A Manifesto for transformative action on HIV among Black communities in Canada.

Black communities bear a hugely disproportionate share of Canada's HIV epidemic. Black persons annually represent up to one quarter of new diagnoses, while in contrast, diagnoses have been falling among white Canadians for the past two decades. There has been a notable lack of urgency and serious debate about why the trend persists and what to do about it. For too long, public institutions have reproduced hegemonic white supremacy and profoundly mischaracterized Black life. Consequently, Black communities suffer policies and programs that buttress systemic anti-Black racism, socio-economically disenfranchise Black communities, and in the process marginalize knowledgeable, experienced, and creative Black stakeholders. The Interim Committee on HIV among Black Canadian Communities (ICHBCC) is a group of Black researchers, service providers, and community advocates who came together in early 2022 to interject urgency to the HIV crisis facing Black communities. Specifically, the ICHBCC advocates for self-determined community leadership of research, policies, and programs, backed by access to appropriate resources, to change the trajectory of HIV among Black Canadian communities. In this article, we introduce the wider community to the Black HIV Manifesto that we developed in 2022.

RéSUMé: Les communautés noires constituent une part très disproportionnée de l'épidémie de VIH du Canada. Les personnes noires représentent annuellement jusqu'au quart des nouveaux diagnostics, tandis que les diagnostics diminuent chez les Canadiennes et les Canadiens de race blanche depuis 20 ans. Il y a une absence notable d'urgence et de débat sérieux sur la raison de la persistance de ces tendances et sur ce qu'il faudrait y faire. Pendant trop longtemps, les institutions publiques ont reproduit l'hégémonie de la suprématie blanche et extrêmement mal caractérisé les vies noires. Par conséquent, les communautés noires souffrent de politiques et de programmes qui soutiennent le racisme anti-Noirs systémique, privent les communautés noires de leurs droits socioéconomiques et marginalisent ainsi les connaissances, l'expérience et la créativité des parties prenantes noires. Le comité intérimaire sur le VIH dans les communautés noires du Canada (ICHBCC) est un groupe de chercheurs et de chercheuses, de prestataires de services et de porte-parole communautaires de race noire qui se sont regroupés au début de 2022 pour mettre l'accent sur le caractère urgent de la crise du VIH dans les communautés noires. Plus précisément, l'ICHBCC plaide en faveur d'un leadership communautaire autodéterminé de la recherche, des politiques et des programmes, appuyé par un accès aux ressources nécessaires, pour changer la trajectoire du VIH dans les communautés noires du Canada. Dans cet article, nous présentons à l'ensemble de la communauté le « manifeste noir sur le VIH ¼ que nous avons élaboré en 2022.

Assessment of fluid responsiveness in children using respiratory variations in descending aortic flow.

BACKGROUND: The primary aim of the current study was to investigate the ability of respiratory variations in descending aortic flow, measured with two-dimensional echo at the suprasternal notch (ΔVpeak dAo), to predict fluid responsiveness in anesthetized mechanically ventilated children. In addition, variations in peak descending aortic flow measured with apical transthoracic echo (ΔVpeak LVOT) were examined for the same properties. METHODS: Twenty-seven patients under general anesthesia were investigated in this prospective observational study. Cardiac output, ΔVpeak dAo, and ΔVpeak LVOT were measured at stable conditions after anesthesia induction. The measurements were repeated after a 10 mL kg-1 fluid bolus. Patients were classified as responders if stroke volume index increased by >15% after fluid bolus. The ability of each parameter to predict fluid responsiveness was assessed using receiver operating characteristic curves. RESULTS: Twenty-seven patients were analyzed, mean age and weight 43 months and 16 kg, respectively. Twelve responders and 15 non-responders were identified. ΔVpeak dAo was significantly higher in the responder group (14%, 95% confidence interval [CI]: 12%-17%) compared to the non-responder group (11%, 95% CI: 9%-13%) (p = .04) at baseline. Area under the ROC curve for ΔVpeak dAo and ΔVpeak LVOT was 0.73 (95% CI: 0.52-0.89, p = .02) and 0.56 (0.34-0.78, p = .3), respectively. A baseline level of ΔVpeak dAo of >14% predicted fluid responsiveness with a sensitivity of 58% (95% CI: 28%-85%) and specificity of 73% (95% CI: 45%-92%). CONCLUSION: In mechanically ventilated children, ΔVpeak dAo identified fluid responders with moderate diagnostic power in the current study. ΔVpeak LVOT failed to predict fluid responders in the current study.

