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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%.

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.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

// 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.

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.

In the version of this article originally published, an asterisk was omitted from Fig. 1a. The asterisk has been added to the figure. Additionally, a “NOTCH2” label was erroneously included in Fig. 4a. The label has been removed. The errors have been corrected in the PDF and HTML versions of this article.

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.

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...

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.

Background An integrative analysis was conducted to identify genomic alterations at a pathway level that could predict overall survival (OS) in patients with advanced urothelial carcinoma (UC) treated with platinum-based chemotherapy. Patients and Methods DNA and RNA were extracted from 103 formalin-fixed paraffin embedded (FFPE) invasive high-grade UC samples and were screened for mutations, copy number variation (CNV) and gene expression analysis. Clinical data were available from 85 cases. Mutations were analyzed by mass-spectrometry based on genotyping platform (Oncomap 3) and genomic imbalances were detected by comparative genomic hybridization (CGH) analysis. Regions with threshold of log2 ratio ≥0.4, or ≤0.6 were defined as either having copy number gain or loss and significantly recurrent CNV across the set of samples were determined using a GISTIC analysis. Expression analysis on selected relevant UC genes was conducted using Nanostring. To define the co-occurrence pattern of mutations and CNV, we grouped genomic events into 5 core signal transduction pathways: 1) TP53 pathway, 2) RTK/RAS/RAF pathway, 3) PI3K/AKT/mTOR pathway, 4) WNT/CTNNB1, 5) RB1 pathway. Cox regression was used to assess pathways abnormalities with survival outcomes. Results 35 samples (41%) harbored mutations on at least one gene: TP53 (16%), PIK3CA (9%), FGFR3 (2%), HRAS/KRAS (5%), and CTNNB1 (1%). 66% of patients had some sort of CNV. PIK3CA/AKT/mTOR pathway alteration (mutations+CNV) had the greatest impact on OS (p=0.055). At a gene level, overexpression of CTNNB1 (p=0.0008) and PIK3CA (p=0.02) were associated with shorter OS. Mutational status on PIK3CA was not associated with survival. Among other individually found genomic alterations, TP53 mutations (p=0.07), mTOR gain (p=0.07) and PTEN overexpression (p=0.08) have a marginally significant negative impact on OS. Conclusions Our study suggests that targeted therapies focusing on the PIK3CA/AKT/mTOR pathway genomic alterations can generate the greatest impact in the overall patient population of high-grade advanced UC.

Mutations of the TSH receptor leading to constitutive activation of the cAMP cascade are responsible for the development of hot nodules, if arising in a somatic cell, and nonautoimmune hyperthyroidism, when occurring in a germinal cell. An animal model of constitutive activation of the thyroid cAMP cascade has been obtained by generating transgenic mice expressing the adenosine receptor (Tg-A2aR) under the control of the thyroglobulin promoter. These mice develop huge goiters and die prematurely due to hyperthyroidism induced cardiac failure. To identify new genes involved in the tumorigenic pathway of the thyroid, we designed a protocol using microarray technology to study the differential expression, between normal and transgenic thyroid, of +/-13,000 genes. A total of 360 genes or expressed sequence tags showed a strong modulation with background corrected values of fluorescence superior to 2-fold change. The modulated genes were classified according to their proposed gene ontology functions. Approximately half of them were up-regulated. The function of the majority of these genes in thyroid physiology is still to be determined. Some of them, like IGF-I or IGF binding protein 3 or 5, may play an important role in the development of thyroid nodules through paracrine mechanisms. This study demonstrates the feasibility of sequentially following the cascade of events leading to the formation of benign tumors such as hot thyroid nodule or hyperfunctional goiter.

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

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.

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.

