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XLSX file - 45K, Genes containing breakpoint from translocation in mTNBC

XLSX file - 273K, Somatic genic focal copy number changes in mTNBC

XLSX file - 598K, edgeR differential expression results in mTNBC for select cancer related genes

XLSX file - 607K, Outlier differential expression results in mTNBC for select cancer related genes

XLSX file - 61K, List of somatic point mutations in mTNBC detected by NGS sequencing analysis

XLSX file - 42K, Ingenuity Canonical Pathways results for mTNBC based on edgeR differential expression results

XLSX file - 23K, Mapping statistics for genome sequencing of tumor and germline DNA from mTNBC

XLSX file - 40K, Characteristics of mTNBC study population

PDF file - 1635K, PET/CT images of mTNBC2 breast lesion before and after treatment on combination MEK and AKT inhibitors

XLSX file - 48K, Transcriptome sequencing (RNA-seq) run statistics for mTNBC

PDF file - 135K, Validation of RB1 39bp homozygous deletion in mTNBC1

Additional Longitudinal Analysis of Relapsed and Refractory Childhood Solid Tumors.

Table S1. Extended Cohort Annotations. Table S2. Mutational Signatures. Table S3. GISTIC Copy Number Analysis. Table S4. Somatic Variants in Relapsed/Refractory Childhood Solid Tumors (n=184 Patients with T/N WES). Table S5. Clinvar Pathogenic and Likely Pathogenic Germline Variants in Relapsed/Refractory Childhood Solid Tumors(n=184 Patients with T/N WES). Table S6. Gene Lists. Table S7. Log2(TPM+1) Values for the 2,199 Most Variable Genes Expressed Across the Cohort of Relapsed/Refractory Childhood Solid Tumors with RNA-seq (n=245). Table S8. Differential Expression Analysis of 2,199 Most Variable Genes Among Clusters. Table S9. Genes Filtered for COSMIC, Transcription Factors and Potential Drug Targets from Table S8 with log2FC greater than or equal to 2. Table S10. Differential Expression Analysis Among C3a, C3b, and C3c Subclusters. Table S11. Samples Falling in Clusters C3a-C3c and Genes that are Uniformly Highly Expressed in the Clusters. Table S12. Longitudinal Samples.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment.Significance:Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Background: Children with relapsed CNS tumors, neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. Here we describe a study to determine the feasibility of leveraging genomic profiling results within a molecular tumor board (MTB) to make real-time treatment decisions for children with relapsed/refractory solid tumors. Methods: Subjects were divided to 3 strata: Strata 1: Relapsed/refractory neuroblastoma, Strata 2: Relapsed/refractory CNS tumors, and Strata 3: Relapsed/refractory rare solid tumors. Samples were sent for tumor/normal whole exome (WES) and tumor whole transcriptome sequencing, and the genomic data were used in a MTB to make real-time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every 2 cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of CR, PR, SD, NED, and PD subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. Results: 144 eligible subjects were enrolled with 144 evaluable for safety and 124 evaluable for response. Tumor types included neuroblastoma (n=31), CNS tumors (n=41), and rare tumors (n=72). Sarcomas (n=40) were the most common tumor type in the rare tumor stratum. The average time from biopsy to completion of DNA/RNA sequencing was 10 days (range 2-31 days); to completed analysis and drug prediction report, 17 days (8-41); 23 days (10-66) to MTB decisions; and 38 days (18-146) to initiation of the 4-drug combination agreed upon by the MTB. Treatments were selected on DNA and RNA findings in 19.5% of cases and in 80.5% on RNA alone. Patient benefit was exhibited in 70% of all subjects, 85% of neuroblastoma subjects, 73% of CNS tumor subjects, and 62% of rare tumor subjects. AEs occurred in Conclusions: It has been well-established that comprehensive genomic sequencing is the future of precision medicine. Here, we have demonstrated the feasibility, efficacy, and safety of a comprehensive sequencing model to guide targeted therapy for patients with any relapsed/refractory solid malignancies. Targeted therapy was well tolerated, and the response benefit rate of 70% in these heavily pretreated populations suggests that this treatment could be an effective option for relapsed and refractory pediatric cancers. Citation Format: Giselle L. Saulner Sholler, Jacqueline Kraveka, Genevieve Bergendahl, Elizabeth Lewis, William Ferguson, Abhinav Nagulapally, Karl Dykema, Valerie Brown, Michael Isakoff, Joseph Junewick, Deanna Mitchell, Don Eslin, Virginia Harrod, William Roberts, Javier Oesterheld, Randy Wada, Jawhar Rawwas, Devang Pastakia, Kevin Ginn, Raya Saab, Kevin Bielamowicz, Jason Glover, Eugenia Chang, Gina Hanna, Daniel Enriquez, Tyler Izatt, Rebecca Halperin, Sara Byron, William Hendricks, Jeffrey Trent. Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT089.

