Whole-exome Sequencing Of Tumors Research Articles

Abstract 3828: Genomic and pathway analyses provide insights into the mechanisms underlying malignant peripheral nerve sheath tumors

Abstract Background: Dysfunction of the NF1 gene, which occurs in patients with neurofibromatosis type 1, is associated with an increased risk of developing Malignant Peripheral Neural Sheath Tumor (MPNST). The neurofibromin protein is a negative regulator of RAS function and 1 in 10 neurofibromatosis type 1 patients develop MPNST in their young adult years. Effective chemotherapy treatments for MPNST are not available and the prognosis for unresectable or metastatic tumors is poor. Genome-wide sequencing of human MPNSTs has identified several key genes in MPNST including NF1, CDKN2A, TP53, SUZ12, and EED. Interestingly, high-throughput drug screening of FDA-approved chemotherapy agents demonstrated differential drug sensitivities amongst a panel of MPNST cell lines. Further elucidation of the molecular mechanisms underlying therapeutic responses in MPNST will allow the development of treatments catered to tumor genetics and may improve understanding of other RAS-associated diseases. Methods: We used a combination of genomics, high-throughput drug screening, and pathway analysis to better understand how the oncogenic pathways important for MPNST development may dictate treatment outcomes. We performed whole exome sequencing of tumors from 5 MPNST patients as well as 4 established MPNST cell lines (SNF02.2, SNF10.1, SNF94.3, SNF96.2). In the cell lines, we additionally performed RNA sequencing and high-density SNP arrays. With the cell line models, we used our high-throughput screening platform to assess the efficacy of 54 agents of interest alone or in combination at clinically relevant doses, guided by published pharmacokinetics. The cell lines were further subjected to pathway analysis when subject to specific blockades downstream of RAS at the protein level with the use of immunoblotting and antibody arrays. Results: Our sequencing results indicate alterations in NF1, CDKN2A, TP53, SUZ12, and EED are present in the tumors and cell lines studied. Using cell line models, the predicted genetic alterations resulted in striking and consistent effects on expression at both the RNA and protein level. Within our panel of four cell lines, there was a broad spectrum of NF1 activity, ranging from complete loss to normal expression of full-length transcript. Interestingly, we found that NF1 exhibited a spectrum of loss from unexpressed, to partial expression, and normal expression of the full-length transcript. We found that drug sensitivity to targets downstream of RAS is correlated with the degree of NF1 expression. These findings suggest promising opportunities for developing combination clinical trials that take into account a patients’ genetics in order to improve treatment outcomes. Citation Format: Elliot Kahen, Andrew S. Brohl, Diana X. Yu, Justine Clark, Christopher L. Cubitt, Daniel M. Sullivan, Damon R. Reed. Genomic and pathway analyses provide insights into the mechanisms underlying malignant peripheral nerve sheath tumors. [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 3828.

Abstract LB-287: Combining immunomics and genomics for immunotherapy of refractory and rare cancers

Abstract Introduction: An underlying basis of cancer immunotherapy is the activation of the patient's own immune effector cells to specifically eradicate tumor cells. Different strategies of cancer immunotherapy have shown outstanding success in a number of patients across various tumor types. However, currently only a fraction of patients respond to immunotherapy, and therapy works only in a subset of tumors. To understand why, an integrated analysis of host and tumor factors is required. Fundamental issues in the analysis of key prognostic and predictive components include elucidating the complex interaction between tumors and their microenvironment, determining the type and densities of tumor-infiltrating immune cells, and understanding how the mutational load influences therapy outcome. Sophisticated analysis is required for patients with refractory or rare cancers to explore new therapeutic strategies. We established a systems medicine approach to obtain detailed data about tumor-host interactions, which may provide information on clinical outcome and help identify patients who are most likely to benefit from immunotherapy. Methods and Results: Whole-exome sequencing of tumor and control samples is performed from freshly obtained patient samples. In addition, whole-transcriptome sequencing of tumor tissue is performed if possible. Single nucleotide variations (SNVs) are detected in each tumor. Sequence-based HLA typing and computational epitope prediction methods are used to assess the immunogenicity of each SNV. The grade of immune cell infiltration and the expression of selected immunomodulatory proteins, e.g. PD-1 and PD-L1, is assessed by immunohistochemical stainings of tissue samples. Additionally, cytokine and chemokine profiles are determined in the tumor microenvironment. The resulting data can aid in treatment decision, e.g. PD-1 blocking antibodies can be administered when elevated PD-1 expression is detected. Alternatively, peptide vaccination is considered if a sufficient number of immunogenic mutations is detected. Based on the mutational profiles, mutation specific 29mer peptides and corresponding wild type peptides are produced for ELIspot assays with patient PBMCs to assess mutation-specific T cell reactivity. To assess the changes over time, blood and tissue are obtained during therapy to monitor therapy-induced changes in the periphery and the tumor microenvironment. Conclusion: Here we present our established workflow to systematically analyze tumor-host interactions in cancer patients and show preliminary data of different facets and clinical implications of these complex interactions. To evaluate and validate immunological and pathological characteristics of a tumor and the methods to assess its immunogenicity as well as its complex interplay with the microenvironment, comprehensive translational studies have to be conducted. Eventually, the obtained information will help to identify important subgroups and patients who are most likely to benefit from a certain type of immunotherapy. Citation Format: Zeynep Kosaloglu, Inka Zoernig, Niels Halama, Bruno Koehler, Anne Katrin Berger, Christoph Springfeld, Alexis Ulrich, Martin Schneider, Felix Lasitschka, Hanno Glimm, Stefan Froehling, Benedikt Brors, Dirk Jaeger. Combining immunomics and genomics for immunotherapy of refractory and rare cancers. [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 LB-287.