A Randomized, Double-Blind, Placebo-Controlled Phase 2a Study of Tildrakizumab Efficacy and Safety in Patients With Active Ankylosing Spondylitis.

OBJECTIVE: Tildrakizumab is an anti-interleukin-23p19 monoclonal antibody approved to treat moderate to severe plaque psoriasis. This study evaluated the efficacy and safety of tildrakizumab in patients with ankylosing spondylitis (AS). METHODS: In this randomized, double-blind, parallel-group, multinational trial ( clinicaltrials.gov NCT02980705), patients with active AS, according to modified New York criteria and Bath Ankylosing Spondylitis Disease Activity Index Score ≥4, were randomized 1:1 to tildrakizumab 200 mg or placebo every 4 weeks until week 24. Thereafter, all patients received tildrakizumab 200 mg every 4 weeks until week 48. The primary outcome was proportion of patients achieving 20% improvement from baseline by Assessment in SpondyloArthritis International Society criteria (ASAS20) at week 24. This outcome was analyzed in subgroups defined by prior treatment experience, weight, age, and sex using the full analysis set. Safety was assessed through treatment-emergent adverse events. RESULTS: From December 5, 2017-September 3, 2019, 101 patients (76.2% male, 97% White) enrolled and were randomized to treatment. At week 24, the ASAS20 response rate was 74.0% in patients receiving tildrakizumab 200 mg (n = 50) versus 80.4% in placebo-treated patients (n = 51; treatment difference, -6.31%; 95% confidence interval, -22.34 to 9.71; p = 0.44). No difference in treatment effect by subgroups was observed. Tildrakizumab treatment was generally well tolerated, with no unexpected safety findings. The study was terminated after the week 24 interim analysis due to lack of efficacy. CONCLUSIONS: Tildrakizumab treatment was generally well tolerated but did not improve ASAS20 response rate versus placebo in patients with AS.

Correlation of spinal epidural fat volume with body mass index: a longitudinal study.

PURPOSE: Spinal epidural lipomatosis is abnormal accumulation of normal fat in the epidural space with weight loss suggested as first-line therapy in select symptomatic patients. However, moderate to large longitudinal studies establishing concordant changes between body mass index and epidural fat are lacking. The purpose of this study was to longitudinally assess this relationship. METHODS: We performed an ancillary study of the Habitual Diet and Avocado Trial. Baseline and six-month abdominal MRIs were analyzed for 98 overweight or obese but otherwise healthy subjects. Dorsal epidural fat volumes in the lumbar spine were measured and correlated with changes in body mass index, changes in visceral fat volume, and demographic information. RESULTS: There was a linear relationship between body mass index changes and epidural fat volume changes with a one-point change in body mass index corresponding to a 45 mm3 change in dorsal epidural fat volume (p < 0.001, 95% CI 31.87 to 76.77) as well as between visceral fat volume changes and epidural fat volume changes (regression coefficient 0.51, p < 0.001, 95% CI 0.22 to 0.47). Age was inversely related with subjects older than 45.7 years tending to lose epidural fat (regression coefficient -0.22, p = 0.025, 95% CI -10.43 to -0.72). CONCLUSION: Changes in spinal dorsal epidural fat volume parallel changes in body mass index and visceral fat, supporting weight loss as initial treatment for uncomplicated obesity-associated spinal epidural lipomatosis.

Resident-attending discrepancy rates for two consecutive versus nonconsecutive weeks of overnight shifts.

