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High-grade (grade 3) neuroendocrine neoplasms (G3 NENs) have poor survival outcomes. From a clinical standpoint, G3 NENs are usually grouped regardless of primary site and treated similarly. Little is known regarding the underlying genomics of these rare tumors, especially when compared across different primary sites. We performed whole transcriptome (n = 46), whole exome (n = 40), and gene copy number (n = 43) sequencing on G3 NEN formalin-fixed, paraffin-embedded samples from diverse organs (in total, 17 were lung, 16 were gastroenteropancreatic, and 13 other). G3 NENs despite arising from diverse primary sites did not have gene expression profiles that were easily segregated by organ of origin. Across all G3 NENs, TP53, APC, RB1, and CDKN2A were significantly mutated. The CDK4/6 cell cycling pathway was mutated in 95% of cases, with upregulation of oncogenes within this pathway. G3 NENs had high tumor mutation burden (mean 7.09 mutations/MB), with 20% having >10 mutations/MB. Two somatic copy number alterations were significantly associated with worse prognosis across tissue types: focal deletion 22q13.31 (HR, 7.82; P = 0.034) and arm amplification 19q (HR, 4.82; P = 0.032). This study is among the most diverse genomic study of high-grade neuroendocrine neoplasms. We uncovered genomic features previously unrecognized for this rapidly fatal and rare cancer type that could have potential prognostic and therapeutic implications.

Background: Gastric cancer is a leading cause of cancer morbidity and mortality. Developing information systems which integrate clinical and genomic data may accelerate discoveries to improve cancer prevention, detection, and treatment. To support translational research in gastric cancer, we developed the Gastric Cancer Registry (GCR), a North American repository of clinical and cancer genomics data. Methods: Participants self-enrolled online. Entry criteria into the GCR included the following: (i) diagnosis of gastric cancer, (ii) history of gastric cancer in a first- or second-degree relative, or (iii) known germline mutation in the gene CDH1. Participants provided demographic and clinical information through a detailed survey. Some participants provided specimens of saliva and tumor samples. Tumor samples underwent exome sequencing, whole-genome sequencing, and transcriptome sequencing. Results: From 2011 to 2021, 567 individuals registered and returned the clinical questionnaire. For this cohort 65% had a personal history of gastric cancer, 36% reported a family history of gastric cancer, and 14% had a germline CDH1 mutation. 89 patients with gastric cancer provided tumor samples. For the initial study, 41 tumors were sequenced using next-generation sequencing. The data was analyzed for cancer mutations, copy-number variations, gene expression, microbiome, neoantigens, immune infiltrates, and other features. We developed a searchable, web-based interface (the GCR Genome Explorer) to enable researchers’ access to these datasets. Conclusions: The GCR is a unique, North American gastric cancer registry which integrates clinical and genomic annotation. Impact: Available for researchers through an open access, web-based explorer, the GCR Genome Explorer will accelerate collaborative gastric cancer research across the United States and world.

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell-free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra-deep sequencing for a 61-gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next-generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%.

// Zeus A. Antonello 1,2 , Nancy Hsu 3 , Manoj Bhasin 4 , Giovanni Roti 5 , Mukta Joshi 4 , Paul Van Hummelen 6 , Emily Ye 1,2 , Agnes S. Lo 7 , S. Ananth Karumanchi 7 , Christine R. Bryke 3 and Carmelo Nucera 1,2 1 Laboratory of human thyroid cancers preclinical and translational research, Division of Experimental Pathology, Cancer Research Institute, Cancer Center, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2 Department of Pathology, Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 3 Cytogenetics Laboratory, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 4 Bioinformatic and Systems Biology Unit, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 5 Department of Medicine and Surgery, University of Parma, Parma, Italy 6 Center for Cancer Genome Discovery, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 7 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Correspondence to: Carmelo Nucera, email: // Keywords : papillary thyroid cancer preclinical model, BRAF V600E , chromosome 5, combined therapy with vemurafenib and palbociclib, drug resistance Received : July 14, 2017 Accepted : August 15, 2017 Published : September 24, 2017 Abstract Purpose: Papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAF V600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAF WT/V600E –positive PTC patient-derived cells with P16 -/- (CDKN2A -/- ). Experimental Design: Following treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples. Results: Vemurafenib-resistant cells grow similarly to naive cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naive-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAF WT/V600E -PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAF WT/V600E mutation. Conclusions: Critically, our findings suggest for the first time that targeting BRAF WT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naive radioiodine-refractory BRAF WT/V600E -PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAF V600E inhibitor.

DNA copy number aberrations (CNA) were frequently observed in colorectal cancers (CRC). There is an urgent call for CNA-based biomarkers in clinics, in particular for Stage III CRC, if combined with imaging or pathologic evidence, promise more precise care at the timing. We conducted this Stage III specific biomarker discovery with a cohort of 134 CRCs, and with a newly developed high-efficiency CNA profiling protocol. Specifically, we developed the profiling protocol for tumor-normal matched tissue samples based on low-coverage clinical whole-genome sequencing (WGS). We demonstrated the protocol’s accuracy and robustness by a systematic benchmark with microarray, high-coverage whole-exome and -genome approaches, where the low-coverage WGS-derived CNA segments were highly accordant (PCC>0.95) with those derived from microarray, and they were substantially less variable if compared to exome-derived segments. A lasso-based model and multivariate cox regression analysis identified a chromosome 17p loss, containing the TP53 tumor suppressor gene, that was significantly associated with reduced survival (P=0.0139, HR=1.688, 95% CI = [1.112-2.562]), which was validated by an independent cohort of 187 Stage III CRCs. In summary, the new low-coverage WGS protocol has high sensitivity, high resolution and low cost and the identified 17p-loss is an effective poor prognosis marker for Stage III patients.

// Harshabad Singh 1, 2 , Yang Liu 2 , Xiuli Xiao 3 , Ling Lin 2 , Jaegil Kim 4 , Paul Van Hummelen 2 , Chin-Lee Wu 3 , Adam J. Bass 2 , Philip J. Saylor 1 1 Massachusetts General Hospital Cancer Center, Boston, MA, USA 2 Dana Farber Cancer Institute, Boston, MA, USA 3 Department of Pathology, Massachusetts General Hospital, Boston, MA, USA 4 Broad Institute, Cambridge, MA, USA Correspondence to: Adam J. Bass, email: adam_bass@dfci.harvard.edu Philip J. Saylor, email: psaylor@mgh.harvard.edu Keywords: urachal adenocarcinoma, NF1, whole exome sequencing Received: March 04, 2016 Accepted: March 18, 2016 Published: April 7, 2016 ABSTRACT Urachal adenocarcinoma is a rare bladder malignancy arising from the urachal remnant. Given its rarity and the lack of knowledge about its genetic characteristics, optimal management of this cancer is not well defined. Practice patterns vary and outcomes remain poor. In order to identify the genomic underpinnings of this malignancy, we performed whole exome sequencing using seven tumor/normal pairs of formalin fixed archival specimens. We identified recurrent evidence of MAP-kinase pathway activation as three patients had neurofibromin 1 ( NF1 ) mutations, with one of these patients also harboring an oncogenic KRAS G13D mutation. We also observed recurrent evidence of Wnt/β-catenin pathway activation as three patients had oncogenic mutations in APC or RNF43 . In addition, somatic copy number analysis revealed focal chromosome 12p amplifications in three samples, resembling findings from testicular germ cell tumors. We describe the genomic landscape of this malignancy in our institutional cohort and propose investigation of the therapeutic potential for MAP-K pathway inhibition in the subset of patients who show evidence of its activation.

Rapamycin analogs have reproducible but modest efficacy in endometrial cancer (EC). Identification of molecular biomarkers that predict benefit could guide clinical development.Fixed primary tissue and whole blood were collected prospectively from patients enrolled on GOG 248. DNA was isolated from macro-dissected tumors and blood; next-generation sequence analysis was performed on a panel of cancer related genes. Associations between clinical outcomes [response rate (RR) 20%; progression-free survival (PFS) median 4.9months] and mutations (PTEN, PIK3CA, PIK3R1, KRAS, CTNNB1, AKT1, TSC1, TSC2, NF1, FBXW7) were explored.Sequencing data was obtained from tumors of 55 of the 73 enrolled pts. Mutation rates were consistent with published reports: mutations in PTEN (45%), PIK3CA (29%), PIK3R1 (24%), K-RAS (16%), CTNNB1 (18%) were common and mutations in AKT1 (4%), TSC1 (2%), TSC2 (2%), NF1 (9%) and FBXW7 (4%) were less common. Increased PFS (HR 0.16; 95% CI 0.01-0.78) and RR (response difference 0.83; 95% CI 0.03-0.99) were noted for AKT1 mutation. An increase in PFS (HR 0.46; 95% CI 0.20-0.97) but not RR (response difference 0.00, 95% CI -0.34-0.34) was identified for CTNNB1 mutation. Both patients with TSC mutations had an objective response. There were no statistically significant associations between mutations in PIK3CA, PTEN, PIK3R1, or KRAS and PFS or RR.Mutations in AKT1, TSC1 and TSC2 are rare, but may predict clinical benefit from temsirolimus. CTNNB1 mutations were associated with longer PFS on temsirolimus.

