Comprehensive Characterization of Cancer Driver Genes and Mutations.

Identifying molecular cancer drivers is critical for precision oncology. Multiple advanced algorithms to identify drivers now exist, but systematic attempts to combine and optimize them on large datasets are few. We report a PanCancer and PanSoftware analysis spanning 9,423 tumor exomes (comprising all 33 of The Cancer Genome Atlas projects) and using 26 computational tools to catalog driver genes and mutations. We identify 299 driver genes with implications regarding their anatomical sites and cancer/cell types. Sequence- and structure-based analyses identified >3,400 putative missense driver mutations supported by multiple lines of evidence. Experimental validation confirmed 60%-85% of predicted mutations as likely drivers. We found that >300 MSI tumors are associated with high PD-1/PD-L1, and 57% of tumors analyzed harbor putative clinically actionable events. Our study represents the most comprehensive discovery of cancer genes and mutations to date and will serve as a blueprint for future biological and clinical endeavors.

Inherited DNA-Repair Defects in Colorectal Cancer.

Colorectal cancer (CRC) heritability has been estimated to be around 30%. However, mutations in the known CRC-susceptibility genes explain CRC risk in fewer than 10% of affected individuals. Germline mutations in DNA-repair genes (DRGs) have recently been reported in CRC, but their contribution to CRC risk is largely unknown. We evaluated the gene-level germline mutation enrichment of 40 DRGs in 680 unselected CRC individuals and 27,728 ancestry-matched cancer-free adults. Significant findings were then examined in independent cohorts of 1,661 unselected CRC individuals and 1,456 individuals with early-onset CRC. Of the 680 individuals in the discovery set, 31 (4.56%) individuals harbored germline pathogenic mutations in known CRC-susceptibility genes, and another 33 (4.85%) individuals had DRG mutations that have not been previously associated with CRC risk. Germline pathogenic mutations in ATM and PALB2 were enriched in both the discovery (OR = 2.81 and p = 0.035 for ATM and OR = 4.91 and p = 0.024 for PALB2) and validation (OR = 2.97 and adjusted p = 0.0013 for ATM and OR = 3.42 and adjusted p = 0.034 for PALB2) sets. Biallelic loss of ATM was evident in all individuals with matched tumor profiling. CRC individuals also had higher rates of actionable mutations in the HR pathway, which can substantially increase the risk of developing cancers other than CRC. Our analysis provides evidence for ATM and PALB2 as CRC-risk genes, underscoring the importance of the homologous recombination pathway in CRC. In addition, we identified frequent complete homologous recombination deficiency in CRC tumors, representing a unique opportunity to explore targeted therapeutic interventions such as poly-ADP ribose polymerase inhibitor (PARPi).

CoMut: visualizing integrated molecular information with comutation plots.

MOTIVATION: Large-scale sequencing studies have created a need to succinctly visualize genomic characteristics of patient cohorts linked to widely variable phenotypic information. This is often done by visualizing the co-occurrence of variants with comutation plots. Current tools lack the ability to create highly customizable and publication quality comutation plots from arbitrary user data. RESULTS: We developed CoMut, a stand-alone, object-oriented Python package that creates comutation plots from arbitrary input data, including categorical data, continuous data, bar graphs, side bar graphs and data that describes relationships between samples. AVAILABILITY AND IMPLEMENTATION: The CoMut package is open source and is available at https://github.com/vanallenlab/comut under the MIT License, along with documentation and examples. A no installation, easy-to-use implementation is available on Google Colab (see GitHub).

Evaluating the molecular diagnostic yield of joint genotyping-based approach for detecting rare germline pathogenic and putative loss-of-function variants.

