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Introduction Tumor mutational burden (TMB) has emerged as a clinically relevant biomarker that may be associated with immune checkpoint inhibitor efficacy. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment. Objective Here we describe the in-depth evaluation of bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples from a phase III clinical trial. Methods In the CheckMate 026 clinical trial, TMB was retrospectively assessed in 312 patients with non-small-cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or standard-of-care chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne® CDx assay. Results TMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman’s r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. WES and FoundationOne CDx outputs were highly correlated (Spearman’s r = 0.90). Conclusions Parameter variation can impact TMB calculations, highlighting the need for standardization. Encouragingly, differences between assays could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms, and centers is achievable.

The UK Biobank Exome Sequencing Consortium (UKB-ESC) is a unique private/public partnership between the UK Biobank and eight biopharma companies that will sequence the exomes of all ∼500,000 UK Biobank participants. Here we describe early results from the exome sequence data generated by this consortium for the first ∼200,000 UKB subjects and the key features of this project that enabled the UKB-ESC to come together and generate this data.Exome sequencing data from the first 200,643 UKB enrollees are now accessible to the research community. Approximately 10M variants were observed within the targeted regions, including: 8,086,176 SNPs, 370,958 indels and 1,596,984 multi-allelic variants. Of the ∼8M variants observed, 84.5% are coding variants and include 2,139,318 (25.3%) synonymous, 4,549,694 (53.8%) missense, 453,733 (5.4%) predicted loss-of-function (LOF) variants (initiation codon loss, premature stop codons, stop codon loss, splicing and frameshift variants) affecting at least one coding transcript. This open access data provides a rich resource of coding variants for rare variant genetic studies, and is particularly valuable for drug discovery efforts that utilize rare, functionally consequential variants.Over the past decade, the biopharma industry has increasingly leveraged human genetics as part of their drug discovery and development strategies. This shift was motivated by technical advances that enabled cost-effective human genetics research at scale, the emergence of electronic health records and biobanks, and a maturing understanding of how human genetics can increase the probability of successful drug development. Recognizing the need for large-scale human genetics data to drive drug discovery, and the unique value of the open data access policies and contribution terms of the UK Biobank, the UKB-ESC was formed. This precompetitive collaboration has further strengthened the ties between academia and industry and provided teams an unprecedented opportunity to interact with and learn from the wider research community.

Purpose Hearing loss (HL) is the most common sensory disorder in children. Prompt molecular diagnosis may guide screening and management; especially in syndromic cases when HL is the single presenting feature. Exome sequencing (ES)is an appealing diagnostic tool for HL as the genetic causes are highly heterogeneous. Methods ES was performed on a prospective cohort of 43 probands with HL. Sequence data were analyzed for primary and secondary findings. Capture and coverage analysis was performed for genes and variants associated with HL. Results The diagnostic rate using ES was 37.2% compared to 15.8% with clinical HL panel. Secondary findings were discovered in three patients. For 247 genes associated with HL, 94.7% of the exons were targeted for capture and 81.7% of these exons were covered at 20× or greater. Further analysis of 454 randomly selected HL-associated variants showed 89% were targeted for capture and 75% were covered at a read depth of at least 20×. Conclusion ES has an improved yield to clinical testing and may capture diagnoses not initially considered due to subtle clinical phenotypes. Technical challenges were identified, including inadequate capture and coverage of HL genes. Additional considerations of ES include secondary findings, cost, and turnaround time.

KRAS is the most common oncogenic driver in lung adenocarcinoma (LUAC). We previously reported that STK11/LKB1 (KL) or TP53 (KP) comutations define distinct subgroups of KRAS-mutant LUAC. Here, we examine the efficacy of PD-1 inhibitors in these subgroups. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%), and K-only (28.6%) subgroups (P < 0.001) in the Stand Up To Cancer (SU2C) cohort (174 patients) with KRAS-mutant LUAC and in patients treated with nivolumab in the CheckMate-057 phase III trial (0% vs. 57.1% vs. 18.2%; P = 0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P < 0.001) and overall (P = 0.0015) survival compared with KRASMUT;STK11/LKB1WT LUAC. Among 924 LUACs, STK11/LKB1 alterations were the only marker significantly associated with PD-L1 negativity in TMBIntermediate/High LUAC. The impact of STK11/LKB1 alterations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1–positive non–small cell lung cancer. In Kras-mutant murine LUAC models, Stk11/Lkb1 loss promoted PD-1/PD-L1 inhibitor resistance, suggesting a causal role. Our results identify STK11/LKB1 alterations as a major driver of primary resistance to PD-1 blockade in KRAS-mutant LUAC. Significance: This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets. Cancer Discov; 8(7); 822–35. ©2018 AACR. See related commentary by Etxeberria et al., p. 794. This article is highlighted in the In This Issue feature, p. 781