Abstract 470: Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma

Abstract BRAF/MEK inhibitors and immunotherapies have revolutionized care for patients with advanced melanoma, improving expected median survival from 9 months to 25-30 months, but the majority of patients still die of their disease. Personalized medicine strives to individualize and improve patient care. To individualize treatment decisions in advanced melanoma we analyzed 364 samples from 214 patients. We performed whole exome sequencing (WES) and circulating tumor DNA (ctDNA) analysis, and we developed patient derived xenografts (PDX). WES and targeted sequencing of ctDNA allowed us to predict responses to therapy and to identify and monitor mechanisms of resistance. WES of tumors revealed new therapeutic strategies in BRAF V600 wild-type and BRAF inhibitor-resistant melanoma and we validated these in patient derived xenografts (PDX). Thus, we describe a powerful combination of techniques for personalized medicine to improve the management of melanoma patients. Citation Format: Maria R. Girotti, Gabriela Gremel, Rebecca Lee, Elena Galvani, Dominic Rothwell, Amaya Viros, Amit Kumar Mandal, Kok Haw Jonathan Lim, Grazia Saturno, Simon J Furney, Franziska Baenke, Malin Pedersen, Jane Rogan, Jacqueline Swan, Matthew Smith, Alberto Fusi, Deemesh Oudit, Nathalie Dhomen, Ged Brady, Caroline Dive, Richard Marais. Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma. [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 470.

Integrated tumor and germline whole-exome sequencing identifies mutations in MAPK and PI3K pathway genes in an adolescent with rosette-forming glioneuronal tumor of the fourth ventricle.

The integration of genome-scale studies such as whole-exome sequencing (WES) into the clinical care of children with cancer has the potential to provide insight into the genetic basis of an individual's cancer with implications for clinical management. This report describes the results of clinical tumor and germline WES for a patient with a rare tumor diagnosis, rosette-forming glioneuronal tumor of the fourth ventricle (RGNT). Three pathogenic gene alterations with implications for clinical care were identified: somatic activating hotspot mutations in FGFR1 (p.N546K) and PIK3CA (p.H1047R) and a germline pathogenic variant in PTPN11 (p.N308S) diagnostic for Noonan syndrome. The molecular landscape of RGNT is not well-described, but these data are consistent with prior observations regarding the importance of the interconnected MAPK and PI3K/AKT/mTOR signaling pathways in this rare tumor. The co-occurrence of FGFR1, PIK3CA, and PTPN11 alterations provides further evidence for consideration of RGNT as a distinct molecular entity from pediatric low-grade gliomas and suggests potential therapeutic strategies for this patient in the event of tumor recurrence as novel agents targeting these pathways enter pediatric clinical trials. Although RGNT has not been definitively linked with cancer predisposition syndromes, two prior cases have been reported in patients with RASopathies (Noonan syndrome and neurofibromatosis type 1 [NF1]), providing an additional link between these tumors and the mitogen-activated protein kinase (MAPK) signaling pathway. In summary, this case provides an example of the potential for genome-scale sequencing technologies to provide insight into the biology of rare tumors and yield both tumor and germline results of potential relevance to patient care.

Diagnostic Yield of Clinical Tumor and Germline Whole-Exome Sequencing for Children With Solid Tumors.

Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown. To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors. Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children's hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record. Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations. Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%). Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

Application of Sequencing, Liquid Biopsies, and Patient-Derived Xenografts for Personalized Medicine in Melanoma.

Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.

Genomic features of primary and recurrent anal squamous cell carcinoma.

556 Background: Approximately 7000 cases of anal squamous cell carcinoma (ASCC) are diagnosed annually in the US, and the incidence is increasing. Combination chemoradiotherapy (CRT) is the standard of care for locally-advanced ASCC, and the 5-year survival rate is approximately 70%. Unlike many other tumor types, no large-scale genomic studies have been reported for anal cancer. In order to characterize the mutational landscape of ASCC and identify potential therapeutic targets, we perform comprehensive genomic analysis of a pilot cohort of ASCC cases. Methods: We performed whole exome sequencing of tumor and matched germline DNA from a pilot cohort of twelve patients with locally advanced (Stage II-IVA) ASCC cases treated at Dana-Farber Cancer Institute. All patients received concurrent chemoradiotherapy (CRT): seven ‘responders’ had complete response and no evidence of recurrence with a minimum of one year follow-up, while five ‘non-responders’ had residual or recurrent disease following CRT. For non-responders, both primary and residual/recurrent tumors were analyzed. Results: The overall mutation rate was 3.7 mutations per megabase (Mb). Recurrent mutations in several known cancer genes were observed. Five of twelve cases had a hotspot mutation in FBXW7, including 3 of the 5 non-responders. Known oncogenic mutations were also observed in PIK3CA (3 tumors) and NFE2L2/KEAP1 (3 tumors). Analysis of paired primary and recurrent samples revealed surprising examples of distinct driver mutations in clonally-related tumors. Copy number analysis revealed focal amplifications of chromosome 3q. Conclusions: This study represents one of the first genome-scale analyses in ASCC. The overall mutation rate was similar to other HPV-associated squamous cell carcinomas, and recurrent mutations in several known cancer genes were observed. Analysis of paired primary and residual/recurrent tumors revealed surprising heterogeneity. To extend our findings, we are currently performing a similar analysis in a larger extension cohort of ASCC tumors.

