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Although luminescence is considered a fundamental technique to study molecular properties and functions of structural and bioactive components, its application to foods has been limited. Herein, we discuss how advances in probe characterization and design, instrumentation, and high-resolution techniques provide new approaches to scout undisturbed food matrices and assist in monitoring food safety and food quality attributes during processing and storage. The role of novel techniques in identifying mechanisms, interactions, and structural features responsible for (or conducive to) food stability, nutrient accessibility and desirable textural characteristics in food products is also discussed.

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.

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.

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.

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.

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., Measurement(s)Somatic Mutation AnalysisTechnology Type(s)whole genome sequencing • Whole Exome SequencingFactor Type(s)sequencing platform • sample prepration • library preparation • bioinformatics methodSample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.16713655

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 (15 mg/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.

With the rapid advancement of sequencing technologies in the past decade, 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 errors or 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 benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired 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. Background & Summary The NGS technology has become a powerful tool for precision medicine. More researchers and clinicians are utilizing NGS to identify clinically actionable mutations in cancer patients and to establish targeted therapies for patients based on the patient’s genetic makeup or genetic variants of their tumor1, there is a critical need to have a full understanding of the many different variables affecting the NGS analysis output. The rapid growing number of sample processing protocols, library preparation methods, sequencing platforms, and bioinformatics pipelines to detect mutations in cancer genome, presents great technical challenges for the accuracy and reproducibility of utilizing NGS for cancer genome mutation detections. To investigate how these experimental and analytical elements may affect mutation detection accuracy, recently we carried out a comprehensive benchmarking study using both whole-genome (WGS) and whole-exome sequencing (WES) data sets generated from two well-characterized reference samples: a human breast cancer cell line (HCC1395) and a B lymphocytes cell line (HCC1395BL) derived from the same donor (NBT-RS47789). We generated WGS and WES data using various NGS library preparation protocols, seven NGS platforms at six centers (NBT-A46164B).

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.

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

Background HIV Pre-exposure prophylaxis (PrEP) is an underutilized intervention to prevent HIV infection in Canada. Known barriers to PrEP uptake include lack of awareness, low HIV risk perception, side effects, PrEP not being publicly funded (which is the case in Ontario) and stigma. We aimed to identify barriers to PrEP use and actions that may facilitate PrEP uptake in Ontario and British Columbia. Methods Gay, bisexual and other men who have sex with men 19 years or older living in Ontario and British Columbia, Canada, answered a survey between July 2019 and August 2020. Participants who met Canadian PrEP guideline criteria for PrEP and not already using PrEP indicated which barriers were relevant to them and which actions would make them more likely to start PrEP. We used descriptive statistics and tested differences between Ontario and British Columbia using Chi-square tests for proportions and t-tests or Wilcoxon rank-sum tests for continuous variables. Results Of 1527 survey responses, 260 (184 in Ontario and 76 in British Columbia) who were never PrEP users and met criteria for PrEP were included. In Ontario, the most common barriers were affordability (43%) and concern about side effects (42%). In British Columbia, the most common reasons were concern about side effects (41%) and not feeling at high enough risk (36%). In Ontario, the actions that would most likely encourage the respondent to start PrEP were short waiting time (63%), the healthcare provider informing about their HIV risk being higher than perceived (62%) and a written step-by-step guide (60%). In British Columbia, the actions that would most likely encourage the respondent to start PrEP were short waiting time (68%), people speaking publicly about PrEP (68%) and their healthcare provider counselling about: their HIV risk being higher than perceived (64%), side effects of PrEP (64%) and about how PrEP works (62%). Table. Top reasons for not using PrEP and top actions that might influence the decision to start PrEP stratified by province. (n= 184 in Ontario, n= 76 in British Columbia). Conclusion Concern about side effects and not feeling at high enough risk were common barriers. Short waiting times may increase PrEP uptake. In Ontario, the findings suggested lack of affordability. In British Columbia, actions involving healthcare providers were valued. Disclosures Kevin Woodward, MD FRCPC, Gilead (Independent Contractor) Darrell Tan, MD PhD, Abbvie (Grant/Research Support)Gilead (Grant/Research Support)GlaxoSmithKline (Scientific Research Study Investigator)ViiV Healthcare (Grant/Research Support)

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.

