A pan-cancer mycobiome analysis reveals fungal involvement in gastrointestinal and lung tumors.

Fungal microorganisms (mycobiota) comprise a small but immunoreactive component of the human microbiome, yet little is known about their role in human cancers. Pan-cancer analysis of multiple body sites revealed tumor-associated mycobiomes at up to 1 fungal cell per 104 tumor cells. In lung cancer, Blastomyces was associated with tumor tissues. In stomach cancers, high rates of Candida were linked to the expression of pro-inflammatory immune pathways, while in colon cancers Candida was predictive of metastatic disease and attenuated cellular adhesions. Across multiple GI sites, several Candida species were enriched in tumor samples and tumor-associated Candida DNA was predictive of decreased survival. The presence of Candida in human GI tumors was confirmed by external ITS sequencing of tumor samples and by culture-dependent analysis in an independent cohort. These data implicate the mycobiota in the pathogenesis of GI cancers and suggest that tumor-associated fungal DNA may serve as diagnostic or prognostic biomarkers.

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.

Genomic Landscape of Lynch Syndrome Colorectal Neoplasia Identifies Shared Mutated Neoantigens for Immunoprevention.

BACKGROUND & AIMS: Lynch syndrome (LS) carriers develop mismatch repair-deficient neoplasia with high neoantigen (neoAg) rates. No detailed information on targetable neoAgs from LS precancers exists, which is crucial for vaccine development and immune-interception strategies. We report a focused somatic mutation and frameshift-neoAg landscape of microsatellite loci from colorectal polyps without malignant potential (PWOMP), precancers, and early-stage cancers in LS carriers. METHODS: We generated paired whole-exome and transcriptomic sequencing data from 8 colorectal PWOMP, 41 precancers, 8 advanced precancers, and 12 early-stage cancers of 43 LS carriers. A computational pipeline was developed to predict, rank, and prioritize the top 100 detected mutated neoAgs that were validated in vitro using ELISpot and tetramer assays. RESULTS: Mutation calling revealed >10 mut/Mb in 83% of cancers, 63% of advanced precancers, and 20% of precancers. Cancers displayed an average of 616 MHC-I neoAgs/sample, 294 in advanced precancers, and 107 in precancers. No neoAgs were detected in PWOMP. A total of 65% of our top 100 predicted neoAgs were immunogenic in vitro, and were present in 92% of cancers, 50% of advanced precancers, and 29% of precancers. We observed increased levels of naïve CD8+ and memory CD4+ T cells in mismatch repair-deficient cancers and precancers via transcriptomics analysis. CONCLUSIONS: Shared frameshift-neoAgs are generated within unstable microsatellite loci at initial stages of LS carcinogenesis and can induce T-cell responses, generating opportunities for vaccine development, targeting LS precancers and early-stage cancers.

A full-proteome, interaction-specific characterization of mutational hotspots across human cancers.

Rapid accumulation of cancer genomic data has led to the identification of an increasing number of mutational hotspots with uncharacterized significance. Here we present a biologically informed computational framework that characterizes the functional relevance of all 1107 published mutational hotspots identified in approximately 25,000 tumor samples across 41 cancer types in the context of a human 3D interactome network, in which the interface of each interaction is mapped at residue resolution. Hotspots reside in network hub proteins and are enriched on protein interaction interfaces, suggesting that alteration of specific protein-protein interactions is critical for the oncogenicity of many hotspot mutations. Our framework enables, for the first time, systematic identification of specific protein interactions affected by hotspot mutations at the full proteome scale. Furthermore, by constructing a hotspot-affected network that connects all hotspot-affected interactions throughout the whole-human interactome, we uncover genome-wide relationships among hotspots and implicate novel cancer proteins that do not harbor hotspot mutations themselves. Moreover, applying our network-based framework to specific cancer types identifies clinically significant hotspots that can be used for prognosis and therapy targets. Overall, we show that our framework bridges the gap between the statistical significance of mutational hotspots and their biological and clinical significance in human cancers.

A 3D structural SARS-CoV-2-human interactome to explore genetic and drug perturbations.

