomes crucial in the continuity of care and proper health maintenance as we move through the pandemic.
Our most frequent cardiovascular diagnosis was cardiomyopathy (34%), which is reflective of the management need that first helped start the emerging field of Cardio-Oncology, and is similar to the most frequent cardiovascular diagnosis noted by clinicians from several other leading centers (20–35%) [24, 25]. However, Cardio-Oncology has grown remarkably over the last 10–20 years, with a wide spectrum of cardiovascular diagnoses and indications for referral (Fig. 5b) [8, 14]. Accordingly, at some other leading centers, the most frequent cardiovascular diagnosis or indication for referral has been reported as hypertension [14], arrythmia [17], or comprehensive risk assessment prior to beginning of therapy to optimize cardioprotection [8] in the practice of Preventive Cardio-Oncology [21]. This illustrates an opportunity for growth in our Clinic, to increase the fraction of high-risk patients who undergo comprehensive cardiovascular risk evaluation and management of risk factors prior to administration of cardiotoxic therapy.
Our clinic cares for patients with a range of cancer types (Fig. 5d). Individuals with breast, lung, and hematologic cancers represent a substantial proportion of our patient population, similar to other Cardio-Oncology clinics [8, 14, 22, 26]. The most frequent cancer diagnosis, breast cancer (43%), is consistent with reports from other leading cardio-oncology clinics such as the Mayo Clinic (39.2%) [15] and the Cleveland Clinic in Florida (44.3%) [14]. While hematologic malignancies such as leukemia and lymphoma represented 29% of our patients and was the second most prevalent cancer within our cohort, they comprised the most frequent forms of cancer at other cardio-oncology clinics such as at the Moffitt Cancer Center (31%) [8] and at UCLA (32.70%) [25]. However, the absolute difference was relatively insignificant. Overall, similar to these established cardio-oncology clinics, we receive patients from across a variety of cancers.
A wide breadth of cancer therapeutics is associated with cardiotoxicity [21]. Anthracyclines associate with cardiomyopathy, especially when used with trastuzumab. Targeted therapies (e.g., TKIs) can cause new or worsening of pre-existing hypertension. ICIs are associated with an increased incidence of myocarditis. Some cytotoxic chemotherapeutics, such as cisplatin, increase the risk of venous thromboembolism, and antimetabolites such as fluoropyrimidines have long been associated with a broad range of cardiotoxicities. Radiation therapy is associated with ischemic heart disease, valve dysfunction, conduction abnormalities, pericardial disease, and cardiomyopathy. Patients with cancer who have developed cardiovascular toxicity or who may be at high risk for cardiovascular toxicity should be referred to the Cardio-Oncology clinic for close follow-up.
The most frequently used cancer medication used among our patients was trastuzumab (29%), with the second most frequent being anthracyclines (24%). This was similar to other institutions, with anthracyclines and trastuzumab among the most common cancer drugs in their Cardio-Oncology clinics. Yet, anthracyclines were typically noted more commonly than trastuzumab. The Cleveland Clinic in Florida saw patients most commonly treated with radiation (40%), followed by anthracyclines (26.8%) [14]. The Moffitt Cancer Center most frequently had patients who were treated with anthracyclines (52%), with HER2 targeted therapies representing 27% of the cancer drugs [8]. This difference may reflect a high frequency of patients with HER+ breast cancer in our population (diagnosed by a ratio of HER2 to chromosome 17 signals on dual probe fluorescent in situ hybridization ≥2 or ≥ 6 HER2 signals/cell [27]), as well as the keen attention to a substantial fall in left ventricular ejection fraction or global longitudinal strain as a potential prognostic factor in our patients, per American Society of Echocardiography (ASE) guidelines [28].
All of these patient data distributions were obtained in the context of the Virtual-Hybrid patient flow in our de novo Cardio-Oncology Clinic (Fig. 3), that can be modeled by other future Virtual-Hybrid Cardio-Oncology clinics initiated during the pandemic. Table 1 compares the first few weeks of our de novo C-O clinic setup model with two published manuscripts describing conversion of pre-existing in-person C-O clinics to providing telehealth visits as an option for patients. The table shows similar numbers of patients seen in the initial periods of the clinics, although the numbers in our new clinic went from 0 to 10 in the first 3 weeks, compared to going from up to 40 patients weekly to 11 patients in 2.5 weeks for a group that converted their in-person clinic to a virtual option. Overall, CV diagnoses and cancer types were comparable; distributions of cancer drugs were not reported by the other group. Important differences were noted. Most of our patients in the pandemic were new (90%), given the de novo status of the Cardio-Oncology Clinic, while the converted virtual clinic of another group initially focused on established patients for > 50% of their patient visits. While key personnel were also the same (e.g., physician, advanced practice provider, nurse or nurse coordinator), we also report virtual versions of supportive staffing patterns, including the virtual scheduling and rooming process and pharmacy and lifestyle modification visits. Additionally, trainees have been integrally involved in the establishment of our Cardio-Oncology Clinic, with residents training in program-building, and medical students and fellows training in ambulatory cardio-oncology clinical practice and cardio-oncology critical thinking, respectively. Finally, besides the patient flow (Fig. 3) and de novo nature of our Virtual-Hybrid clinic initiated in the pandemic, our unique contribution may be the virtual resources, compared to the essential “webside manner” [7] or an alternative algorithm [4] for triaging virtual or in-person visits to the physician or advanced practice provider (Table 1).
Table 1 Comparisons Among Clinic Models Described In The Pandemic
Virtual-Hybrid Clinic Telehealth Clinic (14) Triage Clinic (4)
Model Type De Novo Conversion Conversion
Time Frame Compared 3 weeks 2.5 weeks Not reported
Number of Patients 10 11 Not reported
New Visits (%) 90 45 Not reported
Variety of CV Diagnoses Yes Yes Not reported
Variety of CA Types Yes Yes Not reported
Variety of CA Drugs Yes Unknown Not reported
Referrals Yes Unknown Yes
Scheduling Yes Unknown Not reported
Rooming Process Yes Unknown Not Reported
Virtual AA Yes Unknown Not Reported
Virtual MA Yes Unknown Not Reported
Virtual Physician Yes Yes Yes
Virtual APP Yes Unknown Yes
Virtual Pharmacist Yes Unknown Not Reported
Virtual Nurse (Coordinator) Yes Unknown Yes
Unique Contribution Virtual Resources Webside Manner Triage Algorithm
Similar to the formation or conversion of Cardio-Oncology clinics, many protocols for treatment regimens and cancer patients are yet to be standardized. While no standard protocols have been widely adopted at Cardio-Oncology practices, various institutions and writing groups have proposed some approaches (e.g., ASE or ASCO guidelines). We have collaboratively developed institutional algorithms for various cardiovascular toxicities and medications based on existing scientific statements, society guidelines, expert consensus statements, and manuscripts from leading cardio-oncology research institutions. The goal is to adopt, adapt, develop, and continuously update these algorithms, as new literature arises in order to establish best practices and an institutional standard of care.
Conclusion
Starting a new Cardio-Oncology Clinic in the pandemic has its challenges, and yet for our patients can be invaluable. Appropriately competing priorities in the pandemic can limit the scheduling of meetings and gathering of people together in one virtual room to discuss a mutual vision. Gathering resources for patient and clinician education can also be formidable, as can social distancing and obtaining important imaging. However, multiple virtual one-on-one or small group meetings can be beneficial for building institutional relationships. Similarly, virtual visits have risen to the challenge to ensure maintenance of patient care throughout the pandemic. Modifications have also been made to enable safety and distancing during imaging. With the benefit of these adjustments to address the challenge, this report provides a foundation for initiating a cardio-oncology clinic in the pandemic, with virtual resources and tools to equip patients and clinicians.
In the future, we will also lay out a roadmap for initiation of comprehensive cardio-oncology programs with the five pillars of patient care, education, research, community engagement, and innovation in the era of digital transformation accelerated by the pandemic. Novel risk modifiers and risk attenuation methods, such as breast arterial calcification, clonal hematopoiesis of indeterminate potential, and Cardio-Oncology prehabilitation, habilitation, and rehabilitation will also be addressed. Future innovation to implement recommendations from clinical trials across the nation currently underway that utilize mobile health or web-based diet and physical activity interventions and/or seek to determine the impact of cardioprotective pharmacotherapy in Preventive Cardio-Oncology will also be assessed (ClinicalTrials.Gov: NCT01988571, NCT02943590, NCT02562716, NCT01968200, NCT03265574, NCT03760588, NCT03386383, NCT02244411, NCT03223753). Many of these studies incorporate virtual technologies that will be very helpful during and after the pandemic as we continue pursuit of digital transformation.
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Acknowledgements
We are grateful to the following individuals, as well as all of the departments and divisions at Froedtert & MCW that have rallied together to help us establish the hybrid Cardio-Oncology clinic or to see our patients needing subspecialty care, or to partner with us in referrals from the inpatient service, especially the following clinicians: Stacey Gardiner MD, Jason Rubenstein MD, David Ishizawar MD, Jalaj Garg MD, Andrew Rosenblum MD. We are also grateful to Nicole Lohr MD PhD and the clinical sections at the Zablocki Veterans Administration Medical Center that have also helped us establish an affiliate Cardio-Oncology Clinic in tandem at the VAMC in Milwaukee, WI.
Authors’ contributions
SAB conceived of the study, obtained, analyzed and interpreted the data, and made a substantial contribution to the writing of the manuscript; SP made a substantial contribution to the writing of the manuscript; DR made a substantial contribution to the writing of the manuscript; SZ obtained the data and made a substantial contribution to the writing of the manuscript; ML interpreted the data and made a substantial contribution to the writing of the manuscript; TN made a substantial contribution to the writing of the manuscript; BS analyzed the data and made a substantial contribution to the writing of the manuscript; RM made a substantial contribution to the writing of the manuscript; JMac made a substantial contribution to the writing of the manuscript; KD made a substantial contribution to the writing of the manuscript; JMes made a substantial contribution to the writing of the manuscript; DM made a substantial contribution to the writing of the manuscript; JS made a substantial contribution to the writing of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was associated with this work.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Froedtert & MCW Institutional Review Board (ID PRO00038807); HIPAA informed consent was waived for this minimal risk study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests. | TRASTUZUMAB | DrugsGivenReaction | CC BY | 33441188 | 18,793,759 | 2021-01-13 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chest pain'. | A virtual-hybrid approach to launching a cardio-oncology clinic during a pandemic.
BACKGROUND
As cardiovascular disease is a leading cause of death in cancer survivors, the new subspecialty of Cardio-Oncology has emerged to address prevention, monitoring, and management of cardiovascular toxicities to cancer therapies. During the coronavirus disease of 2019 (COVID-19) pandemic, we developed a Virtual-Hybrid Approach to build a de novo Cardio-Oncology Clinic.
METHODS
We conceptualized a Virtual-Hybrid Approach including three arms: information seeking in locations with existing Cardio-Oncology clinics, information gathering at the location for a new clinic, and information sharing to report clinic-building outcomes. A retrospective review of outcomes included collection and synthesis of data from our first 3 months (at pandemic peak) on types of appointments, cancers, drugs, and cardiotoxicities. Data were presented using descriptive statistics.
RESULTS
A de-novo Cardio-Oncology clinic was developed structured from the ground up to integrate virtual and in-person care in a hybrid and innovative model, using the three arms of the Virtual-Hybrid Approach. First, we garnered in-person and virtual preparation through hands-on experiences, training, and discussions in existing Cardio-Oncology Clinics and conferences. Next, we gleaned information through virtual inquiry and niche-building. With partners throughout the institution, a virtual referral process was established for outpatient referrals and inpatient e-consult referrals to actualize a hybrid care spectrum for our patients administered by a multidisciplinary hybrid care team of clinicians, ancillary support staff, and clinical pharmacists. Among the multi-subspecialty clinic sessions, approximately 50% were in Cardio-Oncology, 20% in Preventive Cardiology, and 30% in General Cardiology. In the hybrid model, the Heart & Vascular Center had started to re-open, allowing for 65% of our visits to be in person. In additional analyses, the most frequent cardiovascular diagnosis was cardiomyopathy (34%), the most common cancer drug leading to referral was trastuzumab (29%), and the most prevalent cancer type was breast cancer (42%).
CONCLUSIONS
This Virtual-Hybrid Approach and retrospective review provides guidance and information regarding initiating a brand-new Cardio-Oncology Clinic during the pandemic for cancer patients/survivors. This report also furnishes virtual resources for patients, virtual tools for oncologists, cardiologists, and administrators tasked with starting new clinics during the pandemic, and innovative future directions for this digital pandemic to post-pandemic era.
Introduction
Cardio-Oncology care has been adjusted in the COVID-19 pandemic with limited in-person clinic or hospital visits, increased use of teleconsultation, less frequent imaging, increased reliance on biomarkers, and considerations of differential diagnoses involving COVID-19 when evaluating cancer patients or survivors for possible cardiovascular toxicity [1]. Monitoring and management algorithms have been developed to help guide virtual care [2–4]. In the pandemic, we have changed the way in which we provide healthcare services at our clinics and institutions. This has challenged us to restructure current systems for the safety of our patients.
Various forms of innovation have come to bear in the pandemic, including telemedicine, digital health, artificial intelligence, social media, informatics, big data, and precision medicine [5, 6]. Telemedicine is the primary form of innovation that has been most developed in the pandemic [2, 5, 7]. Social media has been very helpful for dissemination of information, as well as education, and has been integral for creating online groups for support and determining the best ways for proceeding in the pandemic and advocating for our patients and colleagues in this period [5, 6]. In addition, the Doximity social media application has been valuable to practices across the nation, due to its telehealth platform (Doximity Video and Phone; https://www.doximity.com/dialer-video).
Despite the growing need, and allowances made during the pandemic, many centers do not have formal Cardio-Oncology clinics. Starting a new clinic can be challenging. The COVID-19 pandemic has made the process significantly more difficult, with the need to minimize exposure and maximize patient safety.
Currently, limited information is available on how to start a Cardio-Oncology Clinic during a pandemic, albeit given the high risk of morbidity or mortality in COVID-19-positive patients who also have cancer or CVD [8–13]. Several institutions have published on their experiences with starting in-person Cardio-Oncology clinics prior to the pandemic [8, 14–17]. One group has reported on their conversion from existing in-person Cardio-Oncology visits to telemedicine consultations, seeing 11 patients virtually within a few weeks [7]. Many have considered implications of the pandemic on the practice and study of cardio-oncology [2, 4, 5, 18–20], and two groups have suggested models for clinics converting from existing in-person care to televisits [4, 5]. Yet, no groups have directly addressed steps for de novo virtual-hybrid clinic formation within the limitations of the pandemic and without conversion of a pre-existing Cardio-Oncology clinic.
Our report offers a template for other centers to develop their own new Cardio-Oncology clinics during the pandemic. We determined a Virtual-Hybrid Approach to clinic launch, with both virtual and in-person elements of three key arms: information seeking where there are existing Cardio-Oncology Clinics in place, information gathering where the clinic will be built, and information sharing to report on initial patient data demonstrating the success of the launch (Fig. 1). We then performed retrospective chart review to collect and synthesize data on the types of appointments (new versus established, virtual versus in-person), cancers (e.g., breast, prostate, leukemia, lung), cancer drugs, and cardiovascular toxicities (e.g., cardiomyopathy, hypertension) for patients seen virtually or in person in our new Cardio-Oncology clinic at Froedtert Hospital and Medical College of Wisconsin (F&MCW). Here, we will discuss our findings in the context of previous publications on launching Cardio-Oncology Clinics prior to the pandemic. Our results will present distributions of cancer drugs and types, and cardiovascular diagnoses, similar to previous publications on Cardio-Oncology clinic-building. However, we will differentiate and illuminate the techniques that leverage the virtual underpinnings of pandemic clinic-building. We submit that it is feasible to establish a new Cardio-Oncology Clinic for cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy during a pandemic, providing optimal care for new patients in the midst of the need for safety and minimizing exposure. We also propose virtual resources for patients and clinicians and describe innovative future directions in the pandemic and post-pandemic period.
Fig. 1 The Virtual-Hybrid Approach to Cardio-Oncology Clinic-building in the pandemic
Methods
Virtual-hybrid approach
We pursued a Virtual-Hybrid Approach of information seeking, information gathering, and information sharing (Fig. 1). For information seeking, we focused on institutions that already had a Cardio-Oncology clinic in place. Prior to the COVID-19 pandemic, substantial experience was gained at an established in-person Cardio-Oncology clinic at a world-renowned leading hospital. Published manuscripts on pre-pandemic building and operations of Cardio-Oncology clinics at other institutions were reviewed. Extensive networking with leaders of Cardio-Oncology clinics around the United States and in the United Kingdom was accomplished at regional, national, and international meetings in person and on social media in a hybrid approach. These meetings were attended in person pre-COVID-19 and virtually during the pandemic. For information gathering, we addressed the location in which the new Cardio-Oncology clinic would be built. We learned about existing resources in the destination Heart & Vascular Center and existing needs and patients in the destination partner cancer center. We networked with physicians, advanced practice providers (APPs), nurses, service line leaders, and administrators in the Heart & Vascular Center and the cancer center, as well as in primary care and other supporting specialties. The subsequent Cardio-Oncology clinic sessions were intermingled with other subspecialty areas, to optimize availability for patient visits while filling clinic slots and tailoring spectrum of care to emerging patient needs. Cardio-Oncology patient visits were included in multi-subspecialty clinics.