Erythropoietin (EPO) is a neuroprotective cytokine in models of ischemic and nervous system injury, where it reduces neuronal apoptosis and inflammatory cytokines and increases neurogenesis and angiogenesis. EPO also improves cognition in healthy volunteers and schizophrenic patients. We studied the effect of EPO administration on the gene-expression profile in the ischemic cortex of rats after cerebral ischemia at early time points (2 and 6 h). EPO treatment up-regulated genes already increased by ischemia. Hierarchical clustering and analysis of overrepresented functional categories identified genes implicated in synaptic plasticity— Arc , BDNF , Egr1 , and Egr2 , of which Egr2 was the most significantly regulated. Up-regulation of Arc , BDNF , Dusp5 , Egr1 , Egr2 , Egr4 , and Nr4a3 was confirmed by quantitative PCR. We investigated the up-regulation of Egr2 / Krox20 further because of its role in neuronal plasticity. Its elevation by EPO was confirmed in an independent in vivo experiment of cerebral ischemia in rats. Using the rat neuroblastoma B104, we found that wild-type cells that do not express EPO receptor (EPOR) do not respond to EPO by inducing Egr2 . However, EPOR-expressing B104 cells induce Egr2 early upon incubation with EPO, indicating that Egr2 induction is a direct effect of EPO and that EPOR mediates this effect. Because these changes occur in vivo before decreased inflammatory cytokines or neuronal apoptosis is evident, these findings provide a molecular mechanism for the neuroreparative effects of cytokines and suggest a mechanism of neuroprotection by which promotion of a plastic phenotype results in decreased inflammation and neuronal death.

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 and Objectives Mesenchymal stem cells (MSC) are adult stem cells that can be expanded many fold in vitro and have the therapeutic potential to restore the bone marrow microenvironment and support hematopoietic recovery after myeloablative conditioning for hematopoietic stem cell transplantation. Successful homing to the target tissue, such as bone marrow, implies that MSC are able to extravasate after systemic administration. However, the extravasation capacity of MSC and the underlying mechanisms are poorly understood to date. We studied in vitro the capacity of MSC to migrate through bone marrow endothelium.Design and Methods In vitro invasion and transendothelial migration assays were performed. The expression of matrix metalloproteinase (MMP) was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Migration of cells cultured at high or low confluence was compared and differential gene expression in these conditions was analyzed with microarray and real-time RT-PCR. The functional involvement in MSC migration was assessed using neutralizing anti-MMP-2 antibody, MMP-2 short interfering RNA or recombinant tissue inhibitor of metalloproteinase (TIMP-3).Results We demonstrated that MSC can invade reconstituted basement membrane and that bone marrow endothelial cells stimulate this process. We also showed that the transendothelial migration of MSC is at least partially regulated by MMP-2. High culture confluence was found to increase production of the natural MMP-inhibitor TIMP-3 and decrease transendothelial migration of MSC.Interpretation and Conclusions We show that MSC have the potential to migrate through bone marrow endothelium and that this process involves MMP-2. Moreover, the migration of MSC is significantly influenced by the level of culture confluence. Increased culture confluence impairs migration and is related to an upregulation of TIMP-3. The therapeutic use of MSC would benefit from a selection of culture conditions that allow optimal extravasation of these cells.

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

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

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]

To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. Microarray analysis identified 692 genes with significant differences in expression. Of these, 28 appeared to be down-regulated and 12 up-regulated at least 2-fold in SCARKOs compared with controls. For nine of the more than 2-fold down-regulated genes, androgen regulation was confirmed by treatment of wild-type mice with an antiandrogen ( flutamide). Some of them were previously described to be androgen regulated or essential for spermatogenesis. Serine-type protease inhibitors were markedly overrepresented in this down-regulated subgroup. A time study (d 8-20), followed by cluster analysis, allowed identification of distinct expression patterns of differentially expressed genes. Three genes with a pattern closely resembling that of Pem, a prototypical an-drogen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RTPCR and additional analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. Moreover, they suggest that protease inhibitors and other proteins related to tubular restructuring and cell junction dynamics may be controlled in part by androgens.

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

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.

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...