PURPOSE: Recent Accreditation Council for Graduate Medical Education policy changes no longer limit the number of consecutive night shifts allowed for trainees. Few studies have examined radiology resident overnight performance over time. This study aimed to compare significant resident-attending discrepancy rates for residents working 2 nonconsecutive versus consecutive weeks of overnight shifts. The authors hypothesized significantly increased week-two discrepancies in the consecutive group. METHODS: For 2020, a retrospective analysis of significant overnight resident-attending discrepancy rates over a 24-week period using database searches was performed for residents self-selecting 2 nonconsecutive versus consecutive weeks. The nonconsecutive group typically had a 7-day mix of days off and day shifts between their night shift weeks. Paired and unpaired t tests were performed with p < 0.05 considered significant. RESULTS: For the 24 sets of 2 weeks covered by two residents at a time, eight were nonconsecutive and 16 were consecutive. The nonconsecutive group had 75.0% R4 coverage compared to 37.5% for the consecutive group. There were no significant study volume differences between the groups. A total of 27,906 studies (35.3% cross-sectional [CT and MR], 54.9% radiograph plus fluoroscopy, 9.8% US) were performed with 223 discrepancies (0.80%). Overall discrepancies for the nonconsecutive versus consecutive groups were 39/4505 (0.87%) versus 59/9462 (0.62%; p = 0.32) for week one and 46/4732 (1.0%) versus 79/9207 (0.86%; p = 0.60) for week two with no significant differences between the groups by modality. CONCLUSION: Residents self-selecting 2 consecutive weeks of overnight shifts do not have increased resident-attending discrepancy rates compared to 2 nonconsecutive weeks.

Whole genome and exome sequencing reference datasets from a multi-center and cross-platform benchmark study.

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

Appraising clinical applicability of studies: mapping and synthesis of current frameworks, and proposal of the FrACAS framework and VICORT checklist.

BACKGROUND: Not all research findings are translated to clinical practice. Reasons for lack of applicability are varied, and multiple frameworks and criteria exist to appraise the general applicability of epidemiological and clinical research. In this two-part study, we identify, map, and synthesize frameworks and criteria; we develop a framework to assist clinicians to appraise applicability specifically from a clinical perspective. METHODS: We conducted a literature search in PubMed and Embase to identify frameworks appraising applicability of study results. Conceptual thematic analysis was used to synthesize frameworks and criteria. We carried out a framework development process integrating contemporary debates in epidemiology, findings from the literature search and synthesis, iterative pilot-testing, and brainstorming and consensus discussions to propose a concise framework to appraise clinical applicability. RESULTS: Of the 4622 references retrieved, we identified 26 unique frameworks featuring 21 criteria. Frameworks and criteria varied by scope and level of aggregation of the evidence appraised, target user, and specific area of applicability (internal validity, clinical applicability, external validity, and system applicability). Our proposed Framework Appraising the Clinical Applicability of Studies (FrACAS) classifies studies in three domains (research, practice informing, and practice changing) by examining six criteria sequentially: Validity, Indication-informativeness, Clinical relevance, Originality, Risk-benefit comprehensiveness, and Transposability (VICORT checklist). CONCLUSIONS: Existing frameworks to applicability vary by scope, target user, and area of applicability. We introduce FrACAS to specifically assess applicability from a clinical perspective. Our framework can be used as a tool for the design, appraisal, and interpretation of epidemiological and clinical studies.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Pulmonary injury and oxidative stress in rats induced by inhaled sulfur mustard is ameliorated by anti-tumor necrosis factor-α antibody.

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15-30 min later by anti-TNFα antibody (15mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard.

Detection of bone metastases in uterine cancer: How common are they and should PET/CT be the standard for diagnosis?

OBJECTIVES: Osseous metastases (OM) in endometrial cancer (EMCA) are thought to be rare. This study aimed to address the gap in present knowledge by defining the rate of OM in endometrial cancer (EMCA) as stratified by histology and ascertaining the best diagnostic modality for detection. METHODS: 435 consecutive cases of EMCA evaluated in tertiary care setting were reviewed. Clinico-pathologic data were abstracted and analyzed. RESULTS: 18/403 patients were found to have OM (4.6%). Majority were detected by PET/CT (13/18 (72%)), with conventional CT scans missing the diagnoses otherwise made by PET/CT scans in 2/9 patients. Patients with type II EMCA were at higher risk of developing OM compared with patients with type I EMCA; 2/234 patients with type I EMCA (0.85%) developed OM, as compared to 16/167 patients with type II EMCA (9.58%), OR = 12.3. Patients with serous histology had significantly higher odds of developing OM when compared to patients with non-serous histologies (OR 4, p = 0.001, 95% CI 1.54 to 10.76). Kaplan Myer survival function and log-rank analysis showed that the presence of OM was a significant negative prognosticator of survival, with median overall survival (mOS) of 16 months in OM patients vs. mOS undefined in non-OM patients (p < 0.0001). DISCUSSION: Incidence of detected OM was clinically significant, with most cases identified by PET/CT scans. Patients with type II EMCA, and in particular serous histology, were at a significantly higher risk of developing OM. OM when present, is an indicator of aggressive cancer biology and poor prognosis. Further studies are needed to ascertain the mechanism of predisposition to OM formation in serous EMCA and to confirm PET/CT as modality of choice for detection of OM.

ctDNA Predicts Overall Survival in Patients With NSCLC Treated With PD-L1 Blockade or With Chemotherapy.