PURPOSE: Cohort-based germline variant characterization is the standard approach for pathogenic variant discovery in clinical and research samples. However, the impact of cohort size on the molecular diagnostic yield of joint genotyping is largely unknown. METHODS: Head-to-head comparison of the molecular diagnostic yield of joint genotyping in two cohorts of 239 cancer patients in the absence and then in the presence of 100 additional germline exomes. RESULTS: In 239 testicular cancer patients, 4 (7.4%, 95% confidence interval [CI]: 2.1-17.9) of 54 pathogenic variants in the cancer predisposition and American College of Medical Genetics and Genomics (ACMG) genes were missed by one or both computational runs of joint genotyping. Similarly, 8 (12.1%, 95% CI: 5.4-22.5) of 66 pathogenic variants in these genes were undetected by joint genotyping in another independent cohort of 239 breast cancer patients. An exome-wide analysis of putative loss-of-function (pLOF) variants in the testicular cancer cohort showed that 162 (8.2%, 95% CI: 7.1-9.6) pLOF variants were only detected in one analysis run but not the other, while 433 (22.0%, 95% CI: 20.2-23.9%) pLOF variants were filtered out by both analyses despite having sufficient sequencing coverage. CONCLUSION: Our analysis of the standard germline variant detection method highlighted a substantial impact of concurrently analyzing additional genomic data sets on the ability to detect clinically relevant germline pathogenic variants.

A phase 2 trial of buparlisib in patients with platinum-resistant metastatic urothelial carcinoma.

BACKGROUND: The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway frequently is activated in patients with urothelial carcinoma (UC). In the current study, the authors performed a phase 2 study evaluating the efficacy of the pan-isoform class I PI3K inhibitor buparlisib in patients with platinum-refractory metastatic UC. METHODS: Two cohorts were recruited: an initial genetically unselected cohort and a subsequent expansion cohort of patients with PI3K/Akt/mTOR pathway-altered tumors. The primary endpoint was the 2-month progression-free survival rate. A rate of ≥80% was considered promising using a Simon 2-stage minimax design. Secondary endpoints included safety and correlation of markers of PI3K pathway activation with outcome. RESULTS: Six of 13 evaluable patients within the initial cohort demonstrated stable disease and 1 demonstrated a partial response, which was below the cutoff of 9 patients required to proceed to stage 2. Three of the patients with stable disease and the patient with a partial response harbored somatic TSC1 alterations. Four patients subsequently were recruited onto an expansion cohort: 3 patients with TSC1 alterations and 1 patient with a PIK3CA-activating mutation. No patient achieved disease control at 8 weeks and accrual was halted. Of the 19 patients evaluable for toxicity, 17 demonstrated treatment-related toxicities, 2 of whom had to discontinue therapy. CONCLUSIONS: Buparlisib was found to demonstrate modest activity in patients with metastatic UC whose tumors harbored TSC1 loss of function alterations; however, this was not a robust predictor of response to buparlisib. The pattern of genetic coalterations likely influences drug sensitivity. Given the modest clinical activity and substantial toxicity of buparlisib, future trials of PI3K inhibitors in patients with UC should focus on isoform-selective PI3K inhibitors in genomically selected patients. LAY SUMMARY: The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway frequently is upregulated in patients with metastatic urothelial carcinoma (UC). This trial explored buparlisib, an inhibitor of the pathway, in patients with heavily pretreated metastatic UC. Although the drug was found to have modest efficacy, with 6 patients experiencing stable disease and 1 patient achieving a partial response at 8 weeks on therapy, significant side effects also were observed. Patients with specific genetic alterations responded to treatment. Further studies of PI3K pathway inhibition are warranted using newer agents that have superior toxicity profiles and are more selective inhibitors of the pathway.

Detection of Pathogenic Variants With Germline Genetic Testing Using Deep Learning vs Standard Methods in Patients With Prostate Cancer and Melanoma.