432 Background: Gastric cancer (GC) is the fifth leading cancer diagnosis and third frequent cause of cancer death globally. GC results from a cascade of molecular and genetic changes owing to environmental and genetic factors. While there are known risks to GC, more is to be learned about its development so to establish effective screens for early stages of cancer. Methods: The Gastric Cancer Registry (GCR) was built to investigate GC and discover informative genomic biomarkers. The GCR comprises medical, family, and social history and genomics data from patients with GC, a family history of GC, and/or a known mutation in the gene CDH1. Samples of saliva and gastric tumors are collected when available. The GCR allows us to leverage several genome sequencing datasets to construct a complete molecular landscape of GC. Early analysis of GC tissue includes whole exome sequencing (WES) to identify mutations, whole genome sequencing (WGS) for copy number variation, and RNA sequencing (RNAseq) for expression profiling. Results are used to inform of clonal neoantigens, microbiome, and immune cell populations. Saliva will be analyzed with linked reads sequencing to unearth germline mutations not picked up in standard clinical gene panels. Results: Datasets from 455 patients and samples from 159 patients have been collected. In a pilot study, we pinpointed specific mutations using WES and revealed extensive changes in genome copy number involving clinically actionable genes through WGS. Most tumors had a high degree of genomic instability and exhibited candidate markers for treatment decisions and response. Additionally, we found that RNAseq revealed tumor subgroups through gene expression signatures. Conclusions: The GCR is an informative resource enabling the identification of biomarkers for GC. With the GCR we are integrating expertise in translational cancer genomics with molecular biology, statistics, and bioinformatics to build a platform for discovery and the development of tools that will ultimately improve the detection, treatment, and prevention of GC.

624 Background: Grade 3 neuroendocrine neoplasms (G3 NENs), if poorly differentiated, have a median survival of only 10-19 months. Little is known regarding their underlying genomics. Methods: We applied multiomics analysis to 46 cases of G3 NEN that included copy number analysis, whole exome, and transcriptomic sequencing. Results: Of the 46 unique cases, 17 were lung, 16 gastroenteropancreatic (GEP), 13 other; 5 well-differentiated, 39 poorly differentiated and 2 mixed. Using a multivariate Cox model, we found histology characteristics (including differentiation, Ki67 and mitotic index) did not correlate with changes in overall survival (OS). The clinical variables that did correlate with OS included: number of lines of treatment (hazard ratio for death [HR], 0.72; p < 0.05), GEP primary site (HR, 5.36; p < 0.005), and non-resected primary tumor (HR, 14.52; p < 0.001). Two copy number changes were associated with worse prognosis: focal deletion 22q13 (HR, 10.23; p < 0.005), and arm amplification 19q (HR, 7.09; p < 0.01). The median OS of the top quartile compared to the rest for 22q13 deletion carriers was 9.9 months vs. 24 months, and for 19q amplification carriers was 8.7 months vs. 36.7 months. We estimated a median tumor mutation burden (TMB) of 3.7 mutations/Mb, with 20% (8/40) of patients showing high TMB ( > 10 mutations/Mb). The top five mutated genes were USH2A, RB1, APC, TP53, and MUC16. We also observed high transcriptomic similarity across all NENs regardless of their site of origin. Conclusions: We identified two copy number changes that can serve as predictive biomarkers in G3 NENs, as they confer an increased risk of death by as high as 10x to the carriers. Further, G3 NENs are characterized by a distinct group of somatic mutations, and a significant number have high tumor mutation burden. Lastly, G3 NENs across different organs were relatively homogeneous in expression profile.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Background. Patients with meningiomas have widely divergent clinical courses. Some entirely recover following surgery alone, while others have relentless tumor recurrences. This clinical conundrum is exemplified by rhabdoid meningiomas, which are designated in the World Health Organization Classification of Tumours as high grade, despite only a subset following an aggressive clinical course. Patient management decisions are further exacerbated by high rates of interobserver variability, biased against missing possibly aggressive tumors. Objective molecular determinants are needed to guide classification and clinical decision making. Methods. To define genomic aberrations of rhabdoid meningiomas, we performed sequencing of cancer-related genes in 27 meningiomas from 18 patients with rhabdoid features and evaluated breast cancer [BRCA]1-associated protein 1 (BAP1) expression by immunohistochemistry in 336 meningiomas. We assessed outcomes, germline status, and family history in patients with BAP1-negative rhabdoid meningiomas. Results. The tumor suppressor gene BAP1, a ubiquitin carboxy-terminal hydrolase, is inactivated in a subset of high-grade rhabdoid meningiomas. Patients with BAP1-negative rhabdoid meningiomas had reduced time to recurrence compared with patients with BAP1-retained rhabdoid meningiomas (Kaplan-Meier analysis, 26 mo vs 116 mo, P < .001; hazard ratio 12.89). A subset of patients with BAP1-deficient rhabdoid meningiomas harbored germline BAP1 mutations, indicating that rhabdoid meningiomas can be a harbinger of the BAP1 cancer predisposition syndrome. Conclusion. We define a subset of aggressive rhabdoid meningiomas that can be recognized using routine laboratory tests. We implicate ubiquitin deregulation in the pathogenesis of these high-grade malignancies. In addition, we show that familial and sporadic BAP1-mutated rhabdoid meningiomas are clinically aggressive, requiring intensive clinical management.

// Malak S. Abedalthagafi 1 , Parker H. Merrill 1 , Wenya Linda Bi 2 , Robert T. Jones 1 , Marc L. Listewnik 3 , Shakti H. Ramkissoon 1 , Aaron R. Thorner 4,5 , Ian F. Dunn 2 , Rameen Beroukhim 4,6,9 , Brian M. Alexander 7 , Priscilla K. Brastianos 4,6,8 , Joshua M. Francis 4,6 , Rebecca D. Folkerth 1 , Keith L. Ligon 1,4 , Paul Van Hummelen 4,5 , Azra H. Ligon 3 and Sandro Santagata 1,9 1 Department of Pathology, Division of Neuropathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA 2 Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA 3 Clinical Cytogenetics Laboratory, Center for Advanced Molecular Diagnostics, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA 4 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 5 Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA, USA 6 Broad Institute of MIT and Harvard, Cambridge, MA, USA 7 Department of Radiation Oncology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA 8 Department of Neuro-Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 9 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Correspondence: Sandro Santagata, email: // Keywords : meningioma, angiomatous, polysomy, aCGH, molecular diagnostics, Next Generation Sequencing Received : September 14, 2014 Accepted : September 24, 2014 Published : September 25, 2014 Abstract Meningiomas are a diverse group of tumors with a broad spectrum of histologic features. There are over 12 variants of meningioma, whose genetic features are just beginning to be described. Angiomatous meningioma is a World Health Organization (WHO) meningioma variant with a predominance of blood vessels. They are uncommon and confirming the histopathologic classification can be challenging. Given a lack of biomarkers that define the angiomatous subtype and limited understanding of the genetic changes underlying its tumorigenesis, we compared the genomic characteristics of angiomatous meningioma to more common meningioma subtypes. While typical grade I meningiomas demonstrate monosomy of chromosome 22 or lack copy number aberrations, 13 of 14 cases of angiomatous meningioma demonstrated a distinct copy number profile – polysomies of at least one chromosome, but often of many, especially in chromosomes 5, 13, and 20. WHO grade II atypical meningiomas with angiomatous features have both polysomies and genetic aberrations characteristic of other atypical meningiomas. Sequencing of over 560 cancer-relevant genes in 16 cases of angiomatous meningioma showed that these tumors lack common mutations found in other variants of meningioma. Our study demonstrates that angiomatous meningiomas have distinct genomic features that may be clinically useful for their diagnosis.

Cervical cancer is the second leading cause of cancer deaths among women worldwide. The objective of this study was to describe the most common oncogenic mutations in cervical cancers and to explore genomic differences between the 2 most common histologic subtypes: adenocarcinoma and squamous cell carcinoma.A high-throughput genotyping platform, termed Oncomap, was used to interrogate 80 cervical tumors for 1250 known mutations in 139 cancer genes. Samples were analyzed using a mass spectrometry-based genotyping platform and were validated using orthogonal chemistry. Epidermal growth factor receptor (EGFR) mutations were further validated by massive parallel sequencing. Human papilloma virus (HPV) genotyping also was performed.Validated mutations were detected in 48 of 80 tumors (60%) examined. The highest mutation rates were in the genes phosphatidylinositol 3-kinase, catalytic subunit α (PIK3CA) (31.3%); Kirsten rat sarcoma viral oncogene homolog (KRAS) (8.8%); and EGFR (3.8%). PIK3CA mutation rates did not differ significantly between adenocarcinomas and squamous cell carcinomas (25% vs 37.5%, respectively; P = .33). In contrast, KRAS mutations were identified only in adenocarcinomas (17.5% vs 0%; P = .01), and a novel EGFR mutation was detected only in squamous cell carcinomas (0% vs 7.5%; P = .24). There were no associations between HPV-16 or HPV-18 and somatic mutations or overall survival. In adjusted analyses, PIK3CA mutations were associated with shorter survival (67.1 months vs 90.3 months; hazard ratio, 9.1; 95% confidence interval, 2.8-29.5 months; P.001).Cervical cancers harbor high rates of potentially targetable oncogenic mutations. In addition, cervical squamous cell carcinoma and adenocarcinoma have distinct molecular profiles, suggesting that clinical outcomes may be improved with the use of more tailored treatment strategies, including PI3K and MEK inhibitors.

