Assessment of genomic alterations in non-syndromic von Hippel-Lindau: Insight from integrating somatic and germline next generation sequencing genomic data.

Von Hippel-Lindau (VHL) syndrome is a hereditary cancer genetic condition associated with inactivating pathogenic alterations in the VHL tumor suppressor gene located at 3p (short arm of chromosome 3). Classic features of VHL include clear cell renal cell carcinoma, hemangioblastomas of the brain, spinal cord, and retina, pheochromocytoma, pancreatic cysts, and neuroendocrine tumors. Two sets of genomic information may be available from patients with VHL: the germline data showing the constitutional genetic profile and somatic profile obtained from patient tumor(s). Here we present both somatic and germline dataset from heterozygous carriers of germline VHL variants who exhibit non-syndromic VHL phenotypes. This data description article accompanies the paper "Pathogenicity of VHL variants in families with non-syndromic von Hippel-Lindau phenotypes: an integrated evaluation of germline and somatic genomic results'' by Huma Q. Rana, Diane R. Koeller, Alison Schwartz, Danielle K. Manning, Katherine A. Schneider, Katherine M. Krajewski, Toni K. Choueiri, Neal I. Lindeman, Judy E. Garber, Arezou A. Ghazani. We provide next generation sequencing (NGS) data obtained from DNA from tumors (renal cancer, bladder cancer, and cerebral hemangioblastoma) of three VHL carriers. The somatic dataset was analyzed for single nucleotide variants (SNVs) and copy number variants (CNVs) in 447 cancer genes, and structural variation (SVs) in 191 regions across 60 genes for rearrangements. We also present germline raw NGS data and analyzed SNV and CNV data in exonic regions of 133 hereditary cancer genes obtained from the peripheral blood of two VHL carriers.

Next-generation sequencing informs diagnosis and identifies unexpected therapeutic targets in lung squamous cell carcinomas.

OBJECTIVES: Potentially targetable genomic alterations have been identified in lung squamous cell carcinoma (LUSC), but none have yet translated into effective therapy. We examined potential benefits of next generation sequencing (NGS) in a cohort of consecutive LUSC patients with emphasis on distinctions between smokers and light/never smokers and implications for clinical trial enrollment. METHODS: We retrospectively evaluated results from an internally developed NGS assay (OncoPanel) targeting ∼300 genes with a mean overall target coverage of >200x for consecutive LUSC seen at our institution over 30 months. RESULTS: Tissue was obtained from 172 patients for targeted NGS. 42 (24 %) samples were insufficient for testing. Median age of tested patients was 66, including 87 % moderate/heavy versus 13 % light/never smokers; 66 % were stage IIIB or IV. Of 130 patients with evaluable NGS results, 49 (38 %) had at least 1 alteration qualifying for enrollment to a LungMAP treatment arm (PIK3CA, MET, FGFR family, cell cycle, or homologous recombination pathways) or for an approved therapy or other clinical trial (e.g. EGFR sensitizing mutations, MET exon 14 splice mutations, TSC1/2 mutation, or microsatellite instability). Therapeutic targets were enriched in light/never smokers (47 % vs 35 % moderate/heavy smokers). Unexpectedly, genomic features suggested an alternative diagnosis (metastatic cutaneous squamous carcinoma; mesothelioma) in 7 patients, including 35 % of never/light smokers. CONCLUSION: NGS in a real-world LUSC cohort yields potentially targetable genomic alterations informing clinical trial enrollment and approved therapies and critical diagnostic insights. Our findings strongly support current guidelines recommending mutational profiling of LUSC arising in light/never smoking patients; the utility of sequencing in smokers with LUSC appears to be limited to identification of research targets.

Improved outcomes in PI3K-pathway-altered metastatic HPV oropharyngeal cancer.

While it has been recognized that human papillomavirus-associated (HPV-associated) oropharyngeal cancer (OPC) portends an improved prognosis, distinct patterns of disease recurrence have emerged. Molecular characterization of this subset of HPV patients remains unexplored. We evaluated 52 metastatic HPV+ OPC patients from our institution and paired massively parallel sequencing data with clinical parameters and survival outcomes in 81% of patients. Genomic data were then compared with 2 molecularly defined, curable HPV+ cohorts. Metastatic HPV+ OPC patients with pulmonary-only metastases demonstrated worse outcomes. Nonexclusive somatic alterations in KMT2D and PIK3CA were most frequent, with PRKDC alterations occurring at higher frequency when compared with all sequenced HPV+ OPC patients. PI3K pathway alterations were associated with improved outcomes among metastatic HPV+ OPC patients. We demonstrate subtle differences in the mutational landscape between curable and metastatic HPV+ OPC populations, with a trend towards more frequent DNA repair protein alterations in the latter. We demonstrate improved outcomes when PI3K pathway alterations are present in these patients. We provide molecular insights for this important HPV+ subgroup that have significant therapeutic implications.