Importance: Less than 10% of patients with cancer have detectable pathogenic germline alterations, which may be partially due to incomplete pathogenic variant detection. Objective: To evaluate if deep learning approaches identify more germline pathogenic variants in patients with cancer. Design, Setting, and Participants: A cross-sectional study of a standard germline detection method and a deep learning method in 2 convenience cohorts with prostate cancer and melanoma enrolled in the US and Europe between 2010 and 2017. The final date of clinical data collection was December 2017. Exposures: Germline variant detection using standard or deep learning methods. Main Outcomes and Measures: The primary outcomes included pathogenic variant detection performance in 118 cancer-predisposition genes estimated as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The secondary outcomes were pathogenic variant detection performance in 59 genes deemed actionable by the American College of Medical Genetics and Genomics (ACMG) and 5197 clinically relevant mendelian genes. True sensitivity and true specificity could not be calculated due to lack of a criterion reference standard, but were estimated as the proportion of true-positive variants and true-negative variants, respectively, identified by each method in a reference variant set that consisted of all variants judged to be valid from either approach. Results: The prostate cancer cohort included 1072 men (mean [SD] age at diagnosis, 63.7 [7.9] years; 857 [79.9%] with European ancestry) and the melanoma cohort included 1295 patients (mean [SD] age at diagnosis, 59.8 [15.6] years; 488 [37.7%] women; 1060 [81.9%] with European ancestry). The deep learning method identified more patients with pathogenic variants in cancer-predisposition genes than the standard method (prostate cancer: 198 vs 182; melanoma: 93 vs 74); sensitivity (prostate cancer: 94.7% vs 87.1% [difference, 7.6%; 95% CI, 2.2% to 13.1%]; melanoma: 74.4% vs 59.2% [difference, 15.2%; 95% CI, 3.7% to 26.7%]), specificity (prostate cancer: 64.0% vs 36.0% [difference, 28.0%; 95% CI, 1.4% to 54.6%]; melanoma: 63.4% vs 36.6% [difference, 26.8%; 95% CI, 17.6% to 35.9%]), PPV (prostate cancer: 95.7% vs 91.9% [difference, 3.8%; 95% CI, -1.0% to 8.4%]; melanoma: 54.4% vs 35.4% [difference, 19.0%; 95% CI, 9.1% to 28.9%]), and NPV (prostate cancer: 59.3% vs 25.0% [difference, 34.3%; 95% CI, 10.9% to 57.6%]; melanoma: 80.8% vs 60.5% [difference, 20.3%; 95% CI, 10.0% to 30.7%]). For the ACMG genes, the sensitivity of the 2 methods was not significantly different in the prostate cancer cohort (94.9% vs 90.6% [difference, 4.3%; 95% CI, -2.3% to 10.9%]), but the deep learning method had a higher sensitivity in the melanoma cohort (71.6% vs 53.7% [difference, 17.9%; 95% CI, 1.82% to 34.0%]). The deep learning method had higher sensitivity in the mendelian genes (prostate cancer: 99.7% vs 95.1% [difference, 4.6%; 95% CI, 3.0% to 6.3%]; melanoma: 91.7% vs 86.2% [difference, 5.5%; 95% CI, 2.2% to 8.8%]). Conclusions and Relevance: Among a convenience sample of 2 independent cohorts of patients with prostate cancer and melanoma, germline genetic testing using deep learning, compared with the current standard genetic testing method, was associated with higher sensitivity and specificity for detection of pathogenic variants. Further research is needed to understand the relevance of these findings with regard to clinical outcomes.

Somatic structural variants drive distinct modes of oncogenesis in melanoma.

The diversity of structural variants (SVs) in melanoma and how they impact oncogenesis are incompletely known. We performed harmonized analysis of SVs across melanoma histologic and genomic subtypes, and we identified distinct global properties between subtypes. These included the frequency and size of SVs and SV classes, their relation to chromothripsis events, and the impact on cancer-related genes of SVs that alter topologically associated domain (TAD) boundaries. Following our prior identification of double-stranded break repair deficiency in a subset of triple-wild-type cutaneous melanoma, we identified MRE11 and NBN loss-of-function SVs in melanomas with this mutational signature. Experimental knockouts of MRE11 and NBN, followed by olaparib cell viability assays in melanoma cells, indicated that dysregulation of each of these genes may cause sensitivity to PARP inhibitors in cutaneous melanomas. Broadly, harmonized analysis of melanoma SVs revealed distinct global genomic properties and molecular drivers, which may have biological and therapeutic impact.

Integrative Analysis of Germline Rare Variants in Clear and Non-clear Cell Renal Cell Carcinoma.