Methyl isocyanate (MIC, “Bhopal agent”) is a highly reactive, toxic industrial chemical. Inhalation of high levels (500–1000 ppm) of MIC vapor are almost uniformly fatal. No therapeutic interventions other than supportive care have been described that can delay the onset of illness or death owing 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 (LC(90−100)). 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.

We characterized two reference samples for NGS technologies: a human triple-negative breast cancer cell line and a matched normal cell line. Leveraging several whole-genome sequencing (WGS) platforms, multiple sequencing replicates, and orthogonal mutation detection bioinformatics pipelines, we minimized the potential biases from sequencing technologies, assays, and informatics. Thus, our “truth sets” were defined using evidence from 21 repeats of WGS runs with coverages ranging from 50X to 100X (a total of 140 billion reads). These “truth sets” present many relevant variants/mutations including 193 COSMIC mutations and 9,016 germline variants from the ClinVar database, nonsense mutations inBRCA1/2and missense mutations inTP53andFGFR1.Independent validation in three orthogonal experiments demonstrated a successful stress test of the truth set. We expect these reference materials and “truth sets” to facilitate assay development, qualification, validation, and proficiency testing. In addition, our methods can be extended to establish new fully characterized reference samples for the community.

Study Design Retrospective review with telephone follow-up. Background Acute spondylolytic injuries have a dramatic impact on the young athlete. Excellent short-term clinical outcomes have been observed, but not enough is known about long-term clinical outcomes. Objectives (1) To report long-term clinical outcomes for patients diagnosed with acute spondylolysis, and (2) to assess the prognostic ability of retrospective variables for long-term outcomes. Methods Patients from 2010 through 2013 were retrospectively reviewed to identify patients and to obtain demographic, baseline, and short-term outcomes. Long-term follow-up data were collected by telephone from patients diagnosed with acute spondylolysis to assess recurrence rate of low back pain, perceived outcome, pain, and functional ability. Patients were categorized as having a good or poor long-term outcome based on these measures. Logistic regression analysis was performed to assess the prognostic ability of the retrospective variables for long-term outcomes 3.4 years (range, 1.5-5.6 years) after treatment. Results One hundred twenty-one (71.6%) patients completed the follow-up questionnaire (48 female; mean age at baseline, 14.4 years). At follow-up, 81 (66.9%) patients were able to maintain their same or a higher level of sport. Recurrence of significant symptoms was reported by 55 (45.5%) patients, with 41 (33.9%) requiring medical treatment. The final logistic regression model revealed that female sex, adverse reaction during care, and multilevel injury were significant predictors of poor long-term outcome (R2 = 0.22). Conclusion Although excellent short-term outcomes were noted, 42% of patients reported a poor outcome at long-term follow-up. Female sex, multilevel injury, and experiencing an adverse reaction during care were significant predictors of poor long-term clinical outcome for patients diagnosed with acute spondylolysis. Level of Evidence Prognosis, level 4. Registered January 15, 2015 at www.clinicaltrials.gov (NCT02332200). J Orthop Sports Phys Ther 2016;46(12):1029-1036. Epub 8 Nov 2016. doi:10.2519/jospt.2016.7028.

Early breast cancer diagnosis is typically followed by surgical resection to remove the tumor, after which adjuvant therapy is administered for up to five years, or until relapse and presentation of metastatic disease. Archived tissue from the primary tumor is the initial source of material for NGS applications, in particular the identification of genomic driver mutations. In some cases, core needle biopsies may be collected at relapse or during later lines of therapy, but this occurs in a minority of patients and typically samples only one metastatic site. NGS on DNA from fresh biopsies is useful for real time assessment of genomic drivers, or to identify resistance mechanisms that may arise on prior therapies. In contrast to tissue, blood collection for NGS of ctDNA is more straightforward than obtaining fresh biopsies and has many conceptual uses over the natural history of breast cancer. Pending development of sensitive and specific methodologies, ctDNA could be used for screening and early detection. Analysis after surgery may be useful in detecting residual tumor DNA and identifying patients at high risk of relapse. Finally, in the metastatic setting ctDNA could be used to identify genomic drivers, dissect heterogeneity of disease, and in longitudinal monitoring to detect resistance mutations. This article is protected by copyright. All rights reserved