Over 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publically available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment called the Public Repository of Xenografts (PRoXe; www.proxe.org). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional and proteomic biomarkers in both treatment-naïve and relapsed/refractory disease.

Changes in DNA copy number, i.e., somatic CNVs, are common genetic aberrations in cancers. The effects of CNV include alteration in gene dosage across large segments of the cancer genome affecting the expression of cancer driver genes by amplifications, or cancer suppressor genes by deletions. In addition, CNVs are markers of underlying rearrangements within or between chromosomes and there is increasing evidence supporting a greater role for CNVs in developing and maintaining neoplastic cell population diversity. Copy number aberrations can be estimated from next generation sequencing data, with high sensitivity and genomic resolution by sequencing the whole genome (WGS). For this study, we demonstrated that high quality CNV calls can be extracted in a fast and cost-effective way from low-coverage whole genome sequencing. Novaseq S2 flowcells (Illumina Inc) enables to obtain an average coverage of 3-4x per sample after pooling up to 96 samples per flowcell. We examined three different copy number detection tools (CNVkit, BicSeq, and seqCBS) from paired tumor and normal WGS using microarray data as a reference. Pearson correlations were computed between the reference and CNVs from the WGS in two fashion; i) segment based and ii) gene based. The segment based comparison used sliding window of 100 K bp while gene based comparison used segments at the gene level. We found high correlations between microarray and WGS segments. The highest correlations were obtained by CNVkit, ranging from 0.964 to 0.985 (SD: 0.973 - 0.007) and BicSeq, ranging from 0.963 to 0.986 (SD: 0.975 - 0.008). These results open the prospect of assessing large cancer cohorts of hundreds of samples at a reasonable cost. We are planning to apply this method to a large cohort of Stage III colon cancer patients and determine the clinical relevance of CNVs for survival. Citation Format: HoJoon Lee, Li Charlie Xia, Stephanie Greer, John Bell, Sue M. Grimes, Christina Wood Bouwens, Giwon Shin, Billy TC Lau, Lucas Johnson, Noemi Andor, Kenneth Day, Mickey Miller, Helaman Escobar, Lincoln Nadauld, Hanlee P. Ji, Paul Van Hummelen. High-quality CNV segments from low-coverage whole genome sequencing from FFPE cancer biopsies based on an evaluation of multiple CNV tools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 438.

Formalin-fixed, paraffin-embedded (FFPE) storage is a universally-adopted, cost-effective and long-term solution for tissue storage. Given the abundance of FFPE clinical samples, they provide a good source of tissue for genome sequencing studies. However, the extraction of nucleic acids from FFPE tissues yield highly fragmented DNA of variable quality. The poor quality and low yield make it difficult to generate adequate sequencing libraries for genomic studies. For a population cancer genome study, we optimized a procedure for whole-genome sequencing on FFPE tumor and normal tissue of colorectal cancer patients. This pilot study aimed to first identify changes in DNA copy number using low-pass sequencing, with the additional goal of later adding structural variant and gene fusion detection and single nucleotide variant analysis with deeper sequencing. We processed, sequenced and analyzed 193 patients. These FFPE samples had varying storage characteristics. We optimized the initial extraction quality thresholds, DNA extraction and library preparation to generate high quality sequencing data. We identified an initial sample quality and library threshold to hold the best correlation for sequencing quality. Optimal yield and DNA size from extracted FFPE was achieved by using Promega's LEV DNA FFPE Extraction Kit. Library prep optimization involved using mechanical shearing of DNA to increase overall library size for structural variant detection as well as using unique dual index adapters to identify index swapping artifacts during sequencing. An additional cleanup was also performed post-fragmentation to increase overall library size. Overall, we successfully developed a robust protocol for conducting whole genome sequencing on FFPE tumor samples. Citation Format: Matthew Kubit, Christina Wood-Bouwens, Sue Grimes, John Bell, GiWon Shin, Billy Lau, Mickey Miller, Kenneth Day, Helaman Escobar, Hanlee Ji, Lincoln Nadauld, Paul Van Hummelen. High-throughput whole-genome sequencing of formalin fixed, paraffin-embedded tissues from colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4331.

e15588Background: Colorectal cancer is the third most common malignancy worldwide, and a major contributor to cancer-related deaths annually. National and international guidelines for the managemen...

Background Meningiomas are the most common primary intracranial tumor in adults. Identification of SMO and AKT1 mutations in meningiomas has raised the possibility of targeted therapies for some patients. The frequency of such mutations in clinical cohorts and the presence of other actionable mutations in meningiomas are important to define. Methods We used high-resolution array-comparative genomic hybridization to prospectively characterize copy-number changes in 150 meningiomas and then characterized these samples for mutations in AKT1, KLF4, NF2, PIK3CA, SMO, and TRAF7. Results Similar to prior reports, we identified AKT1 and SMO mutations in a subset of non-NF2-mutant meningiomas (ie, ∼9% and ∼6%, respectively). Notably, we detected oncogenic mutations in PIK3CA in ∼7% of non-NF2-mutant meningiomas. AKT1, SMO, and PIK3CA mutations were mutually exclusive. AKT1, KLF4, and PIK3CA mutations often co-occurred with mutations in TRAF7. PIK3CA-mutant meningiomas showed limited chromosomal instability and were enriched in the skull base. Conclusion This work identifies PI3K signaling as an important target for precision medicine trials in meningioma patients.

Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model.We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6).We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67).Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure.Deutsche Krebshilfe, Terry Fox Research Institute.

Brain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed wholeexome sequencing of 86 matched brain metastases, primary tumors and normal tissue. In all clonally related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found potentially clinically informative alterations in the brain metastases not detected in the matched primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were genetically homogenous. Distal extracranial and regional lymph node metastases were highly divergent from brain metastases. We detected alterations associated with sensitivity to PI3K/AKT/mTOR, CDK, and HER2/EGFR inhibitors in the brain metastases. Genomic analysis of brain metastases provides an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors, regional lymph nodes, or extracranial metastases., Massachusetts Institute of Technology. Department of Biology, National Institutes of Health (U.S.) (National Human Genome Research Institutes of Health Large-scale Sequencing and Analysis Center. Grant U54 HG003067)

Viruses are a major contributor to oncogenesis, causing 10-15% of human cancers. Molecular pathways involved in malignant transformation are frequently activated by genetic alterations, including but not limited to, somatic mutations, copy number aberrations, structural variants, and oncoviruses. Precision cancer medicine aims to classify tumors by site, histology, and molecular tests to determine an “individualized” profile of cancer alterations. However, clinical tests for these various alterations are sequential, time consuming, and use a lot of material, which is often quite limited (e.g., biopsies). Moreover, tests for the presence of viral sequence are generally performed separately to tests (such as massively parallel sequencing) to detect human genomic alterations. Here we present a hybrid capture and massively parallel sequencing approach to detect viral infection concurrently with targeted genomic analysis, which may decrease assay costs, increase sensitivity and scalability, and detect many types of alterations, thereby providing a more complete tumor genetic profile all from a single sample. We have created a custom hybrid capture probeset for targeted Illumina sequencing to determine whether oncoviruses are present in tissue samples and also determine if the virus has integrated into the host’s genome. We have created both ‘detection’ and ‘integration’ baits for several oncoviruses, including polyomaviruses, human papilloma viruses, Epstein-Barr virus, human cytomegalovirus, Kaposi sarcoma herpesvirus, human T-lymphotropic virus, and hepatitis B virus. To distinguish between different strains of a single virus, strain-specific detection baits were created to bind to variable regions of viral genomes. The integration bait was designed to bind to regions of the viral genomes that are commonly integrated into the human genome. This baitset can also be combined with other capture panels targeting oncogenes to simultaneously determine infection and integration statuses, as well as somatic mutations, copy number and structural variants. To detect virus presence, reads were aligned to a hybrid reference of both the human, and targeted virus genomes. Viral integration status and integration loci were determined by leveraging discordant read pairs that aligned to both the human genome and a viral genome. We have tested our techniques on tissue samples that were infected with either Merkel Cell Polyomavirus or Epstein-Barr virus, as determined using quantitative polymerase chain reaction (qPCR) or immunohistochemistry (IHC) techniques, and have successfully detected these viruses and identified viral integration loci. Overall, this viral hybrid capture probeset provides the ability to simultaneously determine a tissue sample’s infection and viral integration status alongside other somatic genomic analyses, saving both time and sample material. Citation Format: Robert T. Burns, Samuel S. Hunter, Matthew D. Ducar, Aaron R. Thorner, James A. Decaprio, Paul Van Hummelen, Alexander Frieden, Anwesha Nag, Haley A. Coleman, Michael K. Slevin, Andrea Clapp, Samantha D. Drinan, Suzanne R. McShane, Edwin Thai, Priyanka Shivdasani, Joshua Bohannon, Johann Hoeftberger, Reuben Jacobs, Bruce M. Wollison, Neil A. Patel, Monica D. Manam, Phani Davineni, Matthew Meyerson, Laura E. MacConaill. Oncovirus detection and integration analysis from human tumor samples using targeted massively parallel sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2017-562

Importance Lung cancer is the leading cause of cancer death in the United States in all ethnic and racial groups. The overall death rate from lung cancer is higher in black patients than in white patients. Objective To compare the prevalence and types of somatic alterations between lung cancers from black patients and white patients. Differences in mutational frequencies could illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients’ treatment. Design, Setting, and Participants Tumor specimens were collected from Baptist Cancer Center (Memphis, Tennessee) over the course of 9 years (January 2004-December 2012). Genomic analysis by massively parallel sequencing of 504 cancer genes was performed at Dana-Farber Cancer Institute (Boston, Massachusetts). Overall, 509 lung cancer tumors specimens (319 adenocarcinomas; 142 squamous cell carcinomas) were profiled from 245 black patients and 264 white patients. Main Outcomes and Measures The frequencies of genomic alterations were compared between tumors from black and white populations. Results Overall, 509 lung cancers were collected and analyzed (273 women [129 black patients; 144 white patients] and 236 men [116 black patients; 120 white patients]). Using 313 adenocarcinomas and 138 squamous cell carcinomas with genetically supported ancestry, overall mutational frequencies and copy number changes were not significantly different between black and white populations in either tumor type after correcting for multiple hypothesis testing. Furthermore, specific activating alterations in members of the receptor tyrosine kinase/Ras/Raf pathway including EGFR and KRAS were not significantly different between populations in lung adenocarcinoma. Conclusions and Relevance These results demonstrate that lung cancers from black patients are similar to cancers from white patients with respect to clinically actionable genomic alterations and suggest that clinical trials of targeted therapies could significantly benefit patients in both groups.