Homologous recombination repair deficiency (HRD) is a common driver of tumorigenesis that carries therapeutic implications, particularly in breast, ovarian, prostate, and pancreatic cancers. The influence of genetic ancestry on the incidence of HRD in these tumors remains to be defined. Previous studies have reported a higher incidence of variants of unknown significance (VUS) in individuals of non-European ancestry. Here, we evaluated the frequency of HR-deficiency within a diverse cohort of 566 patients with breast, prostate, ovarian, or pancreatic cancer. Genetic ancestry was estimated from constitutional whole exome sequencing data using a single nucleotide polymorphism weight model containing five ancestral populations: European, Native American, African, East Asian, and South Asian. Frequencies of clinical genetic testing, germline variants in HR-genes, and tumor HRD were compared between individuals of admixed Native American and European ancestry (n=82) and those of European ancestry (n=378). Admixed Native American/European individuals had a younger age at diagnosis compared to individuals of European ancestry (median 52.4 years (range, 20.8-83.6) vs 62.9 years (range, 20.9-87.9), p Citation Format: Sara A. Byron, Guangfa Zhang, Kimber Slater, Jiaming Zhang, Tyler Izatt, Bryce Turner, Xiao-Yu Xia, Rick A. Kittles, Jonathan J. Keats, Jeffrey M. Trent, Nicholas J. Schork, Lorna Rodriguez-Rodriguez. Influence of patient ancestry on homologous recombination repair deficiency in cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C039.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. Significance: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

10006 Background: The major genomic profiling studies that have helped define the molecular landscapes of pediatric cancers have typically focused on untreated pediatric cancers at diagnosis. Despite improvements in overall survival for childhood cancers, patients with treatment-refractory or relapsed (R/R) solid tumors face a poor prognosis. The genomic underpinnings of R/R disease are less well-characterized. Here, we describe the integrated genomic and transcriptomic analysis of 250 R/R solid tumors from 202 children profiled within precision medicine studies (NCT01355679, NCT01802567, NCT02162732) conducted by the Beat Childhood Cancer Consortium. Methods: Tumor-normal whole-exome and tumor mRNA sequencing was performed by Ashion Analytics (Phoenix, Arizona), a CAP-accredited, CLIA-certified laboratory, or within the research setting at TGen. Longitudinal tumor samples were sequenced for 20 patients. Variant calling included single nucleotide variants, indels, copy number alterations, and fusions. Integrated genomic and transcriptomic research analysis included microsatellite instability assessment, immunogenomic profiling, and functional gene set enrichment analysis. Results: Forty-six tumor types were represented, grouped into four general categories: sarcomas (36.1%; n = 73), neuroblastomas (29.2%; n = 59), CNS tumors (23.3%; n = 47), and other rare tumors (11.4%; n = 23). For patients with whole exome sequencing data, 78.3% (n = 144/184) of tumors bore a somatic alteration in at least one known cancer gene. Over one-third (39.1%; 72/184) of the cohort bore oncogenic fusions and/or oncogenic/likely-oncogenic hotspot mutations in a known cancer gene. Pathognomonic fusions were identified in 25% (46/184) of tumors, occurring most frequently in sarcomas. Pathogenic or likely pathogenic germline variants were identified in 8.7% (16/184) of patients. Microsatellite instability was detected in five different tumor types. Despite nearly all tumors (94%, 173/184) having at least one predicted strong binding neoantigen, over a quarter of tumors lacked transcript expression of these neoantigens or exhibited low MHC class I expression. Further, a subset of tumors showed elevated expression of the co-inhibitory immune checkpoint molecule PDL1. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology tumor clusters associated with immune signaling, development, and cellular signaling pathways. Longitudinal analysis revealed temporal heterogeneity pointing to the importance of re-biopsy at relapse for targeted treatment planning. Conclusions: Together, these data suggest R/R childhood solid tumors exhibit shared molecular features that are reflective of underlying biology, demonstrating the importance of comprehensive profiling to inform molecularly-guided treatment of R/R disease.

Chromosome 1q41-q42 deletions have recently been associated with a recognizable neurodevelopmental syndrome of early childhood (OMIM 612530). Within this group, a predominant phenotype of developmental delay (DD), intellectual disability (ID), epilepsy, distinct dysmorphology, and brain anomalies on magnetic resonance imaging/computed tomography has emerged. Previous reports of patients with de novo deletions at 1q41-q42 have led to the identification of an evolving smallest region of overlap which has included several potentially causal genes including DISP1, TP53BP2, and FBXO28. In a recent report, a cohort of patients with de novo mutations in WDR26 was described that shared many of the clinical features originally described in the 1q41-q42 microdeletion syndrome (MDS). Here, we describe a novel germline FBXO28 frameshift mutation in a 3-year-old girl with intractable epilepsy, ID, DD, and other features which overlap those of the 1q41-q42 MDS. Through a familial whole-exome sequencing study, we identified a de novo FBXO28 c.972_973delACinsG (p.Arg325GlufsX3) frameshift mutation in the proband. The frameshift and resulting premature nonsense mutation have not been reported in any genomic database. This child does not have a large 1q41-q42 deletion, nor does she harbor a WDR26 mutation. Our case joins a previously reported patient also in whom FBXO28 was affected but WDR26 was not. These findings support the idea that FBXO28 is a monogenic disease gene and contributes to the complex neurodevelopmental phenotype of the 1q41-q42 gene deletion syndrome.

DNA focused panel sequencing has been rapidly adopted to assess therapeutic targets in advanced/refractory cancer. Integrated Genomic Profiling (IGP) utilising DNA/RNA with tumour/normal comparisons in a Clinical Laboratory Improvement Amendments (CLIA) compliant setting enables a single assay to provide: therapeutic target prioritisation, novel target discovery/application and comprehensive germline assessment. A prospective study in 35 advanced/refractory cancer patients was conducted using CLIA-compliant IGP. Feasibility was assessed by estimating time to results (TTR), prioritising/assigning putative therapeutic targets, assessing drug access, ascertaining germline alterations, and assessing patient preferences/perspectives on data use/reporting. Therapeutic targets were identified using biointelligence/pathway analyses and interpreted by a Genomic Tumour Board. Seventy-five percent of cases harboured 1–3 therapeutically targetable mutations/case (median 79 mutations of potential functional significance/case). Median time to CLIA-validated results was 116 days with CLIA-validation of targets achieved in 21/22 patients. IGP directed treatment was instituted in 13 patients utilising on/off label FDA approved drugs (n = 9), clinical trials (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in five patients (two partial response, three stable disease). Although barriers to broader application exist, including the need for wider availability of therapies, IGP in a CLIA-framework is feasible and valuable in selection/prioritisation of anti-cancer therapeutic targets.