Author(s): Deodhar, Atul; Gensler, Lianne S; Sieper, Joachim; Clark, Michael; Calderon, Cesar; Wang, Yuhua; Zhou, Yiying; Leu, Jocelyn H; Campbell, Kim; Sweet, Kristen; Harrison, Diane D; Hsia, Elizabeth C; van der Heijde, Desiree | Abstract: ObjectiveTo evaluate the efficacy and safety of ustekinumab in 3 randomized, placebo-controlled studies in patients with axial spondyloarthritis (SpA). Studies 1 and 2 included patients with radiographic axial SpA (anti-tumor necrosis factor [anti-TNF]-naive patients and patients with inadequate response or intolerance to anti-TNF, respectively); study 3 patients had nonradiographic axial SpA.MethodsIn all 3 studies, patients were randomly assigned (1:1:1) to receive subcutaneous ustekinumab at 45 mg or 90 mg or placebo up to 24 weeks, after which placebo-treated patients were rerandomized to receive ustekinumab at 45 mg or 90 mg. The primary end point in studies 1 and 2 was the proportion of patients who met the Assessment of SpondyloArthritis international Society criteria for 40% improvement in disease activity (achieved an ASAS40 response). The primary end point in study 3 was the proportion of patients who achieved an ASAS20 response. Other disease activity and safety measures were also evaluated. A week 24 analysis of study 1 was preplanned to determine continuation of studies 2 and 3.ResultsFor study 1, the primary and major secondary end points were not met, and the study was discontinued. As a result, studies 2 and 3 were prematurely discontinued before they were fully enrolled. For all 3 studies, neither ustekinumab dose group demonstrated clinically meaningful improvement over placebo on key efficacy end points. The proportion of patients experiencing adverse events in the ustekinumab groups was consistent with that in previous studies.ConclusionIn these 3 placebo-controlled trials, efficacy of ustekinumab in the treatment of axial SpA was not demonstrated. The safety profile was consistent with that of studies in other indications.

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 O(2) 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 post exposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 seconds; 125 ppm MIC: 790 ± 62 seconds; 250 ppm: 676 ± 28.0 seconds; 500 ppm: 581 ± 175 seconds). 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.

Background Overall survival (OS) is the most relevant endpoint for evaluation of checkpoint inhibitors (CPI). Identification of early surrogates of OS is important to inform early clinical efficacy signals and treatment decisions. Here, we evaluated multiple metrics of circulating tumor DNA (ctDNA) for association with OS in the Ph3 OAK study, which demonstrated clinically significant OS benefit in ≥ 2L NSCLC patients treated with atezolizumab vs docetaxel. Methods Plasma from 94 patients taken at baseline and at subsequent cycles of therapy every three weeks (C2D1, C3D1, and C4D1) were analyzed retrospectively for ctDNA with the AVENIO ctDNA Surveillance Kit* (Jiang et al., 2018). Mutations detected in matched PBMC DNA were excluded in correlation analyses with clinical outcomes (Yaung et al., 2019). ctDNA was measured by allele frequency (AF) or mutant molecules per milliliter (MMPM), and summarized across multiple mutations within a sample by median, mean or maximum. Concordance between these per-sample metrics and PFS/OS were assessed using C index, which is equivalent to AUC under a Receiver Operating Characteristic (ROC) curve. *For Research Use Only; Not for use in diagnostic procedures. Results Using absolute and relative change from baseline of these metrics showed various levels of association with OS for both treatment arms (atezolizumab arm C index average 0.63, range 0.47 -0.76 and docetaxel arm C index average 0.50, range 0.39 - 0.61). As noted, maximal MMPM at C3D1 or 6 weeks had comparable association with OS in both treatment arms (atezolizumab arm C index = 0.74 and docetaxel arm C index = 0.73). Dichotomized at a median of 8.75 maximal MMPM, KM curves showed the 1-year survival rate in the atezolizumab arm among MMPM low vs high patients was 0.81 vs 0.43; and in the docetaxel arm was 0.7 vs 0.07. Associations with PFS tended to be weaker than those for OS. Conclusions Overall ctDNA levels were associated with OS in 2L+ NSCLC. ctDNA may be a promising non-invasive blood based biomarker in the metastatic setting of NSCLC and warrants further studies to demonstrate its utility in clinical development. Clinical trial identification OAK (NCT02008227). Legal entity responsible for the study Roche. Funding Roche. Disclosure D.R. Gandara: Advisory / Consultancy: AstraZeneca, Celgene, CellMax Life, FujiFilm, Roche-Genentech, Guardant Health, Inviata, IO Biotech, Lilly, Liquid Genomics, Merck, Samsung Bioepis, Pfizer; Research grant / Funding (institution), Research grants : Bristol-Myers Squib, Roche-Genentech, Novartis, Merck. W. Zou: Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. J. Jiang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. S. Yaung: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. F. Fuhlbruck: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. J. Wu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. M. Peterson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. J. Palma: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. M. Ballinger: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. E. Peters: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. D. Shames: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche. N. Patil: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche.