Emergence of new viral agents is driven by evolution of interactions between viral proteins and host targets. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV-1 arose in part through rapid evolution along the interface between the spike protein and its human receptor ACE2, leading to increased binding affinity. To facilitate broader exploration of how pathogen-host interactions might impact transmission and virulence in the ongoing COVID-19 pandemic, we performed state-of-the-art interface prediction followed by molecular docking to construct a three-dimensional structural interactome between SARS-CoV-2 and human. We additionally carried out downstream meta-analyses to investigate enrichment of sequence divergence between SARS-CoV-1 and SARS-CoV-2 or human population variants along viral-human protein-interaction interfaces, predict changes in binding affinity by these mutations/variants and further prioritize drug repurposing candidates predicted to competitively bind human targets. We believe this resource ( http://3D-SARS2.yulab.org ) will aid in development and testing of informed hypotheses for SARS-CoV-2 etiology and treatments.

Sequencing at lymphoid neoplasm susceptibility loci maps six myeloma risk genes.

Inherited genetic risk factors play a role in multiple myeloma (MM), yet considerable missing heritability exists. Rare risk variants at genome-wide association study (GWAS) loci are a new avenue to explore. Pleiotropy between lymphoid neoplasms (LNs) has been suggested in family history and genetic studies, but no studies have interrogated sequencing for pleiotropic genes or rare risk variants. Sequencing genetically enriched cases can help discover rarer variants. We analyzed exome sequencing in familial or early-onset MM cases to identify rare, functionally relevant variants near GWAS loci for a range of LNs. A total of 149 distinct and significant LN GWAS loci have been published. We identified six recurrent, rare, potentially deleterious variants within 5 kb of significant GWAS single nucleotide polymorphisms in 75 MM cases. Mutations were observed in BTNL2, EOMES, TNFRSF13B, IRF8, ACOXL and TSPAN32. All six genes replicated in an independent set of 255 early-onset MM or familial MM or precursor cases. Expansion of our analyses to the full length of these six genes resulted in a list of 39 rare and deleterious variants, seven of which segregated in MM families. Three genes also had significant rare variant burden in 733 sporadic MM cases compared with 935 control individuals: IRF8 (P = 1.0 × 10-6), EOMES (P = 6.0 × 10-6) and BTNL2 (P = 2.1 × 10-3). Together, our results implicate six genes in MM risk, provide support for genetic pleiotropy between LN subtypes and demonstrate the utility of sequencing genetically enriched cases to identify functionally relevant variants near GWAS loci.

What happens in the long term: Uptake of cancer surveillance and prevention strategies among at-risk relatives with pathogenic variants detected via cascade testing.

BACKGROUND: Cascade genetic testing for hereditary cancer syndromes offers affected relatives the opportunity to pursue cancer screening and risk-reducing surgery and thus reduces morbidity and mortality. The purpose of this study was to measure the long-term utilization of targeted cancer prevention and quality of life among at-risk relatives offered clinician-facilitated cascade genetic testing. METHODS: In a pilot study, at-risk relatives of patients with a hereditary cancer syndrome were contacted directly by the clinical team and offered telephone genetic counseling and genetic testing via an at-home, mailed saliva kit. Two-year follow-up results evaluating the use of targeted cancer prevention strategies and the quality of life for enrolled relatives were reported. Quality-of-life was measured with validated surveys, and scores were compared to the time of initial contact by the Wilcoxon signed-rank test. RESULTS: Ninety-five at-risk relatives were enrolled in the initial pilot study, and 72 (76%) participated in the 2-year follow-up; 57 of these (79%) had completed genetic testing. Twenty-five of those 57 relatives (44%) were found to harbor an inherited pathogenic variant. Guideline-based cancer surveillance was recommended to 18 relatives; 13 (72%) completed at least one recommended screening, and six (33%) completed all recommended screenings. Risk-reducing surgery was recommended to 10 relatives; four (40%) completed a total of eight procedures. Quality-of-life surveys demonstrated low levels of anxiety, depression, distress, and uncertainty. CONCLUSIONS: The 2-year follow-up of the original pilot study revealed that clinician-facilitated cascade testing resulted in genetically targeted cancer screening and prevention with preserved quality of life. These results, to be confirmed by larger randomized controlled trials, suggest that medical systems should consider supporting clinician-facilitated cascade testing programs.

Recurrent Frameshift Neoantigen Vaccine Elicits Protective Immunity With Reduced Tumor Burden and Improved Overall Survival in a Lynch Syndrome Mouse Model.