IntroductionTumor mutational burden (TMB) has emerged as a clinically relevant biomarker that may be associated with immune checkpoint inhibitor efficacy. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment.ObjectiveHere we describe the in-depth evaluation of bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples from a phase 3 clinical trial.MethodsIn the CheckMate 026 clinical trial, TMB was retrospectively assessed in 312 patients with non-small cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or standard-of-care chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne®CDx assay.ResultsTMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman’s r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. WES and FoundationOne CDx outputs were highly correlated (Spearman’s r = 0.90).ConclusionsParameter variation can impact TMB calculations, highlighting the need for standardization. Encouragingly, differences between assays could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms, and centers is achievable.Key PointsTumor mutational burden (TMB) is a clinically relevant biomarker for efficacy of immunotherapy in patients with cancerVariations in TMB assessment parameters can shift the final TMB value. Harmonization and standardization are important to the successful clinical implementation of TMB testingTMB values assessed by different methods are highly correlated. Harmonization of TMB testing in patients with cancer is therefore achievable

Summary Durable responses and encouraging survival have been demonstrated with immune checkpoint inhibitors in small-cell lung cancer (SCLC), but predictive markers are unknown. We used whole exome sequencing to evaluate the impact of tumor mutational burden on efficacy of nivolumab monotherapy or combined with ipilimumab in patients with SCLC from the nonrandomized or randomized cohorts of CheckMate 032. Patients received nivolumab (3 mg/kg every 2 weeks) or nivolumab plus ipilimumab (1 mg/kg plus 3 mg/kg every 3 weeks for four cycles, followed by nivolumab 3 mg/kg every 2 weeks). Efficacy of nivolumab ± ipilimumab was enhanced in patients with high tumor mutational burden. Nivolumab plus ipilimumab appeared to provide a greater clinical benefit than nivolumab monotherapy in the high tumor mutational burden tertile.

Background: Metastatic or surgically unresectable urothelial cancer (UC) is a disease with high unmet need. Nivolumab monotherapy demonstrated clinical benefit in patients with platinum-resistant UC (CheckMate 275; NCT02387996). Because not all patients respond to nivolumab therapy, identifying biomarkers for response is of critical importance. In the CheckMate 275 cohort, higher tumor mutational burden (TMB) was associated with longer overall survival (OS) (1). Previous studies have shown that certain mutational signatures, combinations of mutation types arising from specific mutagenesis processes, were associated with mutational burden and clinical outcome in UC (2,3). We hypothesized that specific mutational signatures may be associated with response to nivolumab in CheckMate 275. Methods: Whole exome sequencing data and clinical annotations for 139 archival, tumor-normal paired samples from patients enrolled in the CheckMate 275 cohort were collected and analyzed. Sequencing reads were processed and somatic variants called as described previously (4). Percentage of mutations across 30 mutational signatures were generated using deconstructSigs, which infers signature activity with given known signatures (5). Association of the most prevalent mutational signatures with TMB, previously known clinical biomarkers, and clinical efficacy (OS, progression free survival [PFS], and objective response [OR]) were examined. Results: We identified age-related (signature 1), endogenous mutagenesis-related APOBEC (signatures 2 and 13) and UV-induced (signature 7) as the most prevalent mutational signatures in UC. When examining their correlations with TMB, signature 1 showed negative correlation, signatures 2 and 13 showed high positive correlation, and signature 7 showed low correlation. Higher signature 2 scores were associated with better OS, but were poor predictors for PFS and OR. Overall, while signature 2 added predictive value over previously known clinical biomarkers such as PD-L1 expression, serum hemoglobin level, and presence of liver metastasis, it did not improve on the predictive performance of TMB. Conclusions: An APOBEC-related mutational signature (signature 2) was associated with OS in patients with advanced UC treated with nivolumab. This signature was correlated with TMB and did not improve upon TMB as a predictive biomarker. Our work demonstrates the importance of including TMB as a covariate when analyzing mutational signatures. References: 1. Galsky MD et al. Ann Oncol 2017;28(suppl 5). Abstract 848PD 2. Glaser AP et al. Oncotarget 2018;9:4537-48 3. Roberts SA et al. Nat Genet 2013;45:970-6 4. Carbone DP et al. NEJM 2017;376:2415-26 5. Forbes SA et al. Nucleic Acids Res 2017;45:D777-83 Citation Format: G. Celine Han, Peter M. Szabo, Abdel Saci, Alice M. Walsh, Natallia Kalinava, Ariella Sasson, Bruce Fischer, Matthew D. Galsky, Padmanee Sharma. Mutational signatures as biomarkers of response to nivolumab in metastatic bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4890.