Retrospective study design
For quantitative data, we pursued a retrospective observational study to determine the distribution of outpatient visits in the first 3 months of our virtual-hybrid Cardio-Oncology Clinic. We reviewed data from charts of patients (all were 18 years of age or older) who received outpatient care from the new Director of Cardio-Oncology at F&MCW between April 15, 2020 and July 17, 2020 to determine which of these patients were considered to be in Cardio-Oncology, Preventive Cardiology, or General Cardiology. We collated all three to determine the percentage of patients seen in Cardio-Oncology, compared to the other two specialties. Preventive Cardiology was collated as a partner clinic to help build the Preventive Cardio-Oncology component of the Cardio-Oncology Clinic, to help apply established principles for prevention. From among the multi-subspecialty clinic sessions, we determined the proportion of patients who were specifically cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy and thereby seen in the Cardio-Oncology Clinic. Next, we identified the distribution of cardiovascular toxicities in cancer patients or survivors seen in the Cardio-Oncology Clinic. We also evaluated the spectrum of cancer drugs received by cancer patients or survivors seen in the Cardio-Oncology Clinic. In addition, we summarized the types of cancers in patients seen in the Cardio-Oncology Clinic. Finally, we assessed the frequency of virtual visits during the course of the pandemic over our first 3 months for patients seen in the Cardio-Oncology Clinic. This retrospective review was approved by the F&MCW IRB; HIPAA informed consent was waived for this minimal risk study, which did not involve any form of intervention and was conducted in compliance with good clinical and research practice. The team designed and carried out the study with reliance on virtual communication tools.
Data collection and analysis
Data gathering, management, and analysis were conducted at F&MCW. We collected patient-related, disease-related, treatment-related, and outcome-related data, particularly patient sex, type of appointment (new versus established, virtual versus in-person), type of cancer (e.g., breast, prostate, leukemia, lung), type of cancer drug, and type of cardiovascular toxicity (e.g., cardiomyopathy, hypertension). In order to minimize any risk of breaching patient confidentiality, all data collection occurred on institutional-based computing environments with de-identified data used for analyses. There were no alternative procedures for the subjects as this is a retrospective review of data that are not amenable to prospective collection and review. Descriptive graphs or tables of patient-, disease-, treatment-, and outcome-related variables distributions were prepared, with no comparisons made needing statistical tests.
Results
Virtual preparation
Preparation for starting the Cardio-Oncology clinic followed a Virtual-Hybrid Approach (Fig. 1, left). Five overarching factors employing virtual communication methods emerged to ensure the successful launching of the clinic. Team and individual experience and exposure to various areas of interest in Cardio-Oncology were achieved and assessed before and during the pandemic. Far-reaching connections to experts and potential collaborators in the field were developed and exercised. Close contact with the institution launching this clinic was important to determine the resources available; these resources dictated the strategy and potential outcomes of the clinic. Importantly, the expectations of others for the Cardio-Oncology clinic were determined and incorporated. Finally, recognition of the limitations that exist at the destination institution guided care and goal setting.
Virtual inquiry
Before initiating the Cardio-Oncology clinic in the destination institution, existing structures, patient base, and needs in the Heart & Vascular Center as well as the Cancer Center were evaluated, adhering to pandemic protocols (Fig. 1, middle). Pre-existing building blocks for the planned Cardio-Oncology clinic were assessed, and the partner Preventive Cardiology clinic was investigated. We also evaluated characteristics of the cancer center patient population to best position the clinic for success.
Virtual niche-building
Five main aspects of niche-building were pursued. Partnerships with Vascular and Cancer Center physicians, advanced practice providers, and service line leaders were developed to initiate and grow the clinic (Fig. 1, middle). The Cardio-Oncology team and clinic flexibility were demonstrated through openness to taking quicksteps. Presentations were made at Grand Rounds and rounds across the institution in Cardiology, Hematology/Oncology, Radiation Oncology, Surgical Oncology, Internal Medicine, and Family Medicine to promote the clinic capabilities. Collaborative solutions for problems facing fields complementing Cardio-Oncology developed trust and collaboration. Teamwork was developed by leveraging diversity of perspectives and virtual communication technologies, to establish effective patient care despite COVID-19 limitations.
Hybrid care Spectrum
The hybrid F&MCW Cardio-Oncology Clinic was initiated and established in the outpatient setting, in close partnership with the Preventive Cardiology Clinic, Cancer Center, and inpatient Cardiology Consult and Hematology/Oncology teams (Fig. 1, right).
Initial and subsequent visits have been completed in person or by video, with phone visits also available for virtual return visits if patients without adept and available smartphone use have limited ability to appear in person. Virtual patient visits over our first 3 months occurred with the use of telemedicine platforms integrated with Epic (via MyChart for patients and Haiku/Canto for clinicians), or using the Doximity video call function. Patients with in-person appointments are screened appropriately on arrival for signs or symptoms of COVID-19 or exposure, following institutional protocols. Wearing masks is required of all patients, and each patient can be accompanied by a family member; some choose to also wear gloves or face shields. There is sufficient room for maintaining social distancing in the clinic waiting room and hallways.
Innovation
Current innovation in the clinic also includes Virtual Clinician Tools and Virtual Patient Resources (Fig. 2). For clinicians, the links for an AHA CME course on Novel Concepts, Current Debates and Treatment Considerations in Cardio-Oncology, an online Cardio-Oncology Compendium hosting risk assessment clinical decision aids, Cardio-Oncology Drug Regimen and Acronym Databases, and UPTODATE access for reviewing Cardio-Oncology drug information are supplied. For patients, the video from the International Cardio-Oncology Society explaining the Cardio-Oncology subspecialty, American College of Cardiology (ACC) mobile health (mHealth) CardioSmart education app and website, Cancer Heart Talk mini-podcast series accessed via SoundCloud app and website, Cardio-Oncology Frequently Asked Questions, and ChemoCare website are provided for patient-facing Cardio-Oncology and heart anatomy and physiology education, engagement, and awareness. Virtual Resources for Preventive Cardio-Oncology are also made available to our patients. These include the American Heart Association (AHA) Physical Activity Recommendations, AHA Life’s Simple 7 Webpages, American Society For Preventive Cardiology Online Coaching Webpages in partnership with Intervent, and the Become An Ex Smoking Cessation Support Webpages in partnership with Mayo Clinic. The resources are provided in the Epic patient portal MyChart, and more ways to make the resources accessible to a broad and diverse patient population are in development. Future innovation in the clinic will explore contemporary initiatives connecting patients and their safely guarded data with their permission with wearable devices, health information technology, informatics, artificial intelligence, personalized medicine, and additional mobile health (mHealth) applications.
Fig. 2 Virtual patient and clinician education and resources. Available online as PDF with hot links in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual visit infrastructure and timeline
There was no pre-existing Cardio-Oncology program at the time of launching our de novo Virtual/Hybrid Cardio-Oncology Clinic. The newly recruited Director of Cardio-Oncology was tasked with the responsibility of launching the new clinic, with support from the Heart & Vascular Center clinic administrators, medical director, and Cardiology Division and Department of Medicine leadership. Prior to opening the Cardio-Oncology Clinic, the Heart & Vascular Center initiated virtual conversion then additionally collaborated with Inception Health (MCW’s innovation lab company) over the course of 4 weeks to iteratively develop the clinical informatics infrastructure for virtual visits. The video visits were designed to function using clinician’s personal smartphones, iPads, and tablets, with direct web browser video links from the electronic health record mobile application. Direct video calls through the Doximity mobile application were also approved. Existing Inception Health personnel re-allocated their time in order to adopt and maintain responsibility for the virtual component of all ambulatory clinics across the health system, in partnership with medical and administrative directors of each clinic area, such as the Heart & Vascular Center. No additional costs or hires were pursued to facilitate the development of the virtual visit infrastructure and timeline. Existing resources and personnel were re-allocated to virtual visit design to enable building the virtual clinics in the Heart & Vascular Center. To assist clinicians and billing compliance colleagues, note templates were created for video and phone visits to indicate patient informed consent for virtual visits due to the pandemic, as well as to capture limited appropriate physical examinations, in addition to the amount of time spent on records review and real-time medical counseling.
Initial consults were electronically triaged by either a cardiologist or a cardiology fellow supervised by a cardiologist. Each triage team determined which consults would be appropriate as virtual video visits, versus in-person to occur once the Heart and Vascular Center started re-opening routine physical visits, or whether patients needed to be evaluated urgently in person. During the first week of operation, the brand-new Cardio-Oncology Clinic started entirely virtually with only video and phone visits. As the Heart and Vascular Center re-opened for physical patient visits the following week, from week 2 through the remainder of the first 3 months the Cardio-Oncology Clinic had both virtual and in-person visits integrated throughout each clinic session weekly, based on whether patients were new and whether they had smart device or computer functionality available.
Virtual referral network and process
Cardio-Oncology patient assessment begins within a referral network before the patient arrives at a Cardio-Oncology clinic. Consequently, forming a virtual referral network and enacting a user-friendly virtual referral process was a key component of building the Cardio-Oncology clinic during the pandemic. All referral patterns and networks for our de novo Cardio-Oncology Clinic were built from the ground up. Initial referrals were from within our health system; this quickly expanded to consults from outside of our health system encompassing the entire state. Patients were referred to our Clinic by clinicians or by self-referrals. Some of our local patients connected to us after being introduced to us by their clinicians in other states or through family members in other states who learned about us from their own clinicians or community-based Cardiology society outreach events.
Referrals across the institution and outside of our health system have come to us from the Divisions of Hematology and Oncology, Internal Medicine, Family Medicine, Surgical Oncology/Breast Clinic, and Survivorship Clinics. From the cancer center’s perspective, there may be many “triggers” that would warrant a Cardio-Oncology referral. For example, an abnormal ECG, an abnormal echocardiogram, cardiovascular symptoms, previous cardiovascular history (e.g., coronary artery disease, hypertension, cardiomyopathy) particularly in a patient who previously underwent treatment or is beginning new treatment with cardiotoxic neoplastic medications or radiation therapy and is at high risk of cardiovascular toxicity, or those in preparation for stem cell transplant, or oncologic surgery. Referral protocols were determined based on standard practice, discussions with colleagues in Medical and Radiation Oncology, Hematology, Bone Marrow Transplant, Surgical Oncology/Breast Clinic, Children’s Hospital, Radiation Oncology, Primary Care, and updated literature reviews.
Cancer survivors are at a higher risk than the general population for cardiovascular morbidity and mortality. If a cancer survivor needing to be evaluated is already under the care of a cardiologist, the referring provider can reach out to their cardiologist for guidance on the appropriate CV surveillance. If they do not already have a cardiologist, a Cardio-Oncology consult should be requested. The Cardio-Oncology consult can be placed using a direct Cardio-Oncology button within the universally available Cardiovascular Consult order panel. Referrers can also place a General Cardiology consult and mention the Cardio-Oncology physician by name as requested by the clinician or patient. An E-Consult functionality is also being implemented for those patients who need to be assessed sooner than the next available appointment, or for those patients who may not need a full Cardio-Oncology evaluation, or if referring providers are uncertain. The e-consult can also be placed as a second opinion requested by the inpatient Cardiology Consult team.
The inpatient Cardiology Consult service will continue to directly address inpatient consults from the inpatient hematology/oncology services. The inpatient Cardiology Consult service can collaborate with the Cardio-Oncology Clinic via formal Cardio-Oncology E-consults in the electronic health record Epic if a specific focused question arises regarding Cardio-Oncology relevant to the care of individual currently hospitalized patients that have already been formally evaluated by the inpatient Cardiology Consult service. After a patient has been formally evaluated by the inpatient Cardiology Consult service, if the patient is appropriate for outpatient follow up in the Cardiology clinic with Cardio-Oncology, this should be communicated to the primary Hematology/Oncology service. If appropriate at the time of consultation, the inpatient cardiology consult service can make the follow-up appointment. Oftentimes, this patient population remains in the inpatient setting for several weeks. If this is the case, the Cardiology clinic phone number and clinician information should be provided to the primary service to do so prior to the patient being discharged from the hospital.
Virtual-hybrid multidisciplinary team
It is important to develop a multidisciplinary team and initially focus on allocation of pre-existing resources. Accordingly, some roles among our Cardio-Oncology clinic personnel are shared with other subspecialties. Our virtual-hybrid multidisciplinary Cardio-Oncology Clinic personnel include physicians, a nurse practitioner (NP), a nurse, a research support specialist, medical assistants, pharmacists, administrative assistants, and administrators. All personnel with pre-existing in-person roles and practices re-allocated a portion of their time to the development and practice of virtual visits.
Our clinic and partners consist of board-certified Cardiologists with special training in various cardiac subspecialties (e.g., cardio-oncology, preventive cardiology, heart failure and transplant, electrophysiology, interventional cardiology), who collaborate closely with our cancer experts. Our physicians together specialize in the prevention, diagnosis, and treatment of heart and vascular disorders resulting from side effects of cancer therapy. Our comprehensive team of advanced practice providers, nurses, and pharmacists work alongside our physicians to care for patients from the moment of cancer diagnosis through life’s survivorship journey. The NP typically sees established patients when needed to follow up on imaging, intervention, or diagnostic and management plans, and may also see select new patients. In complex cases, the NP discusses the care of established patients with both the cardio-oncologist and the referring clinician. The nurse assists with patient triage and communications (including addressing patient requests and queries), liaises closely with the nurse practitioner and pharmacists, and educates patients on Cardio-Oncology using virtual materials. Our clinical pharmacists function at the highest level of their advanced training, similar to all clinic personnel, and assist with medication education, review, titration, discussion, and prescription, particularly for heart failure, hypertension, hyperlipidemia, and smoking cessation, as well as commenting on potential drug interactions.
For Preventive Cardio-Oncology, we additionally partner with our dietitians and exercise physiologists to help advise our patients on nutrition and exercise plans, as well as our colleagues in cardiopulmonary stress testing where applicable. Further, in the pandemic, we provide patients with free online coaching options for lifestyle modification (Fig. 2). We also direct patients to AHA webpages with guidance on pursuing ideal cardiovascular health.
Virtual-hybrid patient flow
Once a referral is placed by the designated order buttons in the electronic health record, central schedulers or the Cardio-Oncology Clinic administrative assistant schedule the new patient for a video or in-person visit (Fig. 3). The clinic administrative assistant works closely with our health professionals in our interdisciplinary advanced subspecialty clinic to gather relevant clinical reports and history pertinent to patient appointments. Virtual medical assistants contact patients a few days before their appointments to confirm and troubleshoot virtual connectivity. On the appointment day, medical assistants then ‘room’ patients for virtual or in-person visits by preparing patients for their medical visits (including reviewing medications and in-person or at-home virtual vital signs), and also rechecking virtual connectivity for video visits. The clinician then completes the visit virtually or in-person and introduces the patient to the range of electronic resources available. Following the visit, the clinical administrative assistant arranges follow-up testing and appointments.
Fig. 3 Virtual-Hybrid Patient Flow Chart. Admin = Administrative; MA = Medical Assistant; MD = Medical Doctor; NP = Nurse Practitioner
Virtual risk assessment
Baseline risk assessment and follow-up start with oncology and primary care [21]. Asymptomatic low risk patients with low-risk treatment plans can have continued assessment and follow-up by oncology and primary care in partnership. Patients who have symptoms, are at high risk based on their history, or are planned for high-risk treatment plans should be referred to Cardio-Oncology for prevention, monitoring, and management recommendations. Recommendations should adhere to society expert consensus, scientific statements, and guidelines for prevention, surveillance, and survivorship, and optimize CVD risk and medications [21]. A putative risk score based on medication-related and patient-related risk factors can be used to guide monitoring and management recommendations for most Cardio-Oncology patients [22], and can be used in a virtual clinical decision aid (https://tinyurl.com/CardioOncCDA) (Fig. 4). Specific risk scores are also available for adults treated with anthracyclines, trastuzumab, or other drugs, or for adult survivors of childhood cancers [9–13].
