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First clinical trial: Azienda Ospedaliera Sant’Andrea, Rome | EMERGENCY | In the first clinical trial performed at the Department of Emergency Medicine at Azienda Ospedaliera Sant’Andrea, Rome, analyses were conducted on urine samples immediately after collection, in parallel with the MBS AST-POCT and the reference culture method for bacterial load assessment, and AST, performed by the hospital clinical microbiology laboratory.During this trial the impact of urine samples freezing on MBS AST-POCT results was also assessed. | PMC10321614 | |
Urine collection | MSU, STERILE | Urine samples were provided by the hospital staff after collection in the morning. Midstream urine (MSU) samples or catheter specimens were collected using disposable, sterile containers, kept refrigerated for maximum 2 hours and split into 10 aliquots of 1 ml each: 4 were immediately analyzed by the hospital laboratory and with the MBS AST POCT, 3 were frozen at -80°C using sterile glycerol at a final 15% (vol/vol) concentration, while the other 3 were further divided in 100 μl aliquots, frozen at -80°C using sterile glycerol at a final 15% (vol/vol) concentration, and analyzed within 7 days, to assess the impact of sample freezing or in case of the need of further verification analysis. | PMC10321614 | |
Hospital laboratory tests | UTI | Urine culture was the reference method used for confirmation of UTI suspicion. Bacterial identification and AST were performed using the VITEK | PMC10321614 | |
Antimicrobial Susceptibility Testing using the AST-POCT | bacteriuria | UTI | The MBS AST-POCT analysis is performed in ready to use disposable vials containing the lyophilized growth medium used for the detection of UTI [For the MBS AST-POCT analysis, urine samples were tested in parallel using antibiotic-free and antibiotic-supplemented vials. Samples (20 μl) were manually injected in the rehydrated vials (final volume 10 ml) and incubated in a dedicated thermostatic optical reader, the MBS multireader, at 37°C for 26 hours. At the end of the analysis, vials were sterilized with sodium dichloroisocyanurate by pressing their cap and then were disposed of as “Non-Hazardous Waste” [The MBS AST-POCT is a colorimetric system that allows a semi-quantitative assessment of viable bacteria concentration. Different from other culture-based methods, it measures the enzymatic activity associated with bacterial metabolism, allowing results to be obtained in short time. Vials change color from blue to yellow in times that are inversely related to the bacterial concentration in the sample.The AST POCT results were obtained by comparing the time taken by the antibiotic-free vial and the antibiotic-supplemented vials to change color. Regarding antibiotic free vials, bacteriuria, i.e. presence of ≥ 10The vials used in the study were provided by MBS Diagnostics ltd (London, UK) and produced by Sclavo Diagnostics spa (Sovicille, SI, Italy) in compliance with requirements set in the IVDD (EU In Vitro Diagnostic Directive) and following the Quality Control System confirmed by ISO 9001 and ISO 13485 certifications. Five independent production batches of vials were used throughout the two trials. | PMC10321614 |
Evaluation of the impact of sample freezing on MBS AST-POCT results | In order to assess the impact of freezing MBS AST-POCT results, analyses were performed only on frozen culture-positive samples analyses, i.e., on samples for which urine culture and AST results were made available by the hospital’s laboratory. Analyses were performed on a total of 10 urine samples. For each sample aliquots (100 μl each) were stored at -80°C and stored up to 7 days. Analyses were performed in duplicate on each sample after 2, 5 and 7 days from collection at the Science Department of Roma Tre University, Rome. Considering the dilution factor, due to the addition of glycerol, 40 μl of the frozen aliquots were analyzed with MBS AST-POCT and results were compared with those previously obtained for the same fresh samples. | PMC10321614 | ||
Second clinical trial: Istituto Dermopatico dell’Immacolata, Rome | In the second trial, at Istituto Dermopatico dell’Immacolata, the MBS AST-POCT analyses were performed only on culture-positive samples, i.e., on samples for which urine culture and AST results were made available by the hospital’s laboratory. Analyses were performed on frozen samples, allowing the possibility to expand the pool pf patients tested and to obtain statistically significant results. | PMC10321614 | ||
Urine collection | STERILE | Urine samples were collected by medical staff in the morning in the outpatient clinic or provided by patients themselves following self-sampling of first morning midstream clean-catch urine specimens. Samples after collection were kept refrigerated for maximum 2 hours and split into 10 aliquots of 1 ml each: 5 were sent to the hospital laboratory while the other 5 were frozen at -80°C using sterile glycerol at a final 15% (vol/vol) concentration. The latter samples were used for antibiotic susceptibility assessment with the MBS AST-POCT after central laboratory results were available, and in all cases within 48 hours. Supplementary aliquots were stored at -80°C in case of the need of further verification analysis. | PMC10321614 | |
Hospital laboratory tests | Urine culture, bacterial identification and AST were performed as previously described for the first clinical trial. | PMC10321614 | ||
Antimicrobial Susceptibility Testing using the AST-POCT | The MBS AST-POCT analyses were carried out not later than 48 hours from collection, on frozen aliquots of urine samples. This allowed to perform analyses only on patients confirmed as positive by the hospital laboratory after urine culture, and consequently undergoing antibiotic susceptibility analysis, and not on every patient received at the hospital. Analyses were performed as previously described. | PMC10321614 | ||
Ex-post verification analysis | STERILE | In case of discordance between the results obtained with the reference method, performed by the central laboratory within the hospital, and the MBS AST POCT, a verification analysis was performed at the Science Department of Roma Tre University, Rome, strictly within one or two days, to investigate the source of discordance. Analyses were repeated with both reference method and MBS AST POCT using aliquots of urine samples stored at -80°C using sterile glycerol at a final 15% (vol/vol) concentration maximum after 7 days from collection. The reference method chosen for ex-post analysis was the Kirby-Bauer diffusion disk test, as the traditional and most widely used approach [ | PMC10321614 | |
Results and discussion | UTI | The MBS AST POCT is a device with a simple operating protocol that can be used by healthcare staff at the patient’s bedside, and has been developed with the aim to provide fast and reliable information concerning the effectiveness of first-line antibiotics, thus helping clinicians choose the best treatment.Standard AST provides results in at least 48 hours, and, for this reason, antibiotic therapy following UTI diagnosis is usually started on empirical basis, possibly leading to incorrect treatment and ultimately participating in the rise in bacterial antibiotic resistance. In order to improve treatment, providing a more rational and effective antibiotic administration, a reduction in analytical time is desirable, allowing medical staff to base therapy on evidence-based information. This would prevent from either exploiting new and expensive antibiotics unnecessarily or using ineffective ones and lead to more effective treatment and faster resolution.In this work, the accuracy of antimicrobial susceptibility assessment using the MBS AST POCT was investigated. Two different clinical settings were taken into account, both a first-aid and an outpatient clinical framework, reflecting two very different patient pools. | PMC10321614 | |
Patients’ characteristics | EMERGENCY | A total of 349 patients were enrolled in the study: 101 patients attending the Department of Emergency Medicine of Azienda Ospedaliera Sant’Andrea, rome, in the first trial, 248 attending the outpatient clinic at Istituto Dermopatico dell’Immacolata, in the second trial. Of the patients enrolled in the first trial, antibiotic susceptibility testing was performed with both methods for 13 positive culture patients; among these, 7 (54%) were admitted with an ongoing antibiotic therapy (last antibiotic uptake within 12 hours before urine sampling) and the most frequently used antibiotics were metronidazole (43%), ciprofloxacin (29%), cefepime (14%), piperacillin-tazobactam (14%) and vancomycin (14%), used in combination only in one case. Of the patients enrolled in the second trial antibiotic susceptibility testing was performed with both methods for 84 culture-positive patients. Among these only 1 patient was admitted with an ongoing antibiotic therapy with ciprofloxacin ( | PMC10321614 | |