Extramedullary disease (EMD) in Multiple Myeloma (MM) is characterized by the detection of monoclonal plasma cells outside the bone marrow niche, and is frequently associated with poor prognosis. Here we describe novel genomic events leading to drug refractory disease in a heavily pretreated 37-year-old IgG-kappa MM patient presenting with progressive, multi-drug refractory EMD. For the first time we report an acquired truncating mutation of Cereblon (CRBN) as well as point mutations in proteasome subunit G2 and the glucocorticoid receptor as an explanation for drug resistance. Initial myeloma treatment for the patient occurred over multiple years and included the immunomodulatory drugs (IMiDs) thalidomide and lenalidomide, the proteasome inhibitor bortezomib, cortiosteroids, radiation, one autologous and two allogeneic transplantations. She experienced extramedullary relapse, presenting as an extensive neck mass and smaller soft tissue nodules in the upper left triceps. The most recent therapy immediately prior to genomic sequencing was hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) incorporating alkylating agent cyclophosphamide with transient minor response. The patient was then enrolled in a pilot study utilizing next generation sequencing (NGS) to identify novel markers and potential therapeutic targets. Samples were acquired with patient consent in compliance with Mayo Clinic Institutional Review Board. For this study we completed array comparative genomic hybridization, whole exome, whole genome (insert = 1 kb) and RNA sequencing (RNASeq) of a biopsy taken from the neck mass to thoroughly interrogate the tumour genome of this patient. The presence of mutations of interest was evaluated by capillary sequencing in an expanded cohort of 25 CD138+ MM samples with low CRBN expression. The neck mass pathology confirmed sheets of atypical plasma cells, kappa light chain restriction, CD138+, CD20− and CD45−. Array comparative genomic hybridization revealed multiple copy number abnormalities, most notably del1(p13.2–34.2), monosomy 13 and monosomy X. Therapy subsequent to biopsy for genome sequencing was with pomalidomide and dexamethasone without response. Unfortunately, the patient succumbed to her disease in less than the 12 weeks required at the time for sequencing and data analysis. Sequencing revealed a highly disturbed genome (Figure 1) consisting of 4 somatic insertions/deletions, 38 intra-chromosomal rearrangements, and 35 translocations, including the high risk marker and initiating tumour event t(14;16). Furthermore, 271 nonsynonymous, somatic point mutations were detected in genes including KRAS, PIK3CA, ATM, and NFKB2 (Table I). Importantly, a Q99* truncating mutation as well as a R283K point mutation were observed in CRBN, that we recently demonstrated as essential for the anti-MM action of IMiDs (Zhu, et al 2011). To our knowledge this is the first documented mutation of Cereblon in a primary myeloma sample. Additional sequencing of CRBN in the expanded cohort of 25 patients revealed a synonymous mutation in only one sample. Figure 1 Circos plot depicting genome wide somatic variants, rearrangements and copy number changes derived from next generation sequencing. Numbers with circles around them indicate the following: 1) somatic single nucleotide variation (SNV), 2) location of SNV ... Table I Summary of clinically relevant single nucleotide variations We also observed in the patient biopsy a potentially clinically relevant nonsynonymous point mutation in proteasome assembly chaperone 2, PSMG2 (E171K). PSMG2 is a proteasome assembly protein involved in mammalian 20S proteasome maturation (Hirano, et al 2005). Mutations in proteasome assembly components contribute to proteasome inhibitor resistance (Keats, et al 2007), possibly explaining this patient’s bortezomib-refractory disease. Capillary sequencing of PSMG2 in our expanded cohort revealed no mutations, although exonic deletion of PSMG2 has also been reported in myeloma (Walker, et al 2012). The last nonsynonymous point mutation associated with drug resistance was identified in NR3C1 (G369A), a glucocorticoid receptor. Mutation of NR3C1 has been associated with resistance to steroid therapy (Bray and Cotton 2003), which this patient received and proved refractory. No NR3C1 mutations were identified in our expanded cohort and none have been previously reported in other myeloma genomes (Chapman, et al 2011, Walker, et al 2012). Mutations in NR3C1 have however been described in the glucocorticoid resistant MM.1R cell line (Moalli, et al 1992). Patients with low NR3C1 expression levels who received thalidomide demonstrated better progression-free survival and overall survival than those with low NR3C1 who did not receive thalidomide (Heuck, et al 2012). While these mutations suggest causality of drug-refractory disease, they do not identify pathways that can be exploited with targeted therapies. Additional mutations were observed in pathways for which targeted therapies are available. This patient had mutations in KRAS (G12C) and in ATM (T1985I), both of which affect the signalling of MEK downstream, thus making MEK a therapeutic target of interest in this patient. While there are no approved MEK inhibitors available for MM treatment, more than 100 trials are currently investigating MEK inhibitors, of which three of these trials are being conducted in MM patients (www.clinicaltrials.gov). The patient tumour also contained canonical, activating mutations in PIK3CA (E542K). Interestingly, one study demonstrated that 64% of PIK3CA mutations occur in exon 9, where codon 542 is located. Moreover, 19% of patients with PIK3CA mutations also presented with KRAS mutations, of which 9% are G12C (Janku, et al 2012), found in our patient. The PI3K pathway is vitally important as it regulates downstream targets, such as AKT and MTOR, which are responsible for cell proliferation, growth, survival and metastasis (Bartholomeusz and Gonzalez-Angulo 2012). In addition, a number of clinical trials are currently investigating PIK3 inhibitors (www.clinicaltrials.gov). In summary, this is the first description of CRBN mutations in a primary myeloma sample and furthermore of a “triple negative” MM patient possessing mutations probably contributing to resistance to all three major drug classes utilized in MM therapy. These mutations were not replicated in our validation cohort of 25 patients with low level CRBN expression and functional data have not yet been obtained, thus further investigation is necessary to better understand the mutation frequency and the functional significance of mutation in these genes. In summary, our approach utilizing comprehensive next generation sequencing not only identified mutations suggestive of the patient’s refractory disease, but also revealed unforeseen therapeutic options highlighting the importance of this technology in advancing individualized medicine.