ERBB2-directed therapy is now a routine component of therapy for ERBB2-amplified metastatic gastroesophageal adenocarcinomas. However, there is little knowledge of the mechanisms by which these tumors develop acquired resistance to ERBB2 inhibition. To investigate this question we sought to characterize cell line models of ERBB2-amplified gastroesophageal adenocarcinoma with acquired resistance to ERBB2 inhibition. We generated lapatinib-resistant (LR) subclones from an initially lapatinib-sensitive ERBB2-amplified esophageal adenocarcinoma cell line, OE19. We subsequently performed genomic characterization and functional analyses of resistant subclones with acquired lapatinib resistance. We identified a novel, acquired Src E527K mutation in a subset of LR OE19 subclones. Cells with this mutant allele harbour increased Src phosphorylation. Genetic and pharmacologic inhibition of Src resensitized these subclones to lapatinib. Biochemically, Src mutations could activate both the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways in the lapatinib-treated LR OE19 cells. Ectopic expression of Src E527K mutation also was sufficient to induce lapatinib resistance in drug-naïve cells. These results indicate that pathologic activation of Src is a potential mechanism of acquired resistance to ERBB2 inhibition in ERBB2-amplified gastroesophageal cancer. Although Src mutation has not been described in primary tumor samples, we propose that the Src hyperactivation should be investigated in the settings of acquired resistance to ERBB2 inhibition in esophageal and gastric adenocarcinoma.

Massively parallel sequencing (MPS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimens are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Indeed, it can be challenging to extract sufficient amounts of starting material from fresh-frozen paraffin embedded (FFPE) samples for library construction, and frequently samples are not processed because they do not reach minimum requirements. Therefore, it is of great importance to improve currently available protocols to process low yield FFPE-derived DNA for MPS. This will allow for the inclusion of more tumor samples to be sequenced per cohort, resulting in increased ability to identify genomic factors involved in tumorigenesis. Here, we optimized the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to successfully perform library construction from low-yield FFPE and fresh frozen samples. The kit normally requires an input of at least 100 ng of DNA. However, we were able to modify the protocol to prepare libraries from as little as 10 ng of starting material by optimizing the ratio of adapter:input DNA and SPRI-clean-up, performing low-volume reactions, and using IDT universal blockers during hybrid capture. Additional library enrichment PCR was not required. Library yields were sufficient for downstream hybrid capture and sequencing, and sequencing metrics were comparable to samples that were prepped using the manufacturer's recommendations. Experiments are underway to demonstrate that library complexity remains unchanged. Citation Format: Aaron R. Thorner, Liuda Ziaugra, Matthew D. Ducar, Ling Lin, Angelica Laing, Haley A. Coleman, Suzanne R. McShane, Andrea Clapp, Rachel R. Paquette, Bruce M. Wollison, Johann Hoeftberger, Neil A. Patel, Samuel S. Hunter, Monica D. Manam, Laura E. MacConaill, William C. Hahn, Matthew L. Meyerson, Paul van Hummelen. Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3644.

2069Background: It is not known whether GBM responds differently to radiation based on tumor cell-intrinsic factors. Newly developed PDCLs of GBM have potential to illuminate individual patient functional response to treatment at a scale not previously possible. We therefore assessed whether intrinsic differences in radiosensitivity are present in a large panel of clinically and genomically annotated GBM PDCLs. Methods: PDCLs were established from newly diagnosed (n = 25) and recurrent (n = 10) GBM patients. Whole exome sequencing and whole genome array comparative genomic hybridization was performed on the PDCLs, and patient outcomes data was collected. High-throughput functional radiation screening was performed across all patients at varying doses. Response was calculated at 9 days after treatment based on ATP measurement using Cell Titer Glo to assess growth and recovery. Proliferation was integrated as a function of dose for each cell line (AUC). AUC was correlated with genomic candidate factors and ...

Of pediatric solid tumors, as many as 10% of tumors are categorized as rare. Many of these rare tumors lack standard effective known therapy. The ability to identify vulnerabilities for many rare tumors has been significantly limited by the lack of in vitro and in vivo models. Furthermore, current approaches to study such vulnerabilities are usually limited to a specific compound or target. Our objectives were 1) to develop a platform to collect tumor samples and generate in vitro models and 2) to develop systematic and orthogonal approaches focused on currently known druggable cancer targets to identify vulnerabilities in these difficult to treat cancers. We have developed a proof of concept cell line from a patient who succumbed to progressive undifferentiated sarcoma treated on an aggressive multi-therapy regimen. This cell line, in its early passages, has novel gene fusions that match that of the primary tumor. Furthermore, even at early passages, this cell line was amenable to high throughput functional screens. Using a targeted pooled shRNA screen (employing matched seed controls) and an analogous CRISPR screen we identified dependencies to XPO1 and CDK4. In parallel, compounds against these targets were identified in a small molecule compound screen. These targetable dependencies were further validated in vivo with a micro-dosing device. These observations identify new targets in this rare malignancy. Furthermore, this suggests that the interrogation of patient derived cell lines facilitates the identification of testable therapeutic approaches. Citation Format: Andrew L. Hong, Glenn S. Cowley, Yuen-Yi Tseng, Jaime H. Cheah, Oliver Jonas, Mihir B. Doshi, Bryan D. Kynnap, Coyin Oh, Stephanie Meyer, Paul Clemons, Michael Burger, Francisca Vazquez, Barbara Weir, Gregory V. Kryukov, Alanna Church, Alma Imamovic, Aviad Tsherniak, Craig Bielski, Brian Crompton, Elizabeth Mullen, Charles Roberts, Carlos Rodriguez-Galindo, Katherine A. Janeway, Kimberly Stegmaier, Paul van Hummelen, Robert Langer, Levi A. Garraway, Stuart L. Schreiber, David E. Root, Jesse S. Boehm, William C. Hahn. Developing a functional genomics platform to interrogate rare pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B38.

Knowledge of “actionable” somatic genomic alterations present in each tumor (e.g., point mutations, small insertions/deletions, and copy-number alterations that direct therapeutic options) should facilitate individualized approaches to cancer treatment. However, clinical implementation of systematic genomic profiling has rarely been achieved beyond limited numbers of oncogene point mutations. To address this challenge, we utilized a targeted, massively parallel sequencing approach to detect tumor genomic alterations in formalin-fixed, paraffin-embedded (FFPE) tumor samples. Nearly 400-fold mean sequence coverage was achieved, and single-nucleotide sequence variants, small insertions/deletions, and chromosomal copynumber alterations were detected simultaneously with high accuracy compared with other methods in clinical use. Putatively actionable genomic alterations, including those that predict sensitivity or resistance to established and experimental therapies, were detected in each tumor sample tested. Thus, targeted deep sequencing of clinical tumor material may enable mutation-driven clinical trials and, ultimately, “personalized” cancer treatment. Significance: Despite the rapid proliferation of targeted therapeutic agents, systematic methods to profile clinically relevant tumor genomic alterations remain underdeveloped. We describe a sequencing-based approach to identifying genomic alterations in FFPE tumor samples. These studies affirm the feasibility and clinical utility of targeted sequencing in the oncology arena and provide a foundation for genomics-based stratification of cancer patients. Cancer Discovery; 2(1); 82–93. ©2011 AACR. Read the Commentary on this article by Corless and Spellman, p. 23 This article is highlighted in the In This Issue feature, p. 1

BackgroundGastric cancer is one of the leading cancer types in incidence and mortality, especially in Asia. In order to improve survival, identification of a catalogue of molecular alterations underlying gastric cancer is a critical step for developing and designing genome-directed therapies.Methodology/principal findingsThe Center for Cancer Genome Discovery (CCGD) at the Dana-Farber Cancer Institute (DFCI) has adapted a high-throughput genotyping platform to determine the mutation status of a large panel of known cancer genes. The mutation detection platform, termed OncoMap v4, interrogates 474 "hotspot" mutations in 41 genes that are relevant for cancer. We performed OncoMap v4 in formalin-fixed paraffin-embedded (FFPE) tissue specimens from 237 gastric adenocarcinomas. Using OncoMap v4, we found that 34 (14.4%) of 237 gastric cancer patients harbored mutations. Among mutations we screened, PIK3CA mutations were the most frequent (5.1%) followed by p53 (4.6%), APC (2.5%), STK11 (2.1%), CTNNB1 (1.7%), and CDKN2A (0.8%). Six samples harbored concomitant somatic mutations. Mutations of CTNNB1 were significantly more frequent in EBV-associated gastric carcinoma (P = 0.046). Our study led to the detection of potentially druggable mutations in gastric cancer which may guide novel therapies in subsets of gastric cancer patients.Conclusions/significanceUsing high throughput mutation screening platform, we identified that PIK3CA mutations were the most frequently observed target for gastric adenocarcinoma.