Patients’ characteristics. | PMC10321614 | |||
Hospital antibiotic susceptibility testing results | In the first clinical trial, at Azienda Ospedaliera Sant’Andrea, Rome, antibiotic susceptibility testing was routinely performed by the hospital laboratory for 13 positive urine cultures, and results were provided after 48–96 hours. Among the 7 patients undergoing antibiotic therapy (54%), AST results confirmed that 53% of the infecting microorganisms were resistant to previously administered antibiotics while only 23.5% were susceptible. In 23.5% of the cases the antibiotics used in therapy were not tested in AST.In the second clinical trial, at Istituto Dermopatico dell’Immacolata, Rome, antibiotic susceptibility testing was performed by hospital laboratory for 84 positive urine cultures, and results were provided after 48 hours. Among these patients, only 1 was undergoing antibiotic therapy. | PMC10321614 | ||
MBS AST POCT antibiotic susceptibility test results | In the first clinical trial analyses with the MBS AST POCT were performed on all patients admitted in the ED immediately after urine collection. Given the small patient pool in this first trial, following data should be considered a proof of concept for the possible application of the MBS AST POCT for the preliminary susceptibility profiling of uroflora in an easier though reliable way, meaning directly from urine samples. The tested antibiotics were amoxicillin-clavulanic acid and levofloxacin.Considering levofloxacin, in 9 cases color change occurred in the reaction vial containing the antibiotic, meaning the infecting bacteria resulted resistant (69%), while in 4 cases no color change occurred in the antibiotic supplemented reaction vial, and infecting bacteria were recorded as susceptible (31%). Considering amoxicillin-clavulanic acid, 11 samples resulted resistance (85%), while 2 samples susceptibility (15%). Agreement was reached for 12 out of 13 samples analyzed (92%), as all MBS AST POCT results were in agreement with those of hospital laboratory, except one. Given such discordance, this sample underwent verification analysis.In the second clinical trial analyses were performed only on culture positive samples. This permitted to expand the pool of tested patients, allowing a more solid statistical analysis and a deeper understanding of MBS AST POCT potential. The tested antibiotics were amoxicillin-clavulanic acid, ciprofloxacin and trimethoprim-sulfamethoxazole: 57 samples were screened for susceptibility to amoxicillin-clavulanic acid, 80 to ciprofloxacin and 72 to trimethoprim-sulfamethoxazole respectively.Considering amoxicillin-clavulanic acid, 36 samples resulted resistant (63%) and 21 (37%) susceptible; for ciprofloxacin results were respectively 34 (42.5%) resistant and 46 (57.5%) susceptible; for trimethoprim-sulfamethoxazole 27 samples were resistant (37.5%) and 45 susceptible (62.5%). Agreement was reached for a total of 193 samples out of 209 (92%), with 16 results (8%) showing discordance between the AST-POCT and the reference method. Following operating protocol, such samples underwent verification analysis.All MBS AST POCT results were obtained within 12 hours. In particular, the average detection time to asses resistance to antibiotics was of 5 hours and results were obtained in less than 6 hours in 71% of cases ( | PMC10321614 | ||
Detection time of resistant bacteria with MBS AST POCT. | bacteriuria | UTI | Histograms show detection frequencies (%) of MBS AST POCT resistant results at different time intervals.This represents an important issue to consider when looking at the overall performance of the MBS method for diagnosis and management of UTI.Previous studies, focused on the evaluation of the MBS POCT accuracy in performing bacterial load assessment for UTI detection, demonstrated that the MBS POCT was able to detect the presence of significant bacteriuria in maximum 5 hours [Taken together these results confirm the potential of the method: its adoption in routine clinical practice could provide, in short time, important information to the medical staff that, once the presence of UTI has been confirmed, could exclude ineffective antibiotics, and perform an evidence-based choice among treatment options, ultimately leading to more effective treatment and faster resolution. The fast response of the method could indeed reduce the adoption of empirical treatment and avoid the misuse and overuse of antibiotics, considered the major problem contributing to the emergence of resistant bacteria.Moreover, its use in combination with other rapidly detected biomarkers as procalcitonin, high levels of which have been positively correlated to culture positive patients [ | PMC10321614 |
Ex-post verification analysis | During the first trial, discordance between the results obtained with reference method and MBS AST POCT was observed for 1 patient out of 13 patients analyzed for both levofloxacin and amoxicillin-clavulanic acid. In this case, with the MBS AST POCT, the infecting bacteria resulted resistant to both the tested antibiotic, whereas the hospital report testified such bacteria as susceptible. Ex-post verification was thus carried out on frozen urine samples. The MBS AST POCT results were coherent with those obtained previously, confirming the presence of bacteria, resistant to both of the tested antibiotics. The Kirby-Bauer diffusion disk test showed the presence of two distinct bacterial strains displaying different susceptibility to both antibiotics, one being susceptible and one resistant. Because of the presence of a resistant strain and the ultimate ineffectiveness of the antibiotic in preventing bacteria multiplication in urine, the sample was considered resistant.In the second trial, discordant AST results were obtained for 16 patients, samples of which underwent verification analysis according to the previously described operating protocol. Of these, 9 discordant results were obtained for amoxicillin-clavulanic acid. After ex-post verification, agreement between MBS AST POCT and reference method’s results was achieved for 5 samples, confirming MBS AST POCT initial results, and such data were then used for statistical analysis; in the 4 remaining cases, discordance between results persisted, thus MBS AST POCT results were included into statistical analysis as either false positive or false negative results. Considering ciprofloxacin, 3 discordant results were obtained, but verification analysis confirmed agreement for all samples, which led to complete agreement between MBS AST POCT and reference method’s results. Lastly, for trimethoprim-sulfamethoxazole, discordant results between methods were obtained for 4 samples. After ex-post verification, agreement was achieved for 2 samples (3%), while for the other 2 samples no agreement was obtained (3%), so these results were included into statistical analysis either as false positive or false negative.The overall performance of the MBS AST POCT is showed in Tables | PMC10321614 | ||
Antibiotic susceptibility test results obtained with both MBS AST POCT. | The Antibiotic Susceptibility Test results obtained with MBS AST POCT were compared to those of the reference method, for the four antibiotics of interest. Data considered for analysis have been gathered following ex-post verification in case of discordant results. | PMC10321614 | ||
Performance of MBS POCT antibiotic susceptibility test. | infection | INFECTION, POLYMICROBIAL INFECTION, POSITIVE | The performance of MBS POCT Antibiotic Susceptibility Test was compared with reference method’s antibiogram’s for the antibiotics of interest, considering the patients enrolled in both studies. Accuracy, sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) have been calculated according to Friedman et al [*(nA key issue brought to light by ex post verification analyses is the bias associated with selection of bacterial colonies for antibiogram profiling. Indeed, in case of polymicrobial infections, more than one bacterial strain is found in urine samples participating in the infection, possibly displaying different susceptibility to antibiotics. After plating, usually, only few colonies undergo further analysis, giving clinicians incomplete information about uropathogens resistance profile. It is not infrequent that prescribed therapy results ineffective. Antibiotic susceptibility assessment using the MBS AST POCT relies on culture, in the presence of antibiotics, of urine sample itself, meaning no colony selection occurs, taking into account all bacterial strains within the sample. In this way, the MBS AST POCT evaluates the antibiotic susceptibility of the overall bacterial pool, avoiding operator’s subjective interpretation of results. | PMC10321614 |