Background and objective: Previous germline studies on renal cell carcinoma (RCC) have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification, which might have led to an inaccurate estimation of genetic risk. Here, we aim to analyze the major germline drivers of RCC risk and clinically relevant but underexplored germline variant types. Methods: We first characterized germline pathogenic variants (PVs), cryptic splice variants, and copy number variants (CNVs) in 1436 unselected RCC patients. To evaluate the enrichment of PVs in RCC, we conducted a case-control study of 1356 RCC patients ancestry matched with 16 512 cancer-free controls using approaches accounting for population stratification and histological subtypes, followed by characterization of secondary somatic events. Key findings and limitations: Clear cell RCC patients (n = 976) exhibited a significant burden of PVs in VHL compared with controls (odds ratio [OR]: 39.1, p = 4.95e-05). Non-clear cell RCC patients (n = 380) carried enrichment of PVs in FH (OR: 77.9, p = 1.55e-08) and MET (OR: 1.98e11, p = 2.07e-05). In a CHEK2-focused analysis with European participants, clear cell RCC (n = 906) harbored nominal enrichment of low-penetrance CHEK2 variants-p.Ile157Thr (OR: 1.84, p = 0.049) and p.Ser428Phe (OR: 5.20, p = 0.045), while non-clear cell RCC (n = 295) exhibited nominal enrichment of CHEK2 loss of function PVs (OR: 3.51, p = 0.033). Patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset than patients without germline PVs (mean: 46.0 vs 60.2 yr, p < 0.0001), and more than half had secondary somatic events affecting the same gene (n = 10/15, 66.7%). Conversely, CHEK2 PV carriers exhibited a similar age of onset to patients without germline PVs (mean: 60.1 vs 60.2 yr, p = 0.99), and only 30.4% carried somatic events in CHEK2 (n = 7/23). Finally, pathogenic germline cryptic splice variants were identified in SDHA and TSC1, and pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. Conclusions and clinical implications: This analysis supports the existing link between several RCC risk genes and RCC risk manifesting in earlier age of onset. It calls for caution when assessing the role of CHEK2 due to the burden of founder variants with varying population frequency. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants. Patient summary: In this study, we carefully compared the frequency of rare inherited mutations with a focus on patients' genetic ancestry. We discovered that subtle variations in genetic background may confound a case-control analysis, especially in evaluating the cancer risk associated with specific genes, such as CHEK2. We also identified previously less explored forms of rare inherited mutations, which could potentially increase the risk of kidney cancer.

Dual Immune Checkpoint Inhibition in Patients With Aggressive Thyroid Carcinoma: A Phase 2 Nonrandomized Clinical Trial.

Importance: Aggressive thyroid carcinoma, including radioiodine refractory (RAIR) differentiated thyroid carcinoma (DTC), medullary thyroid carcinoma (MTC), and anaplastic thyroid carcinoma (ATC), are associated with significant morbidity and mortality and have limited therapeutic options. Distinct immune profiles have been identified in thyroid cancer subtypes suggesting they may be susceptible to immune checkpoint inhibition. Objective: To evaluate the efficacy of anti-programmed cell death 1 nivolumab and anti-cytotoxic lymphocyte-associated protein 4 ipilimumab in patients with aggressive thyroid carcinoma. Design, Setting, and Participants: This phase 2 nonrandomized clinical trial enrolled patients with RAIR DTC in a single center from October 2017 to May 2019, with exploratory cohorts in MTC and ATC. The data were analyzed between June 2021 and September 2023. Intervention: Intravenous nivolumab, 3 mg/kg, every 2 weeks and ipilimumab, 1 mg/kg, every 6 weeks until disease progression, intolerable adverse events, or a maximum duration of 2 years. Main Outcomes and Measures: The primary end point of the study was objective response rate (ORR) in RAIR DTC, which was scored according to RECIST (Response Evaluation Criteria in Solid Tumours), version 1.1. Key secondary end points included safety, progression-free survival, overall survival, and biomarker analyses. Results: A total of 51 patients were registered, and 49 patients were evaluable for analysis. The median (range) age was 65 years (30-88 years), and 25 participants (51%) were female. ORR in the DTC cohort was 9.4% (3/32 [95% CI, 2.8%-28.5%]), with all partial responses in either oncocytic carcinoma (2/6 [33.0%]) or poorly differentiated thyroid carcinoma (1/5 [20.0%]). Clinical benefit rates were 62.5% (20/32) in the overall DTC cohort, including 83.3% (5/6) in oncocytic carcinoma and 40% (2/5) in poorly differentiated thyroid carcinoma. ORR in the exploratory ATC cohort was 30.0% (3/10 [95% CI, 6.7%-65.2%]), with a clinical benefit rates of 50.0% (5/10). No responses were observed in the exploratory MTC cohort. The safety profile was similar to prior reports with dual immune checkpoint inhibition (pruritus, rash, diarrhea, fatigue, and elevation of lipase and liver enzymes). The presence of NRAS tumor genetic sequence variations, but not BRAF V600E, was associated with worse outcomes. Conclusions and Relevance: This phase 2 nonrandomized clinical trial reported clinical activity of dual immune checkpoint inhibition in aggressive thyroid cancer. The study did not meet its end point in the primary population of RAIR DTC and does not support further investigation in non-biomarker-selected DTC. However, the signal observed in ATC may merit further evaluation. Trial Registration: ClinicalTrials.gov Identifier: NCT03246958.