Frameshift events predict anti-PD-1/L1 response in head and neck cancer.

Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.

Validation of OncoPanel: A Targeted Next-Generation Sequencing Assay for the Detection of Somatic Variants in Cancer.

CONTEXT: - The analysis of somatic mutations across multiple genes in cancer specimens may be used to aid clinical decision making. The analytical validation of targeted next-generation sequencing panels is important to assess accuracy and limitations. OBJECTIVE: - To report the development and validation of OncoPanel, a custom targeted next-generation sequencing assay for cancer. DESIGN: - OncoPanel was designed for the detection of single-nucleotide variants, insertions and deletions, copy number alterations, and structural variants across 282 genes with evidence as drivers of cancer biology. We implemented a validation strategy using formalin-fixed, paraffin-embedded, fresh or frozen samples compared with results obtained by clinically validated orthogonal technologies. RESULTS: - OncoPanel achieved 98% sensitivity and 100% specificity for the detection of single-nucleotide variants, and 84% sensitivity and 100% specificity for the detection of insertions and deletions compared with single-gene assays and mass spectrometry-based genotyping. Copy number detection achieved 86% sensitivity and 98% specificity compared with array comparative genomic hybridization. The sensitivity of structural variant detection was 74% compared with karyotype, fluorescence in situ hybridization, and polymerase chain reaction. Sensitivity was affected by inconsistency in the detection of FLT3 and NPM1 alterations and IGH rearrangements due to design limitations. Limit of detection studies demonstrated 98.4% concordance across triplicate runs for variants with allele fraction greater than 0.1 and at least 50× coverage. CONCLUSIONS: - The analytical validation of OncoPanel demonstrates the ability of targeted next-generation sequencing to detect multiple types of genetic alterations across a panel of genes implicated in cancer biology.

Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features Accounts for More Than Half of "Carcinomas" Harboring RAS Mutations.

BACKGROUND: Molecular testing of thyroid nodules is increasingly being utilized to guide clinical management decisions. RAS mutations are the most frequent mutations detected in the context of an indeterminate fine-needle aspiration (FNA) diagnosis. The term "noninvasive follicular thyroid neoplasm with papillary-like nuclear features" (NIFTP) was recently introduced to promote conservative management of tumors previously classified as noninvasive follicular variant of papillary thyroid carcinoma (FVPTC). This change in terminology was based on the indolent clinical behavior of these tumors and their molecular profile, which includes frequent RAS mutations. The aim of this study was to determine the percentage of RAS-mutant "carcinomas" that would now be classified as NIFTPs. METHODS: A search was performed for cases with known activating RAS mutations in a database of 199 thyroid carcinomas that underwent molecular characterization as part of Profile:Oncopanel between July 2013 and July 2015. Cases of FVPTC were re-reviewed to identify tumors that now would be categorized as NIFTP. Preceding FNA diagnoses were recorded, and cases with an indeterminate FNA result (defined as a diagnosis of atypia/follicular lesion of undetermined significance, suspicious for follicular neoplasm, or suspicious for malignancy) were identified. RESULTS: A total of 27 RAS-mutant thyroid tumors were identified. Fifteen (56%) cases had an NRAS mutation, nine (33%) had an HRAS mutation, and three (11%) had a KRAS mutation. Twenty-four (89%) cases had a preceding FNA, 19 (79%) of which had an indeterminate FNA diagnosis. The surgical resection specimen demonstrated FVPTC in 20 (74%) cases, classical type PTC in two (7%), solid variant of PTC in one (4%), and follicular thyroid carcinoma in four (15%). Of the 20 FVPTCs, 16 (80%) would now be classified as NIFTP. NIFTPs accounted for 59% of RAS-mutant carcinomas overall and 63% of RAS-mutant carcinomas with a prior indeterminate FNA diagnosis. CONCLUSION: NIFTPs accounted for more than half of RAS-mutant "carcinomas" in this cohort. In cases where clinical and sonographic data support a low-risk phenotype, these results suggest that a lobectomy should be considered as the initial surgical approach for a nodule with an indeterminate FNA diagnosis and a RAS mutation.

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing.