Whole-exome sequencing (WES) of penile squamous cell carcinoma (PSCC) to identify multiple recurrent mutations.

484 Background: Molecular alterations and drivers of PSCC, an orphan malignancy, remain unclear. The Cancer Genome Atlas is not studying PSCC and the Catalogue of Somatic Mutations in Cancer has performed targeted analyses only. We report WES of PSCC tumors from a group of patients (pts). Methods: Freshfrozen macrodissected PSCC tumor tissue and adjacent normal tissue samples were procured from the Cooperative Human Tissue Network. DNA was isolated from tissue sections by phenol chloroform extraction. Exome capture was performed with the Agilent SureSelect clinical research exome kit and whole exome-seq was done on the Illumina HiSeq2500 with paired end 100bp chemistry. Raw sequence data in Fastq format were aligned to human reference genome and quantified, and compared by using a local instance of Galaxy (galaxy.uabgrid.uab.edu). These data were analyzed for mutations (SNPs) analysis, by Partek Genomic Suite/Flow(PGS, Partek, St. Louis, MO) for variance calling against human reference genome (hg19) as referenced to dbSNP; and copy number variants (cnv) by FishingCNV tool together with picard tools/samtools/GATK). We focused on missense mutations and amplifications among ≥ 2 tumor samples but not in normal samples as they may cause upregulation of gene/protein function, which may be therapeutically actionable. Results: PSCC tumors were available from 11 patients and adjacent normal tissue from 3 patients. The 10 most common genes with > 4 missense mutations among ≥ 2 tumor samples overall were the following in decreasing order of frequency: MUC4, HLA-DPA1, MUC16, XIRP2, SSPO, TTN, FCGBP, PABPC3, ALPK2 and MKI67. The top upstream transcriptional regulators were PIH1D3, PRDM5, PTK2, Coup-Tf and NBEAL2. When examining candidate actionable genes, recurrent missense alterations were seen in PIK3C2A and PIK3C2G. Additional analysis will study alterations in functional domains and cnv. Conclusions: WES identified a relatively high mutation burden in PSCC withrecurrent missense mutations in multiple genes, notably including the PI3K gene among potentially actionable genes. Validation of these findings and further study of downstream effects is required.

Abstract IA16: Clinical genomics for children with solid tumors: Current realities and future opportunities

Abstract Genome-scale sequencing methods such as whole exome sequencing (WES) have provided significant insight into the pathogenesis of cancer. However, experience with the use of these tests in the clinical care of cancer patients remains limited. Sequencing of tumor and matched normal samples can reveal multiple types of results with implications for clinical practice. The identification of somatic (tumor-specific) mutations has the potential to offer diagnostic and prognostic information and inform selection of therapies. Detection of germline mutations in cancer susceptibility genes may prompt further genetic testing and guide cancer surveillance strategies for both the patient and family members. Germline mutations may also explain non-cancer phenotypes, predict drug responses, or provide reproductive counseling information for parents. The goal of the BASIC3 (Baylor College of Medicine Advancing Sequencing into Childhood Cancer Care) study is to determine the clinical impact of incorporating clinical tumor and constitutional WES into the care of children with newly diagnosed solid tumors. This study follows pediatric patients with newly diagnosed CNS and non-CNS solid tumors at Texas Children's Cancer Center for two years after performing CLIA-certified WES of blood and frozen tumor samples. Results are deposited into the electronic health record and disclosed to families by their oncologist and a genetic counselor. The potential impact of tumor exome findings on clinical decision-making is assessed through review of the medical record over the two year follow-up period as well as through surveys of the oncologists regarding prioritization of treatment options in the hypothetical event of tumor recurrence before and after receiving tumor exome results. Preferences of patient families and oncologists for reporting this complex information are obtained by interviews and audio recording of the WES result disclosure visits. Since the study opened in August 2012, more than 210 subjects have been enrolled (~80% of potentially eligible patients), representing the expected distribution of both CNS and non-CNS tumors. WES results have been reported for 170 subjects, revealing potentially-clinically relevant germline and somatic mutations in cancer genes known to be related to pediatric solid tumors as well as others known to be mutated primarily in adult cancer patients. Data will be presented regarding the diagnostic yield of combined tumor and germline WES for children with newly-diagnosed solid tumors. These results demonstrate the feasibility of routine tumor WES in the pediatric oncology clinic and a significant level of parental interest in receiving WES results and have significant implications for the treatment of children with relapsed and refractory solid tumors and the design of clinical trials using precision oncology approaches for these patients. Further analyses of the clinical utility of the WES data and the preferences of oncologists and parents for reporting of these results are under study. The BASIC3 study is a Clinical Sequencing Exploratory Research (CSER) program project supported by NHGRI/NCI 1U01HG006485. Citation Format: D. William Parsons, Angshumoy Roy, Yaping Yang, Tao Wang, Sarah Scollon, Katie Bergstrom, Robin A. Kerstein, Stephanie Gutierrez, Abhishek Bavle, Frank Y. Lin, Dolores H. López-Terrada, Federico A. Monzon, Jed G. Nuchtern, Uma Ramamurthy, Amy L. McGuire, Susan G. Hilsenbeck, Jeffrey G. Reid, Donna M. Muzny, David A. Wheeler, Stacey L. Berg, Murali M. Chintagumpala, Christine M. Eng, Richard A. Gibbs, Sharon E. Plon. Clinical genomics for children with solid tumors: Current realities and future opportunities. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA16.