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies., Wellcome Trust (London, England) (Core Award 090532/Z/09/Z), Wellcome Trust (London, England) (Senior Investigator Award 095552/Z/11/Z ), Wellcome Trust (London, England) (WT095908), Wellcome Trust (London, England) (WT109497), Wellcome Trust (London, England) (WT098051), Wellcome Trust (London, England) (WT086084/Z/08/Z), Wellcome Trust (London, England) (WT100956/Z/13/Z ), Wellcome Trust (London, England) (WT097307), Wellcome Trust (London, England) (WT0855322/Z/08/Z ), Wellcome Trust (London, England) (WT090770/Z/09/Z ), Wellcome Trust (London, England) (Major Overseas program in Vietnam grant 089276/Z.09/Z), Medical Research Council (Great Britain) (grant G0801823), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I02593X/1), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I021213/1), Zhongguo ke xue ji shu qing bao yan jiu suo. Office of 863 Programme of China (2012AA02A201), National Basic Research Program of China (2011CB809201), National Basic Research Program of China (2011CB809202), National Basic Research Program of China (2011CB809203), National Natural Science Foundation of China (31161130357), Shenzhen Municipal Government of China (grant ZYC201105170397A), Canadian Institutes of Health Research (grant 136855), Quebec Ministry of Economic Development, Innovation, and Exports (PSR-SIIRI-195), Germany. Bundesministerium für Bildung und Forschung (0315428A), Germany. Bundesministerium für Bildung und Forschung (01GS08201), Germany. Bundesministerium für Bildung und Forschung (BMBF-EPITREAT grant 0316190A), Deutsche Forschungsgemeinschaft (Emmy Noether Grant KO4037/1-1), Beatriu de Pinos Program (2006 BP-A 10144), Beatriu de Pinos Program (2009 BP-B 00274), Spanish National Institute for Health (grant PRB2 IPT13/0001-ISCIII-SGEFI/FEDER), Japan Society for the Promotion of Science (fellowship number PE13075), Marie Curie Actions Career Integration (grant 303772), Fonds National Suisse del la Recherche, SNSF, Scientifique (31003A_130342), National Center for Biotechnology Information (U.S.) (U54HG3067), National Center for Biotechnology Information (U.S.) (U54HG3273), National Center for Biotechnology Information (U.S.) (U01HG5211), National Center for Biotechnology Information (U.S.) (U54HG3079), National Center for Biotechnology Information (U.S.) (R01HG2898), National Center for Biotechnology Information (U.S.) (R01HG2385), National Center for Biotechnology Information (U.S.) (RC2HG5552), National Center for Biotechnology Information (U.S.) (U01HG6513), National Center for Biotechnology Information (U.S.) (U01HG5214), National Center for Biotechnology Information (U.S.) (U01HG5715), National Center for Biotechnology Information (U.S.) (U01HG5718), National Center for Biotechnology Information (U.S.) (U01HG5728), National Center for Biotechnology Information (U.S.) (U41HG7635), National Center for Biotechnology Information (U.S.) (U41HG7497), National Center for Biotechnology Information (U.S.) (R01HG4960), National Center for Biotechnology Information (U.S.) (R01HG5701), National Center for Biotechnology Information (U.S.) (R01HG5214), National Center for Biotechnology Information (U.S.) (R01HG6855), National Center for Biotechnology Information (U.S.) (R01HG7068), National Center for Biotechnology Information (U.S.) (R01HG7644), National Center for Biotechnology Information (U.S.) (DP2OD6514), National Center for Biotechnology Information (U.S.) (DP5OD9154), National Center for Biotechnology Information (U.S.) (R01CA166661), National Center for Biotechnology Information (U.S.) (R01CA172652), National Center for Biotechnology Information (U.S.) (P01GM99568), National Center for Biotechnology Information (U.S.) (R01GM59290), National Center for Biotechnology Information (U.S.) (R01GM104390), National Center for Biotechnology Information (U.S.) (T32GM7790), National Center for Biotechnology Information (U.S.) (R01HL87699), National Center for Biotechnology Information (U.S.) (R01HL104608), National Center for Biotechnology Information (U.S.) (T32HL94284), National Center for Biotechnology Information (U.S.) (HHSN268201100040C), National Center for Biotechnology Information (U.S.) (HHSN272201000025C), Lundbeck Foundation (grant R170-2014-1039, Simons Foundation (SFARI award SF51), National Science Foundation (U.S.) (Research Fellowship DGE-1147470)

