Number Of Non-synonymous Somatic Mutations Research Articles

Abstract 4982: The somatic mutation signature detected in gynecological mucosal melanoma

Abstract Background: Gynecological Mucosal Melanoma(GMM) is aggressive disease with a poor prognosis, where few studies focusing on its target drug and mutation signatures. Cancer as outcome of DNA mutagenesis process and can be inferred from somatic mutations signature by analyzing the genome sequence. Due to the rapid development of next-generation sequencing (NGS) and molecularly driven targeted therapy, discovering different mutation signature in cancer plays an important role in precision medicine. Methods: The study was conducted at a high-volume melanoma center, the Peking University Cancer Hospital. We enrolled female patients with melanoma (n = 22) from 2017 to 2021. Subsequently, whole-exome sequencing performed on the Formalin-fixed paraffin-embedded (FFPE) specimens from 22 GMM patients and match normal tissues of patients. Tumor mutational burden (TMB) was measured as the number of nonsynonymous somatic mutations of whole-exome sequence coding region. Mutation signature detection base pair of six substitution subtypes: C>A, C>G, C>T, T>A, T>C, and T>G. NMF can be used to decompose matrix A into two nonnegative matrices. Results: In all mutations of 22 GMM patients, we screen two mutation signatures. Signature 1 showed a strong correlation [0.77;cosine similarity value (CSV)] to the signature 3 from the COSMIC database, which represents failure of DNA double-strand break-repair by homologous recombination, which predominated in 55%(12/22) GMM patients. Signature 2 showed a strong correlation to Cosmic signature 1 (CSV = 0.791), which is has been found in all cancer types correlated with age cancer diagnosis. Patient-18 did not any treatment in our hospital, follow-up was lost. Kaplan-Meier revealed that Signature 2 was associated with longer overall survival in GMM patients and later recurrence or progression. Conclusions: This study aimed to identify prognostic markers and the development of targeted therapy for the treatment of GMM. Citation Format: Zhongwu Li, Lianyi Shen, Siyao Liu. The somatic mutation signature detected in gynecological mucosal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4982.

Abstract 1068: Tumor infiltrating lymphocytes (TIL) recognize a unique and diverse number of non-synonymous somatic mutations in patients with metastatic breast cancer

Abstract Introduction: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can mediate long-term durable regression in patients with metastatic melanoma, a type of cancer which is characterized by a high number of mutated genes and pronounced lymphocytic infiltrate. In contrast, metastatic breast cancer expresses far fewer somatic mutations and may contain lower levels of TIL. This pilot study investigated the ability to identify immunogenic non-synonymous somatic mutations which can serve as targets for adoptively transferred autologous T cells into patients with metastatic breast cancer, refractory to other treatments. Methods: WES and RNAseq were performed on metastatic lesions resected in the Surgery branch, NCI, NIH from patients with metastatic breast cancer. Non-synonymous somatic mutations were identified and, in parallel, TIL from the same lesions were grown ex vivo in the presence of IL-2. The somatic mutations were tested for recognition by the autologous TIL using previously described functional assays, involving tandem mini-genes (TMG), long peptides or predicted minimal peptide approaches. For some patients, the mutation-reactive T cells were sorted by FACS and the respective TCR was identified by single cell sequencing. Results: Metastatic tumor lesions from 37 patients with breast cancer were studied and all of them were found to contain and express non-synonymous somatic mutations (range: 4-1788, median: 102). TIL were successfully grown ex vivo from the tumors of all patients. Following functional assays (IFN-γ secretion and 4-1BB/OX-40 upregulation), it was found that autologous TIL recognized at least one (range: 1-11, median: 3) mutated product in 25 of 37 patients (68%) tested. Seventy-five percent of those were recognized by CD4+ and 25% by CD8+ T cells. All identified immunogenic tumor mutations (n=88) were unique and not shared by other patients. Sixty-four unique TCR pairs were isolated from the reactive cells and verified to recognize 30 immunogenic tumor mutations (range 1-7 TCRs/neoantigen). Conclusions: Patients with metastatic breast cancer contain a substantial number of non-synonymous somatic mutations and mutation-reactive TIL were found in the majority of those patients. The immunogenicity of tumor-specific mutations in metastatic breast cancer can be the platform for a highly personalized adoptive T cell transfer of selected TIL or mutation-reactive TCR transduced T cells targeting those tumor mutated genes. Citation Format: Nikolaos Zacharakis, Peter Kim, Paul Robbins, Jared Gartner, Todd Prickett, Victoria Hill, Biman Paria, Steven Feldman, Stephanie Goff, Steven Rosenberg. Tumor infiltrating lymphocytes (TIL) recognize a unique and diverse number of non-synonymous somatic mutations in patients with metastatic breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1068.

Plasma cell free PD-L1 RNA expression correlated with tissue PD-L1 immunohistochemical staining and tumor mutation burden in non-small cell lung cancer.

e15049 Background: Programmed death ligand-1 (PD-L1) protein expression by immunohistochemical staining (IHC) correlates with response to immune checkpoint inhibitor (ICI) therapies in non-small cell lung cancer (NSCLC). Tumor mutational burden (TMB) is another immune biomarker of ICI response in NSCLC. Clinical studies indicate tissue PD-L1 protein expression and TMB are independent yet complementary. Both are limited by tissue acquisition and potential heterogeneity. Plasma cell free PD-L1 RNA (cfRNA) levels and tissue protein PD-L1 expression and TMB was analyzed and correlated in patients with advanced NSCLC. Methods: Patients with advanced NSCLC underwent complementary plasma and tissue next generation sequencing (NGS) with immune biomarkers prior to treatment. Plasma PD-L1 cfRNA was assessed by the Circulogene proprietary direct-on-specimen enrichment technology NGS platform. Correlating tissue testing was performed by the Caris Molecular Intelligence platform with the anti-PD-L1 22C3 IHC antibody and TMB measuring the total number of non-synonymous somatic mutations per megabase. Results: 107 patients with advanced NSCLC were evaluated with simultaneous plasma and tissue NGS testing. 17% (95% confidence interval [CI] 10-24%) patients plasma PD-L1 positive. 48.5% (CI 38-57%) tissue IHC PD-L1 positive. 7 of 18 plasma PD-L1 positive patients were tissue PD-L1 negative. 48% (CI 39-58%) total patients TMB ≥ 10 mutations per megabase (TMB high). Correlating TMB high in 72% (CI 50-94%) of plasma PD-L1 positive and 56% (CI 42-69%) of tissue PD-L1 positive patients. TMB high in 49% (CI 37-59%) plasma PD-L1 negative and 50% (CI 35-65%) tissue PD-L1 negative patients. Conclusions: Although less frequent than tissue PD-L1 protein expression, plasma PD-L1 cfRNA expression correlated with a higher association of tissue TMB high findings than tissue PD-L1 positive patients. There was not any TMB high difference between plasma PD-L1 negative and tissue PD-L1 negative patients. Over one-third of plasma PD-L1 positive patients were tissue PD-L1 negative. Clinical correlation with immune checkpoint inhibitor therapies is ongoing.