Impact of sample freezing on MBS AST-POCT results | macrohematuria | Analyses on frozen samples were performed only on culture-positive samples, i.e., on samples for which urine culture and AST results were made available by the hospital’s laboratory. Analyses were performed after 2, 5 and 7 days. Results, in terms of susceptibility/resistance to the tested antibiotics, were always in agreement with those obtained on fresh samples (data not shown) meaning that freezing urine samples did not significantly affect the response of the MBS AST-POCT within a week. Despite the MBS AST-POCT has been conceived as near-patient testing, this result has been very important to optimize operating procedures, namely helping to exclude samples unsuitable for AST POCT analysis (e.g., evident macrohematuria) and to investigate and clarify cases in which discordant results were obtained from reference method and MBS AST POCT analysis [ | PMC10321614 | |
Conclusions | uropathogens | EMERGENCY | In this paper we evaluated the performance of a new Point Of Care Test (POCT) for the rapid prediction of antimicrobial susceptibility of uropathogens. 349 patients were enrolled in two open-label, monocentric, non-interventional clinical trials in partnership with an Emergency Medicine ward and the Day Hospital of two large healthcare facilities in Rome, Italy. The pool of patients and antibiotic tested was contained, therefore the study can be considered as a preliminary investigation that has, however, provided promising results.Overall, the MBS AST POCT displayed high accuracy (greater than 90%), and PPV (greater than 95%) with all the tested antibiotics, and provided results in less time compared to traditional methods, especially for resistant bacteria, that were detected within 12 hours from urine collection.Based on these evidences, the use of such a POCT in routine clinical practice could have an important impact, allowing an accurate evaluation of susceptibility/resistance of the infecting bacteria to selected antibiotics, though reducing analytical time and labor and diminishing analytical and management costs. Yielding results within a working day would indeed allow clinicians to promptly treat patients without the need of empiric antibiotic therapy, thus ultimately, helping to counteract the rise of antibiotic resistance.The device provides important information about the efficacy of antibiotics of interest directly testing urine samples and thus taking into account the overall urinary bacterial population, therefore preventing important data loss. The method itself does not provide the identification of pathogenic agents, however this can be easily achieved immediately after the analysis, using positive vials as bacterial cultures, ready to be further analyzed with standard techniques.Moreover, the MBS AST POCT could prove useful in the assessment of local antimicrobial susceptibility patterns providing a tool for the rapid screening of urine samples and allowing to build an antibiotic susceptibility database within the hospital even in low-resource settings.The authors gratefully acknowledge Dr. Andreas Bergmann (Sphingotec GmbH, 16761 Hennigsdorf, Germany), Dr. Metod Miklus, Dr. Robert Hellmich and Dr. Paul Scigalla (Oncoprevent GmbH, 16761 Hennigsdorf, Germany), for helpful discussions.Dr. Vincenzo Ziparo passed away before the submission of the final version of this manuscript. Dr. Giovanni Antonini accepts responsibility for the integrity and validity of the data collected and analyzed. | PMC10321614 |
Informed consent | Informed consent was obtained from all individual participants included in the study. | PMC10321614 | ||
Ethical approval | All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (from Ethical Committee of Azienda Ospedaliera Sant’Andrea on 2019/01/14; from Ethical Committee of Istituto Dermopatico dell’Immacolata on 2017/05/25) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. | PMC10321614 | ||
References | PMC10321614 | |||
Subject terms | glioblastoma, toxicities, pembrolizumab | ADENOVIRUS, VIRUS, INFILTRATION, DISEASE, SECONDARY, GLIOBLASTOMA | Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2–20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1–69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7–13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05–0.87). A total of 56.2% (95% CI 41.1–70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).A safety and efficacy trial of a single intratumoral dose of the oncolytic adenovirus DNX-2401 followed by intravenous anti-PD-1 pembrolizumab in patients with recurrent glioblastoma shows an encouraging clinical benefit rate and 12 months overall survival. | PMC10287560 |
Main | high-grade malignant gliomas, cancers, Glioblastoma | ADENOVIRUS, CANCERS, GLIOBLASTOMA, PRIMARY BRAIN TUMOR | Glioblastoma is the most common and lethal adult primary brain tumor. The standard of care treatment for newly diagnosed patients includes surgical resection followed by concomitant chemoradiotherapy and adjuvant temozolomideWhile immune checkpoint blockade by anti-PD1 or anti-PD-L1 antibodies have improved outcomes with objective responses in a variety of other cancers, including those in the brain such as metastatic melanomaDNX-2401 (tasadenoturev; Delta-24-RGD) is a conditionally replicative oncolytic adenovirus engineered to treat high-grade malignant gliomas | PMC10287560 |
Results | PMC10287560 | |||
Patient demographics and baseline characteristics | A total of 49 patients from 13 of the 15 participating institutions were enrolled between 28 September 2016 and 17 January 2019 (Fig. | PMC10287560 | ||
Safety | Cancer | ADVERSE EVENT, ADVERSE EVENTS, EVENT, ADVERSE EVENT, EVENTS, CANCER | Forty-eight of 49 (98%) patients were treated with one dose of DNX-2401 after a standard biopsy, which was then followed by pembrolizumab starting 7 days later. One patient enrolled in the first dose cohort received 5 × 10An overview of adverse events (AEs) in the study is summarized in Extended Data Tables AEs that were considered to be related to treatment are summarized in Table Summary of AEs related to treatmentAE denotes adverse event. SAE denotes serious adverse event. Shown are AEs and SAEs with greater than 5% frequency. Overall frequency refers to patients reporting at least one treatment related AE. Each patient is included once using the highest-grade event. Events were graded according to National Cancer Institute-Common Terminology Criteria for Adverse Events, version 4.03. | PMC10287560 |
Survival analyses | SECONDARY | The secondary efficacy endpoint of 12 month survival was met. The 12 month overall survival was 52.7% (95% CI 40.1–69.2%) in the intent-to-treat population and 53.1% (95% CI 36.8–67.0%) in patients who received the declared dose of DNX-2401 (Fig. | PMC10287560 | |
Exploratory associations | cerebral edema, tumor, tumors | DISEASE PROGRESSION, TUMOR, DISEASE, CEREBRAL EDEMA, TUMORS | We considered that concurrent use of medications may have impacted outcomes. Physicians were permitted to use low-dose bevacizumab or corticosteroids to address cerebral edema in this trial. Baseline corticosteroid use and corticosteroid use throughout the study were not statistically associated with outcomes, though use of corticosteroids throughout the study approached the threshold for statistical significance in some instances (Extended Data Table We also considered that variability in intrinsic patient and tumor factors might be associated with differences in outcomes of patients. To characterize potential biomarkers of treatment response, we obtained gene expression data on 38 patients with biopsy specimens available before treatment. We divided tumors from this study into three tumor microenvironment subtypes (TMETen patients also had biopsy specimens at the time of disease progression after treatment allowing for a biological assessment of matched-pair tissues. Of these ten patients, one initially had a partial response to treatment before progression, while the other nine patients did not demonstrate objective responses (three patients with progressive disease as best response and six patients with initially stable disease as best response). Comparing gene expression profiles at disease progression after treatment to those at baseline before treatment revealed several differentially expressed genes (Extended Data Fig. We performed immunophenotypic characterization of tumors before and after treatment by blinded immunohistochemical and multiplex immunofluorescence analysis. Patients with TMECertain pathogenic mutations are potentially associated with prognosis and specific response to checkpoint inhibition in glioblastomaAnti-adenovirus antibodies were measured by direct immunofluorescence assay in the serum of patients before treatment and throughout the course of the trial. All patients were seropositive for IgG antibodies against adenoviral hexon protein before treatment with DNX-2401, and in general, anti-adenovirus IgG levels increased within 2 months post treatment, with levels sustained longest in patients treated with 5 × 10 | PMC10287560 |