Fig. 4 Virtual Cardio-Oncology clinical decision aid (CDA). Risk assessment (https://tinyurl.com/CardioOncCDA) to guide monitoring and management recommendations regarding development of cardiomyopathy for most Cardio-Oncology patients; a CDA specifically for women with early breast cancer is also available in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual management algorithms
Evidence-based management algorithms have been selected or developed as adjunctive resources for inpatient teams. They are available online in a virtual collection for use in the inpatient setting by the inpatient Cardiology Consult service or hematology/oncology teams to assist with diagnosis and treatment of cardiovascular toxicities from cancer therapies or cancer itself. The algorithms cover cardiomyopathy from anthracyclines or trastuzumab, planned chemotherapy with pre-existing cardiomyopathy, neurohormonal therapy or dexrazoxane for cardioprotection, myocarditis, persistent malignant pericardial effusion, hypertension, surveillance after radiation therapy or drugs that cause ischemia, malignant pericardial effusion, and other salient topics frequently encountered.
Virtual community engagement
The local, regional, national, and international community was virtually engaged via social media posts on Twitter (using #MCWCardioOnc on @DrBrownCares or @PrevCardioOnc), podcasts hosted by the MCW CTSI (available on iTunes, Google, and Apple podcast platforms), Heart Success podcast series, and Cancer Heart Talk brief 15-min mini-podcast series (available on SoundCloud). Perspectives were also published for international community engagement in the Women Heart Alliance newsletter, as well as on the AHA Early Career Blog, ACC Women in Cardiology Blog, CardioOncTrain.Com Blog, and PrevCardioOnc.Com Blog. Virtual continuing medical education (CME) presentations were also given at the Wisconsin state ACC annual conference meeting, Midwest ACC annual conference meeting, Southeast ACC annual conference meeting, Brazilian Cardio-Oncology Symposium, and the Ohio State Cardio-Oncology CME conference, then subsequently at the AHA and ACC annual national scientific sessions.
Distribution of patient data
In our multi-subspecialty clinic visits (virtual and in-person integrated and combined; n = 182; 136 new and 47 returns), approximately 50% of patient visits were in Cardio-Oncology, 20% were in Preventive Cardio-Oncology, and 30% were in General Cardiology (Fig. 5a). Overall among Cardio-Oncology visits, 65% were in person, consistent with early and safe clinic re-opening in a hybrid model, with 19% by video and 16% by phone, with the fraction by phone decreasing over time as patients and clinic personnel became more adept with troubleshooting video. Of new patients, 77% were in person, and the remainder by video. No Cardio-Oncology patients presenting in person developed any signs or symptoms concerning for COVID-19.
Fig. 5 Initial Clinic-Building Outcomes Using the Virtual-Hybrid Approach. a Distribution of Cardio-Oncology, Preventive Cardio-Oncology, and General Cardiology patients seen in our multi-subspecialty clinic visits. b Distribution of cardiovascular diagnosis or indication for referral. c Distribution of cancer drugs. d Distribution of cancer types
The most frequent cardiovascular diagnosis or indication for referral was cardiomyopathy (34%) (Fig. 5b). Other diagnoses included decrease in global longitudinal strain, diastolic congestive heart failure, hypertension, myocarditis, dyspnea, chest pain, palpitations, survivorship, risk assessment, and pre-bone marrow transplant, among other cardiovascular diagnoses or visit indications. The most frequent cancer drug was trastuzumab (29%) (Fig. 5c), managed according to a novel algorithm developed in our de novo Virtual-Hybrid Cardio-Oncology Clinic based on the recent publication indicating the safety of continuation of trastuzumab for left ventricular ejection fraction of 40% or greater [23] (Fig. 6). The second most frequent cancer drug was anthracycline (24%). Other drugs included, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), endocrine therapies, and investigational therapeutics, among others. The most frequent cancer type in our clinic was breast cancer (42%) (Fig. 5d). These trends in cardiovascular diagnosis or indication and cancer drugs or types were similar in assessments of virtual visits alone, with the most frequent being cardiomyopathy (43%), trastuzumab (41%), and breast cancer (44%), respectively. The findings of similar cardiovascular and cancer distributions in virtual versus in-person visits indicated an optimal qualitative return on resource and personnel investment.
Fig. 6 Algorithm for Continuation of Trastuzumab Therapy with Mild LV Dysfunction
Imaging and medication titration
In our clinic, a distribution of cardiovascular diagnoses determines the imaging needed for each patient (Fig. 5b). Therefore, a number of imaging modalities are useful to our patients (e.g., echocardiography, computed tomography with or without angiography, magnetic resonance imaging, coronary angiography, myocardial perfusion imaging). Our most frequently used imaging modality is echocardiography. The frequency of obtaining echocardiograms has depended on each patient’s condition and cancer treatment. A substantial portion of patients coming to us on trastuzumab have needed an echocardiogram every 1–3 months, depending on the extent of adverse effects on left ventricular ejection fraction (LVEF) or strain In these patients, medication titration has occurred approximately every 2 weeks, and for very symptomatic patients with volume overload, they have often been seen weekly..
In our management algorithms, early referral prior to the onset of symptoms has been emphasized, especially in cancer patients or survivors with a history of cardiovascular disease, cardiotoxic neoplastic agents, or a high risk of cardiovascular toxicity. This has provided an opportunity for us to assess and discuss ways to optimize the benefit to risk ratio of continuing with the current cancer treatment plan, and more importantly how and when to put cardioprotective measures in place to facilitate safe cancer therapy. Such discussions have also resulted in closer monitoring. Some conditions have warranted proceeding to other modalities of non-invasive imaging, such as cardiac MRI if myocarditis is suspected. For cases in which coronary artery disease is suspected, our patients undergo functional assessment of their coronaries with a stress test or anatomical assessment with a coronary CT scan or invasive coronary angiography. In our practice, only exercise stress tests were halted due to the pandemic. Every other form of imaging including rest and stress echo, as well as MRI and nuclear medicine have remained readily available for those with cardiovascular toxicities or individuals considered to be at moderate or high risk. This allowed us to adhere to pre-pandemic imaging recommendations tailored during the pandemic to limit imaging if possible to those who are at higher risk for cardiovascular toxicities or who have already been diagnosed with these adverse effects [1–4] (e.g., Fig. 6).
Discussion
The COVID-19 pandemic has inevitably compelled leaders of healthcare clinics to rethink and restructure approaches to deliver optimal care for patients. Our brand-new Cardio-Oncology clinic has been built to thrive in this new pandemic landscape by utilizing virtual technology as one of the key components of our clinic-building and care model since its inception. While existing clinics have reinvented their operations through the uptake of technology, our clinic has been able to capitalize on this resource to deliver virtual-hybrid care from the start. Virtual communication has proved useful to coordinate referral networks and care among providers within a multidisciplinary team across different clinics and departments. We see a variety of cancer patients, types, and drugs (Fig. 5), and our distribution results are generally congruent with reports from other leading cardio-oncology clinics [8, 14, 15, 17, 22].
Various methods have been developed for risk assessment to help guide providers and patients in determining the appropriate guidelines for care. We offer the use of virtual risk assessment tools such as the computed risk scores based on medication- and patient-related risk factors [22] (Fig. 4) (https://tinyurl.com/CardioOncCompendium), as well as recommendations for establishing cross-provider partnerships to continuously evaluate risk [21]. Other online databases containing useful information and guidelines are readily accessible and can help guide clinical practices. We encourage use of these virtual tools, which can further facilitate collaborative Cardio-Oncology care in the pandemic. Our conversations with international colleagues have suggested additional utility of these virtual tools beyond the pandemic. The online resources can be very helpful in settings where clinical practitioners work alone without support from nurses, pharmacists, nutrition specialists, or exercise physiologists.
Virtual-hybrid care has extended the care team’s capabilities for delivering and maintaining patient education and follow-up. The internet continues to be a robust resource, containing a wealth of health information that is easily accessible to the general population. Various mobile applications and electronic devices have also been developed in recent years to educate, track, and manage patients’ health and lifestyles. While these tools provide patients with greater accessibility and independence, they also create a valuable opportunity for healthcare providers to further engage patients. In a virtual-hybrid model, this becomes increasingly important, as patients may frequently transition between virtual and in-person visits. Forming care partnerships with patients through these virtual information and health-tracking resources becomes crucial in the continuity of care and proper health maintenance as we move through the pandemic.
Our most frequent cardiovascular diagnosis was cardiomyopathy (34%), which is reflective of the management need that first helped start the emerging field of Cardio-Oncology, and is similar to the most frequent cardiovascular diagnosis noted by clinicians from several other leading centers (20–35%) [24, 25]. However, Cardio-Oncology has grown remarkably over the last 10–20 years, with a wide spectrum of cardiovascular diagnoses and indications for referral (Fig. 5b) [8, 14]. Accordingly, at some other leading centers, the most frequent cardiovascular diagnosis or indication for referral has been reported as hypertension [14], arrythmia [17], or comprehensive risk assessment prior to beginning of therapy to optimize cardioprotection [8] in the practice of Preventive Cardio-Oncology [21]. This illustrates an opportunity for growth in our Clinic, to increase the fraction of high-risk patients who undergo comprehensive cardiovascular risk evaluation and management of risk factors prior to administration of cardiotoxic therapy.
Our clinic cares for patients with a range of cancer types (Fig. 5d). Individuals with breast, lung, and hematologic cancers represent a substantial proportion of our patient population, similar to other Cardio-Oncology clinics [8, 14, 22, 26]. The most frequent cancer diagnosis, breast cancer (43%), is consistent with reports from other leading cardio-oncology clinics such as the Mayo Clinic (39.2%) [15] and the Cleveland Clinic in Florida (44.3%) [14]. While hematologic malignancies such as leukemia and lymphoma represented 29% of our patients and was the second most prevalent cancer within our cohort, they comprised the most frequent forms of cancer at other cardio-oncology clinics such as at the Moffitt Cancer Center (31%) [8] and at UCLA (32.70%) [25]. However, the absolute difference was relatively insignificant. Overall, similar to these established cardio-oncology clinics, we receive patients from across a variety of cancers.
A wide breadth of cancer therapeutics is associated with cardiotoxicity [21]. Anthracyclines associate with cardiomyopathy, especially when used with trastuzumab. Targeted therapies (e.g., TKIs) can cause new or worsening of pre-existing hypertension. ICIs are associated with an increased incidence of myocarditis. Some cytotoxic chemotherapeutics, such as cisplatin, increase the risk of venous thromboembolism, and antimetabolites such as fluoropyrimidines have long been associated with a broad range of cardiotoxicities. Radiation therapy is associated with ischemic heart disease, valve dysfunction, conduction abnormalities, pericardial disease, and cardiomyopathy. Patients with cancer who have developed cardiovascular toxicity or who may be at high risk for cardiovascular toxicity should be referred to the Cardio-Oncology clinic for close follow-up.
The most frequently used cancer medication used among our patients was trastuzumab (29%), with the second most frequent being anthracyclines (24%). This was similar to other institutions, with anthracyclines and trastuzumab among the most common cancer drugs in their Cardio-Oncology clinics. Yet, anthracyclines were typically noted more commonly than trastuzumab. The Cleveland Clinic in Florida saw patients most commonly treated with radiation (40%), followed by anthracyclines (26.8%) [14]. The Moffitt Cancer Center most frequently had patients who were treated with anthracyclines (52%), with HER2 targeted therapies representing 27% of the cancer drugs [8]. This difference may reflect a high frequency of patients with HER+ breast cancer in our population (diagnosed by a ratio of HER2 to chromosome 17 signals on dual probe fluorescent in situ hybridization ≥2 or ≥ 6 HER2 signals/cell [27]), as well as the keen attention to a substantial fall in left ventricular ejection fraction or global longitudinal strain as a potential prognostic factor in our patients, per American Society of Echocardiography (ASE) guidelines [28].
All of these patient data distributions were obtained in the context of the Virtual-Hybrid patient flow in our de novo Cardio-Oncology Clinic (Fig. 3), that can be modeled by other future Virtual-Hybrid Cardio-Oncology clinics initiated during the pandemic. Table 1 compares the first few weeks of our de novo C-O clinic setup model with two published manuscripts describing conversion of pre-existing in-person C-O clinics to providing telehealth visits as an option for patients. The table shows similar numbers of patients seen in the initial periods of the clinics, although the numbers in our new clinic went from 0 to 10 in the first 3 weeks, compared to going from up to 40 patients weekly to 11 patients in 2.5 weeks for a group that converted their in-person clinic to a virtual option. Overall, CV diagnoses and cancer types were comparable; distributions of cancer drugs were not reported by the other group. Important differences were noted. Most of our patients in the pandemic were new (90%), given the de novo status of the Cardio-Oncology Clinic, while the converted virtual clinic of another group initially focused on established patients for > 50% of their patient visits. While key personnel were also the same (e.g., physician, advanced practice provider, nurse or nurse coordinator), we also report virtual versions of supportive staffing patterns, including the virtual scheduling and rooming process and pharmacy and lifestyle modification visits. Additionally, trainees have been integrally involved in the establishment of our Cardio-Oncology Clinic, with residents training in program-building, and medical students and fellows training in ambulatory cardio-oncology clinical practice and cardio-oncology critical thinking, respectively. Finally, besides the patient flow (Fig. 3) and de novo nature of our Virtual-Hybrid clinic initiated in the pandemic, our unique contribution may be the virtual resources, compared to the essential “webside manner” [7] or an alternative algorithm [4] for triaging virtual or in-person visits to the physician or advanced practice provider (Table 1).
Table 1 Comparisons Among Clinic Models Described In The Pandemic
Virtual-Hybrid Clinic Telehealth Clinic (14) Triage Clinic (4)
Model Type De Novo Conversion Conversion
Time Frame Compared 3 weeks 2.5 weeks Not reported
Number of Patients 10 11 Not reported
New Visits (%) 90 45 Not reported
Variety of CV Diagnoses Yes Yes Not reported
Variety of CA Types Yes Yes Not reported
Variety of CA Drugs Yes Unknown Not reported
Referrals Yes Unknown Yes
Scheduling Yes Unknown Not reported
Rooming Process Yes Unknown Not Reported
Virtual AA Yes Unknown Not Reported
Virtual MA Yes Unknown Not Reported
Virtual Physician Yes Yes Yes
Virtual APP Yes Unknown Yes
Virtual Pharmacist Yes Unknown Not Reported
Virtual Nurse (Coordinator) Yes Unknown Yes
Unique Contribution Virtual Resources Webside Manner Triage Algorithm
Similar to the formation or conversion of Cardio-Oncology clinics, many protocols for treatment regimens and cancer patients are yet to be standardized. While no standard protocols have been widely adopted at Cardio-Oncology practices, various institutions and writing groups have proposed some approaches (e.g., ASE or ASCO guidelines). We have collaboratively developed institutional algorithms for various cardiovascular toxicities and medications based on existing scientific statements, society guidelines, expert consensus statements, and manuscripts from leading cardio-oncology research institutions. The goal is to adopt, adapt, develop, and continuously update these algorithms, as new literature arises in order to establish best practices and an institutional standard of care.
Conclusion
Starting a new Cardio-Oncology Clinic in the pandemic has its challenges, and yet for our patients can be invaluable. Appropriately competing priorities in the pandemic can limit the scheduling of meetings and gathering of people together in one virtual room to discuss a mutual vision. Gathering resources for patient and clinician education can also be formidable, as can social distancing and obtaining important imaging. However, multiple virtual one-on-one or small group meetings can be beneficial for building institutional relationships. Similarly, virtual visits have risen to the challenge to ensure maintenance of patient care throughout the pandemic. Modifications have also been made to enable safety and distancing during imaging. With the benefit of these adjustments to address the challenge, this report provides a foundation for initiating a cardio-oncology clinic in the pandemic, with virtual resources and tools to equip patients and clinicians.
In the future, we will also lay out a roadmap for initiation of comprehensive cardio-oncology programs with the five pillars of patient care, education, research, community engagement, and innovation in the era of digital transformation accelerated by the pandemic. Novel risk modifiers and risk attenuation methods, such as breast arterial calcification, clonal hematopoiesis of indeterminate potential, and Cardio-Oncology prehabilitation, habilitation, and rehabilitation will also be addressed. Future innovation to implement recommendations from clinical trials across the nation currently underway that utilize mobile health or web-based diet and physical activity interventions and/or seek to determine the impact of cardioprotective pharmacotherapy in Preventive Cardio-Oncology will also be assessed (ClinicalTrials.Gov: NCT01988571, NCT02943590, NCT02562716, NCT01968200, NCT03265574, NCT03760588, NCT03386383, NCT02244411, NCT03223753). Many of these studies incorporate virtual technologies that will be very helpful during and after the pandemic as we continue pursuit of digital transformation.
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Acknowledgements
We are grateful to the following individuals, as well as all of the departments and divisions at Froedtert & MCW that have rallied together to help us establish the hybrid Cardio-Oncology clinic or to see our patients needing subspecialty care, or to partner with us in referrals from the inpatient service, especially the following clinicians: Stacey Gardiner MD, Jason Rubenstein MD, David Ishizawar MD, Jalaj Garg MD, Andrew Rosenblum MD. We are also grateful to Nicole Lohr MD PhD and the clinical sections at the Zablocki Veterans Administration Medical Center that have also helped us establish an affiliate Cardio-Oncology Clinic in tandem at the VAMC in Milwaukee, WI.