MR1 in combination with tumor mutational burden and PD-1/PD-L1 expression as a potentially novel clinical predictor for T cell exhaustion and immune checkpoint inhibitor response.

3109 Background: Immune checkpoint inhibitors (ICIs) restore T cell function by reversing T cell exhaustion. Variable response to ICIs warrants the development of precise predictive biomarkers, which is challenging due to difficulty in capturing the interplay of factors involved with tumor cell immune recognition. High intratumoral expression of MR1, the MHC-I related protein basally expressed on cancer cells, may drive T cell exhaustion through presentation of cancer-specific antigens. Here, we construct a database to study the relationship between MR1, tumor mutational burden (TMB), the PD-1/PD-L1 axis and T cell exhaustion across 8,975 sequenced tumors and 27 cancer types. Methods: RNA Seq by expectation maximization (RSEM) values from the TCGA were collected and normalized along with expression data for markers of interest (Table). TMB was defined as the number of non-synonymous somatic mutations per sample. For each cancer, 5 cohorts were created based on ascending mean expression levels of MR1, PD-1, PD-L1, and increasing TMB. For each cancer, an “immunogenicity score” for these factors was computed, and its relationship with T cell exhaustion signatures was assessed via linear regression. Data is presented as adjusted R2 and p-value. Results: While PD-1 and T cell exhaustion marker expression were correlated across cancers, the “immunogenicity score” (IS) correlated with exhaustion markers specifically in cancers with FDA-approved ICIs. Excluding MR1 from the score weakened the correlation with EOMES and TBET expression (Table). Each component of the score analyzed independently failed to show a statistically significant correlation for both EOMES and TBET expression. Conclusions: In this cross-cancer analysis, we support the hypothesis that presentation of metabolic intermediates in cancer cells via MR1 may drive T cell exhaustion. Also, the novel “immunogenicity score”, which incorporates MR1 into standard biomarkers for response to ICIs may convey the global picture of cancer cell recognition by the immune system and warrants further investigation as a tool for predicting clinical response. [Table: see text]

Reduced Antigen Presentation May Contribute to Immunomodulatory Drug Resistance in Multiple Myeloma

Introduction: Maintenance therapy with the immunomodulatory drug (IMiD) lenalidomide improves progression-free survival (PFS) and overall survival among patients with multiple myeloma (MM). It has been suggested that IMiDs enhance immune-mediated anti-tumor responses through increased activation and proliferation of T cells and natural killer (NK) cells as well as inhibition of regulatory T cells. Despite the effectiveness of this therapy, nearly all patients relapse for reasons that remain uncharacterized. We sought to elucidate mechanisms of resistance to lenalidomide maintenance through investigation of mutations in cell-free DNA (cfDNA) and identification of changes in the innate and adaptive immune system by single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs). While MM is a cancer of plasma cells that primarily reside in the bone marrow, we focused on the peripheral blood which offers a comprehensive view of multiple sites of disease including extramedullary locations commonly found at the time of relapse. Methods: Twenty-four patients with MM and no minimal residual disease by 10-color flow cytometry and IGHVnext-generation sequencing (Invivoscribe) after induction therapy with or without an autologous stem cell transplant were selected for analysis. All patients were enrolled in NCT02538198 at Memorial Sloan Kettering Cancer Center. Peripheral blood was collected serially including timepoints prior to lenalidomide maintenance and after disease progression. Plasma and PBMCs were isolated by Ficoll-Paque density gradient centrifugation. cfDNA was extracted from plasma and targeted next-generation sequencing using a 70-gene panel of recurrently mutated genes in myeloma was performed using the QIAseq Targeted DNA kit (QIAGEN). DNA sequencing was also performed using the same gene panel on granulocytes to exclude germline variants. On PBMC samples, 5' gene expression and T and B cell receptor single-cell sequencing were performed using the Chromium Single Cell V(D)J kit (10X Genomics). Analysis of single-cell sequencing was performed on the top 100 principal components and visualized by Uniform Manifold Approximation and Projection (UMAP). A regression-based classifier was trained to automatically assign clustered cells into 26 immune cell phenotypes using lists of genes reported to be uniquely expressed by each cell type. Results: cfDNA sequencing was performed on 12 patients characterized as durable responders (PFS > 57 months) and 12 early progressors (median PFS 25 months). Prior to maintenance therapy, no significant difference was observed in the number of cfDNA mutations between these groups. The number of non-synonymous somatic mutations in cfDNA tended to increase over time and was significantly higher among patients taking lenalidomide for more than 2 years compared to patients at the time of relapsed disease (mean number of mutations was 7 in responders versus 3 in progressors).ZNF292was among the genes most significantly mutated in responders compared to progressors. ZNF292encodes a zinc finger protein involved in RNA binding and has previously been identified as a potential source of tumor-associated antigens in patients with monoclonal gammopathy of undetermined significance. To identify changes in immune cell phenotype and function that associate with disease progression, we performed single-cell RNA sequencing on PBMCs from the 12 progressors before maintenance and at the time of relapse. The median number of cells sequenced per PBMC sample was 3,132. Over 90% of cells could automatically be classified into a specific immune cell type by their gene expression. We observed the frequency of NK cells and plasmablasts were significantly increased at the time progression on lenalidomide while monocytes, dendritic cells, and basophils were decreased. Conclusion: Mechanisms of resistance among patients receiving treatment with IMiDs is not well known. Using targeted sequencing of cfDNA, we identify patients responding to maintenance tended to have a higher mutational load in cfDNA including genes previously reported to encode tumor-associated antigens. Additionally, we show that a decline in antigen-presenting dendritic cells is more common at the time of disease progression. Taken together, these results suggest reduced neoantigen production and a decline in antigen presentation may contribute to IMiD resistance. Disclosures Landgren: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Green:Celgene: Consultancy; Cellectar Biosciences: Research Funding; Seattle Genetics: Research Funding; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; GSK: Consultancy.