PURPOSE: Identification of predictors for overall survival (OS) allows timely detection of clinical efficacy signals and therefore facilitates treatment decisions. We assessed the association between circulating tumor DNA (ctDNA) metrics and the primary end point of OS in a subset of previously treated patients with locally advanced or metastatic non-small-cell lung cancer, who underwent atezolizumab or docetaxel treatment in the open-label randomized phase III OAK trial. MATERIALS AND METHODS: Plasma from 94 patients at baseline and at subsequent cycles of therapy every 3 weeks was analyzed retrospectively for ctDNA. ctDNA was measured by allele frequency and mutant molecules per milliliter (MMPM). Concordance between various per-sample metrics and clinical outcome were assessed using C index. RESULTS: Of all the ctDNA metrics tested, the association of median MMPM at 6 weeks with OS in patients treated with atezolizumab or docetaxel had a C index > 0.7. The OS hazard ratios relative to high ctDNA above median MMPM within each arm were 0.28 (95% CI, 0.11 to 0.75) for atezolizumab and 0.19 (95% CI, 0.08 to 0.48) for docetaxel. For patients who had ctDNA median MMPM levels of < 4.79, the median survival time was more than 17 months in docetaxel-treated patients and the median survival time was not reached in the atezolizumab-treated patients. CONCLUSION: ctDNA MMPM levels measured at 6 weeks post-treatment are associated with OS in advanced non-small-cell lung cancer. Our results suggest that ctDNA has the potential for a noninvasive early liquid biopsy predictor for OS that warrants further studies to demonstrate its utility in clinical development.

Progressive Lung Injury, Inflammation, and Fibrosis in Rats Following Inhalation of Sulfur Mustard.

Sulfur mustard (SM) inhalation causes debilitating pulmonary injury in humans which progresses to fibrosis. Herein, we developed a rat model of SM toxicity which parallels pathological changes in the respiratory tract observed in humans. SM vapor inhalation caused dose (0.2-0.6 mg/kg)-related damage to the respiratory tract within 3 days of exposure. At 0.4-0.6 mg/kg, ulceration of the proximal bronchioles, edema and inflammation were observed, along with a proteinaceous exudate containing inflammatory cells in alveolar regions. Time course studies revealed that the pathologic response was biphasic. Thus, changes observed at 3 days post-SM were reduced at 7-16 days; this was followed by more robust aberrations at 28 days, including epithelial necrosis and hyperplasia in the distal bronchioles, thickened alveolar walls, enlarged vacuolated macrophages, and interstitial fibrosis. Histopathologic changes were correlated with biphasic increases in bronchoalveolar lavage (BAL) cell and protein content and proliferating cell nuclear antigen expression. Proinflammatory proteins receptor for advanced glycation end product (RAGE), high-mobility group box protein (HMGB)-1, and matrix metalloproteinase (MMP)-9 also increased in a biphasic manner following SM inhalation, along with surfactant protein-D (SP-D). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), inflammatory proteins implicated in mustard lung toxicity, and the proinflammatory/profibrotic protein, galectin (Gal)-3, were upregulated in alveolar macrophages and in bronchiolar regions at 3 and 28 days post-SM. Inflammatory changes in the lung were associated with oxidative stress, as reflected by increased expression of heme oxygenase (HO)-1. These data demonstrate a similar pathologic response to inhaled SM in rats and humans suggesting that this rodent model can be used for mechanistic studies and for the identification of efficacious therapeutics for mitigating toxicity.

CaRMS at 50: Making the match for medical education.

Entry into postgraduate medical training in Canada is facilitated through a national application and matching system which establishes matches between applicants and training programs based on each party's stated preferences. Health human resource planning in Canada involves many factors, influences, and decisions. The complexity of the system is due, in part, to the fact that much of the decision making is dispersed among provincial, territorial, regional, and federal jurisdictions, making a collaborative national approach a challenge. The national postgraduate application and matching system is one of the few aspects of the health human resources continuum that is truly pan-Canadian. This article examines the evolution of the application and matching system over the past half century, the values that underpin it, and CaRMS' role in the process.