Authors’ contributions
SAB conceived of the study, obtained, analyzed and interpreted the data, and made a substantial contribution to the writing of the manuscript; SP made a substantial contribution to the writing of the manuscript; DR made a substantial contribution to the writing of the manuscript; SZ obtained the data and made a substantial contribution to the writing of the manuscript; ML interpreted the data and made a substantial contribution to the writing of the manuscript; TN made a substantial contribution to the writing of the manuscript; BS analyzed the data and made a substantial contribution to the writing of the manuscript; RM made a substantial contribution to the writing of the manuscript; JMac made a substantial contribution to the writing of the manuscript; KD made a substantial contribution to the writing of the manuscript; JMes made a substantial contribution to the writing of the manuscript; DM made a substantial contribution to the writing of the manuscript; JS made a substantial contribution to the writing of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was associated with this work.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Froedtert & MCW Institutional Review Board (ID PRO00038807); HIPAA informed consent was waived for this minimal risk study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests. | TRASTUZUMAB | DrugsGivenReaction | CC BY | 33441188 | 18,793,759 | 2021-01-13 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Death'. | A virtual-hybrid approach to launching a cardio-oncology clinic during a pandemic.
BACKGROUND
As cardiovascular disease is a leading cause of death in cancer survivors, the new subspecialty of Cardio-Oncology has emerged to address prevention, monitoring, and management of cardiovascular toxicities to cancer therapies. During the coronavirus disease of 2019 (COVID-19) pandemic, we developed a Virtual-Hybrid Approach to build a de novo Cardio-Oncology Clinic.
METHODS
We conceptualized a Virtual-Hybrid Approach including three arms: information seeking in locations with existing Cardio-Oncology clinics, information gathering at the location for a new clinic, and information sharing to report clinic-building outcomes. A retrospective review of outcomes included collection and synthesis of data from our first 3 months (at pandemic peak) on types of appointments, cancers, drugs, and cardiotoxicities. Data were presented using descriptive statistics.
RESULTS
A de-novo Cardio-Oncology clinic was developed structured from the ground up to integrate virtual and in-person care in a hybrid and innovative model, using the three arms of the Virtual-Hybrid Approach. First, we garnered in-person and virtual preparation through hands-on experiences, training, and discussions in existing Cardio-Oncology Clinics and conferences. Next, we gleaned information through virtual inquiry and niche-building. With partners throughout the institution, a virtual referral process was established for outpatient referrals and inpatient e-consult referrals to actualize a hybrid care spectrum for our patients administered by a multidisciplinary hybrid care team of clinicians, ancillary support staff, and clinical pharmacists. Among the multi-subspecialty clinic sessions, approximately 50% were in Cardio-Oncology, 20% in Preventive Cardiology, and 30% in General Cardiology. In the hybrid model, the Heart & Vascular Center had started to re-open, allowing for 65% of our visits to be in person. In additional analyses, the most frequent cardiovascular diagnosis was cardiomyopathy (34%), the most common cancer drug leading to referral was trastuzumab (29%), and the most prevalent cancer type was breast cancer (42%).
CONCLUSIONS
This Virtual-Hybrid Approach and retrospective review provides guidance and information regarding initiating a brand-new Cardio-Oncology Clinic during the pandemic for cancer patients/survivors. This report also furnishes virtual resources for patients, virtual tools for oncologists, cardiologists, and administrators tasked with starting new clinics during the pandemic, and innovative future directions for this digital pandemic to post-pandemic era.
Introduction
Cardio-Oncology care has been adjusted in the COVID-19 pandemic with limited in-person clinic or hospital visits, increased use of teleconsultation, less frequent imaging, increased reliance on biomarkers, and considerations of differential diagnoses involving COVID-19 when evaluating cancer patients or survivors for possible cardiovascular toxicity [1]. Monitoring and management algorithms have been developed to help guide virtual care [2–4]. In the pandemic, we have changed the way in which we provide healthcare services at our clinics and institutions. This has challenged us to restructure current systems for the safety of our patients.
Various forms of innovation have come to bear in the pandemic, including telemedicine, digital health, artificial intelligence, social media, informatics, big data, and precision medicine [5, 6]. Telemedicine is the primary form of innovation that has been most developed in the pandemic [2, 5, 7]. Social media has been very helpful for dissemination of information, as well as education, and has been integral for creating online groups for support and determining the best ways for proceeding in the pandemic and advocating for our patients and colleagues in this period [5, 6]. In addition, the Doximity social media application has been valuable to practices across the nation, due to its telehealth platform (Doximity Video and Phone; https://www.doximity.com/dialer-video).
Despite the growing need, and allowances made during the pandemic, many centers do not have formal Cardio-Oncology clinics. Starting a new clinic can be challenging. The COVID-19 pandemic has made the process significantly more difficult, with the need to minimize exposure and maximize patient safety.
Currently, limited information is available on how to start a Cardio-Oncology Clinic during a pandemic, albeit given the high risk of morbidity or mortality in COVID-19-positive patients who also have cancer or CVD [8–13]. Several institutions have published on their experiences with starting in-person Cardio-Oncology clinics prior to the pandemic [8, 14–17]. One group has reported on their conversion from existing in-person Cardio-Oncology visits to telemedicine consultations, seeing 11 patients virtually within a few weeks [7]. Many have considered implications of the pandemic on the practice and study of cardio-oncology [2, 4, 5, 18–20], and two groups have suggested models for clinics converting from existing in-person care to televisits [4, 5]. Yet, no groups have directly addressed steps for de novo virtual-hybrid clinic formation within the limitations of the pandemic and without conversion of a pre-existing Cardio-Oncology clinic.
Our report offers a template for other centers to develop their own new Cardio-Oncology clinics during the pandemic. We determined a Virtual-Hybrid Approach to clinic launch, with both virtual and in-person elements of three key arms: information seeking where there are existing Cardio-Oncology Clinics in place, information gathering where the clinic will be built, and information sharing to report on initial patient data demonstrating the success of the launch (Fig. 1). We then performed retrospective chart review to collect and synthesize data on the types of appointments (new versus established, virtual versus in-person), cancers (e.g., breast, prostate, leukemia, lung), cancer drugs, and cardiovascular toxicities (e.g., cardiomyopathy, hypertension) for patients seen virtually or in person in our new Cardio-Oncology clinic at Froedtert Hospital and Medical College of Wisconsin (F&MCW). Here, we will discuss our findings in the context of previous publications on launching Cardio-Oncology Clinics prior to the pandemic. Our results will present distributions of cancer drugs and types, and cardiovascular diagnoses, similar to previous publications on Cardio-Oncology clinic-building. However, we will differentiate and illuminate the techniques that leverage the virtual underpinnings of pandemic clinic-building. We submit that it is feasible to establish a new Cardio-Oncology Clinic for cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy during a pandemic, providing optimal care for new patients in the midst of the need for safety and minimizing exposure. We also propose virtual resources for patients and clinicians and describe innovative future directions in the pandemic and post-pandemic period.
Fig. 1 The Virtual-Hybrid Approach to Cardio-Oncology Clinic-building in the pandemic
Methods
Virtual-hybrid approach
We pursued a Virtual-Hybrid Approach of information seeking, information gathering, and information sharing (Fig. 1). For information seeking, we focused on institutions that already had a Cardio-Oncology clinic in place. Prior to the COVID-19 pandemic, substantial experience was gained at an established in-person Cardio-Oncology clinic at a world-renowned leading hospital. Published manuscripts on pre-pandemic building and operations of Cardio-Oncology clinics at other institutions were reviewed. Extensive networking with leaders of Cardio-Oncology clinics around the United States and in the United Kingdom was accomplished at regional, national, and international meetings in person and on social media in a hybrid approach. These meetings were attended in person pre-COVID-19 and virtually during the pandemic. For information gathering, we addressed the location in which the new Cardio-Oncology clinic would be built. We learned about existing resources in the destination Heart & Vascular Center and existing needs and patients in the destination partner cancer center. We networked with physicians, advanced practice providers (APPs), nurses, service line leaders, and administrators in the Heart & Vascular Center and the cancer center, as well as in primary care and other supporting specialties. The subsequent Cardio-Oncology clinic sessions were intermingled with other subspecialty areas, to optimize availability for patient visits while filling clinic slots and tailoring spectrum of care to emerging patient needs. Cardio-Oncology patient visits were included in multi-subspecialty clinics.
Retrospective study design
For quantitative data, we pursued a retrospective observational study to determine the distribution of outpatient visits in the first 3 months of our virtual-hybrid Cardio-Oncology Clinic. We reviewed data from charts of patients (all were 18 years of age or older) who received outpatient care from the new Director of Cardio-Oncology at F&MCW between April 15, 2020 and July 17, 2020 to determine which of these patients were considered to be in Cardio-Oncology, Preventive Cardiology, or General Cardiology. We collated all three to determine the percentage of patients seen in Cardio-Oncology, compared to the other two specialties. Preventive Cardiology was collated as a partner clinic to help build the Preventive Cardio-Oncology component of the Cardio-Oncology Clinic, to help apply established principles for prevention. From among the multi-subspecialty clinic sessions, we determined the proportion of patients who were specifically cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy and thereby seen in the Cardio-Oncology Clinic. Next, we identified the distribution of cardiovascular toxicities in cancer patients or survivors seen in the Cardio-Oncology Clinic. We also evaluated the spectrum of cancer drugs received by cancer patients or survivors seen in the Cardio-Oncology Clinic. In addition, we summarized the types of cancers in patients seen in the Cardio-Oncology Clinic. Finally, we assessed the frequency of virtual visits during the course of the pandemic over our first 3 months for patients seen in the Cardio-Oncology Clinic. This retrospective review was approved by the F&MCW IRB; HIPAA informed consent was waived for this minimal risk study, which did not involve any form of intervention and was conducted in compliance with good clinical and research practice. The team designed and carried out the study with reliance on virtual communication tools.
Data collection and analysis
Data gathering, management, and analysis were conducted at F&MCW. We collected patient-related, disease-related, treatment-related, and outcome-related data, particularly patient sex, type of appointment (new versus established, virtual versus in-person), type of cancer (e.g., breast, prostate, leukemia, lung), type of cancer drug, and type of cardiovascular toxicity (e.g., cardiomyopathy, hypertension). In order to minimize any risk of breaching patient confidentiality, all data collection occurred on institutional-based computing environments with de-identified data used for analyses. There were no alternative procedures for the subjects as this is a retrospective review of data that are not amenable to prospective collection and review. Descriptive graphs or tables of patient-, disease-, treatment-, and outcome-related variables distributions were prepared, with no comparisons made needing statistical tests.
Results
Virtual preparation
Preparation for starting the Cardio-Oncology clinic followed a Virtual-Hybrid Approach (Fig. 1, left). Five overarching factors employing virtual communication methods emerged to ensure the successful launching of the clinic. Team and individual experience and exposure to various areas of interest in Cardio-Oncology were achieved and assessed before and during the pandemic. Far-reaching connections to experts and potential collaborators in the field were developed and exercised. Close contact with the institution launching this clinic was important to determine the resources available; these resources dictated the strategy and potential outcomes of the clinic. Importantly, the expectations of others for the Cardio-Oncology clinic were determined and incorporated. Finally, recognition of the limitations that exist at the destination institution guided care and goal setting.
Virtual inquiry
Before initiating the Cardio-Oncology clinic in the destination institution, existing structures, patient base, and needs in the Heart & Vascular Center as well as the Cancer Center were evaluated, adhering to pandemic protocols (Fig. 1, middle). Pre-existing building blocks for the planned Cardio-Oncology clinic were assessed, and the partner Preventive Cardiology clinic was investigated. We also evaluated characteristics of the cancer center patient population to best position the clinic for success.
Virtual niche-building
Five main aspects of niche-building were pursued. Partnerships with Vascular and Cancer Center physicians, advanced practice providers, and service line leaders were developed to initiate and grow the clinic (Fig. 1, middle). The Cardio-Oncology team and clinic flexibility were demonstrated through openness to taking quicksteps. Presentations were made at Grand Rounds and rounds across the institution in Cardiology, Hematology/Oncology, Radiation Oncology, Surgical Oncology, Internal Medicine, and Family Medicine to promote the clinic capabilities. Collaborative solutions for problems facing fields complementing Cardio-Oncology developed trust and collaboration. Teamwork was developed by leveraging diversity of perspectives and virtual communication technologies, to establish effective patient care despite COVID-19 limitations.
Hybrid care Spectrum
The hybrid F&MCW Cardio-Oncology Clinic was initiated and established in the outpatient setting, in close partnership with the Preventive Cardiology Clinic, Cancer Center, and inpatient Cardiology Consult and Hematology/Oncology teams (Fig. 1, right).
Initial and subsequent visits have been completed in person or by video, with phone visits also available for virtual return visits if patients without adept and available smartphone use have limited ability to appear in person. Virtual patient visits over our first 3 months occurred with the use of telemedicine platforms integrated with Epic (via MyChart for patients and Haiku/Canto for clinicians), or using the Doximity video call function. Patients with in-person appointments are screened appropriately on arrival for signs or symptoms of COVID-19 or exposure, following institutional protocols. Wearing masks is required of all patients, and each patient can be accompanied by a family member; some choose to also wear gloves or face shields. There is sufficient room for maintaining social distancing in the clinic waiting room and hallways.
Innovation
Current innovation in the clinic also includes Virtual Clinician Tools and Virtual Patient Resources (Fig. 2). For clinicians, the links for an AHA CME course on Novel Concepts, Current Debates and Treatment Considerations in Cardio-Oncology, an online Cardio-Oncology Compendium hosting risk assessment clinical decision aids, Cardio-Oncology Drug Regimen and Acronym Databases, and UPTODATE access for reviewing Cardio-Oncology drug information are supplied. For patients, the video from the International Cardio-Oncology Society explaining the Cardio-Oncology subspecialty, American College of Cardiology (ACC) mobile health (mHealth) CardioSmart education app and website, Cancer Heart Talk mini-podcast series accessed via SoundCloud app and website, Cardio-Oncology Frequently Asked Questions, and ChemoCare website are provided for patient-facing Cardio-Oncology and heart anatomy and physiology education, engagement, and awareness. Virtual Resources for Preventive Cardio-Oncology are also made available to our patients. These include the American Heart Association (AHA) Physical Activity Recommendations, AHA Life’s Simple 7 Webpages, American Society For Preventive Cardiology Online Coaching Webpages in partnership with Intervent, and the Become An Ex Smoking Cessation Support Webpages in partnership with Mayo Clinic. The resources are provided in the Epic patient portal MyChart, and more ways to make the resources accessible to a broad and diverse patient population are in development. Future innovation in the clinic will explore contemporary initiatives connecting patients and their safely guarded data with their permission with wearable devices, health information technology, informatics, artificial intelligence, personalized medicine, and additional mobile health (mHealth) applications.
Fig. 2 Virtual patient and clinician education and resources. Available online as PDF with hot links in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual visit infrastructure and timeline
There was no pre-existing Cardio-Oncology program at the time of launching our de novo Virtual/Hybrid Cardio-Oncology Clinic. The newly recruited Director of Cardio-Oncology was tasked with the responsibility of launching the new clinic, with support from the Heart & Vascular Center clinic administrators, medical director, and Cardiology Division and Department of Medicine leadership. Prior to opening the Cardio-Oncology Clinic, the Heart & Vascular Center initiated virtual conversion then additionally collaborated with Inception Health (MCW’s innovation lab company) over the course of 4 weeks to iteratively develop the clinical informatics infrastructure for virtual visits. The video visits were designed to function using clinician’s personal smartphones, iPads, and tablets, with direct web browser video links from the electronic health record mobile application. Direct video calls through the Doximity mobile application were also approved. Existing Inception Health personnel re-allocated their time in order to adopt and maintain responsibility for the virtual component of all ambulatory clinics across the health system, in partnership with medical and administrative directors of each clinic area, such as the Heart & Vascular Center. No additional costs or hires were pursued to facilitate the development of the virtual visit infrastructure and timeline. Existing resources and personnel were re-allocated to virtual visit design to enable building the virtual clinics in the Heart & Vascular Center. To assist clinicians and billing compliance colleagues, note templates were created for video and phone visits to indicate patient informed consent for virtual visits due to the pandemic, as well as to capture limited appropriate physical examinations, in addition to the amount of time spent on records review and real-time medical counseling.
Initial consults were electronically triaged by either a cardiologist or a cardiology fellow supervised by a cardiologist. Each triage team determined which consults would be appropriate as virtual video visits, versus in-person to occur once the Heart and Vascular Center started re-opening routine physical visits, or whether patients needed to be evaluated urgently in person. During the first week of operation, the brand-new Cardio-Oncology Clinic started entirely virtually with only video and phone visits. As the Heart and Vascular Center re-opened for physical patient visits the following week, from week 2 through the remainder of the first 3 months the Cardio-Oncology Clinic had both virtual and in-person visits integrated throughout each clinic session weekly, based on whether patients were new and whether they had smart device or computer functionality available.