Abstract P5-12-02: PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC)

Abstract Purpose: To date no biomarker has been identified that predicts response to ICI in mTNBC. This study aimed to explore if tumor genomic alterations correlate with efficacy of PD-1/PD-L1 inhibition in patients (pts) with mTNBC. Methods: Demographic, treatment response, and long-term outcome data were collected on patients with mTNBC treated at Dana-Farber Cancer Institute (DFCI) under several clinical trials incorporating PD-1/PD-L1 inhibitors, given as monotherapy or combined with chemotherapy (CT). Pts included in this analysis had available results of targeted exon sequencing performed using Oncopanel, our institutional gene sequencing panel, on archival tumor tissue. TMB was calculated by determining the number of non-synonymous somatic mutations that occur per megabase of exonic sequence data across all genes on the panel. High TMB was defined as 310 mutations/megabase. TMB and gene alterations were correlated with objective response rate (ORR) per RECIST 1.1, progression-free (PFS) and overall survival (OS). Results: A total of 50 pts with mTNBC were included in this analysis. At baseline, the median age was 55.9 years (31.8–75.9), 60% had ECOG 0 and 40% had ECOG 1, 72% had visceral metastasis, and 46% had received 31 prior lines of systemic therapy in the metastatic setting. While 26% of pts received monotherapy [pembrolizumab (n=7, NCT02447003); atezolizumab (n=6; NCT01375842)], 74% received combination with CT [pembrolizumab plus eribulin (n=31; NCT02513472); atezolizumab plus nab-paclitaxel (n=6; NCT01633970)]. PTEN alterations were present in 30% of pts (mutations = 7; one copy number loss = 7; two copy number loss = 1). Median follow-up was 14 months (1–40). Pts with tumors harboring PTEN alterations had lower ORR (7% vs 57%; P<0.001), shorter median PFS (2.3 vs 6.3 months; P=0.027), and shorter median OS (8.1 vs 20.1 months; P=0.012) compared to pts without PTEN alterations. The median TMB was 6.6 mut/Mb (1.2–50.8), and 23% of pts had a high TMB. While high TMB was not associated with higher ORR (P=0.56), it was associated with better median PFS (16.5 vs 2.4 months; P=0.017), and better median OS (not reached vs 13.5 months; P=0.026). Both PTEN status and TMB remained significantly associated with PFS in the multivariable model. Only PTEN status remained significantly associated with OS in the multivariable analysis with the same covariables. Ongoing analysis to better understand if these predictors are specific for predicting benefit to immunotherapy and/or a marker of chemotherapy resistance will be presented at the symposium. Conclusion: PTEN genomic alterations and TMB may impact benefit from PD-1/PD-L1 inhibitors largely administered with chemotherapy in mTNBC. These observations warrant prospective validation and may inform the importance of stratifying pts according to these characteristics in future randomized studies with ICI. Table 1.Multivariable analysis for PFS Hazard ratioConfidence Intervalp-valueCombination therapy0.420.16 – 1.130.009Visceral metastasis1.310.63 – 2.770.46Previous lines of therapy1.020.09 – 0.700.85ECOG 12.11.06 – 1.280.034PTEN altered3.741.65 – 8.440.002Hypermutated tumors0.850.75 – 0.970.011 Citation Format: Barroso-Sousa R, Tyekucheva S, Pernas-Simon S, Exman P, Jain E, Garrido-Castro AC, Hughes M, Bychkovsky B, Di Lascio S, Umeton R, Files J, Lindeman NI, MacConaill LE, Hodi FS, Krop IE, Dillon D, Winer EP, Wagle N, Lin NU, Mittendorf EA, Tolaney SM. PTEN alterations and tumor mutational burden (TMB) as potential predictors of resistance or response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-02.

P2.09-11 TMB Estimated with Targeted NGS in Early Stage Squamous Cell Carcinoma: Correlation with PD-L1 Expression and Lymphocyte Density

It has been proposed that a combination of assays may refine the prediction of response to checkpoint inhibitors, with tumour mutation burden (TMB) being lately the strongest biomarker associated with efficacy. Although some targeted next generation sequencing (NGS) assays provide a good estimate of whole exome sequencing TMB, they are only available throughout referral laboratories. We sought to investigate the possibility of profiling TMB in-house with a commercially available targeted NGS assay, and to correlate the results with survival, the status of TP53, PD-L1 overexpression and tumor-infiltrating lymphocytes (TILs). The study included samples from 40 patients diagnosed with early-stage lung squamous cell carcinoma. PD-L1 immunohistochemistry (IHC) was performed with two clones (SP263 and SP142, Ventana Medical Systems). CD8+ TILs were scored with a digital algorithm. The status of TP53 (exons 4–10) was investigated with direct sequencing. Ion TorrentTM OncomineTM Tumor Mutation Load Assay (ThermoFisher Scientific), a targeted NGS assay which covers 1.7Mb across 409 cancer driver genes, was used to assess TMB. NGS was performed on genomic DNA from FFPE tumor samples using the Ion S5TM system. The results were analyzed with the Ion ReporterTM software. According to the manufacturer’s instructions, TMB was calculated based on the number of non-synonymous somatic mutations, after removing polymorphisms and known or predicted driver mutations from all the variants. We investigated the correlation between TMB and PD-L1 expression, lymphocyte density, TP53 status and survival. TMB data was available for all 40 patients. The mean and median number of mutations was 13 and 11, respectively. CD8+/mm2 TILs within the peritumoral stromal compartment ranged from 312 to 4793 and within the intraepithelial compartment ranged from 17 to 2002. Sixty percent and 32% of cases were positive in tumor cells with SP263 or SP142, respectively (1% cut-off). Immune cells PD-L1 expression (1% cut-off) was observed in 92% of samples with both clones. No correlation was found between PD-L1 expression or lymphocyte density and TMB. Tumors with TP53 mutations showed non-significant higher numbers of mutations than those wild-type (p=0.075). Accordingly, there was a similar trend for overall survival using the median number of mutations as a cut-off (p=0.137, HR, 5.29). It is feasible to use targeted NGS to estimate TMB. In our pilot study, there was no obvious correlation with other immunooncology predictive biomarkers. Funding: AES 2017/Feder. ISCIII: PI17/01001