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

Institutional implementation of clinical tumor profiling on an unselected cancer population.

BACKGROUND. Comprehensive genomic profiling of a patient's cancer can be used to diagnose, monitor, and recommend treatment. Clinical implementation of tumor profiling in an enterprise-wide, unselected cancer patient population has yet to be reported. METHODS. We deployed a hybrid-capture and massively parallel sequencing assay (OncoPanel) for all adult and pediatric patients at our combined cancer centers. Results were categorized by pathologists based on actionability. We report the results for the first 3,727 patients tested. RESULTS. Our cohort consists of cancer patients unrestricted by disease site or stage. Across all consented patients, half had sufficient and available (>20% tumor) material for profiling; once specimens were received in the laboratory for pathology review, 73% were scored as adequate for genomic testing. When sufficient DNA was obtained, OncoPanel yielded a result in 96% of cases. 73% of patients harbored an actionable or informative alteration; only 19% of these represented a current standard of care for therapeutic stratification. The findings recapitulate those of previous studies of common cancers but also identify alterations, including in AXL and EGFR, associated with response to targeted therapies. In rare cancers, potentially actionable alterations suggest the utility of a "cancer-agnostic" approach in genomic profiling. Retrospective analyses uncovered contextual genomic features that may inform therapeutic response and examples where diagnoses revised by genomic profiling markedly changed clinical management. CONCLUSIONS. Broad sequencing-based testing deployed across an unselected cancer cohort is feasible. Genomic results may alter management in diverse scenarios; however, additional barriers must be overcome to enable precision cancer medicine on a large scale. FUNDING. This work was supported by DFCI, BWH, and the National Cancer Institute (5R33CA155554 and 5K23CA157631).

KRAS and NKX2-1 Mutations in Invasive Mucinous Adenocarcinoma of the Lung.

INTRODUCTION: Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood. METHODS: We apply targeted next-generation sequencing to characterize the mutational profiles of 21 invasive mucinous adenocarcinomas, mixed mucinous/nonmucinous adenocarcinomas, and adenocarcinomas with mucinous features of the lung and validate key findings on 954 additional lung adenocarcinomas from our institution and 514 lung adenocarcinomas from The Cancer Genome Atlas. RESULTS: Sequencing identifies pathogenic mutations in the oncogenes Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), erb-b2 receptor tyrosine kinase 2 (ERBB2), and anaplastic lymphoma receptor tyrosine kinase (ALK) and recurrent mutations in tumor protein p53 (TP53), serine/threonine kinase 11 (STK11), NK2 homeobox 1 (NKX2-1), and SET domain containing 2 (SETD2). In the combined discovery and validation cohorts, we identify nine neoplasms with distinct molecular and pathological features. All are invasive mucinous adenocarcinomas or mixed mucinous/nonmucinous adenocarcinomas with mutations of KRAS and frameshift or nonsense mutations of NKX2-1. Immunohistochemical analysis shows that these neoplasms are associated with altered differentiation states, including loss of expression of the pulmonary marker thyroid transcription factor 1 (also called Nkx2.1) and expression of gastrointestinal markers. CONCLUSIONS: These findings describe recurrent NKX2-1 mutations in invasive mucinous adenocarcinomas of the lung and support NKX2-1 as a lineage-specific tumor suppressor gene in lung carcinogenesis.

MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression.

PURPOSE: Non-small-cell lung cancers (NSCLCs) harboring mutations in MET exon 14 and its flanking introns may respond to c-Met inhibitors. We sought to describe the clinical, pathologic, and genomic characteristics of patients with cancer with MET exon 14 mutations. PATIENTS AND METHODS: We interrogated next-generation sequencing results from 6,376 cancers to identify those harboring MET exon 14 mutations. Clinical characteristics of MET exon 14 mutated NSCLCs were compared with those of NSCLCs with activating mutations in KRAS and EGFR. Co-occurring genomic mutations and copy number alterations were identified. c-Met immunohistochemistry and real-time polymerase chain reaction to detect exon 14 skipping were performed where sufficient tissue was available. RESULTS: MET exon 14 mutations were identified in 28 of 933 nonsquamous NSCLCs (3.0%) and were not seen in other cancer types in this study. Patients with MET exon 14-mutated NSCLC were significantly older (median age, 72.5 years) than patients with EGFR-mutant (median age, 61 years; P < .001) or KRAS-mutant NSCLC (median age, 65 years; P < .001). Among patients with MET exon 14 mutations, 68% were women, and 36% were never-smokers. Stage IV MET exon 14-mutated NSCLCs were significantly more likely to have concurrent MET genomic amplification (mean ratio of MET to chromosome 7, 4.3) and strong c-Met immunohistochemical expression (mean H score, 253) than stage IA to IIIB MET exon 14-mutated NSCLCs (mean ratio of MET to chromosome 7, 1.4; P = .007; mean H score, 155; P = .002) and stage IV MET exon 14-wild-type NSCLCs (mean ratio of MET to chromosome 7, 1.2; P < .001; mean H score, 142; P < .001). A patient whose lung cancer harbored a MET exon 14 mutation with concurrent genomic amplification of the mutated MET allele experienced a major partial response to the c-Met inhibitor crizotinib. CONCLUSION: MET exon 14 mutations represent a clinically unique molecular subtype of NSCLC. Prospective clinical trials with c-Met inhibitors will be necessary to validate MET exon 14 mutations as an important therapeutic target in NSCLC.