Biomarkers of pembrolizumab efficacy in advanced anal squamous cell carcinoma: analysis of a phase II clinical trial and a cohort of long-term responders.

BACKGROUND: Recent trials suggest that programmed cell death 1 (PD-1)-directed immunotherapy may be beneficial for some patients with anal squamous cell carcinoma and biomarkers predictive of response are greatly needed. METHODS: This multicenter phase II clinical trial (NCT02919969) enrolled patients with metastatic or locally advanced incurable anal squamous cell carcinoma (n=32). Patients received pembrolizumab 200 mg every 3 weeks. The primary endpoint of the trial was objective response rate (ORR). Exploratory objectives included analysis of potential predictive biomarkers including assessment of tumor-associated immune cell populations with multichannel immunofluorescence and analysis of circulating tumor tissue modified viral-human papillomavirus DNA (TTMV-HPV DNA) using serially collected blood samples. To characterize the clinical features of long-term responders, we combined data from our prospective trial with a retrospective cohort of patients with anal cancer treated with anti-PD-1 immunotherapy (n=18). RESULTS: In the phase II study, the ORR to pembrolizumab monotherapy was 9.4% and the median progression-free survival was 2.2 months. Despite the high level of HPV positivity observed with circulating TTMV-HPV DNA testing, the majority of patients had low levels of tumor-associated CD8+PD-1+ T cells on pretreatment biopsy. Patients who benefited from pembrolizumab had decreasing TTMV-HPV DNA scores and a complete responder's TTMV-HPV DNA became undetectable. Long-term pembrolizumab responses were observed in one patient from the trial (5.3 years) and three patients (2.5, 6, and 8 years) from the retrospective cohort. Long-term responders had HPV-positive tumors, lacked liver metastases, and achieved a radiological complete response. CONCLUSIONS: Pembrolizumab has durable efficacy in a rare subset of anal cancers. However, despite persistence of HPV infection, indicated by circulating HPV DNA, most advanced anal cancers have low numbers of tumor-associated CD8+PD-1+ T cells and are resistant to pembrolizumab.

Integrative Analysis of Germline Rare Variants in Clear and Non-Clear Cell Renal Cell Carcinoma.