Abstract PD1-15: The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers

Abstract Introduction:Pathogenic and/or founder germline variants in the ataxia-telangiectasia mutated (ATM) gene confer an increased breast cancer (BC) risk. The protein kinase ATM plays a central role inDNA double-strand break-repair and in the activation of downstream targets such as p53 and BRCA1. We sought to define the repertoire of somatic genetic alterations of BCs from patients with pathogenic germline ATM mutations and whether somatic loss of heterozygosity (LOH) of ATM would be present in these cancers. Methods: 21 BCs from ATM germline mutation carriers were microdissected. Tumor and normal DNA samples were subjected to whole-exome sequencing (WES, n=12) or massively parallel sequencing targeting all coding regions and selected intronic and regulatory regions of 410 key cancer genes (n=9). Somatic mutations, copy number alterations, cancer cell fractions, large-scale state transitions (LSTs) and mutational signatures were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to assess LOH of the wild-type allele of ATM. Results: Of the patients included in this study, 71%, 24% and 5% of cases harbored ATM missense (all but one p.V2424G), frame-shift and nonsense germline mutations, respectively. All tumors were ER-positive and four (19%) were HER2-positive. The median age of the patients was 46 years (32–79 years). Our analyses revealed biallelic inactivation of ATM through LOH of the wild-type allele in 16 of 21 cases (76%), and second somatic ATM mutations were not found. The median number of non-synonymous somatic mutations was 38 (range 15-113) and 2 (range 0-8)in tumors subjected to WES and targeted sequencing, respectively. The repertoire of somatic genetic alterations of ATM-associated BCs was found to be heterogeneous, including clonal PIK3CA mutations (24%), GATA3 mutations (19%), FANCI amplifications (19%) and CCND1 amplifications (14%). Importantly, however, no somatic mutations affecting TP53 were found. Analysis of the WES data revealed that 5 (42%) ATM-associated BCs displayed high LST scores, all of which harbored bi-allelic ATM inactivation. In contrast to BRCA1- and BRCA2-associated BCs, which frequently display the mutational signature 3 associated with defective homologous recombination DNA repair, the ATM-associated BCs studied displayed the ageing mutational signature (i.e. signature 1). Comparison of the mutational profiles of the ATM--associated BCs subjected to WES (n=12) with those of BRCA1- (n=11) and BRCA2-associated (n=10) BCs from The Cancer Genome Atlas revealed that TP53 was more frequently mutated in BCs from BRCA1 germline mutation carriers (0% vs 72%, P<0.001), while no differences with BRCA2-associated BCs were found. Conclusion: ATM-associated BCs frequently display bi-allelic ATM inactivation through LOH of the wild-type allele and a subset of these cases displayed high levels of LSTs. These findings suggest that at least in a subset of ATM-associated BCs, biallelic inactivation of ATM rather than a dominant negative effect of the germline mutation may be the mechanism of inactivation of this tumor suppressor gene. The repertoire of somatic genetic alterations of ATM-associated BCs is heterogeneous, with a noticeable lack of TP53 somatic mutations. Citation Format: Weigelt B, Bi R, Kumar R, James PA, Thorne H, Couch FJ, Eccles DM, Blows F, Geyer FC, Li A, Selenica P, Lim RS, Blecua P, Shen R, Wen H, Robson ME, Reis-Filho JS, Chenevix-Trench G. The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-15.

Abstract PD6-03: Distribution of microsatellite instability, tumor mutational load, and PD-L1 status in molecularly profiled invasive breast cancer

Abstract Background: Recent data indicate a promising response to immune check point blockade (ICB) in patients with breast cancer. Pembrolizumab, a humanized monoclonal antibody against programmed death 1 (PD-1) receptor and one of several ICB agents in development, was given an FDA approval for all MSI (microsatellite instability)-high solid tumors. MSI incidence in breast cancer is not fully elucidated. Other biomarkers being explored in possible relationship to ICB activity include PD-1 ligand (PD-L1) status and tumor mutational load (TML). In this study, we aimed to explore the incidence of these biomarkers in invasive breast cancers. Methods: A retrospective data analysis of patients profiled by commercial next-generation sequencing (NGS) at Caris Life Sciences was performed. MSI results were either high, stable, or equivocal. MSI was calculated by comparing repeat-insertions or deletions across over 7,000 microsatellite sequences in the patient sample to the hg19 reference genome. Samples with repeat variances in more than 45 microsatellites were classified as MSI-High PD-L1 expression was evaluated using immunohistochemical analysis (IHC), with clone SP-142 (Roche Diagnostics). A sample was considered positive if there was >5% membranous staining of tumor cells. Tumor mutational load was calculated as a total number of non-synonymous somatic mutations identified per megabase of the genome coding area with high being greater than or equal to 17. Results: A total of 9,627 breast cancer cases were queried from the Caris Life Sciences database. The mean age (±SD) was 56.8 ± 12.4 years (range 20-90). The tumor distribution was 60.7% hormone receptor (HR) positive (ER and/or PR) and HER2 negative, 9.5% HER2 positive (with HR negative or positive), and 29.8% triple negative (negative for ER, PR and HER2). Of all cases, there were 5,203 tested for PD-L1 status, 354 (6.8%, 95% CI 6.2-7.5%) were positive. Of 1,440 tumors tested for MSI status, 15 (1.04%, 95% CI 0.58-1.71%) were either high (8) or equivocal (7), the rest were MSI-low. Tumor mutational load (TML) was available on 1,766 tumors, of which 55 (3.1%, 95% CI 2.4-4.0%) were high. Seven out of the 8 MSI-high tumors were also TML-high. Four out of the 8 MSI-high breast cancers were triple negative. Conclusion: In this large dataset of molecularly profiled breast cancer, MSI was observed in about 1% of the breast tumors tested. Overall, modest positivity of TML, PD-L1, and MSI of all invasive breast cancers was observed. The percentage of patients that had at least one of these biomarkers that may confer responsiveness to ICB is planned and will be further stratified by subtype. MSI-high breast cancers mostly overlapped with those that were TML-high. Future research is needed to show the clinical utility of these biomarkers in response to ICB. Updated data will be presented. Citation Format: Obeid E, Ellerbrock A, Handorf E, Goldstein L, Gatalica Z, Arguello D, Swain SM, Isaacs C, Vacirca J, Tan A, Schwartzberg L. Distribution of microsatellite instability, tumor mutational load, and PD-L1 status in molecularly profiled invasive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-03.