Background: Older adults and women have historically been underrepresented in randomized controlled trials of cardiology. Recent temporal evolution and factors influencing representation are incompletely investigated. We aimed to contrast age and female representation in the most influential randomized controlled trials in cardiology of the last 20 years to population prevalence and to assess the study factors affecting representation. Methods and Results: Using Web of Science, we selected the 25 most-cited cardiology articles each year between 1996 and 2015, and extracted mean age, percentage of women, funding source, sample size, disease condition, intervention type, and exclusion criteria. The outcomes were the evolution of the mean age and the percentage of women over time. Protocol design elements and year of publication were assessed as predictors of outcomes in multivariable regressions. A total of 500 studies were analyzed, where the mean age was 62.6±7.4 years and the median percentage of women was 28.6% (22.2–40.5). Compared with population prevalence derived from National Health and Nutrition Examination Survey 2015–2016, gaps in representation were apparent and more pronounced for coronary artery disease (−5.0 years; −27.2% women) and heart failure (−6.0 years; −25.4% women). The mean age (0.15 year per year; 95% confidence interval, 0.04–0.26) and percentage of women (+0.29% per year; 95% confidence interval, 0.07–0.50), slightly but significantly increased over time. Private funding, small sample size, and exclusions pertaining to maximal age, atrial fibrillation, and diabetes mellitus were associated with a decreased mean age in multivariable linear regressions. Age and life expectancy exclusions were associated with lower female percentage. Conclusions: Although age and female representation increased over time, the modest trends are unlikely to resolve the persistently wide gaps with actual populational prevalence, especially for coronary artery disease and HF. Representation is modulated by the cardiovascular condition studied and some modifiable protocol elements.

Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing.Two NGS assays - a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) - were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated.In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays.This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.

We explore anion-induced interface fluctuations near protein–water interfaces using coarse-grained representations of interfaces as proposed by Willard and Chandler (J. Phys. Chem. B2010, 114, 1954−195820055377). We use umbrella sampling molecular dynamics to compute potentials of mean force along a reaction coordinate bridging the state where the anion is fully solvated and one where it is biased via harmonic restraints to remain at the protein–water interface. Specifically, we focus on fluctuations of an interface between water and a hydrophobic region of hydrophobin-II (HFBII), a 71 amino acid residue protein expressed by filamentous fungi and known for its ability to form hydrophobically mediated self-assemblies at interfaces such as a water/air interface. We consider the anions chloride and iodide that have been shown previously by simulations as displaying specific-ion behaviors at aqueous liquid–vapor interfaces. We find that as in the case of a pure liquid–vapor interface, at the hydrophobic protein–water interface, the larger, less charge-dense iodide anion displays a marginal interfacial stability compared with that of the smaller, more charge-dense chloride anion. Furthermore, consistent with the results at aqueous liquid–vapor interfaces, we find that iodide induces larger fluctuations of the protein–water interface than chloride.

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays.