Meningiomas are the most common primary brain tumor in the US. While many Grade I meningiomas are cured by surgery, 20% recur and are minimally responsive to systemic therapy. Meningiomas of higher grades are particularly challenging to treat and are associated with a poor prognosis. Though the tumor suppressor gene NF2 has been linked to meningiomagenesis, the spectrum of genetic changes in meningiomas remains poorly understood, particularly in the large subset of tumors without NF2 alterations. We performed whole-exome or whole-genome sequencing on 17 meningiomas and focused sequencing using a 645 gene-set on an additional 48 tumors. The majority of meningiomas exhibited simple genomes, with fewer copy-number alterations, mutations, and rearrangements compared to other adult tumors. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors. A subset of NF2-wildtype meningiomas harbored recurrent oncogenic mutations in AKT1 and the Hedgehog pathway regulator SMO. Tumors with these mutations varied histologically from their NF2-mutated counterparts, and exhibited immunohistochemical evidence of pathway activation. These results begin to define the spectrum of genetic alterations in meningiomas. Our findings of known driver oncogenic mutations including AKT1 and SMO have immediate therapeutic potential. Citation Format: Priscilla Brastianos, Peleg M. Horowitz, Sandro Santagata, Robert T. Jones, Aaron McKenna, Gad Getz, Keith Ligon, Paul Van Hummelen, Matt Ducar, Alina Raza, Ashwini Sunkavalli, Laura MacConaill, Anat Stemmer-Rachamimov, David N. Louis, William C. Hahn, Ian Dunn, Rameen Beroukhim. Genomic sequencing of meningiomas reveals oncogenic SMO and AKT1 mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2013-803

Background Given the high incidence of metastatic esophageal squamous cell carcinoma, especially in Asia, we screened for the presence of somatic mutations using OncoMap platform with the aim of defining subsets of patients who may be potential candidate for targeted therapy. Methods and Materials We analyzed 87 tissue specimens obtained from 80 patients who were pathologically confirmed with esophageal squamous cell carcinoma and received 5-fluoropyrimidine/platinum-based chemotherapy. OncoMap 4.0, a mass-spectrometry based assay, was used to interrogate 471 oncogenic mutations in 41 commonly mutated genes. Tumor specimens were prepared from primary cancer sites in 70 patients and from metastatic sites in 17 patients. In order to test the concordance between primary and metastatic sites from the patient for mutations, we analyzed 7 paired (primary-metastatic) specimens. All specimens were formalin-fixed paraffin embedded tissues and tumor content was >70%. Results In total, we have detected 20 hotspot mutations out of 80 patients screened. The most frequent mutation was PIK3CA mutation (four E545K, five H1047R and one H1047L) (N = 10, 11.5%) followed by MLH1 V384D (N = 7, 8.0%), TP53 (R306, R175H and R273C) (N = 3, 3.5%), BRAF V600E (N = 1, 1.2%), CTNNB1 D32N (N = 1, 1.2%), and EGFR P733L (N = 1, 1.2%). Distributions of somatic mutations were not different according to anatomic sites of esophageal cancer (cervical/upper, mid, lower). In addition, there was no difference in frequency of mutations between primary-metastasis paired samples. Conclusions Our study led to the detection of potentially druggable mutations in esophageal SCC which may guide novel therapies in small subsets of esophageal cancer patients.

5011 Background: Cervical adenocarcinomas (AC) have higher rates of recurrence and distant metastasis, compared with squamous cell carcinomas (SCC), and decreased survival in advanced disease. Yet, tailored treatments for cervical cancers have not emerged. The aim of this study was to compare the frequency and type of somatic mutations in cervical AC and SCC to identify novel therapeutic targets for both subtypes. Methods: Tumors from 61 patients with cervical cancer (36 AC, 25 SCC) underwent genomic profiling by OncoMap, a multiplexed mass spectrometric genotyping technology that interrogates more than 400 known mutations in 33 cancer genes. Results: Overall, 31/61 (50.8%) tumors harbored candidate mutations, and 6/61 (9.8%) had ≥2 mutations. PIK3CA mutations were present in 21/64 (34.4%) of cervical cancers, with a trend towards higher rates in SCC compared with ACC (21/25 or 48.0% vs. 9/27 or 33.3%, p=0.10). KRAS mutations were present in 6/31 (16.7%) of AC, but none of the SCC (0%; 0/25). EGFR mutations were present in 9/25 (36.0%) of SCC, including G719S, but none of AC (0%; 0/31). Conclusions: The identification of distinct genomic alterations in SCC and AC of the cervix suggest different therapeutic rationales for each subtype. EGFR amplification in cervical SCC has been previously reported, but this is the first identification, to our knowledge, of activating EGFR mutations in cervical SCC. Together, this suggests that EGFR-inhibitors might be useful in selected patients with cervical SCC. Similarly, activating mutations in KRAS and PIK3CA in AC suggest that inhibition of the MAPK pathway (MEK inhibitors) and/or PI3K pathway may be beneficial in this subtype. Future studies should include more comprehensive genomic profiling strategies, such as targeted massively parallel sequencing, to detect multiple types of genomic alterations.