An increasing number of anticancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Recent evidence suggests that the selection of patients whose tumors harbor specific genetic alterations identifies the subset of patients who are most likely to benefit from the use of such agents. As the number of genetic alterations that provide diagnostic and/or therapeutic information increases, the comprehensive characterization of cancer genomes will be necessary to understand the spectrum of distinct genomic alterations in cancer, to identify patients who are likely to respond to particular therapies, and to facilitate the selection of treatment modalities. Rapid developments in new technologies for genomic analysis now provide the means to perform comprehensive analyses of cancer genomes. In this article, we review the current state of cancer genome analysis and discuss the challenges and opportunities necessary to implement these technologies in a clinical setting. Significance: Rapid advances in sequencing technologies now make it possible to contemplate the use of genome scale interrogation in clinical samples, which is likely to accelerate efforts to match treatments to patients. However, major challenges in technology, clinical trial design, legal and social implications, healthcare information technology, and insurance and reimbursement remain. Identifying and addressing these challenges will facilitate the implementation of personalized cancer medicine. Cancer Discovery; 1(4): 297–311. ©2011 AACR.

Background: Epithelial ovarian cancer is the most lethal of all gynecologic malignancies, and high grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. The objective of this study was to determine the frequency and types of point somatic mutations in HGSC using a mutation detection protocol called OncoMap that employs mass spectrometric-based genotyping technology. Methodology/Principal Findings: The Center for Cancer Genome Discovery (CCGD) Program at the Dana-Farber Cancer Institute (DFCI) has adapted a high-throughput genotyping platform to determine the mutation status of a large panel of known cancer genes. The mutation detection protocol, termed OncoMap has been expanded to detect more than 1000 mutations in 112 oncogenes in formalin-fixed paraffin-embedded (FFPE) tissue samples. We performed OncoMap on a set of 203 FFPE advanced staged HGSC specimens. We isolated genomic DNA from these samples, and after a battery of quality assurance tests, ran each of these samples on the OncoMap v3 platform. 56% (113/203) tumor samples harbored candidate mutations. Sixty-five samples had single mutations (32%) while the remaining samples had $2 mutations (24%). 196 candidate mutation calls were made in 50 genes. The most common somatic oncogene mutations were found in EGFR, KRAS, PDGRFa, KIT, and PIK3CA. Other mutations found in additional genes were found at lower frequencies (,3%). Conclusions/Significance: Sequenom analysis using OncoMap on DNA extracted from FFPE ovarian cancer samples is feasible and leads to the detection of potentially druggable mutations. Screening HGSC for somatic mutations in oncogenes may lead to additional therapies for this patient population.

Purpose: We hypothesize that a gene expression profile characteristic for inflammatory breast cancer (IBC), an aggressive form of breast cancer associated with rapid cancer dissemination and poor survival, might be related to tumor aggressiveness in non-IBC (nIBC). Experimental Design: RNA from 17 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 1,157 nIBC samples with survival data of 881 nIBC samples. Samples were classified as IBC-like or nIBC-like. The IBC signature classification was compared with the classifications according to other prognostically relevant gene signatures and clinicopathologic variables. In addition, relapse-free survival (RFS) was compared by the Kaplan-Meyer method. Results: Classification according to the IBC signature is significantly (P < 0.05) associated with the cell-of-origin subtypes, the wound healing response, the invasive gene signature, the genomic grade index, the fibroblastic neoplasm signature, and the 70-gene prognostic signature. Significant associations (P < 0.01) were found between the IBC signature and tumor grade, estrogen receptor status, ErbB2 status, and patient age at diagnosis. Patients with an IBC-like phenotype show a significantly shorter RFS interval (P < 0.05). Oncomine analysis identified cell motility as an important concept linked with the IBC signature. Conclusions: We show that nIBC carcinomas having an IBC-like phenotype have a reduced RFS interval. This suggests that IBC and nIBC show comparable phenotypic traits, for example augmented cell motility, with respect to aggressive tumor cell behavior. This observation lends credit to the use of IBC to study aggressive tumor cell behavior.

Background: Brain metastases are associated with a dismal prognosis. There is a limited understanding of the oncogenic alterations harbored by brain metastases and whether these are shared with their primary tumors. Our objectives were to (1) elucidate the evolutionary patterns leading to brain metastases and (2) identify whether brain metastases are genetically distinct from their primary tumors and other distal metastatic sites. Methods: We performed whole-exome sequencing of 104 matched brain metastases, primary tumors and normal tissue, including 7 cases with spatially and temporally separated brain metastasis sites and 8 cases with additional extracranial disease sites, including regional lymph nodes, and distal metastases. We developed novel computational tools to perform an integrative analysis of somatic mutations and copy-number alterations. This analysis allowed us to estimate the clonal architecture of the primary and metastases, and to reconstruct a phylogenetic tree relating the subclones from each patient. Results: In all related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found clinically actionable driver alterations in the brain metastases that were not detectable in the matched primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were more genetically homogenous and shared nearly all driver alterations detected. Extracranial metastases and regional lymph nodes were highly divergent from brain metastases. Several clinically actionable pathways were enriched in brain metastases. Conclusions: These observations demonstrate that brain metastasis tissue provides an opportunity to identify clinically important driver alterations that may be undetected in single samples of primary tumors, regional lymph nodes, or extracranial metastases. Genetic divergence between primary tumors and brain metastases may underlie some of the difficulties encountered with the combined treatment of systemic disease and brain metastases in patients. When clinically feasible, genomic characterization of brain metastasis tissue should be considered when selecting therapeutic agents for patients with brain metastases. Citation Format: Priscilla K. Brastianos, Scott L. Carter, Sandro Santagata, Daniel Cahill, Amaro Taylor-Weiner, Robert T. Jones, Eliezer Van Allen, Peleg Horowitz, Keith L. Ligon, William T. Curry, Ian F. Dunn, Paul Van Hummelen, Matthew Meyerson, Levi Garraway, Josep Tabernero, Joan Seoane, Stacey Gabriel, Eric S. Lander, Rameen Beroukhim, Tracy T. Batchelor, Jose Baselga, David N. Louis, William C. Hahn, Gad Getz. Genomic characterization of brain metastases reveals branched evolution and metastasis-specific mutations. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR04.

RNA sequencing (RNASeq) provides the ability to comprehensively assay the transcriptome in a high-throughput manner. Current there are a variety of library preparation methodologies for measuring and sequencing the transcriptome depending on (a) the sample source and (b) outcomes of interest. Beyond protocol selection, the requisite computational tools and resources are significant considerations in processing, analyzing and reporting the experimental results. While there are many resources readily available to effectively perform RNA-seq experiments, optimal protocols and analysis tools for the cancer domain remain to be developed. We have developed and characterized a set of protocols and analysis procedures that comprise an RNA-seq pipeline that can effectively be used in a cancer research setting. The analysis pipeline consists of a sequence of functions and tools to process and clean the raw data, generate quality control and summary metrics, and perform secondary analyses that include expression quantification, fusion detection and somatic mutation calling. We applied this pipeline to three different RNAseq strategies (whole-transcriptome, exome, and targeted RNA-seq) and performed an in-depth comparative analysis to investigate the implications of the choice of strategy on the downstream analysis and results. More specifically, we investigated the impact of library preparation methods on the dynamic range and expression profiles, variant calling and fusion detection. While the data indicated that capture-based protocols provided efficient methods for sampling transcripts as compared to whole-transcriptome RNA-seq, there are considerations in its use, particularly for duplicate reads and uncaptured transcripts. We illustrate the implications of these issues on downstream analysis, such as somatic mutation and fusion calling and differential expression. In summary, we have described a RNA-seq analysis platform that provides a varied set of library preparations and analytical components for large-scale clinical or research transcriptomics. Our analysis has characterized the technical features of the different library preparations, providing a necessary understanding of the costs and benefits of each method and the potential effects on the downstream analyses. Citation Format: Ryan P. Abo, Ling Lin, Samuel S. Hunter, Deniz N. Dolcen, Rachel R. Paquette, Angelica Laing, Luc de Waal, Aaron R. Thorner, Matthew D. Ducar, Liuda Ziaugra, William C. Hahn, Matthew L. Meyerson, Laura E. MacConaill, Paul Van Hummelen. Comparative analysis of RNA sequencing methods for characterization of cancer transcriptomics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2015-4867