The purposes of this study were (1) to determine whether the duration of rest before referral to physical therapy (PT) affects the time to make a full return to activity for patients with an acute spondylolysis, (2) to assess the safety of an early referral to PT in patients with an acute spondylolysis.Retrospective chart review.Hospital-based sports medicine clinic.The medical charts of 196 adolescent athletes (mean age = 14.3 ± 1.8 years) with an acute spondylolytic injury met the inclusion criteria and were reviewed.Patients were subgrouped based on physician referral to PT.An aggressive referral group (10 weeks) and a conservative referral group (10 weeks).Duration of rest before clearance to a full return to activity and the frequency of adverse reactions during the course of treatment. Safety was assessed by calculating the risk of experiencing an adverse reaction in each group.Median days to a full return to activity for aggressive referral group (115.5 days, interquartile range 98-150 days) and conservative referral group (140.0 days, interquartile range 114.5-168 days) were significantly different (P = 0.002). Eleven patients had adverse reactions during the course of treatment. The risk of adverse reaction was not statistically significant between groups (P = 0.509).Patients with acute spondylolysis in the aggressive referral group were able to make a full return activity almost 25 days sooner. No differences in the risk of adverse reactions were noted between aggressive and conservative referral groups.

Aims: Individualization of cancer chemotherapy based on the patient’s genetic makeup holds promise for reducing side effects and improving efficacy. However, the relative contribution of genetics to drug response is unknown. Materials & methods: In this study, we investigated the cytotoxic effect of 29 commonly prescribed chemotherapeutic agents from diverse drug classes on 125 lymphoblastoid cell lines derived from 14 extended families. Results: The results of this systematic study highlight the variable role that genetics plays in response to cytotoxic drugs, ranging from a heritability of 0.60 for epirubicin. Conclusion: Putative quantitative trait loci for cytotoxic response were identified, as well as drug class-specific signatures, which could indicate possible shared genetic mechanisms. In addition to the identification of putative quantitative trait locis, the results of this study inform the prioritization of chemotherapeutic drugs with a sizable genetic response component for future investigation. Original submitted 6 April 2011; Revision submitted 1 July 2011

Introduction: Increase in targeted therapies has resulted in the need for a single assay capable of detecting diverse biomarkers indicated for these agents. Comprehensive genomic profiling (CGP) provides such a solution, but due to the complexity and number of assays available today, standardization of validation has become critically important. We present FoundationOne CDx, the first NGS-based comprehensive companion diagnostics (CDx) platform developed and performed in compliance with FDA 21 CFR part 820. The assay interrogates 324 genes, and has CDx indications in five tumor types associated with 17 targeted therapies (Table 1). The versatile assay design will facilitate streamlined development of future CDx indications. Methods: DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB). Results: Clinical validity was established such that the concordance between CGP and approved CDx were statistically non-inferior to that of two runs of approved CDx. For analytical validity, limit of detection (LoD) was at allele frequency 4% for known substitutions and indels. LoD was 16% tumor content for copy number amplifications, 30% for homozygous deletions, 11% for rearrangements, 12% for MSI, and 20% for TMB. Concordance with an orthogonal NGS platform was 94.6% for substitutions and indels. Within-assay reproducibility had PPA 99.4%. Conclusion: Rapid expansion of targeted therapies and CDx has necessitated a new approach and urgency to defining performance standards. We developed a comprehensive CDx assay and demonstrated clinical and analytical validity to support and accelerate using CGP for routine clinical care. Table 1. Companion Diagnostic IndicationsIndicationBiomarkerTherapyNon-small cell lung cancer (NSCLC)EGFR exon 19 deletions and EGFR exon 21 L858R alterationsafatinib, gefitinib, or erlotinibEGFR exon 20 T790M alterationsosimertinibALK rearrangementsalectinib, crizotinib, or ceritinibBRAF V600Edabrafenib in combination with trametinibMelanomaBRAF V600Edabrafenib, vemurafenibBRAF V600E and V600Ktrametinib, cobimetinib, in combination with vemurafenibBreast cancerERBB2 (HER2) amplificationtrastuzumab, ado-trastuzumab-emtansine, or pertuzumabColorectal cancerKRAS wild-type (absence of mutations in codons 12 and 13)cetuximabKRAS and NRAS wild-type (absence of mutations in exons 2, 3, and 4)panitumumabOvarian cancerBRCA1/2 alterationsrucaparib Citation Format: James X. Sun, Yali Li, Coren Milbury, Joel Skoletsky, Christine Burns, Wai-ki Yip, Jun Luo, Ninad Dewal, Adrienne Johnson, Kyle Gowen, Jing Tong, Yuting He, Jie He, Pei Ma, Jared White, Steve Roels, John Truesdell, Eric Peters, Houston Gilbert, Charlie Wu, Erica Schleifman, Johannes Noe, Carl Barrett, Kenneth Thress, Suzanne Jenkins, Julia Elvin, Geoff Otto, Doron Lipson, Jeffrey Ross, Vincent Miller, Philip Stephens, Michael Doherty, Christine Vietz. A clinically validated comprehensive companion diagnostic platform for care of patients with advanced cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4757.