Detection of critical cancer gene mutations in tumor samples may result in improved care, treatment and outcomes for patients. It has become clear that for specific cancer types there is a high association between mutations in a defined set of oncogenes or tumor suppressors some of which are actionable. While it has become common practice to screen for specific mutations in specific cancer types, to date broad mutation screening has not been practical. We report here the first large scale screening initiative between the Dana Farber Cancer Institute and Brigham and Women's Hospital that allows adult cancer patients the option to have their tumor tissue screened for genetic mutations implicated in cancer. OncoMap is a high throughput mutation profiling platform developed to detect 471 genetic mutations across 41 known oncogenes and tumor suppressor genes. The goal is to utilize OncoMap to identify genetic mutations in cancerous tissue across all tumor and tissue types. Because there is no bias as to which cancers are screened for specific mutations our aim is to identify new trends and associations between cancer types, mutational status and patient outcome. OncoMap has been analytically validated under CLIA guidelines as recommended by the Clinical Laboratory Standards Institute. Our findings show that OncoMap detects somatic mutations that are present at an allele frequency of 7.5% with 95% confidence. Precision and reproducibility studies were performed using 30 distinct formalin-fixed paraffin embedded tumor samples from various tissue types known to harbor somatic mutations in KRAS, BRAF or exon 19 deletions in EGFR. Further concordance studies demonstrate that OncoMap shows ninety-six percent sensitivity with respect to detection of codon 12 and 13 mutations in KRAS, detection of BRAF V600E mutations and JAK2 V617F mutations. Using the OncoMap cancer genetic profiling platform we anticipate that ∼10,000 individual tumors from adult cancer patients will be screened on a yearly basis enabling the identification of newly discovered associations between cancer tissues, their mutational status and patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5058. doi:1538-7445.AM2012-5058

27 Background: Analysis of gene mutations in tumor specimens is essential to treat cancers with molecular target agents. Given the high incidence of metastatic esophageal squamous cell carcinoma in East Asia, we screened for the presence of somatic mutations using OncoMap 4.0 with the aim of establishing novel treatment strategy. Methods: We analyzed 87 tissue specimens obtained from 80 patients who were pathologically confirmed of esophageal squamous cell carcinoma and received 5-fluoropyrimidine/platinum-based chemotherapy. OncoMap 4.0 is designed to interrogate >700 mutations in 113 genes. Tumor specimens were prepared from primary cancer sites in 70 patients and from metastatic sites in 17 patients. In order to test the concordance between primary and metastatic sites from the patient for mutations, we analyzed 7 paired (primary-metastatic) specimens. All specimens were formalin-fixed paraffin embedded tissues and tumor content was > 70%. Results: In total, we have detected 23 mutations out of 87 specimens tested. The most frequent mutation was PIK3CA mutation (four E545K, five H1047R and one H1047L) (N=10, 11.5%) followed by MLH1 V384D (N=7,8.0%), TP53(R306, R175H and R273C)(N=3, 3.5%), BRAF V600E (N=1, 1.2%), CTNNB1 D32N (N=1, 1.2%), and EGFR P733L (N=1, 1.2%). Distributions of somatic mutations were not different according to anatomic sites of esophageal cancer (cervical/upper, mid, lower). In addition, there was no difference in mutations between primary-metastasis paired samples. Conclusions: PIK3CA and BRAF mutations can be new targets for novel molecular agents to treat esophageal squamous cell carcinoma.