Similarity and difference in tumor-infiltrating lymphocytes in original tumor tissues and those of in vitro expanded populations in head and neck cancer.

Though adoptive tumor-infiltrating lymphocyte (TIL) therapy has been explored in clinical trials for many years, there is little information for the clonotype composition between TILs in original tumor tissues and TILs that were in vitro expanded and infused to cancer patients. To investigate the similarity/difference in TILs in original tumor tissues and those of in vitro expanded populations in squamous cell carcinoma of head and neck (SCCHN) as well as their correlation with somatic mutations in cancer cells, we performed whole exome analysis, expression profile analysis of immune-related genes, and T cell receptor (TCR) analysis of original TILs and in vitro expanded TILs in 8 surgically-resected HPV-negative fresh tumors with SCCHN. We found an unusually high number of non-synonymous somatic mutations (4290, 1779 and 901 mutations) in three SCCHN tumors, in which we identified mutations in mismatch repair genes, MSH2 or MSH4, or a DNA polymerase gene, POLE. Interestingly, dominant TCR clonotypes of expanded CD8+ TILs derived from these three tumors revealed high similarity to those in original tumors while for remaining tumors with the lower mutational load, we found that T cell clonotypes between TILs in original tumor tissues and those expanded in vitro were almost entirely different. Our findings might provide clinically useful information for identification of tumor-antigen-specific T cell clones that may lead to further improvement of adoptive TIL therapy for SCCHN patients.

Abstract 387: Targeted exome sequences of cancer-related genes in human cancers using amplicon sequencing

Abstract Objectives: Next-generation sequencing technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting somatic cancer genome alterations. Platforms for genomic DNA alterations are more common in clinical practice and include whole genome/exome sequencing analysis. These tests are still very expensive, although the costs are coming down substantially. Here, we aimed to determine the efficacy and advantages of targeted exome sequencing of known cancer-related genes in human cancers using amplicon sequencing. Methods: DNA was extracted from 61 human cancer specimens and their corresponding non-cancerous tissues, including oral squamous cell carcinomas (OSCCs) and multiple myelomas (MMs). Forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel that offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. This platform was designed to be amplification based capture with 15,992 regions (1.6 megabases in total size). Purified DNA libraries were sequenced with 6-8 samples on Ion Proton P1 chip. Sequence reads of tumor and normal samples were aligned to the hg19 genome, and generated BAM files were used to detect somatic mutations (point mutation, insertion and deletion) and copy number variations. Results: Each sample underwent on mean 8.4 million sequencing reads after quality filtering. The mean base coverage depth was 530, and >95% of targeted bases were represented by at least 20 reads. The number of non-synonymous somatic mutations in 47 patients with OSCC ranged from 1 to 21 with a mean of 7.5 (6.4/Mb). The most frequent mutations in OSCC were in TP53 (63.8%), NOTCH1 (25.5%), CDKN2A (19.2%), TAF1L (17.0%), SYNE1 (14.9%) and PIK3CA (8.5%). We also detected a mean of 6.1 (range 3-11) non-synonymous mutations per MM patient. Somatic mutations were found in known MM-associated genes, including TP53 and NRAS. Pathway assessment has shown that somatic aberrations within MM genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of MM patients. Conclusions: This study demonstrates the utility of using a semiconductor-based sequencing to efficiently identify somatic genetic alterations in human cancers. The targeted next-generation sequencing using low amounts of FFPE DNA is a valuable tool for rapid (5 days) and high-throughput genetic testing in research and clinical settings. Citation Format: Yasushi Sasaki, Takafumi Nakagaki, Miyuki Tamura, Hisayo Fukushima, Hiroshi Ikeda, Ryota Koyama, Masashi Idogawa, Takashi Tokino. Targeted exome sequences of cancer-related genes in human cancers using amplicon sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 387. doi:10.1158/1538-7445.AM2017-387

The correlation between mutation burden and disease free survival in patients with lung adenocarcinomas.