Background: Tumor mutational burden (TMB) has emerged as a new biomarker for response to checkpoint inhibitor immunotherapy. Recently, we described a novel assay to calculate TMB from the circulating tumor DNA (ctDNA) in the blood (bTMB) and demonstrated validation to support its application in a prospective phase III trial in 1L NSCLC comparing the anti-PD-L1 agent, atezolizumab, against standard of care chemotherapy. Herein, we demonstrate clinical utility of the bTMB assay across a retrospective study of 794 NSCLC patients from two randomized clinical trials evaluating progression free survival (PFS) and overall survival (OS) between atezolizumab and chemotherapy. Furthermore, we compare TMB between tissue and blood, and evaluate variant-level concordance between the bTMB assay and the CLIA-validated ctDNA assay, FoundationACT (FACT). Methods: Cell free DNA (cfDNA) from plasma was isolated from retrospective clinical samples. The bTMB assay delivered a count of somatic base substitutions in the ctDNA down to 0.5% allele frequency across 394 genes from as little as 1% tumor content, yielding a bTMB score. Tissue TMB was evaluated from formalin-fixed, paraffin embedded specimens using the FoundationOne assay. Results: Patients with bTMB ≥14 mut/Mb were considered high, and demonstrated significant enrichment for both PFS and OS in the phase II POPLAR study evaluating atezolizumab vs. chemotherapy (PFS HR = 0.57, OS HR = 0.56, n=211). Survival results were validated in the larger phase III study, OAK (PFS HR = 0.65, OS HR = 0.64, n=583). Significant enrichment in PFS was observed between bTMB-high and non-high patients receiving atezolizumab, however this effect was not observed for patients receiving chemotherapy. Amongst 259 patients with blood and tissue TMB, overall and positive percent agreement (PPA) in the TMB categorical result were 81.5% and 63.6%, respectively. When bTMB was restricted to the 62 gene FACT assay, the PPA dropped to 17.0%, suggesting a sufficiently sized panel is required to sensitively identify patients with high TMB. When samples were TMB-high in blood, the median percent overlap of TMB variants between blood and tissue was 70%. Blood samples tested in both the bTMB and FACT assays revealed that 93% of variants were detected in both assays across overlapping regions, with ≥99% of variants detected in both platforms with variant allele frequency ≥1.0%. Conclusions: The bTMB assay is analytically validated for high accuracy and precision, and demonstrates a high degree of variant level concordance with the FACT assay. High bTMB is significantly associated with improved survival to atezolizumab vs. chemotherapy in 2L NSCLC, and the survival benefit is specific to atezolizumab and not prognostic. Finally, TMB between blood and tissue is correlated and largely explained by overlapping variants present in both sample types. Citation Format: David Fabrizio, Daniel Lieber, Christine Malboeuf, Jacob Silterra, Emily White, Michael Coyne, Tina Brennan, Jie Ma, Mark Kennedy, Erica Schleifman, Sarah Paul, Yan Li, David Shames, Craig Cummings, Eric Peters, Marcin Kowanetz, Doron Lipson, Geoff Otto. A blood-based next-generation sequencing assay to determine tumor mutational burden (bTMB) is associated with benefit to an anti-PD-L1 inhibitor, atezolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5706.