Intramedullary spinal cord neoplasms represent 2–4 % of central nervous system tumors, of which astrocytic gliomas represent 80 %. Patients presenting with spinal cord astrocytomas span the traditional pediatric and adult age divisions, having an overall age-distribution that is younger than cohorts with supratentorial gliomas. WHO grade I and II astrocytomas have better outcomes that are largely dependent on extent of surgical resection [10], whereas Grade III and IV astrocytomas are less amenable to safe surgical resection, and typically require adjuvant radiation and chemotherapy for treatment. Given the premium on preserving neurologic function during spinal cord surgery, intraoperative frozen section histologic analysis has an important role in driving therapeutic decision-making. However, histologic grading can be challenging in spinal cord astrocytomas because of the often relatively small samples obtained at the time of the surgical procedure. Therefore, grade-defining molecular biomarkers would be particularly useful for the accurate diagnostic classification of these tumors [13]. Recent genome level sequencing studies of supratentorial gliomas revealed discrete genomic alterations that discriminate pilocytic astrocytomas, WHO grade II and III diffuse gliomas, and WHO grade IV glioblastoma (GBM), with notable differences between pediatric [9, 14, 15, 20] and adult [2, 3, 6] patients. To address the hypothesis that genomic alterations could segregate spinal cord astrocytoma histologic grades, we performed sequencing of cancer-related genes in a cohort of 17 tumors. Spinal cord astrocytomas from children and adults were obtained as formalin-fixed, paraffin-embedded (FFPE) specimens from Massachusetts General Hospital, the University of Toronto, and New York University. Central neuropathology review performed by a neuropathologist (DNL) and specimens with clear histologic diagnosis and grading were used for further analysis. The characteristics of the discovery cohort (n = 17 specimens) are listed in Table 1. Targeted sequencing of 560 cancer related genes and 39 translocation events was performed on DNA extracted from these specimens (Supplementary Table 1) [5]. Briefly, DNA was sonicated to achieve an average fragment size of 250 base pairs, size selected and barcoded. Multiplexed pools were hybridized with biotinylated baits (Agilent SureSelect) designed to capture exonic sequences. The captures were sequenced on the Illumina HiSeq 2500 in Rapid Run Mode. Mutation analysis was performed by MuTect [4] and SomaticIndelDetector, copy number variant analysis was performed by ReCapSeg, and rearrangement analysis was performed by BreaKmer [1]. When applicable, statistical comparisons were performed by Chi squared test. Table 1 Baseline characteristics of discovery cohort The most recurrent findings in Grade I spinal cord astrocytomas were a BRAF-KIAA1549 translocation (n = 3/10) and BRAF copy number gain (n = 5/10) (Fig. 1). Additionally, WHO grade I astrocytomas were found to have non-synonymous mutations in NF2, NTRK1, NTRK3, PDGFRA, and TP53 (Supplementary Table 2). WHO grade II astrocytomas were similarly characterized by alterations involved in the MAPK-ERK or PI3K pathways, including BRAF-KIAA1549 translocation (n = 1/3) and BRAF amplification (n = 2/3). For samples with sufficient material, low coverage whole genome sequencing (mean 1× depth) was performed revealing that the BRAF amplification resulted from a chromosome 7 arm level gain in three of these specimens (SA-N101, SA-TL07, and SA-TL17, Supplementary Figure 1). Notably, no specimen in the discovery cohort was characterized by the BRAF V600E mutation. Fig. 1 Exomic characterization of spinal cord astrocytomas reveals that BRAF alterations and the H3F3A K27M mutation segregate histologic grade. Central neuropathology review was performed on the cohort of specimens used in this study (top row). Grade I and ... In addition, we observed that all four Grade III and IV astrocytomas in the discovery cohort shared the H3F3A K27M mutation. Further targeted Sanger sequencing of H3F3A was performed in five additional specimens (validation cohort) and revealed the K27M mutation in 2/3 spinal Grade IV astrocytomas and 0/2 Grade I astrocytomas (Supplementary Figure 2). The age distribution of our findings are consistent with prior observations that H3F3A K27M primarily occurs in pediatric and young adult gliomas [11, 15, 19]. In the aggregate cohort of 22 specimens (discovery and validation cohorts), the presence of H3F3A K27M in Grade III and IV (85.7 %, n = 6/7 specimens) and absence in Grade I and II (n = 0/15 specimens) astrocytomas was a statistically significant difference (p

Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0167-x) contains supplementary material, which is available to authorized users.