IMPORTANCE: RCC encompasses a set of histologically distinct cancers with a high estimated genetic heritability, of which only a portion is currently explained. Previous rare germline variant studies in RCC have usually pooled clear and non-clear cell RCCs and have not adequately accounted for population stratification that may significantly impact the interpretation and discovery of certain candidate risk genes. OBJECTIVE: To evaluate the enrichment of germline PVs in established cancer-predisposing genes (CPGs) in clear cell and non-clear cell RCC patients compared to cancer-free controls using approaches that account for population stratification and to identify unconventional types of germline RCC risk variants that confer an increased risk of developing RCC. DESIGN SETTING AND PARTICIPANTS: In 1,436 unselected RCC patients with sufficient data quality, we systematically identified rare germline PVs, cryptic splice variants, and copy number variants (CNVs). From this unselected cohort, 1,356 patients were ancestry-matched with 16,512 cancer-free controls, and gene-level enrichment of rare germline PVs were assessed in 143 CPGs, followed by an investigation of somatic events in matching tumor samples. MAIN OUTCOMES AND MEASURES: Gene-level burden of rare germline PVs, identification of secondary somatic events accompanying the germline PVs, and characterization of less-explored types of rare germline PVs in RCC patients. RESULTS: In clear cell RCC (n = 976 patients), patients exhibited significantly higher prevalence of PVs in VHL compared to controls (OR: 39.1, 95% CI: 7.01-218.07, p-value:4.95e-05, q-value:0.00584). In non-clear cell RCC (n = 380 patients), patients carried enriched burden of PVs in FH (OR: 77.9, 95% CI: 18.68-324.97, p-value:1.55e-08, q-value: 1.83e-06) and MET (OR: 1.98e11, 95% CI: 0-inf, p-value: 2.07e-05, q-value: 3.50e-07). In a CHEK2-focused analysis with European cases and controls, clear cell RCC patients (n=906 European patients) harbored nominal enrichment of the previously reported low-penetrance CHEK2 variants, p.Ile157Thr (OR:1.84, 95% CI: 1.00-3.36, p-value:0.049) and p.Ser428Phe (OR:5.20, 95% CI: 1.00-26.40, p-value:0.045) while non-clear cell RCC patients (n=295 European patients) exhibited nominal enrichment of CHEK2 LOF germline PVs (OR: 3.51, 95% CI: 1.10-11.10, p-value: 0.033). RCC patients with germline PVs in FH, MET, and VHL exhibited significantly earlier age of cancer onset compared to patients without any germline PVs in CPGs (Mean: 46.0 vs 60.2 years old, Tukey adjusted p-value < 0.0001), and more than half had secondary somatic events affecting the same gene (n=10/15, 66.7%, 95% CI: 38.7-87.0%). Conversely, patients with rare germline PVs in CHEK2 exhibited a similar age of disease onset to patients without any identified germline PVs in CPGs (Mean: 60.1 vs 60.2 years old, Tukey adjusted p-value: 0.99), and only 30.4% of the patients carried secondary somatic events in CHEK2 (n=7/23, 95% CI: 14.1-53.0%). Finally, rare pathogenic germline cryptic splice variants underexplored in RCC were identified in SDHA and TSC1, and rare pathogenic germline CNVs were found in 18 patients, including CNVs in FH, SDHA, and VHL. CONCLUSIONS AND RELEVANCE: This systematic analysis supports the existing link between several RCC risk genes and elevated RCC risk manifesting in earlier age of RCC onset. Our analysis calls for caution when assessing the role of germline PVs in CHEK2 due to the burden of founder variants with varying population frequency in different ancestry groups. It also broadens the definition of the RCC germline landscape of pathogenicity to incorporate previously understudied types of germline variants, such as cryptic splice variants and CNVs.

Genomic Features of Muscle-invasive Bladder Cancer Arising After Prostate Radiotherapy.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is a rare but serious event following definitive radiation for prostate cancer. Radiation-associated MIBC (RA-MIBC) can be difficult to manage given the challenges of delivering definitive therapy to a previously irradiated pelvis. The genomic landscape of RA-MIBC and whether it is distinct from non-RA-MIBC are unknown. OBJECTIVE: To define mutational features of RA-MIBC and compare the genomic landscape of RA-MIBC with that of non-RA-MIBC. DESIGN, SETTING, AND PARTICIPANTS: We identified patients from our institution who received radiotherapy for prostate cancer and subsequently developed MIBC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We performed whole exome sequencing of bladder tumors from RA-MIBC patients. Tumor genetic alterations including mutations, copy number alterations, and mutational signatures were identified and were compared with genetic features of non-RA-MIBC. We used the Kaplan-Meier method to estimate recurrence-free (RFS) and overall (OS) survival. RESULTS AND LIMITATIONS: We identified 19 RA-MIBC patients with available tumor tissue (n = 22 tumors) and clinical data. The median age was 76 yr, and the median time from prostate cancer radiation to RA-MIBC was 12 yr. The median RFS was 14.5 mo and the median OS was 22.0 mo. Compared with a cohort of non-RA-MIBC analyzed in parallel, there was no difference in tumor mutational burden, but RA-MIBCs had a significantly increased number of short insertions and deletions (indels) consistent with previous radiation exposure. We identified mutation signatures characteristic of APOBEC-mediated mutagenesis, aging, and homologous recombination deficiency. The frequency of mutations in many known bladder cancer genes, including TP53, KDM6A, and RB1, as well as copy number alterations such as CDKN2A loss was similar in RA-MIBC and non-RA-MIBC. CONCLUSIONS: We identified unique mutational properties that likely contribute to the distinct biological and clinical features of RA-MIBC. PATIENT SUMMARY: Bladder cancer is a rare but serious diagnosis following radiation for prostate cancer. We characterized genetic features of bladder tumors arising after prostate radiotherapy, and identify similarities with and differences from bladder tumors from patients without previous radiation.