Background: Brain metastases represent an unmet need in current oncologic care. Approximately 8-10% of cancer patients will develop brain metastases, and more than half of these patients will pass away within a few months of their diagnosis. We have a limited understanding of how brain metastases genetically evolve from their primary tumors. Our objectives were to (1) elucidate the genomic evolutionary patterns leading to brain metastases (2) identify whether brain metastases harbor clinically significant genetic differences compared to their primary tumors and other extracranial metastatic sites, and (3) examine the extent of genetic heterogeneity across regionally separated and anatomically distinct sites of brain metastasis. Methods: We subjected 104 matched primary tumor biopsies, brain metastases, and normal tissue to whole exome sequencing, including 20 cases with regionally and anatomically separated brain metastasis sites, regional lymph nodes, and distal extracranial metastases. We performed an integrative analysis of somatic single nucleotide variants and copy-number alterations to reconstruct phylogenetic trees relating the subclones from each patient. We analyzed evolutionary relationships between related cancer samples and annotated phylogenetic trees with clinically significant genetic alterations. Results: Every brain metastasis displayed branched evolution: the brain metastasis and primary tumor shared a common ancestor yet both the primary tumor and brain metastasis continued to evolve independently. We found novel clinically actionable genetic alterations that were exclusive to brain metastases in 56% of cases. The brain metastases were also enriched for several pathways, some pathways specific to a particular histology. Distal extracranial metastases and regional lymph nodes were highly divergent from brain metastases, and in no cases, did we observe an extracranial site that closely resembled the brain metastasis. In contrast, regionally and anatomically separated brain metastasis sites were genetically homogenous and shared nearly all genetic alterations detected. Conclusions: Brain metastases are genetically divergent from primary tumors. Clinically, these observations demonstrate that biopsies of primary tumors fail to capture the heterogeneity with patients with brain metastases, potentially missing clinically actionable mutations in these life-threatening metastases. Notably, regional lymph nodes and distal extracranial metastases were not reliable genetic surrogates for brain metastases. When clinically feasible, characterization of even a single brain metastasis lesion is superior to that of a primary or lymph node biopsy for selection of a targeted therapeutic agent. Citation Format: Priscilla K. Brastianos, Scott L. Carter, Sandro Santagata, Amaro Taylor-Weiner, Robert T. Jones, Eli Van Allen, Keith L. Ligon, Josep Tabernero, Joan Seoane, Elena Martinez-Saez, Daniel Cahill, William T. Curry, Ian F. Dunn, Sun Ha Paek, Paul Van Hummelen, Aaron R. Thorner, Bruce E. Johnson, Nancy U. Lin, Toni K. Choueiri, Michael S. Rabin, Rameen Beroukhim, Anat Stemmer-Rachamimov, Matthew Meyerson, Levi Garraway, Tracy Batchelor, Jose Baselga, David N. Louis, William C. Hahn, Gad Getz. Genomic characterization of brain metastases reveals divergent evolution and metastasis specific mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4727. doi:10.1158/1538-7445.AM2015-4727

Identification of copy number alterations (CNAs) in tumors can assist in determining diagnosis, prognosis, or predicting response to therapy in certain cancer types. Many CNA detection tools have been developed for whole genome sequencing (WGS). These algorithms typically “call” alterations by using significant deviations of the log2 ratio of tumor to normal coverage from the expected diploid log2 ratio. Several factors, however, including reporting pertinent genomic information, hardware and software limitations, and cost, have impeded the routine application of WGS in a clinical setting. Targeted sequencing - hybrid capture and massively parallel sequencing of defined regions (targets) of the genome can overcome some of these challenges and can specifically interrogate, for example, known cancer genes, thus maximizing the “actionability” or utility of the information generated. Though more suited for large-scale clinical testing, targeted sequencing of smaller gene panels with specific clinical reference presents some significant bioinformatic challenges that hinder the application of pre-existing CNA detection tools, resulting in a heavy dependence on manual review in the clinic. In addition, varying quality of DNA, minor variations in laboratory protocols, along with batch effects, are known to confound CNA profiles and are exasperated by the decreased number of sequenced regions of targeted gene panels. These variables alter the log2 ratio, and thus, our ability to call CNAs suffers. We note, that using matched normals may mitigate some deleterious effects, however, to minimize clinical cost and throughput, we tackle these challenges with unmatched tumor-normal pairs. Similar to the closest normal approach used in SNP array analyses, we rely on the assumption that some “normal” (non-tumor) samples experience similar experimental variability that affects the tumor samples. Minimizing maximally informative cost-functions (incorporating both global and local CNA variation), we iteratively investigate different combinations of normal samples which best resemble a given cancer sample. The new set of normals which minimize the cost-function, defines a panel of normals (PON) whose target medians are used to generate the log2 ratio. Along with batch-effect reduction techniques, we then call CNAs with statistically significant thresholds at the target-level. Interestingly, we find that approximately 15 normals are typically needed to minimize CNA profile variation, as opposed to our typical approach of using all available normals (∼60). In addition, we demonstrate that our procedure can improve our sensitivity and specificity from 58±1% to 81±2% and 94±1% to 99.5±0.1%, respectively, on simulated data. We further compared our approach with other published algorithms and found a significant improvement in performance, reducing the time required to manually review CNAs and interpret copy number profiles in the clinic. Citation Format: Bernard Fendler, Ryan Abo, Samuel Hunter, Matthew Ducar, Elizabeth Garcia, Paul Van Hummelen, Neal Lindeman, Laura MacConaill. Identifying copy number alterations from targeted sequencing data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4850. doi:10.1158/1538-7445.AM2015-4850

Identification of structural gene rearrangements is vital for cancer patients as these events can provide definitive diagnoses, prognostic value, and influence the course of treatment. While FISH, karyotype analysis and aCGH array have traditionally been used to identify and confirm the presence of structural variants, the advent of next generation sequencing has enabled genetic testing including detection of multiple structural variants (SVs) from genomic DNA. To this end, we have developed and validated Oncopanel, a cancer-specific targeted next generation sequencing (NGS) assay designed to detect SNVs, indels, and copy number alterations across 300 genes, and 35 clinically actionable or informative SVs. Each rearrangement was specifically targeted by baiting genomic locations frequently identified to contain breakpoints reported in the literature and publicly available databases. Using BreaKmer, an internally developed SV detection tool (Nucleic Acids Res. 2014 Nov 26, doi: 10.1093/nar/gku1211), rearrangements, including the exact breakpoint coordinates and the genes involved in or adjacent to the breakpoint(s), were identified. Here we examine the utility of Oncopanel using genomic DNA to identify structural variants. We report the results of 3,291 cancer patients tested in our personalized cancer medicine program (Profile), a joint venture between Dana-Farber Cancer Institute, Brigham and Women's Hospital, and Boston Children's Hospital. As compared to conventional cytogenetics, FISH analysis, and molecular detection by PCR methods, Oncopanel's overall sensitivity and specificity for SVs was 81.4% and 100%, respectively. Most discordant results were identified in (1) tumors with SVs involving the IGH enhancer regions (60% of discordant results), or (2) in samples with < 20% tumor (25% of discordant results). Several SVs involving the IGH enhancer regions were missed likely due to lack of Oncopanel coverage. Oncopanel was designed to target a finite sequence pool, but due to IGH enhancer region's large size (1.2Mb), only a small portion of this region was specifically interrogated. Inclusion of all possible IGH enhancer sequences would have hampered the effectiveness of SNV, indel and copy number alteration detection for other cancer critical genes. Discrepant Oncopanel and cytogenetic results were also observed in samples with low tumor purity likely due to masking of variant sequences by stromal contamination. In conclusion, we find that Oncopanel has utility to detect structural variants with a sensitivity of 92%, barring detection of rearrangements involving IGH, and a specificity of 100%. Based on the baiting strategy, detection of many rearrangements can also be interrogated in parallel with SNV, indel and CNV detection resulting in reduced sample input requirements and the inclusion of precise information regarding the breakpoints and the class of rearrangement identified. Citation Format: Elizabeth P. Garcia, Azra H. Ligon, Ryan P. Abo, Paola S. Dal Cin, Stanislawa Weremowicz, Priyanka Shivdasani, Phani K. Davineni, Dimity L. Zepf, Matthew D. Ducar, Paul Van Hummelen, Yonghui Jia, Frank C. Kuo, Lynette M. Sholl, Laura E. MacConaill, Neal I. Lindeman. Detection of gene rearrangements using OncoPanel: a targeted next-generation sequencing assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2015-2991