Abstract A113: iVacALL: A personalized peptide-vaccination design platform for pediatric acute lymphoblastic leukemia patients based on patient-individual tumor-specific variants

Abstract We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Standard chemotherapy is a successful treatment in 80% of patients, only about 20% develop a relapse, however these patients have a dismal prognosis. Prevention of relapse after first-line chemotherapy or stem cell transplantation (SCT) is therefore mandatory. Accumulation of somatic mutations is one characteristic feature of malignant cells. These single nucleotide variants (SNVs) can lead to altered amino acid sequences of the translated proteins, which in turn can be presented as antigenic peptides on HLA molecules of the malignant cells. Mutated peptides represent ideal T cell targets as they are true neoantigens, specific for the tumor, and should not have been subject to central tolerance selection mechanisms. A peptide vaccination composed of mutated T cell epitopes specific for individual patient tumors is therefore a promising approach to prevent relapse in high-risk patients. For this purpose we detect nonsynonymous mutations by whole exome and transcriptome sequencing of patient leukemic blasts and normal reference tissue. HLA binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan for the patients' individual HLA type. Whole exome sequencing was performed for 17 c-ALL, 2 cortical T-ALL and 1 pro-B-ALL sample pairs. ALL-specific SNVs, as well as insertions and deletions (InDels) were identified using a comparative bioinformatics pipeline. The determined variants were further validated by deep sequencing in 9/20 patients so far, with an average of 12 (+/- 8) validated mutations per patient. For all patients with validated variants, MHC class I and MHC class II epitopes could be predicted successfully. We applied the platform for 3 patients based on compassionate need and designed individual peptide vaccines. One patient underwent haploidentical SCT with relapsed c-ALL, a second patient received autologous SCT with ependymoma and the third patient got allogeneic SCT with pro-B ALL. In all cases validated mutations could be identified and epitope prediction was performed for MHC I & II binders. The predicted peptides were synthesized and vaccination cocktails were formulated. The vaccination schedule provides 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvant. The vaccination was generally well tolerated. Response to the vaccination was monitored by detection of T cells recognizing the vaccinated peptides occurring over time in peripheral blood of the patients. Monitoring was performed for each vaccination time point by prestimulation with the peptides and subsequent intracellular cytokine staining (ICS) of T cells and FACS analysis. In all 3 patients we could detect a developing CD4+ T cell response against the vaccinated mutated MHC II binding peptides. Whole exome sequencing of pediatric ALL patients is feasible and yields a small amount of validated mutations per patients. However, these few mutations seem sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T cell responses in patients. Moreover, the platform is not limited to ALL/leukemia but can also be applied for solid tumor patients. Citation Format: Armin Rabsteyn, Christina Kyzirakos, Christopher Schröder, Marc Sturm, Christopher Mohr, Mathias Walzer, Ulrike Pflückhahn, Michael Walter, Magdalena Feldhahn, Karoline Laske, Michael Bonin, Martin Ebinger, Stefan Stevanovic, Peter Bauer, Oliver Kohlbacher, Cecile Gouttefangeas, Hans-Georg Rammensee, Rupert Handgretinger, Peter Lang. iVacALL: A personalized peptide-vaccination design platform for pediatric acute lymphoblastic leukemia patients based on patient-individual tumor-specific variants. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A113.