Discovery and Features of an Alkylating Signature in Colorectal Cancer.

Several risk factors have been established for colorectal cancer, yet their direct mutagenic effects in patients' tumors remain to be elucidated. Here, we leveraged whole-exome sequencing data from 900 colorectal cancer cases that had occurred in three U.S.-wide prospective studies with extensive dietary and lifestyle information. We found an alkylating signature that was previously undescribed in colorectal cancer and then showed the existence of a similar mutational process in normal colonic crypts. This alkylating signature is associated with high intakes of processed and unprocessed red meat prior to diagnosis. In addition, this signature was more abundant in the distal colorectum, predicted to target cancer driver mutations KRAS p.G12D, KRAS p.G13D, and PIK3CA p.E545K, and associated with poor survival. Together, these results link for the first time a colorectal mutational signature to a component of diet and further implicate the role of red meat in colorectal cancer initiation and progression. SIGNIFICANCE: Colorectal cancer has several lifestyle risk factors, but the underlying mutations for most have not been observed directly in tumors. Analysis of 900 colorectal cancers with whole-exome sequencing and epidemiologic annotations revealed an alkylating mutational signature that was associated with red meat consumption and distal tumor location, as well as predicted to target KRAS p.G12D/p.G13D.This article is highlighted in the In This Issue feature, p. 2355.

Integrating molecular profiles into clinical frameworks through the Molecular Oncology Almanac to prospectively guide precision oncology.

Tumor molecular profiling of single gene-variant ('first-order') genomic alterations informs potential therapeutic approaches. Interactions between such first-order events and global molecular features (for example, mutational signatures) are increasingly associated with clinical outcomes, but these 'second-order' alterations are not yet accounted for in clinical interpretation algorithms and knowledge bases. We introduce the Molecular Oncology Almanac (MOAlmanac), a paired clinical interpretation algorithm and knowledge base to enable integrative interpretation of multimodal genomic data for point-of-care decision making and translational-hypothesis generation. We benchmarked MOAlmanac to a first-order interpretation method across multiple retrospective cohorts and observed an increased number of clinical hypotheses from evaluation of molecular features and profile-to-cell line matchmaking. When applied to a prospective precision oncology trial cohort, MOAlmanac nominated a median of two therapies per patient and identified therapeutic strategies administered in 47% of patients. Overall, we present an open-source computational method for integrative clinical interpretation of individualized molecular profiles.

Molecular correlates of response to eribulin and pembrolizumab in hormone receptor-positive metastatic breast cancer.

Immune checkpoint inhibitors (ICIs) have minimal therapeutic effect in hormone receptor-positive (HR+ ) breast cancer. We present final overall survival (OS) results (n = 88) from a randomized phase 2 trial of eribulin ± pembrolizumab for patients with metastatic HR+ breast cancer, computationally dissect genomic and/or transcriptomic data from pre-treatment tumors (n = 52) for molecular associations with efficacy, and identify cytokine changes differentiating response and ICI-related toxicity (n = 58). Despite no improvement in OS with combination therapy (hazard ratio 0.95, 95% CI 0.59-1.55, p = 0.84), immune infiltration and antigen presentation distinguished responding tumors, while tumor heterogeneity and estrogen signaling independently associated with resistance. Moreover, patients with ICI-related toxicity had lower levels of immunoregulatory cytokines. Broadly, we establish a framework for ICI response in HR+ breast cancer that warrants diagnostic and therapeutic validation. ClinicalTrials.gov Registration: NCT03051659.

Intrinsic Immunogenicity of Small Cell Lung Carcinoma Revealed by Its Cellular Plasticity.