BACKGROUND & AIMS: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated. METHODS: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination. RESULTS: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly increased FSP-specific adaptive immunity, reduced intestinal tumor burden, and prolonged overall survival. Combination of FSP vaccination with daily naproxen treatment potentiated immune response, delayed tumor growth, and prolonged survival even more effectively than FSP vaccination alone. CONCLUSIONS: Our preclinical findings support a clinical strategy of recurrent FSP neoantigen vaccination for LS cancer immunoprevention.

Naproxen chemoprevention promotes immune activation in Lynch syndrome colorectal mucosa.

OBJECTIVE: Patients with Lynch syndrome (LS) are at markedly increased risk for colorectal cancer. It is being increasingly recognised that the immune system plays an essential role in LS tumour development, thus making an ideal target for cancer prevention. Our objective was to evaluate the safety, assess the activity and discover novel molecular pathways involved in the activity of naproxen as primary and secondary chemoprevention in patients with LS. DESIGN: We conducted a Phase Ib, placebo-controlled, randomised clinical trial of two dose levels of naproxen sodium (440 and 220 mg) administered daily for 6 months to 80 participants with LS, and a co-clinical trial using a genetically engineered mouse model of LS and patient-derived organoids (PDOs). RESULTS: Overall, the total number of adverse events was not different across treatment arms with excellent tolerance of the intervention. The level of prostaglandin E2 in the colorectal mucosa was significantly decreased after treatment with naproxen when compared with placebo. Naproxen activated different resident immune cell types without any increase in lymphoid cellularity, and changed the expression patterns of the intestinal crypt towards epithelial differentiation and stem cell regulation. Naproxen demonstrated robust chemopreventive activity in a mouse co-clinical trial and gene expression profiles induced by naproxen in humans showed perfect discrimination of mice specimens with LS and PDOs treated with naproxen and control. CONCLUSIONS: Naproxen is a promising strategy for immune interception in LS. We have discovered naproxen-induced gene expression profiles for their potential use as predictive biomarkers of drug activity. TRIAL REGISTRATION NUMBER: gov Identifier: NCT02052908.

Achieving universal genetic assessment for women with ovarian cancer: Are we there yet? A systematic review and meta-analysis.

PURPOSE: Several professional organizations recommend universal genetic assessment for people with ovarian cancer as identifying pathogenic variants can affect treatment, prognosis, and all-cause mortality for patients and relatives. We sought to evaluate the literature on genetic assessment for women with ovarian cancer and determine if any interventions or patient characteristics drive utilization of services. METHODS: We searched key electronic databases to identify trials that evaluated genetic assessment for people with ovarian cancer. Trials with the primary aim to evaluate utilization of genetic assessment with or without interventions were included. Eligible trials were subjected to meta-analysis and the moderating influence of health interventions on rates of genetic assessment were examined. RESULTS: A total of 35 studies were included (19 report on utilization of genetic services without an intervention, 7 with an intervention, and 9 with both scenarios). Without an intervention, pooled estimates for referral to genetic counseling and completion of genetic testing were 39% [CI 27-53%] and 30% [CI 19-44%]. Clinician-facilitated interventions included: mainstreaming of genetic services (99% [CI 86-100%]), telemedicine (75% [CI 43-93%]), clinic-embedded genetic counselor (76% [CI 32-95%]), reflex tumor somatic genetic assessment (64% [CI 17-94%]), universal testing (57% [28-82%]), and referral forms (26% [CI 10-53%]). Random-effects pooled proportions demonstrated that Black vs. White race was associated with a lower rate of genetic testing (26%[CI 17-38%] vs. 40% [CI 25-57%]) as was being un-insured vs. insured (23% [CI 18-28%] vs. 38% [CI 26-53%]). CONCLUSIONS: Reported rates of genetic testing for people with ovarian cancer remain well below the goal of universal testing. Interventions such as mainstreaming can improve testing uptake. Strategies aimed at improving utilization of genetic services should consider existing disparities in race and insurance status.

Facilitated cascade testing (FaCT): a randomized controlled trial.