Abstract 04: Impact of whole exome sequencing results on clinical decision making for pediatric solid tumor patients in the hypothetical scenario of tumor relapse: A survey of pediatric oncologists

Abstract Background: The development of molecularly-targeted agents has made it possible to personalize therapy for patients by targeting the specific mutations in their tumor. Pediatric clinical trials utilizing such strategies are being planned but little is known about the opinions of pediatric oncologists regarding the utility of genomic data for guiding treatment decisions. The goals of this study were to (1) characterize those opinions in the context of children with relapsed/refractory solid tumors and (2) assess the potential impact of clinical whole exome sequencing (WES) data on medical decision-making in that context. Methods: As part of the ongoing BASIC3 clinical sequencing study at Texas Children's Cancer Center, clinical germline and tumor (if sample available) WES were performed for unselected newly-diagnosed pediatric CNS and non-CNS solid tumor patients. The primary oncologist for each (n=17) received online surveys for each study patient before and after review of WES reports. The pre- and post-WES surveys asked oncologists to rank options for off-study systemic chemotherapy (of any type) for their patient in the hypothetical scenario of tumor relapse. Oncologists were then asked if they would consider using a molecularly-targeted agent in the context of a clinical trial, and if so, which agents (from a representative list), their rank order and the rationale for those choices. Post-WES surveys also included questions regarding perceived utility of the tumor WES results for patient care. Pre-WES surveys were analyzed for baseline oncologist responses regarding these hypothetical treatment decisions. When available, pre- and post-WES surveys were analyzed as pairs as an initial assessment of the influence of the WES results on the oncologist's choice of therapy. Results: 177/189 (94%) of pre-WES surveys and 111/161 (69%) post-WES surveys were available for analysis. Analysis of pre-exome surveys revealed that oncologists would recommend systemic chemotherapy for 127/177 (72%) patients in the hypothetical event of tumor relapse but would consider a molecularly-targeted agent off-study as their first option in only 8/177 (4%) cases. In contrast, oncologists indicated that they would consider targeted therapies in the context of a clinical trial for 99/177 (56%) patients, most commonly sorafenib (n=21), cixutumumab (n=17), and crizotinib (n=13). There were 26 cases in which somatic mutations were identified in genes categorized as having established or potential clinical relevance, and for which both pre- and post-WES surveys were available. A corresponding targeted agent was ranked for consideration in the hypothetical scenario of relapse on the post-WES survey for 8/26 (31%) of these patients (somatic mutations in MET, JAK2, HRAS, NRAS X 2, ALK, BRAF, KIT), having only been chosen on the pre-WES survey in 2 of those cases. On 8 of 111 (7%) post-WES surveys, the oncologist removed a targeted agent that had been prioritized on the pre-WES survey after no relevant mutation was detected, including the Sonic Hedgehog inhibitor GDC-0449 in 5 cases. Conclusion: Although genomic tests such as WES have the potential to identify molecular targets for therapy in children with relapsed tumors, a survey of pediatric oncologists revealed that most consider such therapies as options only in the context of a clinical trial. These findings support the potential utility of WES in precision oncology approaches as well as the need for clinical trials evaluating the use of integrated genomic testing to guide treatment of children with relapsed solid tumors. Citation Format: Abhishek Bavle, Tao Wang, Frank Y. Lin, Angshumoy Roy, Robin A. Kerstein, Sarah Scollon, Katie Bergstrom, Stephanie Gutierrez, Uma Ramamurthy, Yaping Yang, Christine M. Eng, Richard A. Gibbs, Murali M. Chintagumpala, Susan G. Hilsenbeck, Sharon E. Plon, Stacey L. Berg, D. Williams Parsons. Impact of whole exome sequencing results on clinical decision making for pediatric solid tumor patients in the hypothetical scenario of tumor relapse: A survey of pediatric oncologists. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 04.

Abstract A116: A predictive model to identify immunogenic neoepitopes in patients with melanoma treated with CTLA-4 blockade

Abstract Recent studies have shown that CD8+ T cells that recognize neoantigens presented by tumor cells, but the target specificity of these tumor infiltrating T cells are largely unknown. Developing a predictive model that identifies immunogenic neoepitopes presented by major histocompatibility complex (MHC) class I molecules is critical to identify potential vaccine targets and to improve prediction of response to immune checkpoint inhibitors. We hypothesize that the immunogenic heterogeneity of tumors may explain, in part, differences in clinical response that individual patients derive to checkpoint blockade therapy. We have recently shown that the relative hydrophobicity of T cell receptor (TCR) contact residues is a hallmark of immunogenic MHC class I epitopes, and that this may represent a biochemical basis by which T cells discriminate immunogenic epitopes within the background of self-peptides. Here, we extend this analysis using candidate MHC class I tumor neopeptides from patients with melanoma. Somatic missense mutations were identified from recently published data on whole-exome sequencing of tumors from 11 patients who derived a long-term benefit and 14 patients who derived a minimal or no benefit from ipilimumab or tremelimumab. Mutational burden ranged from 1 to 1,304 mutations per tumor. More than 3,200 potential MHC class I neopeptides were identified based on a predicted binding algorithm (binding affinity, ≤500 nM). Our preliminary results indicate significant differences in hydrophobicity at the anchor residues P2 and P9 (P < 0.001 for both), reflecting MHC binding differences. In addition, there is an increase in hydrophobicity at the TCR contact residues P4 and P7 (P < 0.01 for both) in long-term responders. The overall frequency of mutations in neoepitopes is significantly different at the TCR contact residues P4 and P7 between long-term responders and minimal or no responders (P < 0.01), suggesting genetic immunoediting of these tumors. Based on these results, we have developed a probabilistic Bayesian-based model to predict immunogenic neoepitopes. The model is being applied on the overall pool of neopeptides from each individual patient with melanoma. The sets of predicted immunogenic neoepitopes may serve as potential therapeutic vaccine candidates for patients with melanoma. Citation Format: Diego Chowell, Sri Krishna, Alexandra Snyder, Makarov Vladimir, Timothy A. Chan, Karen S. Anderson. A predictive model to identify immunogenic neoepitopes in patients with melanoma treated with CTLA-4 blockade. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A116.