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861.

Identification of cancer driver genes based on nucleotide context.

Cancer genomes contain large numbers of somatic mutations but few of these mutations drive tumor development. Current approaches either identify driver genes on the basis of mutational recurrence or approximate the functional consequences of nonsynonymous mutations by using bioinformatic scores. Passenger mutations are enriched in characteristic nucleotide contexts, whereas driver mutations occur in functional positions, which are not necessarily surrounded by a particular nucleotide context. We observed that mutations in contexts that deviate from the characteristic contexts around passenger mutations provide a signal in favor of driver genes. We therefore developed a method that combines this feature with the signals traditionally used for driver-gene identification. We applied our method to whole-exome sequencing data from 11,873 tumor-normal pairs and identified 460 driver genes that clustered into 21 cancer-related pathways. Our study provides a resource of driver genes across 28 tumor types with additional driver genes identified according to mutations in unusual nucleotide contexts.

Integrated molecular drivers coordinate biological and clinical states in melanoma.

We performed harmonized molecular and clinical analysis on 1,048 melanomas and discovered markedly different global genomic properties among subtypes (BRAF, (N)RAS, NF1, triple wild-type (TWT)), subtype-specific preferences for secondary driver genes and active mutational processes previously unreported in melanoma. Secondary driver genes significantly enriched in specific subtypes reflected preferential dysregulation of additional pathways, such as induction of transforming growth factor-ß signaling in BRAF melanomas and inactivation of the SWI/SNF complex in (N)RAS melanomas, and select co-mutation patterns coordinated selective response to immune checkpoint blockade. We also defined the mutational landscape of TWT melanomas and revealed enrichment of DNA-repair-defect signatures in this subtype, which were associated with transcriptional downregulation of key DNA-repair genes, and may revive previously discarded or currently unconsidered therapeutic modalities for genomically stratified melanoma patient subsets. Broadly, harmonized meta-analysis of melanoma whole exomes revealed distinct molecular drivers that may point to multiple opportunities for biological and therapeutic investigation.

Genomic Predictors of Good Outcome, Recurrence, or Progression in High-Grade T1 Non-Muscle-Invasive Bladder Cancer.

High-grade T1 (HGT1) bladder cancer is the highest risk subtype of non-muscle-invasive bladder cancer with unpredictable outcome and poorly understood risk factors. Here, we examined the association of somatic mutation profiles with nonrecurrent disease (GO, good outcome), recurrence (R), or progression (PD) in a cohort of HGT1 patients. Exome sequencing was performed on 62 HGT1 and 15 matched normal tissue samples. Both tumor only (TO) and paired analyses were performed, focusing on 95 genes known to be mutated in bladder cancer. Somatic mutations, copy-number alterations, mutation load, and mutation signatures were studied. Thirty-three GO, 10 R, 18 PD, and 1 unknown outcome patients were analyzed. Tumor mutational burden (TMB) was similar to muscle-invasive disease and was highest in GO, intermediate in PD, and lowest in R patients (P = 0.017). DNA damage response gene mutations were associated with higher TMB (P < 0.0001) and GO (P = 0.003). ERCC2 and BRCA2 mutations were associated with GO. TP53, ATM, ARID1A, AHR, and SMARCB1 mutations were more frequent in PD. Focal copy-number gain in CCNE1 and CDKN2A deletion was enriched in PD or R (P = 0.047; P = 0.06). APOBEC (46%) and COSMIC5 (34%) signatures were most frequent. APOBEC-A and ERCC2 mutant tumors (COSMIC5) were associated with GO (P = 0.047; P = 0.0002). pT1b microstaging was associated with a genomic cluster (P = 0.05) with focal amplifications of E2F3/SOX4, PVRL4, CCNE1, and TP53 mutations. Findings were validated using external public datasets. These findings require confirmation but suggest that management of HGT1 bladder cancer may be improved via molecular characterization to predict outcome. SIGNIFICANCE: Detailed genetic analyses of HGT1 bladder tumors identify features that correlate with outcome, e.g., high mutational burden, ERCC2 mutations, and high APOBEC-A/ERCC2 mutation signatures were associated with good outcome.