Background: The overall death rate from lung cancer is higher in African-Americans (AA) compared to Caucasians (CAU). Understanding differences in the prevalence and type of somatic alterations between races may illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients’ treatment. Methods: Formalin-fixed paraffin embedded tumor samples were collected from Baptist Cancer Center, Memphis, TN. DNA was extracted and sonicated to 250 bp following Covaris FFPE DNA Extraction & Purification protocol and further purified using Agencourt AMPure XP beads. The OncoPanel_v2 target enrichment panel (Agilent SureSelect) was used for hybrid capture of 502 cancer-related genes. Samples were pooled and sequenced on an Illumina HiSeq2500 to a mean depth of coverage of 210x. Tumors with >30x sequencing depth over >80% of targeted bases were considered for analysis. MuTect and SomaticIndelDetector were used to identify somatic single nucleotide variants (SNVs) and short insertions or deletions (indels), respectively. As matched normal DNA was not available for these samples, analysis was limited to variants previously observed in tumors as described in the Catalogue of Somatic Mutations in Cancer (COSMIC) database, and variants were excluded if found in the germline dataset of the Exome Sequencing Project (ESP). Results: 510 tumor specimens from 242 Black and 268 White patients (with self-reported race) were analyzed including 320 adenocarcinomas and 142 squamous cell carcinomas. Pathological classification was independently reviewed and confirmed for 374 of 472 cases for an overall concordance rate of 79%. Using principle component analysis (PCA) on germline SNVs, we observed that the biological ancestry was different than the self-reported race for 1.5% of patients. Mutational frequencies for genes with known roles in adenocarcinoma such as KRAS and EGFR were not significantly different between tumors from Black and White patients (Fisher's exact p-value > 0.05). Amplification rates for NKX2-1, MET, MDM2, and MYC and homozygous deletion rates for CDKN2A were also not significantly different between populations. Translocations involving ALK and ROS1 were detected in tumors from Black patients demonstrating that these events are present in both populations. Similarly, mutational frequencies for genes such as PIK3CA and PTEN were not significantly different across populations in squamous cell carcinomas. Conclusions: These results demonstrate that lung cancers from Black patients are more similar to Whites than East Asians with respect to genes such as EGFR, and suggest that clinical trials of targeted therapies could significantly benefit patients in both populations. Citation Format: Joshua Campbell, Christopher Lathan, Lynette Sholl, Matthew Ducar, Mikenah Vega, Ling Lin, Aaron Thorner, Nick Faris, Paul van Hummelen, Raymond Osarogiagbon, Matthew Meyerson, Laura MacConaill. Comparing the mutational landscape of African American and Caucasian lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2015-3886

RNA sequencing (RNA-seq) is a transcriptome profiling technology that provides multiple levels of insight into the genome. In addition to expression levels (transcript abundance), it generates endpoints such as alternative splicing, somatic mutations and rearrangements, which may have functional consequences in cancer. Although somatic mutations are generally identified by DNA sequencing, RNA-seq has the advantage of detecting allele-specific expression affecting a variant allele, as well as functional chimeric transcripts that result from structural rearrangements. Compared to microarray technologies, RNA-seq can provide additional information about novel transcripts. Due to the complexity of the human transcriptome and the variability of gene abundance, the cost of whole transcriptome sequencing to achieve sufficient coverage to detect these types of alterations remains high. To explore the feasibility of a more cost-effective method, we compared the performance of three different RNA-seq methods: whole-transcriptome-, exome-, and targeted RNA-seq, using RNA derived from cancer cell lines and Formaldehyde Fixed-Paraffin Embedded (FFPE) samples. For whole-transcriptome preparation, we used the Illumina TruSeq Stranded mRNA and total RNA kits for cell line and FFPE samples, respectively. Exome-RNAseq was performed using the Illumina Access kit. The libraries from whole-transcriptome RNAseq were subjected to hybridization capture using OncoPanel-an Agilent SureSelect baitset of 500 cancer-related genes. Compared to whole-transcriptome, exome- and targeted-RNA-seq demonstrated (1) higher coding exon coverage and multiplexing capability; (2) reduced rRNA composition to 1%; (3) comparable gene abundance information and (4) over 90% of reads aligned to coding exon regions in FFPE samples, compare to ∼30% when using whole transcriptome method. In conclusion, we demonstrated that exome- and targeted RNA-seq provide a cost-effective way to analyze a subset of the transcriptome. Furthermore, targeted RNA-seq can be highly multiplexed and is therefore amenable for large-scale tumor profiling in clinical or research settings. Citation Format: Ling Lin, Ryan Abo, Deniz Dolcen, Rachel Paquette, Angelica Laing, Luc de Waal, Aaron Thorner, Matthew Ducar, Liuda Ziaugra, Bruce Wollison, Marc Breneiser, William Hahn, Matthew Meyerson, Paul Van Hummelen, Laura MacConaill. Targeted RNA sequencing improves transcript analysis in cancer samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1115. doi:10.1158/1538-7445.AM2015-1115

299 Background: The genomic landscape of high-grade T1 micropapillary bladder tumors (HGT1micropap) is unknown. Clinically, micropapillary bladder cancer is an aggressive and possibly lethal disease. Our main objective was to assess the genomic landscape of HGT1micropap through identifying mutations, insertions/deletions (indels), translocations, and copy number variations (CNVs). Methods: We prospectively identified nine HGT1micropap with 45.4 months of median follow up. Patients were treated in a uniform manner using TUR, BCG, and appropriate follow up. We performed whole exome sequencing using Ilumina Exome _v5 plus translocation. Mutations and indels were called using the Firehose pipeline. CNVs were called using ExomeCNV. We examined the mutational landscape and compared the genomic alterations to TCGA (>T2, n=131)2 and publicly available data on non-muscle invasive bladder tumors (Ta/T1, n=37)1. Results: Within the HGT1micropap, mutations on TP53, KMT2D, TSC1, and ATM were suggested to occur more frequently compared to the NMIBC control group1. FGFR3 was seen at the expected frequency for NMIBC. The mutations of interest are presented in Table 1 with the percentage seen in the other cohorts. Of interest, TSC1 was seen in higher frequency in micropapillary than in the NMIBC or the TCGA cohort2. We did not see any patterns between CNVs and mutations. We also saw two patients with severe chromothripsis. 3 patients had loss of chromosome 9 or 9q without any other severe chromosome alterations. CNV alterations will be presented and compared to MIBC. Conclusions: In this preliminary analysis, our HGT1micropap, showed a mutational landscape more similar to MIBC compared to NMIBC bladder landscape. We did not find any clear driver of the micropapillary histology at the exome level in this limited sample of patient, which may indicate that tumor heterogeneity or epigentic changes may be driving this aggressive phenotype. [Table: see text]

BACKGROUND: Brain metastases are the most common intracranial tumors in adults. We have a limited understanding of how brain metastases evolve from their primary tumor. Our objectives were to (1) elucidate the evolutionary patterns leading to brain metastases and (2) identify whether brain metastases are genetically distinct from their primary tumors. METHODS: We subjected 101 matched primary tumors, brain metastases, and normal tissue to whole exome sequencing. We developed novel computational tools to perform an integrative analysis of somatic mutations and copy-number alterations. This analysis allowed us to estimate the clonal architecture of the primary and metastases, and to reconstruct a phylogenetic tree relating the subclones from each patient. RESULTS: Every brain metastasis likely developed from a single clone. The brain metastasis and primary tumor shared a common ancestor yet both the primary tumor and brain metastasis continued to evolve independently. Subclonal mutations in the metastasis by definition occurred within the brain; these mutations displayed different mutational signatures than those acquired in the primary tumor. These contrasts were most pronounced in cases of lung cancer or melanoma, with tobacco and UV signatures prominent in these primaries and nearly absent from mutations acquired after metastasis. In order to understand the molecular drivers of clonal evolution and metastasis, we annotated each subclone with driver mutations identified using large numbers of cancer samples analyzed by the cancer genome atlas consortium. This produced a detailed portrait of each patient's cancer, with nearly every node in each phylogenetic tree associated with at least one known driver. We found novel drivers, many of which are known actionable targets, that were exclusive to brain metastases. CONCLUSIONS: This study sheds light on the evolutionary mechanisms in brain metastases. Our data suggests that single biopsies of primary tumors and metastases may not be sufficient to identify potential therapeutic targets.

Background: Brain metastases are the most frequently occurring intracranial tumors in adults. Median survival after the diagnosis of a brain metastasis is in the order of a few months. Despite its large burden of disease and devastating clinical sequelae, we continue to have a limited understanding of how brain metastases evolve from their primary tumor. Our objectives were to (1) elucidate the evolutionary patterns leading the brain metastases and (2) identify whether brain metastases are genetically distinct from their matched primary tumors. Materials and Methods: We subjected 101 trios consisting of primary tumor, brain metastasis, and matched normal tissue to whole exome sequencing (WES). To analyze the data, we developed novel computational tools to perform an integrative analysis of somatic single nucleotide variants (SSNVs) and somatic copy-number alterations (SCNAs). This analysis allowed us to estimate the clonal architecture of the primary and metastatic samples from each patient, and to reconstruct a phylogenetic tree relating all of the subclones.Results: Every metastasis developed from a single clone, consistent with a single cell of origin. We did not detect evidence of self-seeding or a multiclonal origin of metastasis. In all cases, we observed a sibling or a branched evolutionary relationship; the brain metastasis and primary tumor share a common ancestor, but there was continued evolution in the primary tumor reflected by fully clonal mutations in the primary biopsy that were not present in the metastasis. When we average over all the phylogenetic trees, 61% of mutations are present in the common ancestor, 24% are unique to the metastasis and 15% are unique to the primary tumor. Subclonal mutations in the metastasis by definition occurred within the brain; these mutations displayed different mutational signatures than those acquired in the primary tumor. These contrasts were most pronounced in cases of lung cancer or melanoma, with tobacco and UV signatures prominent in these primaries and nearly absent from the mutations acquired after metastasis. In order to understand the molecular drivers of clonal evolution and metastasis in our data, we annotated each subclone with driver mutations identified using large numbers of cancer samples analyzed by the cancer genome atlas (TCGA) consortium. This produced a detailed portrait of each patient's cancer, with nearly node in each phylogenetic tree associated with at least one known driver. We found novel drivers, many of which are known actionable targets, in the clonal and subclonal populations within the brain metastases that were not present in the primary tumor. This suggests ongoing evolution within the brain. Similarly, novel subclonal and clonal drivers were detected in the biopsy of the primary tumor that were not present in the metastasis. The brain metastases were enriched for several pathways when compared to their matched primary tumors, some pathways specific to a particular histologic subtype.Conclusions: In this study, we report the latest results of the largest massively parallel sequencing study to date of matched brain metastases and primary tumors. We used intratumoral heterogeneity estimates to elucidate the evolutionary patterns observed in the process of metastasis. This study shifts our understanding of the metastasis paradigm and sheds light on the evolutionary and molecular mechanisms that are critical for brain metastasis. Our data suggests that single biopsies do not capture the heterogeneity within patients. Assessment of the subclonal phylogenetic architecture of primaries and their metastases should be considered when selecting targeted agents for patients with brain metastases. Citation Format: Priscilla K. Brastianos, Scott L. Carter, Sandro Santagata, Amaro Taylor-Weiner, Robert T. Jones, Peleg M. Horowitz, Keith L. Ligon, Joan Seaone, Elena Martinez-Saez, Josep Tabernero, Daniel P. Cahill, Sun-Ha Paek, Ian F. Dunn, Bruce E. Johnson, Toni K. Choueiri, Michael S. Rabin, Eric P. Winer, Nancy U. Lin, Paul Van Hummelen, Anat Stemmer-Rachamimov, Rameen Beroukhim, David L. Louis, Tracy T. Batchelor, Jose Baselga, Gad Getz, William C. Hahn. Genomic characterization of 101 brain metastases and paired primary tumors reveals patterns of clonal evolution and selection of driver mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG03. doi:10.1158/1538-7445.AM2014-NG03