L'entrée dans la formation médicale postdoctorale au Canada est facilitée par un système national de demande et de jumelage qui établit des correspondances entre les candidats et les programmes de formation en fonction des préférences déclarées par chaque partie.La planification des ressources humaines en santé au Canada implique de nombreux facteurs, influences et décisions. La complexité du système est due, en partie, au fait qu'une grande partie du processus décisionnel est réparti entre les compétences provinciales, territoriales, régionales et fédérales, ce qui rend difficile une approche nationale collaborative. Le système national de demande postdoctorale et de jumelage est l'un des rares aspects du continuum des ressources humaines en santé qui est véritablement pancanadien.Cet article examine l'évolution du système d'application et de jumelage au cours du dernier demi-siècle, les valeurs qui le sous-tendent et le rôle du CaRMS dans le processus.

Alleviation of methyl isocyanate-induced airway obstruction and mortality by tissue plasminogen activator.

Methyl isocyanate (MIC, "Bhopal agent") is a highly reactive, toxic industrial chemical. Inhalation of high levels (500-1000 ppm) of MIC vapor is almost uniformly fatal. No therapeutic interventions other than supportive care have been described that can delay the onset of illness or death due to MIC. Recently, we found that inhalation of MIC caused the appearance of activated tissue factor in circulation with subsequent activation of the coagulation cascade. Herein, we report that MIC exposure (500 ppm for 30 min, nose-only) caused deposition of fibrin-rich casts in the conducting airways resulting in respiratory failure and death within 24 h in a rat model (LC90-100 ). We thus investigated the effect of airway delivery of the fibrinolytic agent tissue plasminogen activator (tPA) on mortality and morbidity in this model. Intratracheal administration of tPA was initiated 11 h post MIC exposure and repeated every 4 h for the duration of the study. Treatment with tPA afforded nearly 60% survival at 24 h post MIC exposure and was associated with decreased airway fibrin casts, stabilization of hypoxemia and respiratory distress, and improved acidosis. This work supports the potential of airway-delivered tPA therapy as a useful countermeasure in stabilizing victims of high-level MIC exposure.

Methyl isocyanate inhalation induces tissue factor-dependent activation of coagulation in rats.

Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125-500 ppm) by nose-only vapor inhalation for 30 min. Arterial O2 saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8 hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125 ppm) concentrations, 250 and 500 ppm MIC caused significant declines in blood oxygen saturation (% SpO2) at 1 hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 s; 125 ppm MIC: 790 ± 62 s; 250 ppm: 676 ± 28.0 s; 500 ppm: 581 ± 175 s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8 hr. Similarly, elevated TF activity and protein were detected in plasma 1 hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.

Determination of methyl isopropyl hydantoin from rat erythrocytes by gas-chromatography mass-spectrometry to determine methyl isocyanate dose following inhalation exposure.

Methyl isocyanate (MIC) is an important precursor for industrial synthesis, but it is highly toxic. MIC causes irritation and damage to the eyes, respiratory tract, and skin. While current treatment is limited to supportive care and counteracting symptoms, promising countermeasures are being evaluated. Our work focuses on understanding the inhalation toxicity of MIC to develop effective therapeutic interventions. However, in-vivo inhalation exposure studies are limited by challenges in estimating the actual respiratory dose, due to animal-to-animal variability in breathing rate, depth, etc. Therefore, a method was developed to estimate the inhaled MIC dose based on analysis of an N-terminal valine hemoglobin adduct. The method features a simple sample preparation scheme, including rapid isolation of hemoglobin, hydrolysis of the hemoglobin adduct with immediate conversion to methyl isopropyl hydantoin (MIH), rapid liquid-liquid extraction, and gas-chromatography mass-spectrometry analysis. The method produced a limit of detection of 0.05 mg MIH/kg RBC precipitate with a dynamic range from 0.05-25 mg MIH/kg. The precision, as measured by percent relative standard deviation, was <8.5%, and the accuracy was within 8% of the nominal concentration. The method was used to evaluate a potential correlation between MIH and MIC internal dose and proved promising. If successful, this method may be used to quantify the true internal dose of MIC from inhalation studies to help determine the effectiveness of MIC therapeutics.

Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.