Virtual referral network and process
Cardio-Oncology patient assessment begins within a referral network before the patient arrives at a Cardio-Oncology clinic. Consequently, forming a virtual referral network and enacting a user-friendly virtual referral process was a key component of building the Cardio-Oncology clinic during the pandemic. All referral patterns and networks for our de novo Cardio-Oncology Clinic were built from the ground up. Initial referrals were from within our health system; this quickly expanded to consults from outside of our health system encompassing the entire state. Patients were referred to our Clinic by clinicians or by self-referrals. Some of our local patients connected to us after being introduced to us by their clinicians in other states or through family members in other states who learned about us from their own clinicians or community-based Cardiology society outreach events.
Referrals across the institution and outside of our health system have come to us from the Divisions of Hematology and Oncology, Internal Medicine, Family Medicine, Surgical Oncology/Breast Clinic, and Survivorship Clinics. From the cancer center’s perspective, there may be many “triggers” that would warrant a Cardio-Oncology referral. For example, an abnormal ECG, an abnormal echocardiogram, cardiovascular symptoms, previous cardiovascular history (e.g., coronary artery disease, hypertension, cardiomyopathy) particularly in a patient who previously underwent treatment or is beginning new treatment with cardiotoxic neoplastic medications or radiation therapy and is at high risk of cardiovascular toxicity, or those in preparation for stem cell transplant, or oncologic surgery. Referral protocols were determined based on standard practice, discussions with colleagues in Medical and Radiation Oncology, Hematology, Bone Marrow Transplant, Surgical Oncology/Breast Clinic, Children’s Hospital, Radiation Oncology, Primary Care, and updated literature reviews.
Cancer survivors are at a higher risk than the general population for cardiovascular morbidity and mortality. If a cancer survivor needing to be evaluated is already under the care of a cardiologist, the referring provider can reach out to their cardiologist for guidance on the appropriate CV surveillance. If they do not already have a cardiologist, a Cardio-Oncology consult should be requested. The Cardio-Oncology consult can be placed using a direct Cardio-Oncology button within the universally available Cardiovascular Consult order panel. Referrers can also place a General Cardiology consult and mention the Cardio-Oncology physician by name as requested by the clinician or patient. An E-Consult functionality is also being implemented for those patients who need to be assessed sooner than the next available appointment, or for those patients who may not need a full Cardio-Oncology evaluation, or if referring providers are uncertain. The e-consult can also be placed as a second opinion requested by the inpatient Cardiology Consult team.
The inpatient Cardiology Consult service will continue to directly address inpatient consults from the inpatient hematology/oncology services. The inpatient Cardiology Consult service can collaborate with the Cardio-Oncology Clinic via formal Cardio-Oncology E-consults in the electronic health record Epic if a specific focused question arises regarding Cardio-Oncology relevant to the care of individual currently hospitalized patients that have already been formally evaluated by the inpatient Cardiology Consult service. After a patient has been formally evaluated by the inpatient Cardiology Consult service, if the patient is appropriate for outpatient follow up in the Cardiology clinic with Cardio-Oncology, this should be communicated to the primary Hematology/Oncology service. If appropriate at the time of consultation, the inpatient cardiology consult service can make the follow-up appointment. Oftentimes, this patient population remains in the inpatient setting for several weeks. If this is the case, the Cardiology clinic phone number and clinician information should be provided to the primary service to do so prior to the patient being discharged from the hospital.
Virtual-hybrid multidisciplinary team
It is important to develop a multidisciplinary team and initially focus on allocation of pre-existing resources. Accordingly, some roles among our Cardio-Oncology clinic personnel are shared with other subspecialties. Our virtual-hybrid multidisciplinary Cardio-Oncology Clinic personnel include physicians, a nurse practitioner (NP), a nurse, a research support specialist, medical assistants, pharmacists, administrative assistants, and administrators. All personnel with pre-existing in-person roles and practices re-allocated a portion of their time to the development and practice of virtual visits.
Our clinic and partners consist of board-certified Cardiologists with special training in various cardiac subspecialties (e.g., cardio-oncology, preventive cardiology, heart failure and transplant, electrophysiology, interventional cardiology), who collaborate closely with our cancer experts. Our physicians together specialize in the prevention, diagnosis, and treatment of heart and vascular disorders resulting from side effects of cancer therapy. Our comprehensive team of advanced practice providers, nurses, and pharmacists work alongside our physicians to care for patients from the moment of cancer diagnosis through life’s survivorship journey. The NP typically sees established patients when needed to follow up on imaging, intervention, or diagnostic and management plans, and may also see select new patients. In complex cases, the NP discusses the care of established patients with both the cardio-oncologist and the referring clinician. The nurse assists with patient triage and communications (including addressing patient requests and queries), liaises closely with the nurse practitioner and pharmacists, and educates patients on Cardio-Oncology using virtual materials. Our clinical pharmacists function at the highest level of their advanced training, similar to all clinic personnel, and assist with medication education, review, titration, discussion, and prescription, particularly for heart failure, hypertension, hyperlipidemia, and smoking cessation, as well as commenting on potential drug interactions.
For Preventive Cardio-Oncology, we additionally partner with our dietitians and exercise physiologists to help advise our patients on nutrition and exercise plans, as well as our colleagues in cardiopulmonary stress testing where applicable. Further, in the pandemic, we provide patients with free online coaching options for lifestyle modification (Fig. 2). We also direct patients to AHA webpages with guidance on pursuing ideal cardiovascular health.
Virtual-hybrid patient flow
Once a referral is placed by the designated order buttons in the electronic health record, central schedulers or the Cardio-Oncology Clinic administrative assistant schedule the new patient for a video or in-person visit (Fig. 3). The clinic administrative assistant works closely with our health professionals in our interdisciplinary advanced subspecialty clinic to gather relevant clinical reports and history pertinent to patient appointments. Virtual medical assistants contact patients a few days before their appointments to confirm and troubleshoot virtual connectivity. On the appointment day, medical assistants then ‘room’ patients for virtual or in-person visits by preparing patients for their medical visits (including reviewing medications and in-person or at-home virtual vital signs), and also rechecking virtual connectivity for video visits. The clinician then completes the visit virtually or in-person and introduces the patient to the range of electronic resources available. Following the visit, the clinical administrative assistant arranges follow-up testing and appointments.
Fig. 3 Virtual-Hybrid Patient Flow Chart. Admin = Administrative; MA = Medical Assistant; MD = Medical Doctor; NP = Nurse Practitioner
Virtual risk assessment
Baseline risk assessment and follow-up start with oncology and primary care [21]. Asymptomatic low risk patients with low-risk treatment plans can have continued assessment and follow-up by oncology and primary care in partnership. Patients who have symptoms, are at high risk based on their history, or are planned for high-risk treatment plans should be referred to Cardio-Oncology for prevention, monitoring, and management recommendations. Recommendations should adhere to society expert consensus, scientific statements, and guidelines for prevention, surveillance, and survivorship, and optimize CVD risk and medications [21]. A putative risk score based on medication-related and patient-related risk factors can be used to guide monitoring and management recommendations for most Cardio-Oncology patients [22], and can be used in a virtual clinical decision aid (https://tinyurl.com/CardioOncCDA) (Fig. 4). Specific risk scores are also available for adults treated with anthracyclines, trastuzumab, or other drugs, or for adult survivors of childhood cancers [9–13].
Fig. 4 Virtual Cardio-Oncology clinical decision aid (CDA). Risk assessment (https://tinyurl.com/CardioOncCDA) to guide monitoring and management recommendations regarding development of cardiomyopathy for most Cardio-Oncology patients; a CDA specifically for women with early breast cancer is also available in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual management algorithms
Evidence-based management algorithms have been selected or developed as adjunctive resources for inpatient teams. They are available online in a virtual collection for use in the inpatient setting by the inpatient Cardiology Consult service or hematology/oncology teams to assist with diagnosis and treatment of cardiovascular toxicities from cancer therapies or cancer itself. The algorithms cover cardiomyopathy from anthracyclines or trastuzumab, planned chemotherapy with pre-existing cardiomyopathy, neurohormonal therapy or dexrazoxane for cardioprotection, myocarditis, persistent malignant pericardial effusion, hypertension, surveillance after radiation therapy or drugs that cause ischemia, malignant pericardial effusion, and other salient topics frequently encountered.
Virtual community engagement
The local, regional, national, and international community was virtually engaged via social media posts on Twitter (using #MCWCardioOnc on @DrBrownCares or @PrevCardioOnc), podcasts hosted by the MCW CTSI (available on iTunes, Google, and Apple podcast platforms), Heart Success podcast series, and Cancer Heart Talk brief 15-min mini-podcast series (available on SoundCloud). Perspectives were also published for international community engagement in the Women Heart Alliance newsletter, as well as on the AHA Early Career Blog, ACC Women in Cardiology Blog, CardioOncTrain.Com Blog, and PrevCardioOnc.Com Blog. Virtual continuing medical education (CME) presentations were also given at the Wisconsin state ACC annual conference meeting, Midwest ACC annual conference meeting, Southeast ACC annual conference meeting, Brazilian Cardio-Oncology Symposium, and the Ohio State Cardio-Oncology CME conference, then subsequently at the AHA and ACC annual national scientific sessions.
Distribution of patient data
In our multi-subspecialty clinic visits (virtual and in-person integrated and combined; n = 182; 136 new and 47 returns), approximately 50% of patient visits were in Cardio-Oncology, 20% were in Preventive Cardio-Oncology, and 30% were in General Cardiology (Fig. 5a). Overall among Cardio-Oncology visits, 65% were in person, consistent with early and safe clinic re-opening in a hybrid model, with 19% by video and 16% by phone, with the fraction by phone decreasing over time as patients and clinic personnel became more adept with troubleshooting video. Of new patients, 77% were in person, and the remainder by video. No Cardio-Oncology patients presenting in person developed any signs or symptoms concerning for COVID-19.
Fig. 5 Initial Clinic-Building Outcomes Using the Virtual-Hybrid Approach. a Distribution of Cardio-Oncology, Preventive Cardio-Oncology, and General Cardiology patients seen in our multi-subspecialty clinic visits. b Distribution of cardiovascular diagnosis or indication for referral. c Distribution of cancer drugs. d Distribution of cancer types
The most frequent cardiovascular diagnosis or indication for referral was cardiomyopathy (34%) (Fig. 5b). Other diagnoses included decrease in global longitudinal strain, diastolic congestive heart failure, hypertension, myocarditis, dyspnea, chest pain, palpitations, survivorship, risk assessment, and pre-bone marrow transplant, among other cardiovascular diagnoses or visit indications. The most frequent cancer drug was trastuzumab (29%) (Fig. 5c), managed according to a novel algorithm developed in our de novo Virtual-Hybrid Cardio-Oncology Clinic based on the recent publication indicating the safety of continuation of trastuzumab for left ventricular ejection fraction of 40% or greater [23] (Fig. 6). The second most frequent cancer drug was anthracycline (24%). Other drugs included, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), endocrine therapies, and investigational therapeutics, among others. The most frequent cancer type in our clinic was breast cancer (42%) (Fig. 5d). These trends in cardiovascular diagnosis or indication and cancer drugs or types were similar in assessments of virtual visits alone, with the most frequent being cardiomyopathy (43%), trastuzumab (41%), and breast cancer (44%), respectively. The findings of similar cardiovascular and cancer distributions in virtual versus in-person visits indicated an optimal qualitative return on resource and personnel investment.
Fig. 6 Algorithm for Continuation of Trastuzumab Therapy with Mild LV Dysfunction
Imaging and medication titration
In our clinic, a distribution of cardiovascular diagnoses determines the imaging needed for each patient (Fig. 5b). Therefore, a number of imaging modalities are useful to our patients (e.g., echocardiography, computed tomography with or without angiography, magnetic resonance imaging, coronary angiography, myocardial perfusion imaging). Our most frequently used imaging modality is echocardiography. The frequency of obtaining echocardiograms has depended on each patient’s condition and cancer treatment. A substantial portion of patients coming to us on trastuzumab have needed an echocardiogram every 1–3 months, depending on the extent of adverse effects on left ventricular ejection fraction (LVEF) or strain In these patients, medication titration has occurred approximately every 2 weeks, and for very symptomatic patients with volume overload, they have often been seen weekly..
In our management algorithms, early referral prior to the onset of symptoms has been emphasized, especially in cancer patients or survivors with a history of cardiovascular disease, cardiotoxic neoplastic agents, or a high risk of cardiovascular toxicity. This has provided an opportunity for us to assess and discuss ways to optimize the benefit to risk ratio of continuing with the current cancer treatment plan, and more importantly how and when to put cardioprotective measures in place to facilitate safe cancer therapy. Such discussions have also resulted in closer monitoring. Some conditions have warranted proceeding to other modalities of non-invasive imaging, such as cardiac MRI if myocarditis is suspected. For cases in which coronary artery disease is suspected, our patients undergo functional assessment of their coronaries with a stress test or anatomical assessment with a coronary CT scan or invasive coronary angiography. In our practice, only exercise stress tests were halted due to the pandemic. Every other form of imaging including rest and stress echo, as well as MRI and nuclear medicine have remained readily available for those with cardiovascular toxicities or individuals considered to be at moderate or high risk. This allowed us to adhere to pre-pandemic imaging recommendations tailored during the pandemic to limit imaging if possible to those who are at higher risk for cardiovascular toxicities or who have already been diagnosed with these adverse effects [1–4] (e.g., Fig. 6).
Discussion
The COVID-19 pandemic has inevitably compelled leaders of healthcare clinics to rethink and restructure approaches to deliver optimal care for patients. Our brand-new Cardio-Oncology clinic has been built to thrive in this new pandemic landscape by utilizing virtual technology as one of the key components of our clinic-building and care model since its inception. While existing clinics have reinvented their operations through the uptake of technology, our clinic has been able to capitalize on this resource to deliver virtual-hybrid care from the start. Virtual communication has proved useful to coordinate referral networks and care among providers within a multidisciplinary team across different clinics and departments. We see a variety of cancer patients, types, and drugs (Fig. 5), and our distribution results are generally congruent with reports from other leading cardio-oncology clinics [8, 14, 15, 17, 22].
Various methods have been developed for risk assessment to help guide providers and patients in determining the appropriate guidelines for care. We offer the use of virtual risk assessment tools such as the computed risk scores based on medication- and patient-related risk factors [22] (Fig. 4) (https://tinyurl.com/CardioOncCompendium), as well as recommendations for establishing cross-provider partnerships to continuously evaluate risk [21]. Other online databases containing useful information and guidelines are readily accessible and can help guide clinical practices. We encourage use of these virtual tools, which can further facilitate collaborative Cardio-Oncology care in the pandemic. Our conversations with international colleagues have suggested additional utility of these virtual tools beyond the pandemic. The online resources can be very helpful in settings where clinical practitioners work alone without support from nurses, pharmacists, nutrition specialists, or exercise physiologists.
Virtual-hybrid care has extended the care team’s capabilities for delivering and maintaining patient education and follow-up. The internet continues to be a robust resource, containing a wealth of health information that is easily accessible to the general population. Various mobile applications and electronic devices have also been developed in recent years to educate, track, and manage patients’ health and lifestyles. While these tools provide patients with greater accessibility and independence, they also create a valuable opportunity for healthcare providers to further engage patients. In a virtual-hybrid model, this becomes increasingly important, as patients may frequently transition between virtual and in-person visits. Forming care partnerships with patients through these virtual information and health-tracking resources becomes crucial in the continuity of care and proper health maintenance as we move through the pandemic.
Our most frequent cardiovascular diagnosis was cardiomyopathy (34%), which is reflective of the management need that first helped start the emerging field of Cardio-Oncology, and is similar to the most frequent cardiovascular diagnosis noted by clinicians from several other leading centers (20–35%) [24, 25]. However, Cardio-Oncology has grown remarkably over the last 10–20 years, with a wide spectrum of cardiovascular diagnoses and indications for referral (Fig. 5b) [8, 14]. Accordingly, at some other leading centers, the most frequent cardiovascular diagnosis or indication for referral has been reported as hypertension [14], arrythmia [17], or comprehensive risk assessment prior to beginning of therapy to optimize cardioprotection [8] in the practice of Preventive Cardio-Oncology [21]. This illustrates an opportunity for growth in our Clinic, to increase the fraction of high-risk patients who undergo comprehensive cardiovascular risk evaluation and management of risk factors prior to administration of cardiotoxic therapy.