The ability to rapidly sequence the tumor and germline DNA of an individual holds the eventual promise of revolutionizing our ability to match targeted therapies to tumors harboring the associated genetic biomarkers. Analyzing high throughput genomic data consisting of millions of base pairs and discovering alterations in clinically actionable genes in a structured and real time manner is at the crux of personalized testing. This requires a computational architecture that can monitor and track a system within a regulated environment as terabytes of data are reduced to a small number of therapeutically relevant variants, delivered as a diagnostic laboratory developed test. These high complexity assays require data structures that enable real-time and retrospective ad-hoc analysis, with a capability of updating to keep up with the rapidly changing genomic and therapeutic options, all under a regulated environment that is relevant under both CMS and FDA depending on application. We describe a flexible computational framework that uses a paired tumor/normal sample allowing for complete analysis and reporting in approximately 24 hours, providing identification of single nucleotide changes, small insertions and deletions, chromosomal rearrangements, gene fusions and gene expression with positive predictive values over 90%. In this paper we present the challenges in integrating clinical, genomic and annotation databases to provide interpreted draft reports which we utilize within ongoing clinical research protocols. We demonstrate the need to retire from existing performance measurements of accuracy and specificity and measure metrics that are meaningful to a genomic diagnostic environment. This paper presents a three-tier infrastructure that is currently being used to analyze an individual genome and provide available therapeutic options via a clinical report. Our framework utilizes a non-relational variant-centric database that is scaleable to a large amount of data and addresses the challenges and limitations of a relational database system. Our system is continuously monitored via multiple trackers each catering differently to the diversity of users involved in this process. These trackers designed in analytics web-app framework provide status updates for an individual sample accurate to a few minutes. In this paper, we also present our outcome delivery process that is designed and delivered adhering to the standards defined by various regulation agencies involved in clinical genomic testing.

Background The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy is poor, with median survival of less than one year. The purpose of this study was to identify candidate therapeutically targetable somatic events in mCRC patient samples by whole genome sequencing (WGS), so as to obtain targeted treatment strategies for individual patients. Methods Four patients were recruited, all of whom had received > 2 prior therapy regimens. Percutaneous needle biopsies of metastases were performed with whole blood collection for the extraction of constitutional DNA. One tumor was not included in this study as the quality of tumor tissue was not sufficient for further analysis. WGS was performed using Illumina paired end chemistry on HiSeq2000 sequencing systems, which yielded coverage of greater than 30X for all samples. NGS data were processed and analyzed to detect somatic genomic alterations including point mutations, indels, copy number alterations, translocations and rearrangements. Results All 3 tumor samples had KRAS mutations, while 2 tumors contained mutations in the APC gene and the PIK3CA gene. Although we did not identify a TCF7L2-VTI1A translocation, we did detect a TCF7L2 mutation in one tumor. Among the other interesting mutated genes was INPPL1, an important gene involved in PI3 kinase signaling. Functional studies demonstrated that inhibition of INPPL1 reduced growth of CRC cells, suggesting that INPPL1 may promote growth in CRC. Conclusions Our study further supports potential molecularly defined therapeutic contexts that might provide insights into treatment strategies for refractory mCRC. New insights into the role of INPPL1 in colon tumor cell growth have also been identified. Continued development of appropriate targeted agents towards specific events may be warranted to help improve outcomes in CRC.

Supplemental digital content is available in the text., Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

Purpose New anticancer agents that target a single cell surface receptor, up-regulated or amplified gene product, or mutated gene, have met with some success in treating advanced cancers. However, patients' tumors still eventually progress on these therapies. If it were possible to identify a larger number of targetable vulnerabilities in an individual's tumor, multiple targets could be exploited with the use of specific therapeutic agents, thus possibly giving the patient viable therapeutic alternatives. Experimental Design In this exploratory study, we used next-generation sequencing technologies (NGS) including whole genome sequencing (WGS), and where feasible, whole transcriptome sequencing (WTS) to identify genomic events and associated expression changes in advanced cancer patients. Results WGS on paired tumor and normal samples from nine advanced cancer patients and WTS on six of these patients' tumors was completed. One patient's treatment was based on targets and pathways identified by NGS and the patient had a short-lived PET/CT response with a significant reduction in his tumor-related pain. To design treatment plans based on information garnered from NGS, several challenges were encountered: NGS reporting delays, communication of results to out-of-state participants and their treating oncologists, and chain of custody handling for fresh biopsy samples for Clinical Laboratory Improvement Amendments (CLIA) target validation. Conclusion While the initial effort was a slower process than anticipated due to a variety of issues, we demonstrate the feasibility of using NGS in advanced cancer patients so that treatments for patients with progressing tumors may be improved.

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer. Mol Cancer Ther; 12(1); 104–16. ©2012 AACR.

Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P

Background: The promise of precision medicine is vast and efforts are needed to increase utilization of precision oncology tests. Studies have suggested that mutations in cancer predisposition are more common than previously thought. Herein, we investigated germline alterations in cancer patients undergoing tumor profiling to identify the potential genetic predisposing factors as well as therapeutically relevant germline variants. Methods: Comprehensive genomic profiling was performed on tumor samples from 67 patients from 24 cancer types with ages ranging 23-77 years. We performed targeted exome sequencing of 562 genes in tumor and paired normal DNA which included 116 genes that have been associated with cancer predisposition syndromes. In addition to identifying the somatic changes, we characterized germline mutations in select 116 genes using public databases (ClinVar, Ensembl). Results: Pathogenic germline mutations were found in 17.91% (12/67) of cases, which included 10 unique variants in 8 genes, the majority of which included DNA repair genes (ATM, BRCA2, ERCC4, NBN, PSM2, MUTYH, XPC and AR). Likely pathogenic mutations were found in 23% (16/67) of the cases and 12% (8/67) were predicted pathogenic by SIFT/PolyPhen. In select cases we found existence of a 2nd hit which may have an impact on certain targeted therapeutics such as PARP inhibitors. All of the cases profiled carried either benign or variants of unknown significance (VUS) mutations. Germline findings in cancer susceptibility genes that were concordant with the individual's cancer type included ATM (R2227C) in HER2+ breast cancer patient; BRCA2 missense mutation (K944X) in peritoneal cancer and HER2+ breast cancer; BRCA2 (W1692MfsX3, I2588YfsX60) in patients with pancreatic and breast cancer respectively and PMS2(L729QfsX6) in colorectal cancer (CRC). Germline pathogenic findings in cancer susceptibility genes that were dis-concordant with the individual's cancer type included ERCC4 (R799W) in endometrial cancer; XPC (P334H) in CRC; MUTYH (G393D) in esophageal and CRC; NBN (K219NfsX16) in HER2+ breast cancer and AR (A646D) mutation in CRC. Interestingly, FLT4 (R1324L) mutation which is associated with decreased response and toxicity to sunitinib in renal cell cancer was found across several tumor types including lung adenocarcinoma (n = 3), pancreatic cancer (n = 3), HER2+ breast cancer (n = 2), CRC (n = 2) and n = 1 in gallbladder carcinoma, cholangiocarcinoma and endometrial adenocarcinoma. The comparison of somatic to germline mutation will be presented. Conclusion: Germline data analysis of a small cohort of advanced cancer patients revealed that unselected cancer patients may benefit from germline evaluation which may influence the evaluation and care of the patient or the patient's family members. Even though majority of germline mutations were VUS, several mutations were identified which may be amenable to targeted therapeutic strategies. Citation Format: Subha Krishnan, Gargi D. Basu, Laura Gonzalez-Malerva, Thomas Royce, Tracey White, Janine R. Lobello, Amy King, Kevin Lau, Ahmet Kurdoglu, Matthew Halbert, Jeff Kiefer, Irene Cherni, Jessica Aldrich, Tyler Izatt, Megan Russell, Sara Nasser, John D. Carpten, David W. Craig, Jeffrey Trent, Michael J. Demeure. Germline findings in targeted tumor sequencing using matched normal DNA. [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 4493.

671 Background: The gene encoding the AT-rich interacting domain (ARID1A) is a tumor suppressor and a subunit of the Switch/Sucrose Non fermentable (SWI/SNF) chromatin remodeling complex that regulates gene expression. Additionally, ARID1A functions to maintain genomic integrity by interacting with ATR. Mutations in ARID1A have been observed in a number of cancers. Inactivation of ARID1A has been reported to lead to increased sensitivity to inhibitors of PARP and PI3K/AKT pathway. We evaluated ARID1A alterations in gastrointestinal (GI) cancers to identify new therapeutic options. Methods: Comprehensive genomic profiling was performed on 55 GI cancer samples with 28 metastatic and 27 primary samples. Targeted sequencing was performed on 562 cancer associated genes in paired tumor and blood DNA samples. Results: ARID1A alterations were noted in 20% (11/55) of GI cancers. Alterations included frameshift and nonsense mutations, INDELs and structural breakpoints which are predicted to cause loss of ARID1A function. Tumor tissues harboring ARID1A alterations included 2/8 gastro-esophageal cancers, 2/2 duodenal cancers, 3/29 colorectal (CRC) cancers, 2/2 small bowel cancers, and 2/7 bile duct cancers. Mutations in the C-terminal half of ARID1A, known to interact with ATR and mediate its recruitment to double strand breakpoints was present in 2/11 samples. No pathogenic changes in the DNA repair genes (BRCA1/2, CHEK1/2, ATM, ATR or PALB2) or MMR genes were present in the tumors harboring ARID1A alterations. Interestingly, ARID1A mutations frequently coexisted with alterations leading to activation of the PI3K pathway. We found 45% (5/11) samples with mutated ARID1A had pathogenic alterations in PIK3CA, AKT, NF1, STK11, TSC1 and TSC2 genes which may increase sensitivity to PI3K pathway inhibitors. Conclusions: Targeted sequencing demonstrated that ARID1A is a frequent event in GI cancer. Our results suggest there may be a correlation between samples with ARID1A alteration and PI3K/AKT pathway activation which may lead to therapeutic opportunities with PI3K pathway inhibitors. The presence of ARID1A mutations especially those in the C-terminal half may predict clinical utility of PARP inhibitors in GI cancer.