Discussion | cancers, tumor, Glioblastoma, glioma, glioblastoma, tumors, DNX-2401, retinoblastoma | ADENOVIRUS, CANCERS, RECURRENCE, VIRUS, TUMOR, DISEASE, GLIOBLASTOMA, BRAIN TUMOR, GLIOMA, DELETION, GLIOBLASTOMA, TUMORS, RETINOBLASTOMA | Glioblastoma is a devastating disease, and recurrence of disease is inevitable after initial treatment with radiotherapy and concurrent and adjuvant temozolomide chemotherapy. At progression, treatment options are very limited and of marginal efficacy. Immune checkpoint blockade in other advanced solid cancers such as melanomaDNX-2401 (Delta-24-RGD) is a conditionally replicative oncolytic adenovirus with a 24 base pair deletion in the E1A gene that renders selective replication of the virus in malignant cells with defective retinoblastoma signaling. DNX-2401 also has an RGD peptide insertion into the fiber knob that allows the virus to anchor directly to integrins and improve the infectability of glioblastoma cellsTreatment with oncolytic virus and immune checkpoint blockade combines the initial local effects of the oncolytic virus on the tumor microenvironment with the systemic effects of innate and adaptive immune responses from virus replication and PD-1 inhibitionHere we report the results of a two-part, phase 1/2, multicenter, open-label clinical trial evaluating the safety and efficacy of combined intratumoral delivery of DNX-2401 with systemic pembrolizumab for patients with recurrent glioblastoma treated at 13 institutions in North America. All centers used purpose-built cannulas to standardize the delivery of virus into the tumor, eliminating backflow and ensuring full administration of virus to the tumor. A total of 48 of 49 patients successfully received treatment with DNX-2401 and pembrolizumab.We tested between 5 × 10In total, five patients had objective responses, with two patients showing durable complete responses >45 months and three patients remaining alive at the writing of this manuscript. The objective response rate was 10.4% (90% CI 4.2–20.7%). It is noteworthy that there was one additional patient who received the declared dose of DNX-2401 with complete response at the site of treatment; however, this patient developed a new lesion at a distant site resulting in a classification of progressive disease. This patient remained alive a total of 12.3 months after treatment. In the previous phase 1 trial evaluating DNX-2401 monotherapy in recurrent glioma, there was also one patient with a complete response who developed a distant nodule several years after treatmentThe median overall survival was 12.5 months (10.7–13.5 months) and overall survival at 12 months was 52.7% (95% CI 40.1–69.2%), which was greater than the prespecified threshold of 20% using approved treatment of tumor-treating fields by Novo-TTFWhile the use of bevacizumab may complicate response assessment in trials by inducing changes in contrast enhancement seen on imaging, none of the patients with objective responses received bevacizumab during the study. Moreover, we did not find that baseline corticosteroid use was associated with outcomes in our study, confirming the findings in a prior study evaluating neoadjuvant checkpoint blockade in recurrent glioblastomaWe obtained matched mutational data and gene expression data on tumor specimens from patients, where available. Three of the patients with objective responses (60%) had tumors with mutational burden (TMB) greater than 10 mutations MbUsing gene expression data, we found that objective responses exclusively occurred in patients with moderately inflamed microenvironment, and modest PD-1 expression (TMEThere are limitations to this study that require further investigation. First, this trial did not include a comparator cohort. Further trials to directly compare combination therapy to monotherapy are needed before considering large-scale randomized trials. Second, this trial evaluated a single dose of intratumoral oncolytic virus. Emerging data since the conception of this study have shown some potential benefit with multiple doses of oncolytic virusTo our knowledge, the present study is the first to report on the combined direct delivery of oncolytic viral therapy and systemic checkpoint inhibition for any brain tumor. We identified a safe dose of DNX2401 combined with pembrolizumab with objective and durable responses, including two complete responses, and survival benefit for select patients across multiple institutions. These results are promising and particularly relevant in this population of patients who did not receive repeat resection of tumor and for whom efficacious and nontoxic treatments are entirely lacking. As well, we demonstrate the value that translational analyses and endpoints can add in advancing our understanding of the molecular mechanisms and biomarkers of response and/or resistance to treatment in clinical trial settings. | PMC10287560 |
Methods | PMC10287560 | |||
Patients | glioblastoma, tumor, gliosarcoma | TUMOR, RECURRENCE, DISEASE, GLIOSARCOMA, GLIOBLASTOMA | Adult patients with histologically confirmed glioblastoma or gliosarcoma, presenting with documented failure of previous surgical resection, chemotherapy and/or radiation at first or second recurrence, with a Karnofsky performance score of at least 70, were eligible. All patients were required to have a single contrast-enhancing tumor of at least 1 cm in two planes but no more than 4 cm in any single plane, as assessed by magnetic resonance imaging (MRI). Surgical resection must not have been possible or planned as part of the treatment for their presentation, and the tumor must have been accessible for stereotactic delivery of DNX-2401. Patients with multifocal or bilateral disease were excluded. The full inclusion and exclusion criteria are detailed in | PMC10287560 |
Design | tumor, tumors, DNX-2401 | DISEASE PROGRESSION, TUMOR, TUMORS | To evaluate the safety of combining DNX-2401 with pembrolizumab, we conducted an initial dose-escalation phase to determine a safe dose of DNX-2401 in combination with pembrolizumab and followed by a dose-expansion phase. All patients received a single dose of DNX-2401 by stereotactic injection at the time of standard tumor biopsy followed by 200 mg pembrolizumab infused intravenously at a dose of 200 mg over 30 min every 3 weeks starting 7 days after DNX-2401. Resection of tumors was not permitted. Treatment with pembrolizumab continued for up to 2 years, or until one of the following occurred: disease progression, unacceptable toxic effects or withdrawal of consent. Dose escalation evaluated 5 × 10All patients underwent a stereotactic biopsy to document the presence of tumor tissue before delivery of DNX-2401. Immediately after biopsy, a stereotactic-compatible neuro-ventricular cannula (Alcyone MEMS; ClearPoint SmartFlow) was inserted into the tumor to deliver the precise targeted dose of DNX-2401 via a single micro-tip at a rate of 0.9 ml h | PMC10287560 |
Assessments | bleeding, neutropenia, toxicityMissing, toxicity, toxicities, Cancer | ADVERSE EVENT, BLEEDING, NEUTROPENIA, EVENT, HEMODYNAMIC INSTABILITY, CANCER | Patients were continuously monitored throughout the study for safety as outlined in the schedule of assessments in the study Protocol. AEs and serious AEs were graded according to National Cancer Institute-Common Terminology Criteria for Adverse Events, version 4.03, and their relationship to treatment administered was assessed. For the dose-escalation phase, the dose-limiting toxicity (DLT) window of observation was the first 21 days after initial pembrolizumab infusion. The occurrence of any of the following toxicities is considered a DLT, if judged by the Investigator to be possibly, probably or definitely related to administration of DNX-2401 and pembrolizumab (and not to the administration procedure):Grade 4 nonhematologic toxicity (not laboratory)Grade 4 hematologic toxicity lasting ≥7 daysGrade 3 nonhematologic toxicity (not laboratory) lasting >3 days despite optimal supportive careAny Grade 3 or Grade 4 nonhematologic laboratory value if:Medical intervention is required to treat the subject, orThe abnormality leads to hospitalization, orThe abnormality persists for >1 weekFebrile neutropenia Grade 3 or Grade 4:Grade 3 is defined as ANC <1,000 mmGrade 4 is defined as ANC <1,000 mmThrombocytopenia <25,000 mmA bleeding event that does not result in hemodynamic instability but requires an elective platelet transfusion, orA life-threatening bleeding event which results in urgent intervention and admission to an Intensive Care Unit.Prolonged delay (>2 weeks) in initiating cycle 2 due to treatment-related toxicityMissing >10% of pembrolizumab doses as a result of AE(s) during the first cycleGrade 5 toxicityTreatment response was determined by serial protocolized contrast-enhanced MRI every 4 weeks for 28 weeks, and afterward at an interval of every 8 weeks for the remainder of the treatment period. Patients who completed the treatment phase entered the long-term response and survival follow-up phase of the study for the rest of life, with MRI every 16 weeks. Objective responses were evaluated by the RANO criteria | PMC10287560 |