12000Background: PD-L1 expression has limitations as a biomarker for checkpoint immunotherapy (CI). Prior atezolizumab (atezo) monotherapy studies suggest improved efficacy in high tissue tumor mut...

Because previous preclinical and clinical studies have implicated the endogenous opioid system in major depression and in the neurochemical action of antidepressants, the authors examined how DNA variation in the mu-opioid receptor gene may influence population variation in response to citalopram treatment.A total of 1,953 individuals from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study were treated with citalopram and genotyped for 53 single nucleotide polymorphisms (SNPs) in a 100-kb region of the OPRM1 gene. The sample consisted of Non-Hispanic Caucasians, Hispanic Caucasians, and African Americans. Population stratification was corrected using 119 ancestry informative markers and principal components analysis. Markers were tested for association with phenotypes for general and specific citalopram response as well as remission.Association between one SNP and specific citalopram response was observed. After Bonferroni correction, the strongest finding was the association between the rs540825 SNP and specific response. The rs540825 polymorphism is a nonsynonymous SNP in the final exon of the mu-opioid receptor-1X isoform of the OPRM1 gene, resulting in a histidine to glutamine change in the intracellular domain of the receptor. When Hispanic and Non-Hispanic Caucasians were analyzed separately, similar results in the population-corrected analyses were detected.These results suggest that rates of response to antidepressants and consequent remission from major depressive disorder are influenced by variation in the mu-opioid receptor gene as a result of either an effect on placebo response or true pharmacologic response.

Background Antidepressant response is likely influenced by genetic constitution, but the actual genes involved have yet to be determined. We have carried out a genomewide association study to determine whether common DNA variation influences antidepressant response. Methods Our sample is derived from Level 1 participants in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study, all treated with citalopram. Association for the response phenotype included 883 responders and 608 nonresponders. For the remission phenotype, 743 subjects that achieved remission were compared with 608 nonresponders. We used a subset of single nucleotide polymorphisms (SNPs; n = 430,198) from the Affymetrix 500K and 5.0 Human SNP Arrays, and association analysis was carried out after correcting for population stratification. Results We identified three SNPs associated with response with p values less than 1 × 10 −5 near the UBE3C gene (rs6966038, p = 4.65 × 10 −7 ), another 100 kb away from BMP7 (rs6127921, p = 3.45 × 10 −6 ), and a third that is intronic in the RORA gene (rs809736, p = 8.19 × 10 −6 ). These same SNPs were also associated with remission. Thirty-nine additional SNPs are of interest with p values ≤ .0001 for the response and remission phenotypes. Conclusions Although the findings reported here do not meet a genomewide threshold for significance, the regions identified from this study provide targets for independent replication and novel pathways to investigate mechanisms of antidepressant response. This study was not placebo controlled, making it possible that we are also observing associations to nonspecific aspects of drug treatment of depression.

We report a genome-wide association study (GWAS) of major depressive disorder (MDD) in 1221 cases from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study and 1636 screened controls. No genome-wide evidence for association was detected. We also carried out a meta-analysis of three European-ancestry MDD GWAS data sets: STAR*D, Genetics of Recurrent Early-onset Depression and the publicly available Genetic Association Information Network-MDD data set. These data sets, totaling 3957 cases and 3428 controls, were genotyped using four different platforms (Affymetrix 6.0, 5.0 and 500 K, and Perlegen). For each of 2.4 million HapMap II single-nucleotide polymorphisms (SNPs), using genotyped data where available and imputed data otherwise, single-SNP association tests were carried out in each sample with correction for ancestry-informative principal components. The strongest evidence for association in the meta-analysis was observed for intronic SNPs in ATP6V1B2 (P=6.78 x 10⁻⁷), SP4 (P=7.68 x 10⁻⁷) and GRM7 (P=1.11 x 10⁻⁶). Additional exploratory analyses were carried out for a narrower phenotype (recurrent MDD with onset before age 31, N=2191 cases), and separately for males and females. Several of the best findings were supported primarily by evidence from narrow cases or from either males or females. On the basis of previous biological evidence, we consider GRM7 a strong MDD candidate gene. Larger samples will be required to determine whether any common SNPs are significantly associated with MDD.