8550 Background: Lung cancer is one of the leading causes of cancerous deaths globally. High mutation burden is a special character in lung adenocarcinoma patients. Mutation burden is usually based on the number of non-synonymous mutations implying the instability of genome. We hypothesize genome-wide mutation burden indicates mutation degree and is correlated with prognostic in lung adenocarcinoma. Methods: Whole-exome sequencing was performed on 98 Chinese lung adenocarcinoma patients with tumor and normal tissue to a mean depth of 49.6ⅹ. The total number of non-synonymous somatic mutations was calculated from the sequencing data of each patient. Patients were divided into high mutation burden and low mutation burden groups in accordance with the mean mutation burden and Kaplan-Meier analysis was performed for survival analysis between these two groups. The association between mutation burden and age or smoking status was analyzed by Wilcoxon rank-sum test. Results: Among these 98 patients, the values of mutation burden varied from 5 to 1121 with mean value 161.8, 36 (36.7%) patients with smoking history and 34 (34.7%) patients were older than 65 years; the numbers of patients in I, II, III stage were 19 (19.4%), 16 (16.3%) and 63 (64.3%) respectively. 32 patients were classified into high mutation burden group, the other 66 patients classified into low mutation burden group. Survival analysis showed a significantly longer disease free survival (DFS) in low mutation burden group (p-value = 0.0133).Mutation burden was significantly associated with age ( < 65 vs ≥65, p-value = 0.0208) and smoking status (p-value = 8.67ⅹ10-4). Conclusions: The association between mutation burden and age or smoking status suggested the high risk for mutation burden accumulation. The significant difference of DFS between high mutation burden and low mutation burden groups reveals the potential of mutation burden as one of the prognostic factors in patients with lung adenocarcinomas.

T cell receptor β-chain repertoire analysis reveals the association between neoantigens and tumour-infiltrating lymphocytes in multifocal papillary thyroid carcinoma.

To explore whether a few nonsynonymous somatic mutations could induce activation and proliferation of neoantigen-specific tumour-infiltrating lymphocytes (TILs) in tumours with low mutation rates, we analysed a patient with multifocal papillary thyroid carcinoma (seven noncontiguous cancer foci) to investigate the relationship between neoantigens and TILs. These seven foci had a few or no nonsynonymous somatic mutations; moreover, multiple loci had similar or different spectra of mutations. We used high-throughput sequencing of the rearranged genes in T cell receptor β-chain (TCRβ) to reveal the basic characteristics of T cells in seven tumour foci and matched adjacent normal tissue. We found that in multifocal papillary thyroid carcinoma the number of nonsynonymous somatic mutations was positively associated with oligoclonal TCRβ repertoire, and tumour foci with similar spectra of mutations had higher overlap of TCRβ repertoire. In conclusion, the number of nonsynonymous somatic mutations is small in tumours with low mutation rates but these mutations still play an important role in activating neoantigen-specific TILs.

Abstract 119: Genomic analysis of remnant gastric adenocarcinoma

Abstract Introduction: Prior to pharmaceutical treatment of gastroduodenal ulcer disease, partial gastrectomy was performed for the management of ulcer disease complicated by bleeding (PUD). The proposed mechanism for the development of gastric cancer in this setting is the chronic irritation of the mucosa by duodenal reflux into the gastric remnant. The aim of this study is to characterize the genetic profile of remnant gastric carcinoma (RGC). Material and Methods: Thirty cases of RGC developed in the setting of prior partial gastrectomy for PUD and without neoadjuvant chemotherapy were retrospectively collected from the institution database. Histopathological evaluation was performed to ensure >80% tumor cellularity with matching normal gastric mucosa prior to DNA extraction. Ultra-deep targeted sequencing of the thirty matched pairs was performed using the MSK-IMPACT assay (Integrated Mutation Profiling of Actionable Cancer Targets) to identify mutations and copy number alterations in a common set of 410 cancer-associated genes. MSK-IMPACT data from previously sequenced 45 sporadic gastric carcinomas (GC) was used as comparison. Results: The cohort consisted of 22 (73%) males and 8 (27%) females with the mean age of 73 years. The median interval between PUD and RGC resections was 34.8 years (7-60 years). By Lauren's classification, 60% of the tumors were intestinal, 23% diffuse and 17% mixed. Most of the tumors (47%) were poorly differentiated and stage II (40%). MSK-IMPACT sequencing revealed 299 total somatic alterations occurring in 148 of 410 targeted genes with a median number of non-synonymous somatic mutations and copy number events of 3 and 1 per patient, respectively. Of the 207 total mutations, 27.5% were hotspot COSMIC and 23.2% were loss-of-function mutations. Recurrent genomic events included mutually exclusive mutations in TP53 (33%) and amplifications in MDM2 (26%), alterations in the ERBB family genes (37%), ARID1A (16%), KMT2D (13%), CDH1 (13%), and CDK4 (13%). In addition, actionable alterations in PIK3CA (10%), FGFR1 (4%), PTEN (4%), and concurrent hotspot KRAS mutations and inactivation of p15/p16 (10%) were observed. Compared to the sporadic GC cases, RGC cases presented differences in the landscape and recurrence of alterations. Conclusion: The pathogenesis of RGC is likely associated with the prolonged exposure of the gastric mucosa to gastroduodenal contents. The mean age of patients may also exert effects on the development of the RGC. Based on our sequencing results, RGC may present a genetic profile distinct from that of sporadic gastric tumors. Assessment of these alterations identified 30% of patients with potential therapeutic targets for the treatment of this disease and deserves further investigation. Citation Format: Edaise M. da Silva, Helen H. Won, Dianne Torrence, Daniel Coit, Michael F. Berger, Laura Tang. Genomic analysis of remnant gastric adenocarcinoma. [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 119.

Abstract 05: Genetic heterogeneity in rectal cancer - Identification of subpopulations of tumor cells resistant to neoadjuvant CRT