Endpoints and statistical analyses | toxicity, tumor | DISEASE, TUMOR | The analyses reported in this study were performed according to the statistical analysis plan. All enrolled patients were included in the safety analysis set, and patients were considered evaluable for efficacy if they received at least one dose, or part of one dose, of either study drug, had measurable tumor at baseline and completed the week 4 follow-up visit. Patients who discontinued study participation for any reason other than progressive disease or study treatment-related toxicity before the 4 week visit were not considered evaluable and were replaced; however, they continued to be monitored for safety.The primary safety objective was to evaluate the safety of escalating doses of DNX-2401 and the overall safety of the declared dose of intratumoral DNX-2401 when followed by sequential intravenous administration of pembrolizumab. AEs and serious AEs were summarized for all patients in the study and were considered treatment related if reported as possibly, probably or definitely related to study drug.The primary efficacy objective was to determine the objective response rate, defined as the percentage of patients that had complete or partial responses based on mRANO criteriaStatistical and computations analyses were performed using SAS 9.4 and R 4.1.3. | PMC10287560 |
Study organization and oversight | The study was conducted in compliance with the Protocol at 15 clinical trial sites in the United States and Canada, as well as recognized international standards including the Good Clinical Practice guidelines of the International Conference on Harmonisation and the principles of the Declaration of Helsinki. The Protocol and its amendments were approved by the institutional review board of each participating trial site. Voluntary written informed consent was obtained from every patient before participation in this study. DNX-2401 preparation, handling and administration followed institutional standards for biosafety level 2 agents. | PMC10287560 | ||
Anti-adenovirus antibodies | ADENOVIRUS | Anti-hexon IgG antibody levels were determined before and after treatment by ELISA from patient serum samples according to the manufacturer’s instructions (Adenovirus IgG ELISA Kit; DEIA309; Creative Diagnostics). Absorbance at 450 nm was measured using a Synergy H4 plate reader (BioTek), and concentrations calculated on the basis of a standard curve (Gen 5 software Version 3.0, BioTek). Anti-adenovirus IgG serum concentration increases of fourfold or greater were considered seroconversions. A more stringent threshold of tenfold or greater increases in levels of anti-adenovirus IgG serum concentrations was also tested. | PMC10287560 | |
Targeted mutational sequencing | tumor, Tumor | TUMOR, TUMOR, SOLID TUMOR | Targeted next-generation sequencing was performed on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) pretreatment tumor biopsies available from 28 patients. Tumor samples from 18 subjects were sequenced by NeoGenomics using NeoType Discovery Profile for Solid Tumor. Tumor samples from ten subjects were sequenced by NovoGene using Novogene PM 2.0. | PMC10287560 |
Gene expression profiling and analyses | tumor | DISEASE PROGRESSION, TUMOR | RNA was extracted from FFPE pretreatment tumor biopsies available from 38 patients and analyzed retrospectively on the NanoString nCounter system. For ten patients, there were also tumor biopsy specimens available at the time of disease progression, allowing for an examination of gene expression changes before and after treatment in matched patient samples.The geometric mean of canonical marker genes was used to compute scores for immune cell types | PMC10287560 |
Previously published datasets | high-grade gliomas | Zhao et al. previously published their transcriptomic data in patients receiving anti-PD-1 therapy in high-grade gliomas | PMC10287560 | |
Edema volumetric analysis | edema | EDEMA | Digital Imaging and Communications in Medicine files for study MRIs were imported into Horos (version 3.3.6), and a blinded reviewer used non-motion degraded, axial, FLAIR sequences to segment perilesional FLAIR hyperintense signal. The Horos volume generator function was used to determine the total FLAIR signal volume for each study MRI. Volume of edema at each study MRI was normalized relative to baseline levels. Grouped comparisons were made by calculating the mean normalized edema volume with 95% confidence intervals at the timepoints outlined in the protocol every 4 weeks for 28 weeks and then every 8 weeks thereafter. | PMC10287560 |
IHC | dehydration, tumor | DEHYDRATION, SECONDARY, TUMOR | We performed immunohistochemical analyses for myeloid cell markers (Iba-1, CD68 and CD163) and lymphoid cell markers (CD3, CD4 and CD8) in samples with available tissue before and after treatment in this sample. Staining and subsequent annotation and analyses were performed blinded to clinical status. Slides with 5 µm FFPE tissue sections were rehydrated and a sodium citrate-dihydrate buffer or Tris–EDTA buffer was used for heat-mediated antigen retrieval. A 3% hydrogen peroxide in methanol solution was utilized to block endogenous peroxidase activity. Blocking solution (5% bovine serum albumin in phosphate buffered saline plus 0.1% Triton X-100) was applied to slides for 1 h at room temperature. Subsequently, primary antibodies including anti-CD3 (Agilent, M725401-2, mouse monoclonal, 1:100), anti-IBA1 (Wako, 019-19741, rabbit polyclonal, 1:1,500), anti-CD68 (Agilent, M0514, mouse monoclonal, 1:200), anti-CD4 (abcam, ab133616, rabbit monoclonal, 1:100) and anti-CD8 (abcam, ab93278, rabbit monoclonal, 1:250) were applied overnight at 4 °C in blocking solution. A 1 h incubation with secondary antibody was performed followed by processing with the DAKO polymer-HRP system and DAB peroxidase kit, counterstaining with hematoxylin, dehydration of the tissue and coverslipping. Whole slide images were digitized, and then for each slide tumor versus non tumor content was annotated and representative images were selected. Proportions of stain-positive cells were quantified using HALO (version 3.0311, Indica Labs) software algorithms that were defined to identify cells with either nuclear or cytoplasmic staining as a fraction of all cells. This algorithm was applied to all annotated tissue sections in an unbiased systematic manner, and the density of immunopositivity per square millimeter was recorded for each antibody. PD-L1 protein expression was performed by NeoGenomics Laboratories (NeoGenomics) under the direction of Merck using FFPE tumor biopsy samples according to standard protocols (PD-L1 IHC 22C3 assay). | PMC10287560 |
Multiplex immunofluorescence staining, tissue imaging and cell phenotyping | A validated and standardized multiplex immunofluorescence protocol was developed for simultaneous detection of CD3, CD8, CD11b, CD163, GFAP and DAPI in a single FFPE tissue section. The validation pipeline for the multiplex immunofluorescence protocol has been previously described by our groupMultiplexed immunofluorescence slides were scanned on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Spectral unmixing was performed using inForm software (version 2.4.8, Akoya Biosciences), as described. Image analysis was performed using QuPath and Fiji/ImageJ. Briefly, cells were segmented on the basis of nuclear detection using the StarDist 2D algorithm. A random trees algorithm classifier was trained for each cell marker. Cells were then subclassified as CD3 | PMC10287560 | ||
Reporting summary | Further information on research design is available in the | PMC10287560 | ||
Online content | Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-023-02347-y. | PMC10287560 | ||
Supplementary information |
Supplementary Methods, Tables 1–6 and Figs. 1 and 2.Reporting Summary | PMC10287560 | ||
Extended data | PMC10287560 | |||
Immune cell types and signature markers. | Distribution of scores for immune cell types (top row), immune checkpoint genes and biomarkers (second row), functional orientation markers (third row) and signature scores (last row). Shown are boxplots for each TME subtype. Dots denote individual values. N = 38 independent patient samples in total. Central bars indicate medians, the box defines the upper and lower quartiles of the distribution, and whiskers define the 1.5× IQR. All comparisons have p < 0.05 unless specifically indicated with ns (p > 0.05).