Our clinic cares for patients with a range of cancer types (Fig. 5d). Individuals with breast, lung, and hematologic cancers represent a substantial proportion of our patient population, similar to other Cardio-Oncology clinics [8, 14, 22, 26]. The most frequent cancer diagnosis, breast cancer (43%), is consistent with reports from other leading cardio-oncology clinics such as the Mayo Clinic (39.2%) [15] and the Cleveland Clinic in Florida (44.3%) [14]. While hematologic malignancies such as leukemia and lymphoma represented 29% of our patients and was the second most prevalent cancer within our cohort, they comprised the most frequent forms of cancer at other cardio-oncology clinics such as at the Moffitt Cancer Center (31%) [8] and at UCLA (32.70%) [25]. However, the absolute difference was relatively insignificant. Overall, similar to these established cardio-oncology clinics, we receive patients from across a variety of cancers.
A wide breadth of cancer therapeutics is associated with cardiotoxicity [21]. Anthracyclines associate with cardiomyopathy, especially when used with trastuzumab. Targeted therapies (e.g., TKIs) can cause new or worsening of pre-existing hypertension. ICIs are associated with an increased incidence of myocarditis. Some cytotoxic chemotherapeutics, such as cisplatin, increase the risk of venous thromboembolism, and antimetabolites such as fluoropyrimidines have long been associated with a broad range of cardiotoxicities. Radiation therapy is associated with ischemic heart disease, valve dysfunction, conduction abnormalities, pericardial disease, and cardiomyopathy. Patients with cancer who have developed cardiovascular toxicity or who may be at high risk for cardiovascular toxicity should be referred to the Cardio-Oncology clinic for close follow-up.
The most frequently used cancer medication used among our patients was trastuzumab (29%), with the second most frequent being anthracyclines (24%). This was similar to other institutions, with anthracyclines and trastuzumab among the most common cancer drugs in their Cardio-Oncology clinics. Yet, anthracyclines were typically noted more commonly than trastuzumab. The Cleveland Clinic in Florida saw patients most commonly treated with radiation (40%), followed by anthracyclines (26.8%) [14]. The Moffitt Cancer Center most frequently had patients who were treated with anthracyclines (52%), with HER2 targeted therapies representing 27% of the cancer drugs [8]. This difference may reflect a high frequency of patients with HER+ breast cancer in our population (diagnosed by a ratio of HER2 to chromosome 17 signals on dual probe fluorescent in situ hybridization ≥2 or ≥ 6 HER2 signals/cell [27]), as well as the keen attention to a substantial fall in left ventricular ejection fraction or global longitudinal strain as a potential prognostic factor in our patients, per American Society of Echocardiography (ASE) guidelines [28].
All of these patient data distributions were obtained in the context of the Virtual-Hybrid patient flow in our de novo Cardio-Oncology Clinic (Fig. 3), that can be modeled by other future Virtual-Hybrid Cardio-Oncology clinics initiated during the pandemic. Table 1 compares the first few weeks of our de novo C-O clinic setup model with two published manuscripts describing conversion of pre-existing in-person C-O clinics to providing telehealth visits as an option for patients. The table shows similar numbers of patients seen in the initial periods of the clinics, although the numbers in our new clinic went from 0 to 10 in the first 3 weeks, compared to going from up to 40 patients weekly to 11 patients in 2.5 weeks for a group that converted their in-person clinic to a virtual option. Overall, CV diagnoses and cancer types were comparable; distributions of cancer drugs were not reported by the other group. Important differences were noted. Most of our patients in the pandemic were new (90%), given the de novo status of the Cardio-Oncology Clinic, while the converted virtual clinic of another group initially focused on established patients for > 50% of their patient visits. While key personnel were also the same (e.g., physician, advanced practice provider, nurse or nurse coordinator), we also report virtual versions of supportive staffing patterns, including the virtual scheduling and rooming process and pharmacy and lifestyle modification visits. Additionally, trainees have been integrally involved in the establishment of our Cardio-Oncology Clinic, with residents training in program-building, and medical students and fellows training in ambulatory cardio-oncology clinical practice and cardio-oncology critical thinking, respectively. Finally, besides the patient flow (Fig. 3) and de novo nature of our Virtual-Hybrid clinic initiated in the pandemic, our unique contribution may be the virtual resources, compared to the essential “webside manner” [7] or an alternative algorithm [4] for triaging virtual or in-person visits to the physician or advanced practice provider (Table 1).
Table 1 Comparisons Among Clinic Models Described In The Pandemic
Virtual-Hybrid Clinic Telehealth Clinic (14) Triage Clinic (4)
Model Type De Novo Conversion Conversion
Time Frame Compared 3 weeks 2.5 weeks Not reported
Number of Patients 10 11 Not reported
New Visits (%) 90 45 Not reported
Variety of CV Diagnoses Yes Yes Not reported
Variety of CA Types Yes Yes Not reported
Variety of CA Drugs Yes Unknown Not reported
Referrals Yes Unknown Yes
Scheduling Yes Unknown Not reported
Rooming Process Yes Unknown Not Reported
Virtual AA Yes Unknown Not Reported
Virtual MA Yes Unknown Not Reported
Virtual Physician Yes Yes Yes
Virtual APP Yes Unknown Yes
Virtual Pharmacist Yes Unknown Not Reported
Virtual Nurse (Coordinator) Yes Unknown Yes
Unique Contribution Virtual Resources Webside Manner Triage Algorithm
Similar to the formation or conversion of Cardio-Oncology clinics, many protocols for treatment regimens and cancer patients are yet to be standardized. While no standard protocols have been widely adopted at Cardio-Oncology practices, various institutions and writing groups have proposed some approaches (e.g., ASE or ASCO guidelines). We have collaboratively developed institutional algorithms for various cardiovascular toxicities and medications based on existing scientific statements, society guidelines, expert consensus statements, and manuscripts from leading cardio-oncology research institutions. The goal is to adopt, adapt, develop, and continuously update these algorithms, as new literature arises in order to establish best practices and an institutional standard of care.
Conclusion
Starting a new Cardio-Oncology Clinic in the pandemic has its challenges, and yet for our patients can be invaluable. Appropriately competing priorities in the pandemic can limit the scheduling of meetings and gathering of people together in one virtual room to discuss a mutual vision. Gathering resources for patient and clinician education can also be formidable, as can social distancing and obtaining important imaging. However, multiple virtual one-on-one or small group meetings can be beneficial for building institutional relationships. Similarly, virtual visits have risen to the challenge to ensure maintenance of patient care throughout the pandemic. Modifications have also been made to enable safety and distancing during imaging. With the benefit of these adjustments to address the challenge, this report provides a foundation for initiating a cardio-oncology clinic in the pandemic, with virtual resources and tools to equip patients and clinicians.
In the future, we will also lay out a roadmap for initiation of comprehensive cardio-oncology programs with the five pillars of patient care, education, research, community engagement, and innovation in the era of digital transformation accelerated by the pandemic. Novel risk modifiers and risk attenuation methods, such as breast arterial calcification, clonal hematopoiesis of indeterminate potential, and Cardio-Oncology prehabilitation, habilitation, and rehabilitation will also be addressed. Future innovation to implement recommendations from clinical trials across the nation currently underway that utilize mobile health or web-based diet and physical activity interventions and/or seek to determine the impact of cardioprotective pharmacotherapy in Preventive Cardio-Oncology will also be assessed (ClinicalTrials.Gov: NCT01988571, NCT02943590, NCT02562716, NCT01968200, NCT03265574, NCT03760588, NCT03386383, NCT02244411, NCT03223753). Many of these studies incorporate virtual technologies that will be very helpful during and after the pandemic as we continue pursuit of digital transformation.
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Acknowledgements
We are grateful to the following individuals, as well as all of the departments and divisions at Froedtert & MCW that have rallied together to help us establish the hybrid Cardio-Oncology clinic or to see our patients needing subspecialty care, or to partner with us in referrals from the inpatient service, especially the following clinicians: Stacey Gardiner MD, Jason Rubenstein MD, David Ishizawar MD, Jalaj Garg MD, Andrew Rosenblum MD. We are also grateful to Nicole Lohr MD PhD and the clinical sections at the Zablocki Veterans Administration Medical Center that have also helped us establish an affiliate Cardio-Oncology Clinic in tandem at the VAMC in Milwaukee, WI.
Authors’ contributions
SAB conceived of the study, obtained, analyzed and interpreted the data, and made a substantial contribution to the writing of the manuscript; SP made a substantial contribution to the writing of the manuscript; DR made a substantial contribution to the writing of the manuscript; SZ obtained the data and made a substantial contribution to the writing of the manuscript; ML interpreted the data and made a substantial contribution to the writing of the manuscript; TN made a substantial contribution to the writing of the manuscript; BS analyzed the data and made a substantial contribution to the writing of the manuscript; RM made a substantial contribution to the writing of the manuscript; JMac made a substantial contribution to the writing of the manuscript; KD made a substantial contribution to the writing of the manuscript; JMes made a substantial contribution to the writing of the manuscript; DM made a substantial contribution to the writing of the manuscript; JS made a substantial contribution to the writing of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was associated with this work.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Froedtert & MCW Institutional Review Board (ID PRO00038807); HIPAA informed consent was waived for this minimal risk study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests. | TRASTUZUMAB | DrugsGivenReaction | CC BY | 33441188 | 18,793,759 | 2021-01-13 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dyspnoea'. | A virtual-hybrid approach to launching a cardio-oncology clinic during a pandemic.
BACKGROUND
As cardiovascular disease is a leading cause of death in cancer survivors, the new subspecialty of Cardio-Oncology has emerged to address prevention, monitoring, and management of cardiovascular toxicities to cancer therapies. During the coronavirus disease of 2019 (COVID-19) pandemic, we developed a Virtual-Hybrid Approach to build a de novo Cardio-Oncology Clinic.
METHODS
We conceptualized a Virtual-Hybrid Approach including three arms: information seeking in locations with existing Cardio-Oncology clinics, information gathering at the location for a new clinic, and information sharing to report clinic-building outcomes. A retrospective review of outcomes included collection and synthesis of data from our first 3 months (at pandemic peak) on types of appointments, cancers, drugs, and cardiotoxicities. Data were presented using descriptive statistics.
RESULTS
A de-novo Cardio-Oncology clinic was developed structured from the ground up to integrate virtual and in-person care in a hybrid and innovative model, using the three arms of the Virtual-Hybrid Approach. First, we garnered in-person and virtual preparation through hands-on experiences, training, and discussions in existing Cardio-Oncology Clinics and conferences. Next, we gleaned information through virtual inquiry and niche-building. With partners throughout the institution, a virtual referral process was established for outpatient referrals and inpatient e-consult referrals to actualize a hybrid care spectrum for our patients administered by a multidisciplinary hybrid care team of clinicians, ancillary support staff, and clinical pharmacists. Among the multi-subspecialty clinic sessions, approximately 50% were in Cardio-Oncology, 20% in Preventive Cardiology, and 30% in General Cardiology. In the hybrid model, the Heart & Vascular Center had started to re-open, allowing for 65% of our visits to be in person. In additional analyses, the most frequent cardiovascular diagnosis was cardiomyopathy (34%), the most common cancer drug leading to referral was trastuzumab (29%), and the most prevalent cancer type was breast cancer (42%).
CONCLUSIONS
This Virtual-Hybrid Approach and retrospective review provides guidance and information regarding initiating a brand-new Cardio-Oncology Clinic during the pandemic for cancer patients/survivors. This report also furnishes virtual resources for patients, virtual tools for oncologists, cardiologists, and administrators tasked with starting new clinics during the pandemic, and innovative future directions for this digital pandemic to post-pandemic era.
Introduction
Cardio-Oncology care has been adjusted in the COVID-19 pandemic with limited in-person clinic or hospital visits, increased use of teleconsultation, less frequent imaging, increased reliance on biomarkers, and considerations of differential diagnoses involving COVID-19 when evaluating cancer patients or survivors for possible cardiovascular toxicity [1]. Monitoring and management algorithms have been developed to help guide virtual care [2–4]. In the pandemic, we have changed the way in which we provide healthcare services at our clinics and institutions. This has challenged us to restructure current systems for the safety of our patients.
Various forms of innovation have come to bear in the pandemic, including telemedicine, digital health, artificial intelligence, social media, informatics, big data, and precision medicine [5, 6]. Telemedicine is the primary form of innovation that has been most developed in the pandemic [2, 5, 7]. Social media has been very helpful for dissemination of information, as well as education, and has been integral for creating online groups for support and determining the best ways for proceeding in the pandemic and advocating for our patients and colleagues in this period [5, 6]. In addition, the Doximity social media application has been valuable to practices across the nation, due to its telehealth platform (Doximity Video and Phone; https://www.doximity.com/dialer-video).
Despite the growing need, and allowances made during the pandemic, many centers do not have formal Cardio-Oncology clinics. Starting a new clinic can be challenging. The COVID-19 pandemic has made the process significantly more difficult, with the need to minimize exposure and maximize patient safety.
Currently, limited information is available on how to start a Cardio-Oncology Clinic during a pandemic, albeit given the high risk of morbidity or mortality in COVID-19-positive patients who also have cancer or CVD [8–13]. Several institutions have published on their experiences with starting in-person Cardio-Oncology clinics prior to the pandemic [8, 14–17]. One group has reported on their conversion from existing in-person Cardio-Oncology visits to telemedicine consultations, seeing 11 patients virtually within a few weeks [7]. Many have considered implications of the pandemic on the practice and study of cardio-oncology [2, 4, 5, 18–20], and two groups have suggested models for clinics converting from existing in-person care to televisits [4, 5]. Yet, no groups have directly addressed steps for de novo virtual-hybrid clinic formation within the limitations of the pandemic and without conversion of a pre-existing Cardio-Oncology clinic.
Our report offers a template for other centers to develop their own new Cardio-Oncology clinics during the pandemic. We determined a Virtual-Hybrid Approach to clinic launch, with both virtual and in-person elements of three key arms: information seeking where there are existing Cardio-Oncology Clinics in place, information gathering where the clinic will be built, and information sharing to report on initial patient data demonstrating the success of the launch (Fig. 1). We then performed retrospective chart review to collect and synthesize data on the types of appointments (new versus established, virtual versus in-person), cancers (e.g., breast, prostate, leukemia, lung), cancer drugs, and cardiovascular toxicities (e.g., cardiomyopathy, hypertension) for patients seen virtually or in person in our new Cardio-Oncology clinic at Froedtert Hospital and Medical College of Wisconsin (F&MCW). Here, we will discuss our findings in the context of previous publications on launching Cardio-Oncology Clinics prior to the pandemic. Our results will present distributions of cancer drugs and types, and cardiovascular diagnoses, similar to previous publications on Cardio-Oncology clinic-building. However, we will differentiate and illuminate the techniques that leverage the virtual underpinnings of pandemic clinic-building. We submit that it is feasible to establish a new Cardio-Oncology Clinic for cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy during a pandemic, providing optimal care for new patients in the midst of the need for safety and minimizing exposure. We also propose virtual resources for patients and clinicians and describe innovative future directions in the pandemic and post-pandemic period.
Fig. 1 The Virtual-Hybrid Approach to Cardio-Oncology Clinic-building in the pandemic
Methods
Virtual-hybrid approach
We pursued a Virtual-Hybrid Approach of information seeking, information gathering, and information sharing (Fig. 1). For information seeking, we focused on institutions that already had a Cardio-Oncology clinic in place. Prior to the COVID-19 pandemic, substantial experience was gained at an established in-person Cardio-Oncology clinic at a world-renowned leading hospital. Published manuscripts on pre-pandemic building and operations of Cardio-Oncology clinics at other institutions were reviewed. Extensive networking with leaders of Cardio-Oncology clinics around the United States and in the United Kingdom was accomplished at regional, national, and international meetings in person and on social media in a hybrid approach. These meetings were attended in person pre-COVID-19 and virtually during the pandemic. For information gathering, we addressed the location in which the new Cardio-Oncology clinic would be built. We learned about existing resources in the destination Heart & Vascular Center and existing needs and patients in the destination partner cancer center. We networked with physicians, advanced practice providers (APPs), nurses, service line leaders, and administrators in the Heart & Vascular Center and the cancer center, as well as in primary care and other supporting specialties. The subsequent Cardio-Oncology clinic sessions were intermingled with other subspecialty areas, to optimize availability for patient visits while filling clinic slots and tailoring spectrum of care to emerging patient needs. Cardio-Oncology patient visits were included in multi-subspecialty clinics.
Retrospective study design
For quantitative data, we pursued a retrospective observational study to determine the distribution of outpatient visits in the first 3 months of our virtual-hybrid Cardio-Oncology Clinic. We reviewed data from charts of patients (all were 18 years of age or older) who received outpatient care from the new Director of Cardio-Oncology at F&MCW between April 15, 2020 and July 17, 2020 to determine which of these patients were considered to be in Cardio-Oncology, Preventive Cardiology, or General Cardiology. We collated all three to determine the percentage of patients seen in Cardio-Oncology, compared to the other two specialties. Preventive Cardiology was collated as a partner clinic to help build the Preventive Cardio-Oncology component of the Cardio-Oncology Clinic, to help apply established principles for prevention. From among the multi-subspecialty clinic sessions, we determined the proportion of patients who were specifically cancer patients or survivors with or at risk for cardiovascular toxicity from cancer therapy and thereby seen in the Cardio-Oncology Clinic. Next, we identified the distribution of cardiovascular toxicities in cancer patients or survivors seen in the Cardio-Oncology Clinic. We also evaluated the spectrum of cancer drugs received by cancer patients or survivors seen in the Cardio-Oncology Clinic. In addition, we summarized the types of cancers in patients seen in the Cardio-Oncology Clinic. Finally, we assessed the frequency of virtual visits during the course of the pandemic over our first 3 months for patients seen in the Cardio-Oncology Clinic. This retrospective review was approved by the F&MCW IRB; HIPAA informed consent was waived for this minimal risk study, which did not involve any form of intervention and was conducted in compliance with good clinical and research practice. The team designed and carried out the study with reliance on virtual communication tools.