Abstract One of the benefits of neoadjuvant chemotherapy and radiation therapy (CRT) for the management of rectal cancer is tumor downstaging that can ultimately lead to complete tumor regression (known as complete pathological response - pCR). In a previous study using PET/CT imaging to assess tumor response to CRT, 50% of rectal cancers showed a continuous decrease in metabolic activity (estimated by standard uptake value measurements) between 6 and 12 weeks from CRT completion. However the remaining 50% of the cases showed increased metabolic activity within that period. We reasoned that the increased metabolic activity observed in these later patients could be determined by the clonal expansion of a genetically distinct subpopulation of tumor cells resistant to CRT. To address this question we performed exome sequencing and mutation detection analysis on normal tissue, primary tumor and residual tumor from 7 patients with rectal cancer that exhibited an increase in metabolic activity after CRT. Overall, about 20Gb of unambiguously mapped sequences were generated for each sample resulting in an average fold-coverage of 30X. Captured sequences mapped to the reference human genome were then used for the detection of SNPs and non-synonymous somatic point mutations in all three samples from each patient. Approximately 30,000 single nucleotide variants (SNVs) were identified in each sample and most of these SNVs were common to all samples. As expected, the majority of these common inherited variants (96%) have already been described in dbSNP. To identify non-synonymous somatic mutations occurring in primary and residual tumor samples, we excluded those present in the normal sample and those already described as a known SNP in dbSNP resulting in a mean of 243 SNVs per patient. Noteworthy, a significant number of non-synonymous somatic mutations were exclusively found in the primary and residual tumor samples of each patient, 30 and 32 SNVs on average respectively, revealing a high degree of tumor genetic heterogeneity. Furthermore, we were able to identify non-synonymous somatic mutations that were presented in both samples (mean of 107 SNVs), and for these mutations we determined the mutant allele frequency (number of reads representing the mutation/total number of reads covering the mutated base) in the primary and the residual tumor. We then searched for mutations with significantly different allele frequencies in the two samples, as these mutations would likely represent genetically distinct subpopulations of tumor cells selected during CRT. We were able to identify, on average, 4 SNVs enriched in primary tumor and 37 SNVs enriched in residual tumor per patient. In conclusion, based on exome sequencing of rectal tumors that exhibit incomplete response to CRT and have increased tumor metabolism we were able to identify non-synonymous mutations that may be associated with specific mechanisms of resistance or sensitivity to CRT. Functional analysis of these mutations and mutated genes will be presented, providing new insights into the molecular events influencing response to neoadjuvant CRT in rectal cancer. Citation Format: Fabiana Bettoni, Elisa R. Donnard, Bruna R. S. Correa, Paula F. Asprino, Fernanda C. Koyama, Natália M. Felício, Bruna H. Hessel, Pedro A. F. Galante, Anamaria A. Camargo, Angelita Habr-Gama, Rodrigo O. Perez. Genetic heterogeneity in rectal cancer - Identification of subpopulations of tumor cells resistant to neoadjuvant CRT. [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 05.

Abstract 610: Prospective genomic characterization of small cell lung cancer by targeted next generation sequencing

Abstract INTRODUCTION: Recent studies using next generation sequencing (NGS) technologies have provided insights into the molecular landscape of small cell lung cancer (SCLC), including frequent inactivation of TP53 and RB1. However, molecular profiling of SCLC has been impeded by lack of tissue. We performed prospective genomic profiling of patients with advanced SCLC, demonstrating the ability to test small biopsy specimens, and we report trends associated with clinical characteristics. METHODS: Formalin fixed paraffin embedded surgical resections, core biopsies, and fine needle aspirates from 50 patients with SCLC were subjected to FGFR1 FISH testing, PTEN immunohistochemistry (IHC), and deep-coverage targeted NGS (median 420x) to identify single nucleotide variants, indels, and copy number alterations in a common set of 222 cancer-associated genes. Demographic and clinical data were collected. Genotyping data were stratified by smoking status, stage, and response to therapy. RESULTS: Of the 50 patients with SCLC, 58% had extensive stage disease, and 30% were resistant to first line therapy. We observed diverse genetic profiles in SCLC, with alterations occurring in 202 of 222 targeted genes. The median number of non-synonymous somatic mutations was 7. Of 526 total non-synonymous mutations, 5% were hotspot COSMIC mutations, 25% were loss-of-function, and 47% were G-to-T transversions indicative of tobacco-induced carcinogenesis. Recurrent loss-of-function mutations in RB1 (96%) and TP53 (92%) were observed. Other frequent genomic events included alterations in SOX2 (26%), EPHA5 (22%), CDKN2C (20%), MYCL1 (20%), and PIK3CA (18%). Actionable point mutations in PIK3CA, amplifications of FGFR1, amplifications of MYC, and loss of PTEN were confirmed by independent clinically validated assays. The genotyping data showed differences based on disease stage, response to therapy, and smoking status. Patients with limited disease had a higher median number of mutations detected (9 mut/tumor) than patients with extensive disease (5 mut/tumor). Tumors refractory to chemotherapy were more likely to harbor homozygous deletions including on chromosome 3p, while tumors sensitive to chemotherapy had more diversified amplifications, deletions, and mutations. The median number of mutations detected for never smokers (0 pack years), moderate smokers (<20 pack years), and heavy smokers (20+ pack years) were 3, 4.5, and 8 mut/tumor, respectively (P<0.05). None of the mutations detected in tumors from never smokers were G-to-T transversions. CONCLUSION: Comprehensive molecular profiling is feasible on clinical SCLC specimens. Our study illustrates the molecular diversity and mutational patterns in SCLC. We confirmed previously reported recurring events, and further identified associations between genomic profiles and clinical features including stage, response, and smoking status. Citation Format: Helen H. Won, M. Catherine Pietanza, Lee M. Krug, Anna M. Varghese, Natasha Rekhtman, Lu Wang, William Travis, Paul K. Paik, Gregory J. Riely, Maureen F. Zakowski, Marc Ladanyi, Mark G. Kris, Charles M. Rudin, Michael F. Berger. Prospective genomic characterization of small cell lung cancer by targeted next generation sequencing. [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 610. doi:10.1158/1538-7445.AM2015-610

Studying tumorigenesis through network evolution and somatic mutational perturbations in the cancer interactome.