| PMC10287560 | ||
Extended data | is available for this paper at 10.1038/s41591-023-02347-y. | PMC10287560 | ||
Supplementary information | The online version contains supplementary material available at 10.1038/s41591-023-02347-y. | PMC10287560 | ||
Acknowledgements | BRAIN CANCER | We thank the patients and their families, as well as investigators and research staff at all study sites. This study was supported by DNATrix and Merck & Co., who participated in study design, monitoring and data analysis. The sponsor and authors contributed to data collection and writing of the manuscript and approved the decision to submit the final manuscript for publication. The data from the trial were held and statistically analyzed by Precision for Medicine and checked by authors. Prior work supporting this trial was funded in part by Brain Cancer SPORE support (P50CA127001, F.F.L.). | PMC10287560 | |
Author contributions | Concept and design: all authors. Acquisition, analysis or interpretation of data: F.N., V.P., L.S.Y., J.L., O.S., R.M.A.D., T.N.Y., A.G., P.B., J.R., W.P.M., A.M.S., R.S., A.L.-J., C.E.d.A., M.M.A., J.F., C.G.-M., F.F.L. and G.Z. Drafting of the manuscript: F.N. and G.Z. Critical revision: all authors. Statistical analysis: F.N., G.Z. and J.R. Computational analysis: F.N., V.P., L.S.Y., J.L., R.M.A.D., T.N.Y. and P.B. Administrative, technical or material support: all authors. Supervision: F.F.L. and G.Z. | PMC10287560 | ||
Peer review | PMC10287560 | |||
Data availability | Pseudononymized participant data, including outcomes and relevant reported patient characteristics, are shared as | PMC10287560 | ||
Competing interests | H.C., Brain Cancer, VBI, NewBio, TG, Cancer | BRAIN TUMOR, BRAIN CANCER, BEST, ONCOLOGY, CANCER | A.M.S. has received in-kind (drug) support from BMS, in-kind (ultrasound devices) and research support from Carthera, and in-kind (drug) and research support from Agenus. A.R. is a Medexus consultant (paid), on the Nico Oncology Tissue Advisory Board (unpaid), and has an extramural grant from the Bristol Myers Squibb Diversity in Clinical Trials Program. C.C.C. is a consultant for Clearpoint Neuro and Medtronic, and receives travel reimbursement from GTMedical for lectures. C.G.-M. has license agreements with DNATrix, and is a shareholder of DNATrix. F.F.L. is a patent holder for DNX-2401. H.C. is on the advisory/consult for Best Doctors/Teladoc, Orbus Therapeutics, Bristol Meyers Squibb, Regeneron, Novocure Research Funding (Site PI/Institutional Contract): Newlink Genetics, Plexxikon, Kadmon, Orbus, Merck, DNATrix, Abbvie, Beigene, Forma Therapeutics, GCAR, Array BioPharma, Karyopharm Therapeutics, Nuvation Bio, Bayer, Bristol Meyers Squibb, Sumitomo Dainippon Pharma Oncology, Samus Therapeutics and Erasca. J.F. has license agreements with DNATrix, and is a shareholder of DNATrix. J.M.M. has been a speaker for AstraZeneca, Janssen and TG Therapeutics. J.R. is employed by DNATrix. M.A.V. is consulting/receives honoraria from Chimerix, Midatech and Olympus, their hospital has contracts with Infuseon, Oncosynergy, Celgene, Denovo, Chimerix and NIH, and has patent rights for Cleveland Clinic/Infuseon. M.M.A. has a research grant from DNATrix unrelated to this work. P.U.K. receives consulting fees from Enclear Therapies, Affinia Therapeutics, Biocept, Janssen, Sintetica, Bioclinica/Clario, Novocure, Mirati and Orbus Therapeutics, performs contracted research for Genentech, Novocure, DNAtrix and Orbus Therapeutics, is a scientific consultant for Enclear Therapies (with grant options provided) and has intellectual properties without financial gain of European Patent 3307327, 12 August 2020 and US Patent Pending 15/737,188. R.A. is a consultant for Tactical Therapeutics and Axelar AB. R.S. is a consultant and receives fees from Alpheus Medical, AstraZeneca, Carthera, Celularity, GT Medical, Insightec, Black Diamond, Northwest Therapeutics, Syneos Health (Boston Biomedical) and Varian Medical Systems, is a consultant and their institution receives fees from Boston Scientific, is a consultant and receives no fees from Novocure, is an IP holder for therapeutic ultrasound indications and use, is a Board Member and former president of the European Organisation for Research Treatment of Cancer, and has stock options in Alpheus Medical and Carthera. S.G.N. is an Institutional PI of multicenter trials with BMS, Merck, Strata Oncology and Mirati Therapeutics, and is the Chair of ABIM Med Onc Board. S.K. receives honorarium from Novocure, is reimbursed/has sponsored travel from Novocure, is consulting for Pacific Marine Biotech, Autem Therapeutics, and is management/holds a director position for SKBio Advisory, LLC. T.F.C. is cofounder, major stock holder, consultant and board member of Katmai Pharmaceuticals, holds stock for Erasca, is member of the board and paid consultant for the 501c3 Global Coalition for Adaptive Research, holds stock in Chimerix and receives milestone payments and possible future royalties, is member of the scientific advisory board for Break Through Cancer, is member of the scientific advisory board for Cure Brain Cancer Foundation, and has provided paid consulting services to Blue Rock, Vida Ventures, Lista Therapeutics, Stemline, Novartis, Roche, Sonalasense, Sagimet, Clinical Care Options, Ideology Health, Servier, Jubilant, Immvira, Gan & Lee, BrainStorm, Katmai, Sapience, Inovio, Vigeo Therapeutics, DNATrix, Tyme, SDP, Kintara, Bayer, Merck, Boehinger Ingelheim, VBL, Amgen, Kiyatec, Odonate Therapeutics QED, Medefield, Pascal Biosciences, Bayer, Tocagen, Karyopharm, GW Pharma, Abbvie, VBI, Deciphera, VBL, Agios, Genocea, Celgene, Puma, Lilly, BMS, Cortice, Novocure, Novogen, Boston Biomedical, Sunovion, Insys, Pfizer, Notable labs, Medqia, Trizel, Medscape and has contracts with UCLA for the Brain Tumor Program with Roche, VBI, Merck, Novartis, BMS, AstraZeneca and Servier. The Regents of the University of California (T.F.C. employer) has licensed intellectual property co-invented by T.F.C. to Katmai Pharmaceuticals. V.K.P. receives clinical trial support from Servier, Karyopharm, Samus Therapeutics and Radiomedix, receives research support from Karyopharm, Bexion, SK Lifesciences and J INTS Bio, is a consultant for Servier, Insightec, Novocure, NewBio and Orbus Therapeutics, and has equity in Gilead and Amarin. W.P.M. is a consultant for Century Therapeutics, Ono Therapeutics and Novocure, and receives research funding from Hoffman La Roche, Agios and Orbus Therapeutics. The remaining authors declare no competing interests. | PMC10287560 |
References | PMC10287560 | |||
Subject terms | Weight loss, weight loss, WL | BLIND | Weight loss (WL) differences between isocaloric high-carbohydrate and high-fat diets are generally small; however, individual WL varies within diet groups. Genotype patterns may modify diet effects, with carbohydrate-responsive genotypes losing more weight on high-carbohydrate diets (and vice versa for fat-responsive genotypes). We investigated whether 12-week WL (kg, primary outcome) differs between genotype-concordant and genotype-discordant diets. In this 12-week single-center WL trial, 145 participants with overweight/obesity were identified a priori as fat-responders or carbohydrate-responders based on their combined genotypes at ten genetic variants and randomized to a high-fat (n = 73) or high-carbohydrate diet (n = 72), yielding 4 groups: (1) fat-responders receiving high-fat diet, (2) fat-responders receiving high-carbohydrate diet, (3) carbohydrate-responders receiving high-fat diet, (4) carbohydrate-responders receiving high-carbohydrate diet. Dietitians delivered the WL intervention via 12 weekly diet-specific small group sessions. Outcome assessors were blind to diet assignment and genotype patterns. We included 122 participants (54.4 [SD:13.2] years, BMI 34.9 [SD:5.1] kg/mGenotype patterns may modify diet effects on weight loss, with greater weight loss on genotype-concordant diets. Here, the authors show that with the current ability to genotype participants as fat- or carbohydrate-responders, evidence does not support greater weight loss on genotype-concordant diets. | PMC10562431 |
Introduction | obesity, weight loss, WL | OBESITY, SEVERE OBESITY, INSULIN RESISTANCE, SECONDARY | The 2017–2018 National Health and Nutrition Examination Survey (NHANES) showed that almost 43% of US adults aged 20 and over have obesity, including 9.0% with severe obesity, and another 31% are overweightMany weight loss (WL) strategies emphasize either high-carbohydrate (and low-fat) or high-fat (low-carbohydrate) dietsThe present RCT tested the hypothesis that participants assigned to a diet corresponding to their a priori-determined (fat-responsive or carbohydrate-responsive) genotype would lose more weight over 12 weeks than those assigned to a diet discordant with their genotype. Further, we aimed to analyze those with a fat-responsive genotype (subsequently “fat-responders”) and carbohydrate-responsive genotype (subsequently “carbohydrate-responders”) separately. We hypothesized that (1) fat-responders would lose more weight on the high-fat vs. high-carbohydrate diet and conversely (2) carbohydrate-responders would lose more weight on the high-carbohydrate vs. high-fat diet. A secondary objective of the present RCT was to test the newly-developed genetic risk score to determine fat- and carbohydrate-responsive genotypes that was based on the current state-of-the-art in nutrigenomics. We also aimed to determine associations between baseline insulin levels and homeostatic model assessment for insulin resistance (HOMA-IR) and differential WL between the diets. These analyses were pursued as previous results were mixed with some studies finding that insulin resistance | PMC10562431 |