Abstract IA03: Towards the next clinical option: Experience from a precision cancer medicine trial

Abstract Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low frequency mutations. We describe an evidence-based precision medicine program designed to bring whole exome sequencing (WES) into clinical practice and illustrate how this can be utilized to identify novel predictive biomarkers associated with exceptional systemic response. Metastatic and treatment-resistant cancer patients are prospectively enrolled for paired metastatic tumor and normal WES. Using a comprehensive computational pipeline, point mutations, indels and copy number alterations are detected. Mutations are categorized as Category I-III based on actionability and a report is generated and discussed in tumor board. Patients are followed prospectively for correlation of molecular information with clinical response and patient outcomes. A rapid response functional team follows on key findings to expand actionability based on the WES results. In the first year, 154 tumor-normal pairs from 97 patients were sequenced, with an average coverage of 95X of over 21,000 genes and 16 somatic alterations detected on average per patient. In total, 16 mutations were Category I, 98 were Category II, and 1474 were Category III. Tumor purity ranged from 14 to near 100%. Among unexpected findings, a prostate cancer exceptional responder was identified that harbored a somatic hemizygous deletion of the DNA repair gene FANCA and likely loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. The establishment of a clinical program for whole exome sequencing of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response. Citation Format: Mark A. Rubin. Towards the next clinical option: Experience from a precision cancer medicine trial. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr IA03.

Abstract IA19: Towards the next clinical option: Experience from a precision cancer medicine trial

Abstract Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low frequency mutations. We describe an evidence-based precision medicine program designed to bring whole exome sequencing (WES) into clinical practice and illustrate how this can be utilized to identify novel predictive biomarkers associated with exceptional systemic response. Metastatic and treatment-resistant cancer patients are prospectively enrolled for paired metastatic tumor and normal WES. Using a comprehensive computational pipeline, point mutations, indels and copy number alterations are detected. Mutations are categorized as Category I-III based on actionability and a report is generated and discussed in tumor board. Patients are followed prospectively for correlation of molecular information with clinical response and patient outcomes. A rapid response functional team follows on key findings to expand actionability based on the WES results. In the first year, 154 tumor-normal pairs from 97 patients were sequenced, with an average coverage of 95X of over 21,000 genes and 16 somatic alterations detected on average per patient. In total, 16 mutations were Category I, 98 were Category II, and 1474 were Category III. Tumor purity ranged from 14 to near 100%. Among unexpected findings, a prostate cancer exceptional responder was identified that harbored a somatic hemizygous deletion of the DNA repair gene FANCA and likely loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. The establishment of a clinical program for whole exome sequencing of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response. Citation Format: Mark A. Rubin. Towards the next clinical option: Experience from a precision cancer medicine trial. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr IA19.