Data collection and analysis
Data gathering, management, and analysis were conducted at F&MCW. We collected patient-related, disease-related, treatment-related, and outcome-related data, particularly patient sex, type of appointment (new versus established, virtual versus in-person), type of cancer (e.g., breast, prostate, leukemia, lung), type of cancer drug, and type of cardiovascular toxicity (e.g., cardiomyopathy, hypertension). In order to minimize any risk of breaching patient confidentiality, all data collection occurred on institutional-based computing environments with de-identified data used for analyses. There were no alternative procedures for the subjects as this is a retrospective review of data that are not amenable to prospective collection and review. Descriptive graphs or tables of patient-, disease-, treatment-, and outcome-related variables distributions were prepared, with no comparisons made needing statistical tests.
Results
Virtual preparation
Preparation for starting the Cardio-Oncology clinic followed a Virtual-Hybrid Approach (Fig. 1, left). Five overarching factors employing virtual communication methods emerged to ensure the successful launching of the clinic. Team and individual experience and exposure to various areas of interest in Cardio-Oncology were achieved and assessed before and during the pandemic. Far-reaching connections to experts and potential collaborators in the field were developed and exercised. Close contact with the institution launching this clinic was important to determine the resources available; these resources dictated the strategy and potential outcomes of the clinic. Importantly, the expectations of others for the Cardio-Oncology clinic were determined and incorporated. Finally, recognition of the limitations that exist at the destination institution guided care and goal setting.
Virtual inquiry
Before initiating the Cardio-Oncology clinic in the destination institution, existing structures, patient base, and needs in the Heart & Vascular Center as well as the Cancer Center were evaluated, adhering to pandemic protocols (Fig. 1, middle). Pre-existing building blocks for the planned Cardio-Oncology clinic were assessed, and the partner Preventive Cardiology clinic was investigated. We also evaluated characteristics of the cancer center patient population to best position the clinic for success.
Virtual niche-building
Five main aspects of niche-building were pursued. Partnerships with Vascular and Cancer Center physicians, advanced practice providers, and service line leaders were developed to initiate and grow the clinic (Fig. 1, middle). The Cardio-Oncology team and clinic flexibility were demonstrated through openness to taking quicksteps. Presentations were made at Grand Rounds and rounds across the institution in Cardiology, Hematology/Oncology, Radiation Oncology, Surgical Oncology, Internal Medicine, and Family Medicine to promote the clinic capabilities. Collaborative solutions for problems facing fields complementing Cardio-Oncology developed trust and collaboration. Teamwork was developed by leveraging diversity of perspectives and virtual communication technologies, to establish effective patient care despite COVID-19 limitations.
Hybrid care Spectrum
The hybrid F&MCW Cardio-Oncology Clinic was initiated and established in the outpatient setting, in close partnership with the Preventive Cardiology Clinic, Cancer Center, and inpatient Cardiology Consult and Hematology/Oncology teams (Fig. 1, right).
Initial and subsequent visits have been completed in person or by video, with phone visits also available for virtual return visits if patients without adept and available smartphone use have limited ability to appear in person. Virtual patient visits over our first 3 months occurred with the use of telemedicine platforms integrated with Epic (via MyChart for patients and Haiku/Canto for clinicians), or using the Doximity video call function. Patients with in-person appointments are screened appropriately on arrival for signs or symptoms of COVID-19 or exposure, following institutional protocols. Wearing masks is required of all patients, and each patient can be accompanied by a family member; some choose to also wear gloves or face shields. There is sufficient room for maintaining social distancing in the clinic waiting room and hallways.
Innovation
Current innovation in the clinic also includes Virtual Clinician Tools and Virtual Patient Resources (Fig. 2). For clinicians, the links for an AHA CME course on Novel Concepts, Current Debates and Treatment Considerations in Cardio-Oncology, an online Cardio-Oncology Compendium hosting risk assessment clinical decision aids, Cardio-Oncology Drug Regimen and Acronym Databases, and UPTODATE access for reviewing Cardio-Oncology drug information are supplied. For patients, the video from the International Cardio-Oncology Society explaining the Cardio-Oncology subspecialty, American College of Cardiology (ACC) mobile health (mHealth) CardioSmart education app and website, Cancer Heart Talk mini-podcast series accessed via SoundCloud app and website, Cardio-Oncology Frequently Asked Questions, and ChemoCare website are provided for patient-facing Cardio-Oncology and heart anatomy and physiology education, engagement, and awareness. Virtual Resources for Preventive Cardio-Oncology are also made available to our patients. These include the American Heart Association (AHA) Physical Activity Recommendations, AHA Life’s Simple 7 Webpages, American Society For Preventive Cardiology Online Coaching Webpages in partnership with Intervent, and the Become An Ex Smoking Cessation Support Webpages in partnership with Mayo Clinic. The resources are provided in the Epic patient portal MyChart, and more ways to make the resources accessible to a broad and diverse patient population are in development. Future innovation in the clinic will explore contemporary initiatives connecting patients and their safely guarded data with their permission with wearable devices, health information technology, informatics, artificial intelligence, personalized medicine, and additional mobile health (mHealth) applications.
Fig. 2 Virtual patient and clinician education and resources. Available online as PDF with hot links in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual visit infrastructure and timeline
There was no pre-existing Cardio-Oncology program at the time of launching our de novo Virtual/Hybrid Cardio-Oncology Clinic. The newly recruited Director of Cardio-Oncology was tasked with the responsibility of launching the new clinic, with support from the Heart & Vascular Center clinic administrators, medical director, and Cardiology Division and Department of Medicine leadership. Prior to opening the Cardio-Oncology Clinic, the Heart & Vascular Center initiated virtual conversion then additionally collaborated with Inception Health (MCW’s innovation lab company) over the course of 4 weeks to iteratively develop the clinical informatics infrastructure for virtual visits. The video visits were designed to function using clinician’s personal smartphones, iPads, and tablets, with direct web browser video links from the electronic health record mobile application. Direct video calls through the Doximity mobile application were also approved. Existing Inception Health personnel re-allocated their time in order to adopt and maintain responsibility for the virtual component of all ambulatory clinics across the health system, in partnership with medical and administrative directors of each clinic area, such as the Heart & Vascular Center. No additional costs or hires were pursued to facilitate the development of the virtual visit infrastructure and timeline. Existing resources and personnel were re-allocated to virtual visit design to enable building the virtual clinics in the Heart & Vascular Center. To assist clinicians and billing compliance colleagues, note templates were created for video and phone visits to indicate patient informed consent for virtual visits due to the pandemic, as well as to capture limited appropriate physical examinations, in addition to the amount of time spent on records review and real-time medical counseling.
Initial consults were electronically triaged by either a cardiologist or a cardiology fellow supervised by a cardiologist. Each triage team determined which consults would be appropriate as virtual video visits, versus in-person to occur once the Heart and Vascular Center started re-opening routine physical visits, or whether patients needed to be evaluated urgently in person. During the first week of operation, the brand-new Cardio-Oncology Clinic started entirely virtually with only video and phone visits. As the Heart and Vascular Center re-opened for physical patient visits the following week, from week 2 through the remainder of the first 3 months the Cardio-Oncology Clinic had both virtual and in-person visits integrated throughout each clinic session weekly, based on whether patients were new and whether they had smart device or computer functionality available.
Virtual referral network and process
Cardio-Oncology patient assessment begins within a referral network before the patient arrives at a Cardio-Oncology clinic. Consequently, forming a virtual referral network and enacting a user-friendly virtual referral process was a key component of building the Cardio-Oncology clinic during the pandemic. All referral patterns and networks for our de novo Cardio-Oncology Clinic were built from the ground up. Initial referrals were from within our health system; this quickly expanded to consults from outside of our health system encompassing the entire state. Patients were referred to our Clinic by clinicians or by self-referrals. Some of our local patients connected to us after being introduced to us by their clinicians in other states or through family members in other states who learned about us from their own clinicians or community-based Cardiology society outreach events.
Referrals across the institution and outside of our health system have come to us from the Divisions of Hematology and Oncology, Internal Medicine, Family Medicine, Surgical Oncology/Breast Clinic, and Survivorship Clinics. From the cancer center’s perspective, there may be many “triggers” that would warrant a Cardio-Oncology referral. For example, an abnormal ECG, an abnormal echocardiogram, cardiovascular symptoms, previous cardiovascular history (e.g., coronary artery disease, hypertension, cardiomyopathy) particularly in a patient who previously underwent treatment or is beginning new treatment with cardiotoxic neoplastic medications or radiation therapy and is at high risk of cardiovascular toxicity, or those in preparation for stem cell transplant, or oncologic surgery. Referral protocols were determined based on standard practice, discussions with colleagues in Medical and Radiation Oncology, Hematology, Bone Marrow Transplant, Surgical Oncology/Breast Clinic, Children’s Hospital, Radiation Oncology, Primary Care, and updated literature reviews.
Cancer survivors are at a higher risk than the general population for cardiovascular morbidity and mortality. If a cancer survivor needing to be evaluated is already under the care of a cardiologist, the referring provider can reach out to their cardiologist for guidance on the appropriate CV surveillance. If they do not already have a cardiologist, a Cardio-Oncology consult should be requested. The Cardio-Oncology consult can be placed using a direct Cardio-Oncology button within the universally available Cardiovascular Consult order panel. Referrers can also place a General Cardiology consult and mention the Cardio-Oncology physician by name as requested by the clinician or patient. An E-Consult functionality is also being implemented for those patients who need to be assessed sooner than the next available appointment, or for those patients who may not need a full Cardio-Oncology evaluation, or if referring providers are uncertain. The e-consult can also be placed as a second opinion requested by the inpatient Cardiology Consult team.
The inpatient Cardiology Consult service will continue to directly address inpatient consults from the inpatient hematology/oncology services. The inpatient Cardiology Consult service can collaborate with the Cardio-Oncology Clinic via formal Cardio-Oncology E-consults in the electronic health record Epic if a specific focused question arises regarding Cardio-Oncology relevant to the care of individual currently hospitalized patients that have already been formally evaluated by the inpatient Cardiology Consult service. After a patient has been formally evaluated by the inpatient Cardiology Consult service, if the patient is appropriate for outpatient follow up in the Cardiology clinic with Cardio-Oncology, this should be communicated to the primary Hematology/Oncology service. If appropriate at the time of consultation, the inpatient cardiology consult service can make the follow-up appointment. Oftentimes, this patient population remains in the inpatient setting for several weeks. If this is the case, the Cardiology clinic phone number and clinician information should be provided to the primary service to do so prior to the patient being discharged from the hospital.
Virtual-hybrid multidisciplinary team
It is important to develop a multidisciplinary team and initially focus on allocation of pre-existing resources. Accordingly, some roles among our Cardio-Oncology clinic personnel are shared with other subspecialties. Our virtual-hybrid multidisciplinary Cardio-Oncology Clinic personnel include physicians, a nurse practitioner (NP), a nurse, a research support specialist, medical assistants, pharmacists, administrative assistants, and administrators. All personnel with pre-existing in-person roles and practices re-allocated a portion of their time to the development and practice of virtual visits.
Our clinic and partners consist of board-certified Cardiologists with special training in various cardiac subspecialties (e.g., cardio-oncology, preventive cardiology, heart failure and transplant, electrophysiology, interventional cardiology), who collaborate closely with our cancer experts. Our physicians together specialize in the prevention, diagnosis, and treatment of heart and vascular disorders resulting from side effects of cancer therapy. Our comprehensive team of advanced practice providers, nurses, and pharmacists work alongside our physicians to care for patients from the moment of cancer diagnosis through life’s survivorship journey. The NP typically sees established patients when needed to follow up on imaging, intervention, or diagnostic and management plans, and may also see select new patients. In complex cases, the NP discusses the care of established patients with both the cardio-oncologist and the referring clinician. The nurse assists with patient triage and communications (including addressing patient requests and queries), liaises closely with the nurse practitioner and pharmacists, and educates patients on Cardio-Oncology using virtual materials. Our clinical pharmacists function at the highest level of their advanced training, similar to all clinic personnel, and assist with medication education, review, titration, discussion, and prescription, particularly for heart failure, hypertension, hyperlipidemia, and smoking cessation, as well as commenting on potential drug interactions.
For Preventive Cardio-Oncology, we additionally partner with our dietitians and exercise physiologists to help advise our patients on nutrition and exercise plans, as well as our colleagues in cardiopulmonary stress testing where applicable. Further, in the pandemic, we provide patients with free online coaching options for lifestyle modification (Fig. 2). We also direct patients to AHA webpages with guidance on pursuing ideal cardiovascular health.
Virtual-hybrid patient flow
Once a referral is placed by the designated order buttons in the electronic health record, central schedulers or the Cardio-Oncology Clinic administrative assistant schedule the new patient for a video or in-person visit (Fig. 3). The clinic administrative assistant works closely with our health professionals in our interdisciplinary advanced subspecialty clinic to gather relevant clinical reports and history pertinent to patient appointments. Virtual medical assistants contact patients a few days before their appointments to confirm and troubleshoot virtual connectivity. On the appointment day, medical assistants then ‘room’ patients for virtual or in-person visits by preparing patients for their medical visits (including reviewing medications and in-person or at-home virtual vital signs), and also rechecking virtual connectivity for video visits. The clinician then completes the visit virtually or in-person and introduces the patient to the range of electronic resources available. Following the visit, the clinical administrative assistant arranges follow-up testing and appointments.
Fig. 3 Virtual-Hybrid Patient Flow Chart. Admin = Administrative; MA = Medical Assistant; MD = Medical Doctor; NP = Nurse Practitioner
Virtual risk assessment
Baseline risk assessment and follow-up start with oncology and primary care [21]. Asymptomatic low risk patients with low-risk treatment plans can have continued assessment and follow-up by oncology and primary care in partnership. Patients who have symptoms, are at high risk based on their history, or are planned for high-risk treatment plans should be referred to Cardio-Oncology for prevention, monitoring, and management recommendations. Recommendations should adhere to society expert consensus, scientific statements, and guidelines for prevention, surveillance, and survivorship, and optimize CVD risk and medications [21]. A putative risk score based on medication-related and patient-related risk factors can be used to guide monitoring and management recommendations for most Cardio-Oncology patients [22], and can be used in a virtual clinical decision aid (https://tinyurl.com/CardioOncCDA) (Fig. 4). Specific risk scores are also available for adults treated with anthracyclines, trastuzumab, or other drugs, or for adult survivors of childhood cancers [9–13].
Fig. 4 Virtual Cardio-Oncology clinical decision aid (CDA). Risk assessment (https://tinyurl.com/CardioOncCDA) to guide monitoring and management recommendations regarding development of cardiomyopathy for most Cardio-Oncology patients; a CDA specifically for women with early breast cancer is also available in the CardioOnc Compendium (https://tinyurl.com/CardioOncCompendium)
Virtual management algorithms
Evidence-based management algorithms have been selected or developed as adjunctive resources for inpatient teams. They are available online in a virtual collection for use in the inpatient setting by the inpatient Cardiology Consult service or hematology/oncology teams to assist with diagnosis and treatment of cardiovascular toxicities from cancer therapies or cancer itself. The algorithms cover cardiomyopathy from anthracyclines or trastuzumab, planned chemotherapy with pre-existing cardiomyopathy, neurohormonal therapy or dexrazoxane for cardioprotection, myocarditis, persistent malignant pericardial effusion, hypertension, surveillance after radiation therapy or drugs that cause ischemia, malignant pericardial effusion, and other salient topics frequently encountered.
Virtual community engagement
The local, regional, national, and international community was virtually engaged via social media posts on Twitter (using #MCWCardioOnc on @DrBrownCares or @PrevCardioOnc), podcasts hosted by the MCW CTSI (available on iTunes, Google, and Apple podcast platforms), Heart Success podcast series, and Cancer Heart Talk brief 15-min mini-podcast series (available on SoundCloud). Perspectives were also published for international community engagement in the Women Heart Alliance newsletter, as well as on the AHA Early Career Blog, ACC Women in Cardiology Blog, CardioOncTrain.Com Blog, and PrevCardioOnc.Com Blog. Virtual continuing medical education (CME) presentations were also given at the Wisconsin state ACC annual conference meeting, Midwest ACC annual conference meeting, Southeast ACC annual conference meeting, Brazilian Cardio-Oncology Symposium, and the Ohio State Cardio-Oncology CME conference, then subsequently at the AHA and ACC annual national scientific sessions.