BACKGROUND: Identifying mutation-carrying relatives of patients with hereditary cancer syndromes via cascade testing is an underused first step in primary cancer prevention. A feasibility study of facilitated genetic testing of at-risk relatives of patients with a known pathogenic mutation demonstrated encouraging uptake of cascade testing. PRIMARY OBJECTIVE: Our primary objective is to compare the proportion of genetic testing of identified first-degree relatives of probands with a confirmed BRCA1/2 mutation randomized to a facilitated cascade testing strategy versus standard of care, proband-mediated, information sharing. STUDY HYPOTHESIS: We hypothesize that facilitated cascade testing will drive significantly higher uptake of genetic testing than the standard of care. TRIAL DESIGN: The FaCT (Facilitated Cascade Testing) trial is a prospective multi-institutional randomized study comparing the efficacy of a multicomponent facilitated cascade testing intervention with the standard of care. Patients with a known BRCA1/2 mutation (probands) cared for at participating sites will be randomized. Probands randomized to the standard of care group will be instructed to share a family letter with their first-degree relatives and encourage them to complete genetic testing. First-degree relatives of probands randomized to the intervention arm will receive engagement strategies with a patient navigator, an educational video, and accessible genetic testing services. MAJOR INCLUSION/EXCLUSION CRITERIA: Adult participants who are first-degree relatives of a patient with a BRCA1/2 mutation and have not had prior genetic testing will be included. PRIMARY ENDPOINT: Analyses will assess the proportion of first-degree relatives identified by the proband who complete genetic testing by 6 months in the intervention arm versus the control arm. SAMPLE SIZE: One hundred and fifty probands with a BRCA1/2 mutation will be randomized. Each proband is expected to provide an average of 3 relatives, for an expected 450 participants. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: January 2024. TRIAL REGISTRATION: NCT04613440.

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk.

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance-a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits.

Correction: Toward automation of germline variant curation in clinical cancer genetics.

An update to the original published conflict of interest for author Liying Zhang, PhD. L.Z. received compensation from Future Technology Research LLC (seminar on precision medicine), Roche Diagnostics Asia Pacific, BGI, Illumina (speaking activities at conferences/workshop). L.Z.'s family member has a leadership position and ownership interest of Shanghai Genome Center. This correction has been made.

Toward automation of germline variant curation in clinical cancer genetics.

PURPOSE: Cancer care professionals are confronted with interpreting results from multiplexed gene sequencing of patients at hereditary risk for cancer. Assessments for variant classification now require orthogonal data searches and aggregation of multiple lines of evidence from diverse resources. The clinical genetics community needs a fast algorithm that automates American College of Medical Genetics and Genomics (ACMG) based variant classification and provides uniform results. METHODS: Pathogenicity of Mutation Analyzer (PathoMAN) automates germline genomic variant curation from clinical sequencing based on ACMG guidelines. PathoMAN aggregates multiple tracks of genomic, protein, and disease specific information from public sources. We compared expertly curated variant data from clinical laboratories to assess performance. RESULTS: PathoMAN achieved a high overall concordance of 94.4% for pathogenic and 81.1% for benign variants. We observed negligible discordance (0.3% pathogenic, 0% benign) when contrasted against expert curated variants. Some loss of resolution (5.3% pathogenic, 18.9% benign) and gain of resolution (1.6% pathogenic, 3.8% benign) were also observed. CONCLUSION: Automation of variant curation enables unbiased, fast, efficient delivery of results in both clinical and laboratory research. We highlight the advantages and weaknesses related to the programmable automation of variant classification. PathoMAN will aid in rapid variant classification by generating robust models using a knowledgebase of diverse genetic data ( https://pathoman.mskcc.org).

GeMSTONE: orchestrated prioritization of human germline mutations in the cloud.

Integrative analysis of whole-genome/exome-sequencing data has been challenging, especially for the non-programming research community, as it requires simultaneously managing a large number of computational tools. Even computational biologists find it unexpectedly difficult to reproduce results from others or optimize their strategies in an end-to-end workflow. We introduce Germline Mutation Scoring Tool fOr Next-generation sEquencing data (GeMSTONE), a cloud-based variant prioritization tool with high-level customization and a comprehensive collection of bioinformatics tools and data libraries (http://gemstone.yulab.org/). GeMSTONE generates and readily accepts a shareable 'recipe' file for each run to either replicate previous results or analyze new data with identical parameters and provides a centralized workflow for prioritizing germline mutations in human disease within a streamlined workflow rather than a pool of program executions.

High-depth whole genome sequencing of an Ashkenazi Jewish reference panel: enhancing sensitivity, accuracy, and imputation.