Abstract 930: Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors

Abstract Background: Genetic characterization and monitoring of brain tumors is challenging given the restricted sampling of tumors and the limited abundance of brain tumor-derived circulating tumor DNA (ctDNA) in the plasma. Here, we sought to define whether cerebrospinal fluid (CSF) could serve as a ‘liquid biopsy’ for the genetic characterization of tumor DNA originated from the central nervous system (CNS) of patients (pts) with brain tumors. Methods: CSF, plasma and tumor tissue were obtained from pts with glioblastoma (GBM), brain metastases from breast (BMBC) and lung cancers (BMLC) and leptomeningeal carcinomatosis (LC). Whole exome sequencing was done in CSF and tumor (n = 3) and was coupled with digital PCR for monitoring CSF and plasma ctDNA (n = 6). Targeted capture massively parallel sequencing of 39 samples from 4 BMBC pts, including samples from CSF, plasma and matched metastatic sites obtained at autopsy was performed using two independent platforms (MSK-IMPACT (341 genes) and BC panel (254 genes most frequently mutated in BC)) comprising 488 unique genes. Sequencing was performed on an Illumina HiSeq2000. Single nucleotide variants were defined using MuTect, and indels using Varscan. Copy number alterations were assessed using Varscan2 and GISTIC. Results: Whole exome sequencing of tumor and CSF DNA revealed a high proportion of tumor-derived cell-free DNA in the CSF of 3 pts (1 GBM, 2 BMBC). We identified actionable somatic mutations (EGFR L858R, IDH1 R132H) and also IDH1 mutations associated with molecular diagnosis of proneural GBM. In CSF and plasma ctDNA of 6 pts with GBM, BMBC, BMLC, we observed that the mutations present in the CSF ctDNA, but not plasma ctDNA, and their mutant allele fractions (MAFs) correlated with brain tumor burden. In 3 pts with suspected LC, we compared the results of cytopathologic analysis and CSF ctDNA obtained from samples used for cytopathologic diagnosis, and observed that CSF ctDNA was more robust and sensitive for the diagnosis of LC. The analysis of synchronous CSF ctDNA, plasma ctDNA and intra- and extra-cranial metastases from 4 autopsied BMBC pts showed that CSF ctDNA recapitulated the somatic genetic alterations present in the intra-cranial lesions. We detected similar MAFs for the truncal mutations (RB1, KMT2D, AHNAK2) in both CSF and plasma DNA of BMBC3, a pt with Li-Fraumeni syndrome and a diagnosis of concurrent BMBC and esthesioneuroblastoma; however, mutations in PIK3CB, PAK7, MSH5 found only in the CNS implant of each disease were only detected in the CSF but not in the plasma DNA. Conclusions: Brain tumor-derived ctDNA is abundantly present in the CSF of brain cancer pts and compared to plasma ctDNA, CSF ctDNA is more representative of the brain lesions. Our results demonstrate that massively parallel sequencing can be performed using CSF DNA, allowing for the non-invasive genomic characterization and monitoring of brain lesions. Citation Format: Leticia De Mattos-Arruda, Regina Mayor, Charlotte K. Y. Ng, Britta Weigelt, Francisco Martínez-Ricarte, Davis Torrejon, Mafalda Oliveira, Carolina Raventos, Alexandra Arias, Elena Guerini-Rocco, Elena Martínez-Sáez, Sergio Lois, Oscar Marín, Xavier de la Cruz, Salvatore Piscuoglio, Russell Towers, Ana Vivancos, Vicente Peg, Santiago Ramon y Cajal, Jordi Rodon, Enriqueta Felip, Joan Sahuquillo, Josep Tabernero, Javier Cortes, Jorge S. Reis-Filho, Joan Seoane. Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors. [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 930. doi:10.1158/1538-7445.AM2015-930

Abstract 3893: Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations

Abstract Primary central nervous system lymphoma (PCNSL) is defined as diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system. Although PCNSL cannot be histologically distinguished from extracerebral DLBCL, its prognosis is quite poor. To gain insights into the transforming mechanism of PCNSL, we conducted whole-exome sequencing for 44 PCNSL specimens. Genomic DNA was extracted from tumors and their matched normal samples. Exome fragments were captured by using SureSelectXT Human All ExonV5+lncRNA, and subjected to deep sequencing with the HiSeq 2000 system. There were 8 cases with germinal center B-cell-like (GCB) subtype and 34 cases with non-GCB subtype. Whole-exome sequencing of tumor and paired normal yielded sequencing data with a mean coverage of 156X and 80X, with 98% and 99% of the targeted bases covered by ≥20 independent reads, respectively. Of 17,832 somatic mutations identified in the PCNSL specimens, 11,611 (65%) were nonsynonymous single nucleotide variations. Predominant nucleotide substitution was a C>T transition (57%) especially in the context of the sequence GCG. We also identified 798 insertions/deletions. Interestingly, MYD88 mutations were detected in 35 of 44 patients (79.5%), and PIM1 mutations were detected in 42 patients (95.5%). Also, a number of genetic aberrations were shown to activate the NF-κB pathway. These results suggest that MYD88 and PIM1 mutations have important roles in the development of PCNSL and could be the therapeutic targets of this intractable disorder. Citation Format: Motoo Nagane, Kazutaka Fukumura, Toshihide Ueno, Jeunghun Lee, Yukiko Shishido-Hara, Mitsuaki Shirahata, Kazuhiko Mishima, Koichi Ichimura, Akitake Mukasa, Yoshitaka Narita, Ryo Nishikawa, Hiroyuki Mano. Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 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 3893. doi:10.1158/1538-7445.AM2015-3893

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response.

Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. Feasibility, use of WES for decision making, and identification of novel biomarkers. A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Shedding light on the molecular determinants of response to anti-PD-1 therapy.

Immune checkpoint inhibition targeting the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis has emerged as a very promising therapeutic avenue in the treatment of patients with advanced stage non-small cell lung cancer but only a subset of patients derives clinical benefit from PD-1/PD-L1 inhibitors. Studies to date have reported patients with PDL-1 expressing tumors have a better outcome than those with PDL-1 negative tumors but assays used to identify PD-L1 positive patients has been challenging. Through whole exome sequencing of tumors, investigators have recently described mutational burden in non-small cell lung cancer (NSCLC) was associated with response to pembrolizumab. In two independent patient cohorts, it was reported a high somatic nonsynonymous mutation burden was associated with greater durable clinical benefit, higher objective response rates (ORRs) and a longer progression free survival. In addition clinical efficacy was associated with a molecular smoking signature, certain DNA repair mutations and the burden of neoantigens.