Understanding the relationship between chemical structure and function is a ubiquitous problem within the fields of chemistry and biology. Simulation approaches attack the problem utilizing physics to understand a given process at the particle level. Unfortunately, these approaches are often too expensive for many problems of interest. Informatics approaches attack the problem with empirical analysis of descriptions of chemical structure. The issue in these methods is how to describe molecules in a manner that facilitates accurate and general calculation of molecular properties. Here, we present a novel approach that utilizes aspects of simulation and informatics in order to formulate structure-property relationships. We show how supervised learning can be utilized to overcome the sampling problem in simulation approaches. Likewise, we show how learning can be achieved based on molecular descriptions that are rooted in the physics of dynamic intermolecular forces. We apply the approach to three problems including the analysis of corticosteroid binding globulin ligand binding affinity, identification of formylpeptide receptor ligands, and identification of resveratrol analogues capable of inhibiting activation of transcription factor nuclear factor kappaB.

Targeted resequencing projects can generate massive amount of data in the form of short nucleic acid sequences. These sequences are aligned to a reference genome and stored in BAM (Binary Alignment/Map) files in most cases. A growing challenge for biomedical researchers is to rapidly summarize large BAM files (typically 5 to 25 GB) to evaluate data quality and guide follow-up steps. Here, we present an R package and report template, "bamchop", that retrieves targeted resequencing information from a BAM file and illustrates it in a standardized format. This program is applicable to all resequencing data that investigates selected genomie regions, ranging from a small number of mutation hotspots to a complete human exome. Bamchop reports the sequencing quality and coverage, mapping confidence, strand and nucleic acid bias, and other information about the targeted regions and genomie background. A bamchop report is the product of Sweave framework that requires the LaTeX document preparation system and the R statistical environment. In applying it to a variety of resequencing projects, we determined that bamchop is a powerful tool for both bioinformaticians and bench scientists to conveniently assess their resequencing data. The source code of bamchop is available from https://github.com/CBMi-BiC/bamchop.

BACKGROUND AND AIM: Therapeutic goals for Crohn's disease (CD) have evolved from a mere control of symptoms to the concept of deep remission (DR), including clinical and biomarker remission and mucosal healing (MH). Biologic therapy with anti-TNFα is effective in achieving MH. Yet, CD is a transmural disease, characterized by a progressive bowel damage leading to complications. This is the first pediatric study prospectively evaluating the efficacy of anti-TNFα therapy in inducing clinical remission, MH and transmural healing (TH) in ileal CD. METHODS: Pediatric patients (pts) with ileal CD starting biological therapy with Infliximab or Adalimumab were prospectively enrolled. All pts were Naive to biologics. Clinical activity (Pediatric Crohn's Disease Activity Index, PCDAI), laboratory tests (CRP, ESR), endoscopic activity (simple endoscopic score, SES-CD) and transmural disease assessed by small intestine contrast ultrasonography (SICUS) were evaluated before starting (T0) and after 9-12 months of therapy (T1). Complete MH was defined as a SES-CD of 0-1, partial MH as 50% decrease vs T0. At US the evaluated parameters were: extension of disease (cm), bowel wall thickness >3 mm (BWT), bowel wall vascularity, stratification of the BW (BWS), presence of stricture, fistulae and abscess. Wilcoxon signed rank test was used for pair comparison (T1-T0). RESULTS: 26 pts (mean age 13.3 ± 4, 16 males) were included. The mean PCDAI, ileal SES-CD, CRP, ESR, BWT and disease extension values significantly decreased at T1 (table). Increased bowel wall vascularity was present in 80% of pts at T0 and in 24% at T1 (p