While increasingly large reference panels for genome-wide imputation have been recently made available, the degree to which imputation accuracy can be enhanced by population-specific reference panels remains an open question. Here, we sequenced at full-depth (≥ 30×), across two platforms (Illumina X Ten and Complete Genomics, Inc.), a moderately large (n = 738) cohort of samples drawn from the Ashkenazi Jewish population. We developed a series of quality control steps to optimize sensitivity, specificity, and comprehensiveness of variant calls in the reference panel, and then tested the accuracy of imputation against target cohorts drawn from the same population. Quality control (QC) thresholds for the Illumina X Ten platform were identified that permitted highly accurate calling of single nucleotide variants across 94% of the genome. QC procedures also identified numerous regions that are poorly mapped using current reference or alternate assemblies. After stringent QC, the population-specific reference panel produced more accurate and comprehensive imputation results relative to publicly available, large cosmopolitan reference panels, especially in the range of rare variants that may be most critical to further progress in mapping of complex phenotypes. The population-specific reference panel also permitted enhanced filtering of clinically irrelevant variants from personal genomes.

A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self-renewal.

The intestinal epithelium is the fastest regenerative tissue in the body, fueled by fast-cycling stem cells. The number and identity of these dividing and migrating stem cells are maintained by a mosaic pattern at the base of the crypt. How the underlying regulatory scheme manages this dynamic stem cell niche is not entirely clear. We stimulated intestinal organoids with Notch ligands and inhibitors and discovered that intestinal stem cells employ a positive feedback mechanism via direct Notch binding to the second intron of the Notch1 gene. Inactivation of the positive feedback by CRISPR/Cas9 mutation of the binding sequence alters the mosaic stem cell niche pattern and hinders regeneration in organoids. Dynamical system analysis and agent-based multiscale stochastic modeling suggest that the positive feedback enhances the robustness of Notch-mediated niche patterning. This study highlights the importance of feedback mechanisms in spatiotemporal control of the stem cell niche.

A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.

Importance: Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention. Objective: To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines. Design, Setting, and Participants: From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017. Exposure: Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines. Main Outcomes and Measures: Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing. Results: Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of change to targeted therapy in 38 patients tested (3.7%) and predictive testing in the families of 13 individuals (1.3%), including 6 for whom genetic evaluation would not have been initiated by guideline-based testing. Conclusions and Relevance: In this referral population with selected advanced cancers, universal sequencing of a broad panel of cancer-related genes in paired germline and tumor DNA samples was associated with increased detection of individuals with potentially clinically significant heritable mutations over the predicted yield of targeted germline testing based on current clinical guidelines. Knowledge of these additional mutations can help guide therapeutic and preventive interventions, but whether all of these interventions would improve outcomes for patients with cancer or their family members requires further study. Trial Registration: clinicaltrials.gov Identifier: NCT01775072.

Dissecting disease inheritance modes in a three-dimensional protein network challenges the "guilt-by-association" principle.

To better understand different molecular mechanisms by which mutations lead to various human diseases, we classified 82,833 disease-associated mutations according to their inheritance modes (recessive versus dominant) and molecular types (in-frame [missense point mutations and in-frame indels] versus truncating [nonsense mutations and frameshift indels]) and systematically examined the effects of different classes of disease mutations in a three-dimensional protein interactome network with the atomic-resolution interface resolved for each interaction. We found that although recessive mutations affecting the interaction interface of two interacting proteins tend to cause the same disease, this widely accepted "guilt-by-association" principle does not apply to dominant mutations. Furthermore, recessive truncating mutations in regions encoding the same interface are much more likely to cause the same disease, even for interfaces close to the N terminus of the protein. Conversely, dominant truncating mutations tend to be enriched in regions encoding areas between interfaces. These results suggest that a significant fraction of truncating mutations can generate functional protein products. For example, TRIM27, a known cancer-associated protein, interacts with three proteins (MID2, TRIM42, and SIRPA) through two different interfaces. A dominant truncating mutation (c.1024delT [p.Tyr342Thrfs*30]) associated with ovarian carcinoma is located between the regions encoding the two interfaces; the altered protein retains its interaction with MID2 and TRIM42 through the first interface but loses its interaction with SIRPA through the second interface. Our findings will help clarify the molecular mechanisms of thousands of disease-associated genes and their tens of thousands of mutations, especially for those carrying truncating mutations, often erroneously considered "knockout" alleles.