GE-06 * IDENTIFICATION OF TARGETABLE MUTATIONS IN RARE PEDIATRIC BRAIN TUMORS BY CLINICAL WHOLE EXOME SEQUENCING

INTRODUCTION: Recent genomic studies have provided significant insight into the pathogenesis of pediatric central nervous system (CNS) tumors. However, little is known about the genetic events underlying rare CNS tumor types, which often present significant diagnostic and therapeutic challenges. The BASIC3 clinical exome sequencing study of children with newly-diagnosed tumors offers an opportunity to investigate the genetic basis of these rare CNS tumors. METHODS: Tumor and germline whole exome sequencing (WES) is performed in a CLIA-certified laboratory on fresh-frozen tumor and matched normal blood samples from children with newly-diagnosed CNS and non-CNS solid tumors. Additional research genomic studies, including transcriptome sequencing, are also conducted with specific consent. RESULTS: 67 children with CNS tumors have been enrolled on study to date, including several rare diagnoses. For example, WES of a malignant epithelioid glioneuronal tumor with two known BRAF alterations (BRAF-V600E and KIAA1549-BRAF fusion) also identified a nonsense mutation in TSC1 (p.Q829X), suggesting the theoretical utility of combined BRAF and mTOR inhibition for this patient. WES of a pilocytic astrocytoma with anaplasia with known KIAA1549-BRAF fusion revealed a pathogenic mutation in ACVR1 (p.R206H), which has been previously reported in high grade gliomas and is currently the target of investigational agents. CONCLUSIONS: Tumor WES has the potential to reveal somatic mutations of biological and clinical significance (for both diagnostics and therapeutics) that are not detectable by standard clinical testing in rare pediatric CNS tumors. Further molecular interrogation of both tumor types is ongoing, including evaluation of additional cases, complementary genomic studies including transcriptome sequencing and copy number arrays, and evaluation of intra-tumoral genetic heterogeneity. Orthotopic xenograft models and cell lines derived from the same primary CNS tumors are being generated to allow for additional biologic and pre-clinical studies as indicated.

Abstract IA16: Evaluating the implementation and utility of clinical tumor exome sequencing in the pediatric oncology clinic: Early results of the BASIC3 study

Abstract Advances in sequencing technologies allow for provision of genome-scale data to oncologists and geneticists caring for pediatric cancer patients but current experience with the clinical application of genomic sequencing is limited. The goal of the BASIC3 (Baylor Advancing Sequencing into Childhood Cancer Care) study is to determine the clinical impact of incorporating CLIA-certified tumor and constitutional whole exome sequencing (WES) into the care of children with newly diagnosed solid tumors. This study follows pediatric patients with newly diagnosed CNS and non-CNS solid tumors (target enrollment n=280) at Texas Children's Cancer Center for two years after performing CLIA-certified whole exome sequencing (WES) of blood and frozen tumor samples. Results are deposited into the electronic medical record and disclosed to families by their oncologist and a genetic counselor. The potential impact of tumor exome findings on clinical decision-making is assessed through review of the medical record over the two year follow-up period as well as through surveys of the oncologists regarding prioritization of treatment options in the hypothetical event of tumor recurrence before and after receiving tumor exome results. Preferences of patient families and oncologists for reporting this complex information are obtained by interviews and audiorecording of the exome result disclosure visits. Since the study opened in August 2012, ∼85% of potentially eligible families have consented to enrollment. The first 100 patients comprise a diverse representation of diagnoses, including 32 with CNS tumors (32%) and 68 with non-CNS tumors (68%). Despite limited diagnostic biopsies in many patents, snap-frozen tumor samples adequate for WES were obtained from 84 subjects (84%), including 62/68 non-CNS solid tumors (91%) and 22/32 (69%) CNS solid tumors. Tumor WES results have been reported for the first 55 patients, revealing a median of 9 (range of 0 to 78) protein-altering mutations per tumor and alterations of known cancer genes such as ALK, BRAF, DICER1, KIT, KRAS, NRAS, MET, JAK2, FGFR3, ARID1A, CTNNB1, and TP53. Fourteen of 55 tumors (25%) contained mutations classified as having proven or potential clinical utility. These results demonstrate the feasibility of routine tumor WES in the pediatric oncology clinic and a significant level of parental interest in receiving WES results. Potentially clinically-relevant mutations can be identified in a substantial minority of pediatric solid tumor patients but distinct from the medically actionable mutations seen in adult cancer patients. Data further assessing the clinical utility of the tumor exomes and the preferences of oncologists and parents for reporting of these results are under study. Supported by NHGRI/NCI 1U01HG006485. Citation Format: D. Williams Parsons, Angshumoy Roy, Federico A. Monzon, Dolores H. López-Terrada, Murali M. Chintagumpala, Stacey L. Berg, Susan G. Hilsenbeck, Tao Wang, Robin A. Kerstein, Sarah Scollon, Katie Bergstrom, Richard L. Street, Jr., Laurence B. McCullough, Amy L. McGuire, Uma Ramamurthy, David A. Wheeler, Christine M. Eng, Yaping Yang, Jeff G. Reid, Donna M. Muzny, Richard A. Gibbs, Sharon E. Plon. Evaluating the implementation and utility of clinical tumor exome sequencing in the pediatric oncology clinic: Early results of the BASIC3 study. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA16.