Results | Figure | PMC10562431 | ||
Change in weight and percent body fat during the 12-week intervention. | Results are presented as boxplots for all participants ( | PMC10562431 | ||
Percent weight change and change in body fat and body composition | Similar to absolute weight change, percent weight change (adjusted difference: −0.6% [95% CI: −2.1, 0.9, | PMC10562431 | ||
Change in blood pressure | Changes in resting systolic blood pressure (SBP) and DBP did not differ between genotype-concordant and genotype-discordant diets (SBP adjusted difference: 4.7 mmHg [95% CI: 0.5, 8.8]; DBP adjusted difference: −0.1 mmHg [95% CI: −2.8, 2.5]; Table | PMC10562431 | ||
Change in systolic and diastolic blood pressure during the 12-week intervention. | Results are presented as boxplots for all participants ( | PMC10562431 | ||
Association between insulin levels and HOMA-IR and weight loss | Baseline insulin levels ( | PMC10562431 | ||
Diet adherence | Adherence to the assigned diets is shown in Fig. | PMC10562431 | ||
Adherence to the macronutrient compositions of the respective diet at week 4, week 8, and week 12. | Boxplots showing adherence data for the high-carbohydrate diet ( | PMC10562431 | ||
Session attendance and adverse events | Weekly attendance was similar across the four genotype-diet groups (Supplementary Table | PMC10562431 | ||
Discussion | coronary heart disease, WL | REGRESSION, CORONARY HEART DISEASE | The present RCT determined the participant’s (fat-responsive or carbohydrate-responsive) genotype a priori via a comprehensive genetic risk score based on published and validated effects and tested the effects of a genotype-concordant diet on WL over 12 weeks. We found no difference in WL between individuals on the genotype-concordant vs. genotype-discordant diet. Further, insulin levels or HOMA-IR were not associated with WL. Food cravings tended to decrease among carbohydrate-responders on a high-fat diet compared to those on a high-carbohydrate diet. Finally, fat-responders on a high-carbohydrate diet tended to decrease resting SBP.The lack of significant and clinically meaningful differences in WL (~0.6 kg) between genotype-concordant and genotype-discordant diets aligns with the literatureOur sample consisted of substantially fewer carbohydrate-responders (Future studies could also consider assigning participants to genotype-concordant diets without specific energy intake targets and examine the diet effects not only on WL but also on cardiovascular risk factors. Previously, a low-carbohydrate diet without energy intake target resulted in greater improvements in body composition, blood lipids, and estimated 10-year coronary heart disease risk compared to a low-fat dietFasting insulin levels and HOMA-IR did not predict WL. Previous studies reporting a diet × fasting insulin interaction for WL found lower carbohydrate diets to be superior for individuals with greater insulin resistanceWL can reduce food cravings, particularly for foods restricted on specific dietsAmong fat-responders, a high-carbohydrate diet tended to decrease resting SBP. Nonetheless, these individuals had the highest mean SBP of the 4 genotype-diet groups at baseline. Thus, this effect could be explained, in whole or partially, by regression to the mean. Also, all 4 genotype-diet groups had relatively well-controlled blood pressure, leaving little room for improvement through dietary changes, making the non-significant improvements potentially more meaningful.This trial has some limitations. First, the genetic algorithm to classify individuals as fat- or carbohydrate-responders was created based on published literatureIn conclusion, in this 12-week RCT, there was no difference in WL between individuals with an a priori determined fat- or carbohydrate-responsive genotype on a high-carbohydrate vs. high-fat diet with specific energy targets and the same level of energy restriction across diets. | PMC10562431 |
Methods | PMC10562431 | |||
Design and participants | WL | MINOR | The Personalized Nutrition Study (POINTS, ClinicalTrials.gov identifier: NCT04145466) was a 12-week, single-site, parallel-arm WL trial that was approved by the institutional review board (IRB FWA 00006218) of the Pennington Biomedical Research Center (PBRC, Baton Rouge, LA). Participants were enrolled between October 7, 2020 and September 8, 2021. Participants were identified a priori as carbohydrate-responders and fat-responders based on their combined genotypes at 10 genetic variant loci and randomized to either a high-carbohydrate or high-fat diet, yielding the following groups: (1) fat-responders receiving a high-fat diet, (2) fat-responders receiving a high-carbohydrate diet, (3) carbohydrate-responders receiving a high-fat diet, and (4) carbohydrate-responders receiving a high-carbohydrate diet.Participants were recruited from the community. Eligible participants were 18–75 years old, had a BMI of 27.0–47.5 kg/mThe study included 1 orientation visit, 2 clinic visits (one before and one after the intervention), and 12-weekly intervention sessions. All participants provided written informed consent, and participants who completed the study received a minor compensation of $150. | PMC10562431 |
Genotype determination | Carbohydrate- and fat-responders were identified a priori based on their combined genotypes at the following genetic variants: (1) | PMC10562431 | ||
Intervention | BLIND | After enrollment (Week [W] 0 visit), participants were randomized to either a high-carbohydrate diet (rich in whole-grain foods) or a high-fat diet (rich in unsaturated fats/oils). The high-carbohydrate diet consisted of ~20% of energy from fat and ~65% from carbohydrates, whereas the high-fat diet consisted of ~40% energy from fat and ~45% from carbohydrates. Both diets provided 15% of energy from protein. All participants were assigned an energy intake target that would result in a daily deficit of ~750 kcal and provided with a diet-specific meal plan in 200 kcal increments from 1400 to 2800 kcal/day to self-prepare meals during the intervention period. To facilitate meal plan adherence when preparing or selecting meals, the meal plans included a list of ingredients (and their amounts) for all meals of each day (breakfast, lunch, dinner, and 1 daily snack) and instructions for meal preparation and participants were provided a food scale. Baseline energy requirements were calculated with Mifflin-St. Jeor’s formulasThe PBRC biostatistics department created the randomization sequence using SAS 9.4 statistical software for Windows (SAS Institute, Cary, NC) and uploaded it to REDCap (Research Electronic Data Capture). REDCap used strata for the inaction of genotype and gender. To ensure a relatively equal baseline BMI between the 4 genotype-diet groups, a 1:1 randomization scheme was devised that adjusted for BMI, gender, and genotype. Gender and genotype were used as strata, while BMI was used in an a-priori-created randomization equation. Within each stratum, this equation used block sizes of 6 (for females) and 4 (for males) at the start of the study and ended with block sizes of 4 and 2, respectively, to ensure relative balance of group assignments. Block sizes were assigned during the study by the biostatistician with access only to information about the enrolment progress (percent enrolled).Outcome assessors were blind to diet assignment and genotype patterns. Interventionists administering intervention sessions were blind to genotype patterns but not diet type. Participants were only informed of their genotype (carbohydrate- or fat-responder) once they completed the study.The 12 weekly intervention (group) sessions were diet-specific and had a different focus each week (Supplementary Material). Participants were provided a body weight scale and encouraged to weigh daily throughout the intervention and to send pictures of their weights to their interventionist before each intervention session. With very few exceptions, the first intervention session was conducted in person. Due to the COVID-19 pandemic, almost all subsequent sessions were conducted virtually via webinar (Microsoft Teams). | PMC10562431 | |
Outcome measures | PMC10562431 | |||
Anthropometric data | At W0 and W12, fasting body weight and waist and hip circumference were measured in the PBRC outpatient clinic. Clinic weights were also measured at all intervention visits (though not fasting weights). Further, body fat (%, via bioelectrical impedance analysis [BIA]; X-contact 365, Jawon Medical Co., Ltd, Seoul, South Korea) and blood pressure (after 5 min of seated rest) were measured at W0 and W12. | PMC10562431 | ||