Distribution of patient data
In our multi-subspecialty clinic visits (virtual and in-person integrated and combined; n = 182; 136 new and 47 returns), approximately 50% of patient visits were in Cardio-Oncology, 20% were in Preventive Cardio-Oncology, and 30% were in General Cardiology (Fig. 5a). Overall among Cardio-Oncology visits, 65% were in person, consistent with early and safe clinic re-opening in a hybrid model, with 19% by video and 16% by phone, with the fraction by phone decreasing over time as patients and clinic personnel became more adept with troubleshooting video. Of new patients, 77% were in person, and the remainder by video. No Cardio-Oncology patients presenting in person developed any signs or symptoms concerning for COVID-19.
Fig. 5 Initial Clinic-Building Outcomes Using the Virtual-Hybrid Approach. a Distribution of Cardio-Oncology, Preventive Cardio-Oncology, and General Cardiology patients seen in our multi-subspecialty clinic visits. b Distribution of cardiovascular diagnosis or indication for referral. c Distribution of cancer drugs. d Distribution of cancer types
The most frequent cardiovascular diagnosis or indication for referral was cardiomyopathy (34%) (Fig. 5b). Other diagnoses included decrease in global longitudinal strain, diastolic congestive heart failure, hypertension, myocarditis, dyspnea, chest pain, palpitations, survivorship, risk assessment, and pre-bone marrow transplant, among other cardiovascular diagnoses or visit indications. The most frequent cancer drug was trastuzumab (29%) (Fig. 5c), managed according to a novel algorithm developed in our de novo Virtual-Hybrid Cardio-Oncology Clinic based on the recent publication indicating the safety of continuation of trastuzumab for left ventricular ejection fraction of 40% or greater [23] (Fig. 6). The second most frequent cancer drug was anthracycline (24%). Other drugs included, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), endocrine therapies, and investigational therapeutics, among others. The most frequent cancer type in our clinic was breast cancer (42%) (Fig. 5d). These trends in cardiovascular diagnosis or indication and cancer drugs or types were similar in assessments of virtual visits alone, with the most frequent being cardiomyopathy (43%), trastuzumab (41%), and breast cancer (44%), respectively. The findings of similar cardiovascular and cancer distributions in virtual versus in-person visits indicated an optimal qualitative return on resource and personnel investment.
Fig. 6 Algorithm for Continuation of Trastuzumab Therapy with Mild LV Dysfunction
Imaging and medication titration
In our clinic, a distribution of cardiovascular diagnoses determines the imaging needed for each patient (Fig. 5b). Therefore, a number of imaging modalities are useful to our patients (e.g., echocardiography, computed tomography with or without angiography, magnetic resonance imaging, coronary angiography, myocardial perfusion imaging). Our most frequently used imaging modality is echocardiography. The frequency of obtaining echocardiograms has depended on each patient’s condition and cancer treatment. A substantial portion of patients coming to us on trastuzumab have needed an echocardiogram every 1–3 months, depending on the extent of adverse effects on left ventricular ejection fraction (LVEF) or strain In these patients, medication titration has occurred approximately every 2 weeks, and for very symptomatic patients with volume overload, they have often been seen weekly..
In our management algorithms, early referral prior to the onset of symptoms has been emphasized, especially in cancer patients or survivors with a history of cardiovascular disease, cardiotoxic neoplastic agents, or a high risk of cardiovascular toxicity. This has provided an opportunity for us to assess and discuss ways to optimize the benefit to risk ratio of continuing with the current cancer treatment plan, and more importantly how and when to put cardioprotective measures in place to facilitate safe cancer therapy. Such discussions have also resulted in closer monitoring. Some conditions have warranted proceeding to other modalities of non-invasive imaging, such as cardiac MRI if myocarditis is suspected. For cases in which coronary artery disease is suspected, our patients undergo functional assessment of their coronaries with a stress test or anatomical assessment with a coronary CT scan or invasive coronary angiography. In our practice, only exercise stress tests were halted due to the pandemic. Every other form of imaging including rest and stress echo, as well as MRI and nuclear medicine have remained readily available for those with cardiovascular toxicities or individuals considered to be at moderate or high risk. This allowed us to adhere to pre-pandemic imaging recommendations tailored during the pandemic to limit imaging if possible to those who are at higher risk for cardiovascular toxicities or who have already been diagnosed with these adverse effects [1–4] (e.g., Fig. 6).
Discussion
The COVID-19 pandemic has inevitably compelled leaders of healthcare clinics to rethink and restructure approaches to deliver optimal care for patients. Our brand-new Cardio-Oncology clinic has been built to thrive in this new pandemic landscape by utilizing virtual technology as one of the key components of our clinic-building and care model since its inception. While existing clinics have reinvented their operations through the uptake of technology, our clinic has been able to capitalize on this resource to deliver virtual-hybrid care from the start. Virtual communication has proved useful to coordinate referral networks and care among providers within a multidisciplinary team across different clinics and departments. We see a variety of cancer patients, types, and drugs (Fig. 5), and our distribution results are generally congruent with reports from other leading cardio-oncology clinics [8, 14, 15, 17, 22].
Various methods have been developed for risk assessment to help guide providers and patients in determining the appropriate guidelines for care. We offer the use of virtual risk assessment tools such as the computed risk scores based on medication- and patient-related risk factors [22] (Fig. 4) (https://tinyurl.com/CardioOncCompendium), as well as recommendations for establishing cross-provider partnerships to continuously evaluate risk [21]. Other online databases containing useful information and guidelines are readily accessible and can help guide clinical practices. We encourage use of these virtual tools, which can further facilitate collaborative Cardio-Oncology care in the pandemic. Our conversations with international colleagues have suggested additional utility of these virtual tools beyond the pandemic. The online resources can be very helpful in settings where clinical practitioners work alone without support from nurses, pharmacists, nutrition specialists, or exercise physiologists.
Virtual-hybrid care has extended the care team’s capabilities for delivering and maintaining patient education and follow-up. The internet continues to be a robust resource, containing a wealth of health information that is easily accessible to the general population. Various mobile applications and electronic devices have also been developed in recent years to educate, track, and manage patients’ health and lifestyles. While these tools provide patients with greater accessibility and independence, they also create a valuable opportunity for healthcare providers to further engage patients. In a virtual-hybrid model, this becomes increasingly important, as patients may frequently transition between virtual and in-person visits. Forming care partnerships with patients through these virtual information and health-tracking resources becomes crucial in the continuity of care and proper health maintenance as we move through the pandemic.
Our most frequent cardiovascular diagnosis was cardiomyopathy (34%), which is reflective of the management need that first helped start the emerging field of Cardio-Oncology, and is similar to the most frequent cardiovascular diagnosis noted by clinicians from several other leading centers (20–35%) [24, 25]. However, Cardio-Oncology has grown remarkably over the last 10–20 years, with a wide spectrum of cardiovascular diagnoses and indications for referral (Fig. 5b) [8, 14]. Accordingly, at some other leading centers, the most frequent cardiovascular diagnosis or indication for referral has been reported as hypertension [14], arrythmia [17], or comprehensive risk assessment prior to beginning of therapy to optimize cardioprotection [8] in the practice of Preventive Cardio-Oncology [21]. This illustrates an opportunity for growth in our Clinic, to increase the fraction of high-risk patients who undergo comprehensive cardiovascular risk evaluation and management of risk factors prior to administration of cardiotoxic therapy.
Our clinic cares for patients with a range of cancer types (Fig. 5d). Individuals with breast, lung, and hematologic cancers represent a substantial proportion of our patient population, similar to other Cardio-Oncology clinics [8, 14, 22, 26]. The most frequent cancer diagnosis, breast cancer (43%), is consistent with reports from other leading cardio-oncology clinics such as the Mayo Clinic (39.2%) [15] and the Cleveland Clinic in Florida (44.3%) [14]. While hematologic malignancies such as leukemia and lymphoma represented 29% of our patients and was the second most prevalent cancer within our cohort, they comprised the most frequent forms of cancer at other cardio-oncology clinics such as at the Moffitt Cancer Center (31%) [8] and at UCLA (32.70%) [25]. However, the absolute difference was relatively insignificant. Overall, similar to these established cardio-oncology clinics, we receive patients from across a variety of cancers.
A wide breadth of cancer therapeutics is associated with cardiotoxicity [21]. Anthracyclines associate with cardiomyopathy, especially when used with trastuzumab. Targeted therapies (e.g., TKIs) can cause new or worsening of pre-existing hypertension. ICIs are associated with an increased incidence of myocarditis. Some cytotoxic chemotherapeutics, such as cisplatin, increase the risk of venous thromboembolism, and antimetabolites such as fluoropyrimidines have long been associated with a broad range of cardiotoxicities. Radiation therapy is associated with ischemic heart disease, valve dysfunction, conduction abnormalities, pericardial disease, and cardiomyopathy. Patients with cancer who have developed cardiovascular toxicity or who may be at high risk for cardiovascular toxicity should be referred to the Cardio-Oncology clinic for close follow-up.
The most frequently used cancer medication used among our patients was trastuzumab (29%), with the second most frequent being anthracyclines (24%). This was similar to other institutions, with anthracyclines and trastuzumab among the most common cancer drugs in their Cardio-Oncology clinics. Yet, anthracyclines were typically noted more commonly than trastuzumab. The Cleveland Clinic in Florida saw patients most commonly treated with radiation (40%), followed by anthracyclines (26.8%) [14]. The Moffitt Cancer Center most frequently had patients who were treated with anthracyclines (52%), with HER2 targeted therapies representing 27% of the cancer drugs [8]. This difference may reflect a high frequency of patients with HER+ breast cancer in our population (diagnosed by a ratio of HER2 to chromosome 17 signals on dual probe fluorescent in situ hybridization ≥2 or ≥ 6 HER2 signals/cell [27]), as well as the keen attention to a substantial fall in left ventricular ejection fraction or global longitudinal strain as a potential prognostic factor in our patients, per American Society of Echocardiography (ASE) guidelines [28].
All of these patient data distributions were obtained in the context of the Virtual-Hybrid patient flow in our de novo Cardio-Oncology Clinic (Fig. 3), that can be modeled by other future Virtual-Hybrid Cardio-Oncology clinics initiated during the pandemic. Table 1 compares the first few weeks of our de novo C-O clinic setup model with two published manuscripts describing conversion of pre-existing in-person C-O clinics to providing telehealth visits as an option for patients. The table shows similar numbers of patients seen in the initial periods of the clinics, although the numbers in our new clinic went from 0 to 10 in the first 3 weeks, compared to going from up to 40 patients weekly to 11 patients in 2.5 weeks for a group that converted their in-person clinic to a virtual option. Overall, CV diagnoses and cancer types were comparable; distributions of cancer drugs were not reported by the other group. Important differences were noted. Most of our patients in the pandemic were new (90%), given the de novo status of the Cardio-Oncology Clinic, while the converted virtual clinic of another group initially focused on established patients for > 50% of their patient visits. While key personnel were also the same (e.g., physician, advanced practice provider, nurse or nurse coordinator), we also report virtual versions of supportive staffing patterns, including the virtual scheduling and rooming process and pharmacy and lifestyle modification visits. Additionally, trainees have been integrally involved in the establishment of our Cardio-Oncology Clinic, with residents training in program-building, and medical students and fellows training in ambulatory cardio-oncology clinical practice and cardio-oncology critical thinking, respectively. Finally, besides the patient flow (Fig. 3) and de novo nature of our Virtual-Hybrid clinic initiated in the pandemic, our unique contribution may be the virtual resources, compared to the essential “webside manner” [7] or an alternative algorithm [4] for triaging virtual or in-person visits to the physician or advanced practice provider (Table 1).
Table 1 Comparisons Among Clinic Models Described In The Pandemic
Virtual-Hybrid Clinic Telehealth Clinic (14) Triage Clinic (4)
Model Type De Novo Conversion Conversion
Time Frame Compared 3 weeks 2.5 weeks Not reported
Number of Patients 10 11 Not reported
New Visits (%) 90 45 Not reported
Variety of CV Diagnoses Yes Yes Not reported
Variety of CA Types Yes Yes Not reported
Variety of CA Drugs Yes Unknown Not reported
Referrals Yes Unknown Yes
Scheduling Yes Unknown Not reported
Rooming Process Yes Unknown Not Reported
Virtual AA Yes Unknown Not Reported
Virtual MA Yes Unknown Not Reported
Virtual Physician Yes Yes Yes
Virtual APP Yes Unknown Yes
Virtual Pharmacist Yes Unknown Not Reported
Virtual Nurse (Coordinator) Yes Unknown Yes
Unique Contribution Virtual Resources Webside Manner Triage Algorithm
Similar to the formation or conversion of Cardio-Oncology clinics, many protocols for treatment regimens and cancer patients are yet to be standardized. While no standard protocols have been widely adopted at Cardio-Oncology practices, various institutions and writing groups have proposed some approaches (e.g., ASE or ASCO guidelines). We have collaboratively developed institutional algorithms for various cardiovascular toxicities and medications based on existing scientific statements, society guidelines, expert consensus statements, and manuscripts from leading cardio-oncology research institutions. The goal is to adopt, adapt, develop, and continuously update these algorithms, as new literature arises in order to establish best practices and an institutional standard of care.
Conclusion
Starting a new Cardio-Oncology Clinic in the pandemic has its challenges, and yet for our patients can be invaluable. Appropriately competing priorities in the pandemic can limit the scheduling of meetings and gathering of people together in one virtual room to discuss a mutual vision. Gathering resources for patient and clinician education can also be formidable, as can social distancing and obtaining important imaging. However, multiple virtual one-on-one or small group meetings can be beneficial for building institutional relationships. Similarly, virtual visits have risen to the challenge to ensure maintenance of patient care throughout the pandemic. Modifications have also been made to enable safety and distancing during imaging. With the benefit of these adjustments to address the challenge, this report provides a foundation for initiating a cardio-oncology clinic in the pandemic, with virtual resources and tools to equip patients and clinicians.
In the future, we will also lay out a roadmap for initiation of comprehensive cardio-oncology programs with the five pillars of patient care, education, research, community engagement, and innovation in the era of digital transformation accelerated by the pandemic. Novel risk modifiers and risk attenuation methods, such as breast arterial calcification, clonal hematopoiesis of indeterminate potential, and Cardio-Oncology prehabilitation, habilitation, and rehabilitation will also be addressed. Future innovation to implement recommendations from clinical trials across the nation currently underway that utilize mobile health or web-based diet and physical activity interventions and/or seek to determine the impact of cardioprotective pharmacotherapy in Preventive Cardio-Oncology will also be assessed (ClinicalTrials.Gov: NCT01988571, NCT02943590, NCT02562716, NCT01968200, NCT03265574, NCT03760588, NCT03386383, NCT02244411, NCT03223753). Many of these studies incorporate virtual technologies that will be very helpful during and after the pandemic as we continue pursuit of digital transformation.
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Acknowledgements
We are grateful to the following individuals, as well as all of the departments and divisions at Froedtert & MCW that have rallied together to help us establish the hybrid Cardio-Oncology clinic or to see our patients needing subspecialty care, or to partner with us in referrals from the inpatient service, especially the following clinicians: Stacey Gardiner MD, Jason Rubenstein MD, David Ishizawar MD, Jalaj Garg MD, Andrew Rosenblum MD. We are also grateful to Nicole Lohr MD PhD and the clinical sections at the Zablocki Veterans Administration Medical Center that have also helped us establish an affiliate Cardio-Oncology Clinic in tandem at the VAMC in Milwaukee, WI.
Authors’ contributions
SAB conceived of the study, obtained, analyzed and interpreted the data, and made a substantial contribution to the writing of the manuscript; SP made a substantial contribution to the writing of the manuscript; DR made a substantial contribution to the writing of the manuscript; SZ obtained the data and made a substantial contribution to the writing of the manuscript; ML interpreted the data and made a substantial contribution to the writing of the manuscript; TN made a substantial contribution to the writing of the manuscript; BS analyzed the data and made a substantial contribution to the writing of the manuscript; RM made a substantial contribution to the writing of the manuscript; JMac made a substantial contribution to the writing of the manuscript; KD made a substantial contribution to the writing of the manuscript; JMes made a substantial contribution to the writing of the manuscript; DM made a substantial contribution to the writing of the manuscript; JS made a substantial contribution to the writing of the manuscript. All authors read and approved the final manuscript.
Funding
No funding was associated with this work.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was approved by the Froedtert & MCW Institutional Review Board (ID PRO00038807); HIPAA informed consent was waived for this minimal risk study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests. | TRASTUZUMAB | DrugsGivenReaction | CC BY | 33441188 | 18,793,759 | 2021-01-13 |
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