Whole genome sequencing has rapidly emerged as the preferred method for unbiased genomic interrogation. Leveraging this technology, we have developed a precision medicine clinical research protocol for pediatric, adolescent, and young adult cancer patients. The primary objective of this study is to arrive at genome informed treatment recommendations for individual patients suffering from aggressive and/or worsening disease in a clinically relevant time frame. The second objective is to survey the genomic landscape of newly diagnosed and recurrent/refractory patients. To accomplish this, we have employed an interdisciplinary workflow including a customized bioinformatic analysis pipeline, contextual biological interpretation of the data, and case reviews during weekly molecular tumor board meetings. Here we report the results of having sequenced over 50 patients diagnosed with both common and rare malignancies. Factors affecting the successful identification of therapeutically actionable targets and subsequent intervention are varied. The most consistent findings are the increased mutation burden in patients who have received chemotherapy and/or radiation prior to tissue collection. Lastly, we highlight the realistic opportunities, challenges, and insight gained from an ongoing single institution experience. This abstract is also presented as Poster 27. Citation Format: Troy A. McEachron, Keri B. Zabokrtsky, Aaron F. Sassoon, Sara Nasser, Tyler Izatt, Chad P. Garner, David W. Craig, John D. Carpten, Leonard S. Sender. Precision medicine for newly diagnosed and refractory/recurrent pediatric cancer patients: Lessons learned from “N=1” studies. [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 PR06.

Family history is a significant risk factor for prostate cancer, although the molecular basis for this association is poorly understood. Linkage studies have implicated chromosome 17q21-22 as a possible location of a prostate-cancer susceptibility gene.We screened more than 200 genes in the 17q21-22 region by sequencing germline DNA from 94 unrelated patients with prostate cancer from families selected for linkage to the candidate region. We tested family members, additional case subjects, and control subjects to characterize the frequency of the identified mutations.Probands from four families were discovered to have a rare but recurrent mutation (G84E) in HOXB13 (rs138213197), a homeobox transcription factor gene that is important in prostate development. All 18 men with prostate cancer and available DNA in these four families carried the mutation. The carrier rate of the G84E mutation was increased by a factor of approximately 20 in 5083 unrelated subjects of European descent who had prostate cancer, with the mutation found in 72 subjects (1.4%), as compared with 1 in 1401 control subjects (0.1%) (P=8.5x10(-7)). The mutation was significantly more common in men with early-onset, familial prostate cancer (3.1%) than in those with late-onset, nonfamilial prostate cancer (0.6%) (P=2.0x10(-6)).The novel HOXB13 G84E variant is associated with a significantly increased risk of hereditary prostate cancer. Although the variant accounts for a small fraction of all prostate cancers, this finding has implications for prostate-cancer risk assessment and may provide new mechanistic insights into this common cancer. (Funded by the National Institutes of Health and others.).

Background Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression. Methodology/Principal Findings Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects. Conclusions/Significance This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

126 Background: Breast cancer is a molecularly diverse disease with molecular subtypes defined by ER, PR and HER2 status. Dysregulation of the cell cycle pathway through activation of cyclin D1 and/or inactivation of CDKN2A is a mechanism of tumor progression in breast cancer and drugs targeting CDK4/6 is undergoing investigation in this disease with promising preliminary results. We investigated several key genetic alterations in cell cycle regulatory proteins in advanced breast cancer patient samples to identify potential targets for cell cycle related treatment options. Methods: A comprehensive genomic profiling was performed on 37 breast cancer samples with the majority being metastatic. Testing included targeted exome sequencing of 562 genes in tumor and paired normal DNA. Results: Patients’ ages ranged from 27-66 years. Analysis of cell cycle regulatory genes revealed focal amplification of cyclin D1/D2 or CDKN2A loss in 9/37 (24%) of cases, of which, 45% were hormone receptor (HR) positive, HER2 negative, 33% were HR positive, HER2 positive and 22% were triple negative (TNBC) subtype. Loss of RB1 was analyzed since it negatively regulates response to CDK4/6 inhibitors. Loss of RB1 was noted in 4/37 (10.8%) of cases, all of which were TNBCs. Only a single TNBC with RB1 loss had concurrent CDK4 amplification and loss of CDKN2A. No evidence of CCNE1 amplification was found in any samples. Incidence of CDK4 and CDK6 amplification was found in samples with cyclin D1 amplification or CDKN2A loss. Our cohort included a single male TNBC patient who harbored cyclin D1 amplification as well as CDKN2A loss. Overall, amplification of cyclin D1, cyclin D2, CDK4 and CDK6 was found in 16%, 5.4%, 2.7% and 2.7% respectively. Loss of CDKN2A was noted in 5.4% of tumors profiled. Conclusions: Exome sequencing identified subsets of patients with mutated cell cycle associated genes that may benefit from CDK 4/6 inhibitors. Our data suggests loss of RB1 is more frequent in TNBC subtype which may confer resistance to CDK4/6 inhibitors. Our analysis suggests that in addition to HR positive, HER2 negative patients, a subset of HR positive, HER2 positive patients could be sensitive to CDK4/6 inhibitors due to activation of the cell cycle pathway as evidenced by mutations in key genes.

Background Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers. Methods We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations. Results We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition. Conclusions Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

463 Background: The genomic assessment of cancer has been revolutionized by next-generation sequencing and is increasingly being applied in the clinic to guide therapeutic decision-making. Time to reporting of results, specimen quantity, and analyte quality have constrained initial clinical applications to gene panels and whole exome based strategies. Methods: Patients underwent surgical resection, excisional or core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing and RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients with advanced, treatment-refractory cancers of whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in the CLIA enabled phase. Upon availability of the initial report, identified targets of putative therapeutic relevance were then prioritized by the CGB in 22/27 patients (81%) for subsequent CLIA validation. Eleven patients have been treated with genomically selected therapy with partial response in 3/10 assessed patients. A testicular cancer patient had aberrations in two testes specific genes, a copy number gain in TSSK6 and a novel gene fusion between thyroid hormone receptor associated protein 3 (THRAP3) and Tektin 2 (TEKT2). Additionally, a case of papillary renal cell carcinoma had an amplification of YAP1 and a previously unreported P287T mutation in CCND1, suggesting potential benefit with a CDK4/6 inhibitor. Treatment is ongoing and results will be reported. Conclusions: Integrated whole genome analysis in a CLIA setting is feasible. Integration of SNV, copy number and transcriptional data may allow for selection of putative driver genes to enhance targeted therapy decisions. Barriers for future broader implementation include the need for reduced time from biopsy to report and availability of therapies.