Targeted next-generation sequencing reveals high frequency of mutations in epigenetic regulators across treatment-naïve patient melanomas.

BACKGROUND: Recent developments in genomic sequencing have advanced our understanding of the mutations underlying human malignancy. Melanoma is a prototype of an aggressive, genetically heterogeneous cancer notorious for its biologic plasticity and predilection towards developing resistance to targeted therapies. Evidence is rapidly accumulating that dysregulated epigenetic mechanisms (DNA methylation/demethylation, histone modification, non-coding RNAs) may play a central role in the pathogenesis of melanoma. Therefore, we sought to characterize the frequency and nature of mutations in epigenetic regulators in clinical, treatment-naïve, patient melanoma specimens obtained from one academic institution. RESULTS: Targeted next-generation sequencing for 275 known and investigative cancer genes (of which 41 genes, or 14.9 %, encoded an epigenetic regulator) of 38 treatment-naïve patient melanoma samples revealed that 22.3 % (165 of 740) of all non-silent mutations affected an epigenetic regulator. The most frequently mutated genes were BRAF, MECOM, NRAS, TP53, MLL2, and CDKN2A. Of the 40 most commonly mutated genes, 12 (30.0 %) encoded epigenetic regulators, including genes encoding enzymes involved in histone modification (MECOM, MLL2, SETD2), chromatin remodeling (ARID1B, ARID2), and DNA methylation and demethylation (TET2, IDH1). Among the 38 patient melanoma samples, 35 (92.1 %) harbored at least one mutation in an epigenetic regulator. The genes with the highest number of total UVB-signature mutations encoded epigenetic regulators, including MLL2 (100 %, 16 of 16) and MECOM (82.6 %, 19 of 23). Moreover, on average, epigenetic genes harbored a significantly greater number of UVB-signature mutations per gene than non-epigenetic genes (3.7 versus 2.4, respectively; p = 0.01). Bioinformatics analysis of The Cancer Genome Atlas (TCGA) melanoma mutation dataset also revealed a frequency of mutations in the 41 epigenetic genes comparable to that found within our cohort of patient melanoma samples. CONCLUSIONS: Our study identified a high prevalence of somatic mutations in genes encoding epigenetic regulators, including those involved in DNA demethylation, histone modification, chromatin remodeling, and microRNA processing. Moreover, UVB-signature mutations were found more commonly among epigenetic genes than in non-epigenetic genes. Taken together, these findings further implicate epigenetic mechanisms, particularly those involving the chromatin-remodeling enzyme MECOM/EVI1 and histone-modifying enzyme MLL2, in the pathobiology of melanoma.

Prospective enterprise-level molecular genotyping of a cohort of cancer patients.

Ongoing cancer genome characterization studies continue to elucidate the spectrum of genomic abnormalities that drive many cancers, and in the clinical arena assessment of the driver genetic alterations in patients is playing an increasingly important diagnostic and/or prognostic role for many cancer types. However, the landscape of genomic abnormalities is still unknown for less common cancers, and the influence of specific genotypes on clinical behavior is often still unclear. To address some of these deficiencies, we developed Profile, a prospective cohort study to obtain genomic information on all patients at a large tertiary care medical center for cancer-related care. We enrolled patients with any cancer diagnosis, and, for each patient (unselected for cancer site or type) we applied mass spectrometric genotyping (OncoMap) of 471 common recurrent mutations in 41 cancer-related genes. We report the results of the first 5000 patients, of which 26% exhibited potentially actionable somatic mutations. These observations indicate the utility of genotyping in advancing the field of precision oncology.