Targeted next-generation sequencing (NGS) to capture genes or regions of interest has proven to be a cost-effective alternative to whole genome sequencing (WGS), particularly for cancer research and clinical cancer care. In this context, biologically or clinically relevant selected genes/regions are sequenced to several hundred-fold coverage. However, current algorithms and tools for detecting large structural variants (SVs), such as translocations, fail to achieve either high specificity or sensitivity, due to the fact that most available methods were designed for WGS data, and thus do not take advantage of the reduced size and higher coverage of targeted sequencing to improve SV calling. We developed a novel method, BreaKmer, to detect indels, rearrangements, and translocations from single sample targeted genomic reads. The algorithm extracts mis-mapped single or paired-end NGS reads. From these reads, hypothesized to contain SV breakpoints, contigs are built with a kmer strategy: reads are broken into k-length substrings or kmers, and those occurring in the reference are filtered. The remaining kmers represent sequences containing any sequence variant from the reference, ranging from single nucleotides to larger variants. Contigs are assembled from reads containing sample specific kmers. SVs are called based on alignment of the contigs to the reference sequence. With paired-end (PE) reads, discordantly mapped paired reads are extracted and coupled with SV calls that are made. To demonstrate BreaKmer, we analyzed NGS data from 166 samples enriched using 3 different capture panels (ranging from 305-504 genes). Our dataset contained 25 cancer specimens with known translocation events verified by orthogonal clinical methods and a negative control set of 141 ‘normal’ samples with no known SVs. The samples represented DNA extracted from FFPE, fresh frozen, blood and cell lines. Among 25 samples, 15 had additional replicate samples. Mean target coverage over all the samples was on average 150x. Specimens were barcoded at library preparation and pooled, followed by hybrid-capture targeting cancer genes and sequenced 2x100bp PE. All translocation events from the 25 test samples and their replicates (46) were detected by BreaKmer. An additional 19 translocations were detected among all the samples and their replicates, while no translocation calls were made for the 141 negative control samples. Our novel kmer strategy to detect SVs displayed high sensitivity and specificity. We reliably detected rearrangements of ALK, BCL2-IGH, BCR-ABL1 from lung adenocarcinoma, B-cell lymphoma, and chronic myeloid leukemia samples, respectively; indicating real clinical utility of this algorithm. In addition, our tool effectively detects other SV types - such as indels in FLT3 and KIT, among other genes. Our algorithm thus serves a pressing need for improved SV detection in targeted NGS data, particularly in precision cancer medicine. Citation Format: Ryan P. Abo, Elizabeth P. Garcia, Matthew Ducar, Ravali Adusumilli, Marc Breneiser, Vanessa Rojas-Rudilla, Lynette M. Sholl, Neal I. Lindeman, Matthew L. Meyerson, William C. Hahn, Paul Van Hummelen, Laura E. MacConaill. BreaKmer: Detection of structural rearrangements in targeted next-generation sequencing data using kmers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5321. doi:10.1158/1538-7445.AM2014-5321

Next-generation sequencing (NGS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimen tissues are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Consequently, limited DNA input necessitates amplification during NGS library preparation. Therefore, it is of great importance to amplify the library in an efficient and uniform manner to achieve reproducible amplification of the library fragments and limit GC content bias. Variable GC-bias among libraries is of particular importance for copy number variant (CNV) detection based on sequence coverage. Here, we compare the TruSeq DNA Sample Preparation Kit (Illumina Inc., San Diego, CA) and the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to determine which technique achieves optimal library coverage. The major difference between the methods is the DNA polymerase used for library enrichment. The Kapa HiFi polymerase was engineered to have higher fidelity and increased DNA affinity relative to the Herculase II Fusion DNA Polymerase (Agilent Technologies, Inc., Santa Clara, CA) that we used for library enrichment with the TruSeq kit. Thus, the Kapa HiFi polymerase should more efficiently amplify targets across a larger GC content range and improve sequence coverage uniformity. Relative to TruSeq-prepared libraries, the Kapa-prepared libraries had a larger mean insert size, more uniform coverage distribution, and better coverage of GC-rich regions of the genome. This was observed for DNA extracted from blood, cell lines, and formalin-fixed paraffin embedded (FFPE) tumor samples. More experiments are underway to determine the effect of the improved recovery of high-GC content DNA fragments on CNV detection. Citation Format: Aaron R. Thorner, Ashwini Sunkavalli, Ling Lin, Robert T. Jones, Laura Schubert, Matthew D. Ducar, Ravali Adusumilli, Deniz Nesli Dolcen, Liuda Ziaugra, Jack Lepine, Laura E. MacConaill, William C. Hahn, Matthew Meyerson, Paul Van Hummelen. Reducing GC-bias and improving coverage distribution in Illumina sequencing using the Kapa Biosystems library construction method. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3583. doi:10.1158/1538-7445.AM2014-3583

Formalin fixation followed by paraffin embedding (FFPE) is the most widely used method of tissue preservation and often used in the clinical setting for cancer specimens. However, this process of fixation has negative consequences on the quality of extracted DNA, and inevitably adversely affects downstream analyses such as next generation sequencing (NGS). DNA extraction from FFPE samples requires paraffin disassociation, tissue rehydration and de-crosslinking. Low efficiency in any step will yield low quantities of nucleic acids with incomplete reversal of formaldehyde crosslinks, and mixtures of single and double stranded DNA. In addition, low quality nucleic acids will often result in poor sequencing quality due to inefficient library preparation, lower number of reads with high quality, and variable recovery of sequence reads with high GC-content. Here we describe the use of Adaptive Focused Acoustics technology (Covaris Inc. 2013) to remove paraffin and break down tissue, and we compared this method to traditional xylene-based paraffin removal. We found that Covaris had superior performance overall: (1) DNA yields were 6 times higher, and less specimens failed extraction: this is critically important when dealing with small tumor biopsies. (2) DNA was more readily available for PCR amplification. (3) Mean coverage of DNA fragments with high GC content was higher and more reproducible. (4) Library complexity was higher. In addition, the Covaris protocol required less hands-on time in the lab, and eliminated the need for hazardous organic solvents for paraffin removal. In conclusion, the Covaris FFPE DNA extraction method is very efficient, enhances DNA availability, improves reproducibility and provides higher DNA quality for NGS. Citation Format: Ling Lin, Mikenah Vega, Robert T. Jones, Liuda Ziaugra, Deniz N. Dolcen, Ashwini Sunkavalli, Laura Schubert, Jack R. Lepine, Aaron R. Thorner, Matthew D. Ducar, William C. Hahn, Matthew L. Meyerson, Laura E. MacConaill, Paul Van Hummelen. Improved FFPE DNA extraction for next generation sequencing using adaptive focused acoustics technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4286. doi:10.1158/1538-7445.AM2014-4286

Conclusions: Sensitivity for CIN3+ of p16/Ki-67 from SurePath medium in women with HPV positive LSIL is sufficient that immediate colposcopy is not required for women who are p16/Ki67 negative. Compared to current guidelines for LSIL cytology, p16/ Ki-67 triage could reduce immediate referral to colposcopy by 35%. Unlike p16 alone, which requires morphologic assessment of p16positive cells, p16/Ki-67 evaluation is based on staining alone and may be fully automated. Further studies are required to evaluate p16/Ki-67 for triage of HPV-positive women with NILM and ASC-US. If a negative predictive value of similar magnitude is observed for these women, a substantial and welcome decrement in the total number of colposcopies required will be possible without significant loss of sensitivity for the screening program.

4511 Background: An integrative analysis was conducted to identify genomic alterations and mutational status on the gene pathway level that could predict OS in pts with urothelial cancers (UC) trea...

5515 Background: Although PTEN mutations or loss of PTEN expression are uncommon in endometrial serous carcinomas, approximately half of these tumors harbor PIK3CA mutations and/or amplification of...