Fasting serum glucose and insulin | INSULIN RESISTANCE | Fasting serum glucose and insulin were measured at W0, and HOMA-IR was used to quantify insulin resistance. | PMC10562431 | |
Appetitive traits, food cravings, and food preferences | Appetitive traits were measured with the Eating Inventory (EI) | PMC10562431 | ||
Diet personalization and intervention satisfaction | The Diet Personalization Survey (Supplementary Methods) was completed at W0 and W12, as well as during the intervention session at W6, and the Intervention Satisfaction Survey (Supplementary Methods) was conducted at W12. Data for these surveys were collected and managed using REDCap tools. | PMC10562431 | ||
Diet adherence | As stated above, participants were provided with a kitchen scale and could precisely weigh all ingredients specified in the meal plans for the foods consumed at home. Additional foods that were consumed were weighed and added as well. Adherence to the macronutrient content of the assigned diets was assessed for three 7-day periods throughout the intervention (W4, W8, W12). | PMC10562431 | ||
Statistical analyses | WL | SECONDARY | The distribution of variables was evaluated by visual examination and the Shapiro-Wilk test. The primary outcome was weight change (kg) at 12 weeks. All other measures were secondary endpoints. Changes in outcomes are presented as mean and 95% confidence intervals (CI). We used linear mixed models to determine if changes in outcome variables differed among diets. Covariates in the models included baseline value of the outcome, sex, and race. The mixed-effect model accounted for the correlation of the subject over time, and least-square means based on the estimate from the mixed-effect model were used to test for differences in weight change between diets. To evaluate whether baseline insulin levels and HOMA-IR needed to be included as covariates, their effects on WL were tested using a linear mixed model, adjusted for diet group and other known covariates. Neither baseline insulin levels nor HOMA-IR was significantly associated with WL; hence these variables were not included as covariates. The significance level was set to 0.05 (2-sided). Multiple testing adjustment was performed for secondary outcomes using the Holm-Bonferroni method | PMC10562431 |
Power calculations | RECRUITMENT | The present study planned to obtain data from up to 154 participants in total, and we aimed to complete 32 participants per genotype-diet group (128 participants in total) though we did not limit recruitment to achieve equal numbers of participants in each group. We hypothesized that participants on a genotype-concordant diet would lose more weight than those on a genotype-discordant diet. Based on previous studies | PMC10562431 | |
Reporting summary | Further information on research design is available in the | PMC10562431 | ||
Supplementary information |
Supplementary InformationPeer Review FileReporting Summary | PMC10562431 | ||
Supplementary information | The online version contains supplementary material available at 10.1038/s41467-023-41969-1. | PMC10562431 | ||
Acknowledgements | Obesity, T32DK064584, Digestive, Diabetes | OBESITY, KIDNEY DISEASES, HEART, DIABETES | The center where the research was conducted is supported in part by U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center and by the Nutrition Obesity Research Centers grant P30DK072476 titled “Nutrition and Metabolic Health Through the Lifespan” sponsored by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). C.H. was supported by an NIDDK National Research Service Award (T32DK064584), and J.L.D. was funded by the American Heart Association (Grant # 20POST35210907). The present study was funded by WW International, Inc. (New York, NY, USA). The concept and design of the study were led by the study PI (C.K.M.) and developed in collaboration with five other authors, one of whom is an employee of the sponsor. The sponsor had no role in the execution of the study or statistical analysis. Two sponsor employees are co-authors of the paper and provided editorial comments to the manuscript. | PMC10562431 |
Author contributions | C.K.M. obtained funding for the study. C.K.M., C.H., J.W.A., J.L.D., J.M.O., C.M.C., M.I.C. and G.D.F. designed the study. C.K.M. and C.H. oversaw data acquisition, and S.Y., C.H. and C.K.M. analyzed and interpreted the data. C.H. and C.K.M. drafted the manuscript; all authors provided critical revisions for important intellectual content. C.K.M. was responsible for the overall study supervision, and F.L.G. was responsible for the medical supervision. | PMC10562431 | ||
Peer review | PMC10562431 | |||
Funding | Open Access funding enabled and organized by Projekt DEAL. | PMC10562431 | ||
Data availability | All of the data needed to recapitulate the analysis found within this study can be found in the manuscript, figures and supplementary information. Source data are provided with this paper. Due to privacy reasons, de-identified data from the study cannot be shared publicly but will be available from the corresponding author (christoph.hoechsmann@tum.de) immediately following the publication of the paper upon reasonable request. The study protocol and statistical analysis plan will also be available. | PMC10562431 | ||
Competing interests | G.D.F. and M.I.C. are shareholders and employees at WW International, Inc. (New York, NY, USA). C.K.M. has previously consulted for WW on a fee-for-service basis, with the latest consultation occurring in 2018. All other authors declare no competing interests. | PMC10562431 | ||
References | PMC10562431 | |||
Background
| T1D | TYPE 1 DIABETES | Type 1 diabetes (T1D) is a CD4 | PMC10210318 |
Methods | T1D | DISEASE PROGRESSION | This first-in-human, 24-week, double-blind phase 1b study evaluated the safety of three dosages of IMCY-0098 in adults diagnosed with T1D < 6 months before study start. Forty-one participants were randomized to receive four bi-weekly injections of placebo or increasing doses of IMCY-0098 (dose groups A/B/C received 50/150/450 μg for priming followed by three further administrations of 25/75/225 μg, respectively). Multiple T1D-related clinical parameters were also assessed to monitor disease progression and inform future development. Long-term follow-up to 48 weeks was also conducted in a subset of patients. | PMC10210318 |
Results | death | ADVERSE EVENTS, DISEASE PROGRESSION | Treatment with IMCY-0098 was well tolerated with no systemic reactions; a total of 315 adverse events (AEs) were reported in 40 patients (97.6%) and were related to study treatment in 29 patients (68.3%). AEs were generally mild; no AE led to discontinuation of the study or death. No significant decline in C-peptide was noted from baseline to Week 24 for dose A, B, C, or placebo (mean change − 0.108, − 0.041, − 0.040, and − 0.012, respectively), suggesting no disease progression. | PMC10210318 |
Conclusions | T1D | Promising safety profile and preliminary clinical response data support the design of a phase 2 study of IMCY-0098 in patients with recent-onset T1D. | PMC10210318 | |
Trial registration | IMCY-T1D-001: ClinicalTrials.gov NCT03272269; EudraCT: 2016–003514-27; and IMCY-T1D-002: ClinicalTrials.gov NCT04190693; EudraCT: 2018–003728-35. | PMC10210318 | ||
Supplementary Information | The online version contains supplementary material available at 10.1186/s12916-023-02900-z. | PMC10210318 | ||
Keywords | PMC10210318 | |||
Background
| T1D, hyperglycemia, autoimmune destruction, autoimmune disease | HYPERGLYCEMIA, TYPE 1 DIABETES, AUTOIMMUNE DISEASE, COMPLICATIONS | Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by a loss of self-tolerance to autoantigens expressed by the insulin-producing β-cells in the pancreas. This leads to β-cell destruction, decline in endogenous insulin secretion, and consequent hyperglycemia [Although a unique cause of T1D has not been identified, evidence suggests that T1D results from the interplay of a multigenic background and non-genetic components [Despite the ongoing advances in T1D management, achieving good glycemic targets and preventing complications remains challenging [General immunosuppression, which is partially effective in preserving β-cells from autoimmune destruction in recent-onset T1D, carries the risk of side effects that preclude its long-term use [Imotopes™ are linear synthetic peptides comprising an MHC/HLA class II-restricted T-cell epitope sequence linked to a thiol-disulfide oxidoreductase motif [IMCY-0098 is the Imotope™ developed for T1D. The design of IMCY-0098 was based on previous literature [Figure to show summary of IMCY-0098 structure and the HLA class II epitopes it will bind based on previous literature [Here, we present the results of a first-in-human phase 1b study of Imotope™ technology platform using IMCY-0098 in patients with recent onset T1D across seven European countries (NCT03272269; EudraCT number 2016–003514-27). Alongside safety as a primary objective, multiple clinical response parameters were also examined to potentially inform future trials. | PMC10210318 |
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