Cells govern biological functions through complex biological networks. Perturbations to networks may drive cells to new phenotypic states, for example, tumorigenesis. Identifying how genetic lesions perturb molecular networks is a fundamental challenge. This study used large-scale human interactome data to systematically explore the relationship among network topology, somatic mutation, evolutionary rate, and evolutionary origin of cancer genes. We found the unique network centrality of cancer proteins, which is largely independent of gene essentiality. Cancer genes likely have experienced a lower evolutionary rate and stronger purifying selection than those of noncancer, Mendelian disease, and orphan disease genes. Cancer proteins tend to have ancient histories, likely originated in early metazoan, although they are younger than proteins encoded by Mendelian disease genes, orphan disease genes, and essential genes. We found that the protein evolutionary origin (age) positively correlates with protein connectivity in the human interactome. Furthermore, we investigated the network-attacking perturbations due to somatic mutations identified from 3,268 tumors across 12 cancer types in The Cancer Genome Atlas. We observed a positive correlation between protein connectivity and the number of nonsynonymous somatic mutations, whereas a weaker or insignificant correlation between protein connectivity and the number of synonymous somatic mutations. These observations suggest that somatic mutational network-attacking perturbations to hub genes play an important role in tumor emergence and evolution. Collectively, this work has broad biomedical implications for both basic cancer biology and the development of personalized cancer therapy.

Abstract A133: Genetic heterogeneity in rectal cancer: Identification of subpopulations of tumor cells resistant to neoadjuvant CRT.

Abstract One of the benefits of neoadjuvant chemotherapy and radiation therapy (CRT) for the management of rectal cancer is tumor downstaging that can ultimately lead to complete tumor regression (known as complete pathological response - pCR). In a previous study using PET/CT imaging to assess tumor response to CRT, 50% of rectal cancers showed a continuous decrease in metabolic activity (estimated by standard uptake value measurements) between 6 and 12 weeks from CRT completion. However the remaining 50% of the cases showed increased metabolic activity within that period. We reasoned that the increased metabolic activity observed in these later patients could be determined by the clonal expansion of a genetically distinct subpopulation of tumor cells resistant to CRT. To address this question we performed exome sequencing and mutation detection analysis on normal tissue, primary tumor and residual tumor from one patient with rectal cancer that exhibited an increase in metabolic activity after CRT. Approximately, 15Gb of unambiguously mapped sequences were generated for each normal tissue and residual tumor and 25Gb of mapped sequences were generated for primary tumor. Over 51% of the bases mapped to the targeted regions, resulting in an average fold-coverage of 30X. Captured sequences mapped to the reference human genome were then used for the detection of SNPs and non-synonymous somatic point mutations in all three samples from this patient. Approximately 30,000 single nucleotide variants (SNVs) were identified in each sample and most of the SNVs were common to all samples. As expected, the majority of these common inherited variants (96%) have already been described in dbSNP. To identify non-synonymous somatic mutations occurring in primary and residual tumor samples, we excluded those present in the normal sample and those already described as a known SNP in dbSNP. Noteworthy, a significant number of non-synonymous somatic mutations were exclusively found in the primary and residual tumor samples revealing a high degree of tumor genetic heterogeneity. A total of 80 non-synonymous somatic mutations were present in both samples, and for these mutations we determined the mutant allele frequency (number of reads representing the mutation/total number of reads covering the mutated base) in the primary and the residual tumor. We then searched for mutations with significantly different allele frequencies in the two samples, as these mutations would likely represent genetically distinct subpopulations of tumor cells selected during CRT. Functional analysis of these mutations and mutated genes will be presented, providing new insights into the molecular events influencing response to neoadjuvant CRT in rectal cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A133. Citation Format: Fabiana Bettoni, Elisa R. Donnard, Bruna R. S. Correa, Paula F. Asprino, Fernanda C. Koyama, Natália M. Felício, Bruna H. Hessel, Pedro A. F. Galante, Anamaria A. Camargo, Angelita Habr-Gama, Rodrigo O. Perez. Genetic heterogeneity in rectal cancer: Identification of subpopulations of tumor cells resistant to neoadjuvant CRT. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A133.

Abstract 4872: ICGC PedBrain Tumor - Next-generation sequencing identifies novel subgroup-specific mutations and copy number aberrations in medulloblastoma

Abstract Introduction: The International Cancer Genome Consortium (ICGC) is a worldwide network aiming to provide comprehensive molecular genetic profiles of 50 clinically and societally important tumors. A major focus of the ICGC PedBrain Tumor project is medulloblastoma (MB) - the most common malignant brain tumor in childhood. Importantly, recent work has shown that MB is not a single disease, but is in fact comprised of 4 distinct molecular subgroups (WNT, SHH, Group 3, Group 4). Using an integrative next-generation sequencing-based approach, we have investigated tumorigenic events underlying medulloblastoma, with the aim of identifying novel diagnostic or prognostic markers, and therapeutic targets. Methods: The whole genome of 50 tumor-normal DNA pairs, plus the whole exome or 2,500 target genes in a further 100 sample pairs from different histological and transcriptomic subgroups, was sequenced on an Illumina HiSeq platform. Sequencing-based high-resolution copy-number data was also generated for all cases. Results: Overall, a smaller number of non-synonymous somatic mutations was seen than in most adult malignancies. The number of somatic changes also clearly showed a positive correlation with patient age, suggesting either longer-term selection for more weakly transforming alterations, or the acquisition of additional passenger mutations in tumors which grew over a longer period. Whilst some point mutations and small InDels affected known MB or cancer-related genes (e.g. CTNNB1, PTCH1, MLL2), the vast majority were in genes which have not previously been implicated in medulloblastoma. Furthermore, even the most recurrently altered genes were mutated in <20% of samples overall, although several showed a clear enrichment in distinct subgroups. Pathway analysis revealed chromatin modification as one of the most frequently altered cellular processes, with hits in multiple pathway members. Novel copy-number alterations were also identified. For example, a pattern of catastrophic chromosome shattering (‘chromothripsis’) was observed almost exclusively in the SHH tumor subgroup. Conclusion: Next-generation sequencing of this large tumor cohort has provided a number of new insights into this deadly pediatric tumor. The overriding pattern is one of enormous heterogeneity, but integration with molecular subgrouping shows clear enrichment of certain alterations. This first stage of the ICGC PedBrain project has demonstrated the power of next-gen sequencing approaches to reveal significant new insights, providing a number of novel targets for potential therapeutic intervention. It also, however, indicates the importance of personalised approaches to treatment optimisation, and shows the scale of the coming challenge in terms of functionally validating the huge number of novel mutations. 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 4872. doi:1538-7445.AM2012-4872