Triple negative breast cancer (TNBC), is characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). TNBC comprises about 15 of all breast cancer diagnoses, but ∼25% of deaths. TNBC has a more aggressive clinical course and long-term disease-free survival is poor. As is well known, tumors that develop in women who carry deleterious germline BRCA mutations typically have tumors characteristic of the basal-like TNBC phenotype associated with defects in double stand break repair and homologous recombination. These observations have led to the development of therapeutic strategies aimed at these mechanisms, such as PARP inhibitors, which are in mature stages of drug development for the treatment of a subset of patients diagnosed with mTNBC. Iniparib, although not believed to be a true PARP inhibitor, is believed to act through a similar mechanism of action. We have completed a study of whole genome and transcriptome sequencing of 14 patients with metastatic triple negative breast cancer (mTNBC). One patient who was sequenced had a complete response to a combination including gemcitabine, carboplatin, and iniparib prior to sequencing. Sequencing analysis revealed a collection of somatic mutations and alterations in genes associated with DNA repair and homologous recombination, including DCLRE1C, MEI1, TP53, DDB1, RIF1, ZBTB40, BRIP1, and BRCA2. Furthermore, RNA-seq expression analysis demonstrated significant underexpression of BRCA1. Thus, these alterations may serve as markers that can be predictive of responders from non-responders of treatment regimens including iniparib or possibly PARP inhibitors. To begin to identify a genomic profile indicative of PARP response, tumors and blood of four additional mTNBC patient samples were collected and subjected to whole exome and RNA sequencing. We have also obtained the mutational data of TNBC tumors sequenced by the Cancer Genome Atlas. The genetic profiles of these tumors will be compared to the previously sequence mTNBC sample that responded to iniparib to determine the frequency of these specific events in mTNBC. Through this work we hope to identify a genetic profile that can serve as a possible predictor of response to iniparib or true PARP inhibitors. Citation Format: Shukmei Wong, Joyce A. O'Shaughnessy, Patricia LoRusso, Jessica Aldrich, Ahmet Kurdoglu, Alexis Christoforides, Jennifer Dinh, Tyler Izatt, Shripad Sinari, Angela Baker, Tracy M. Moses, Bodour Salhia, Spyro Mousses, Jeffrey Kiefer, Jeffrey M. Trent, Daniel Von Hoff, Geoffrey Shapiro, Winnie Liang, David W. Craig, John D. Carpten. Whole exome and RNA sequencing reveals genetic alterations in DNA damage and homologous recombination pathways in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2013-2023

Purpose: Pancreatic adenocarcinoma is among the most lethal forms of all malignancies. While significant research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult. The majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to assess global somatic events across multiple pancreatic tumors by using next-generation sequencing technologies (NGS). Experimental Design: We performed whole genome sequencing (WGS) on paired tumor and normal samples from 3 pancreatic adenocarcinoma patients. Additionally, we performed whole transcriptome sequencing (WTS) on 2 of these patients’ tumors for which tumor RNA was available. Results: We generated on average 132 billion mappable bases across all patients using WGS, and identified 140 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. Data from WGS and WTS were integrated and analyzed to identify the most heavily affected pancreatic cancer pathways across all patients. The KRAS signaling pathway was identified as the most significant map. Unbiased global pathway analysis was also performed to identify processes that may be impacted. Consistent with the previous analysis, the most significant pathway from this analysis is also KRAS signaling. This integrated analysis specifically identifies heavily affected pathways in pancreatic cancer and pinpoints genes and processes that may be considered as targets for more effective therapies. Conclusions: While sequencing of additional patients is necessary to strengthen selection of key pathways, we demonstrate the utility of using NGS to understand the processes driving pancreatic adenocarcinoma so that standardized treatments for patients may be improved. 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 5104. doi:1538-7445.AM2012-5104

Colorectal carcinoma is one of the most common cancers in the western world, being the third most common cause of cancer-related morbidity and mortality. The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy, especially with a Kras mutation is poor with median survival of less than one year. The purpose of this study was to identify therapeutically actionable somatic events in mCRC patient samples, so as to obtain select targeted treatment strategies for individual patients. Fresh frozen needle biopsies of liver metastases were collected from three mCRC patients along with whole blood for the extraction of constitutional DNA. All patients had greater than 2 prior regimens and had known K-ras mutations. Whole genome sequencing was performed using Illumina paired-end chemistry on HiSeq2000 sequencing system, which yielded coverage of greater than 30X for both the normal and tumor samples. Somatic genomic alterations (point mutations and indels, copy number alterations, translocations and rearrangements) were analyzed using our custom pipeline for paired analysis. We found that all 3 tumor samples had KRAS mutations, while 2 samples had mutations in the APC gene and the PIK3CA gene. Other genes with therapeutic implications in single tumor samples were INPPL1, INPP4B and SMAD4. No EGFR amplifications were detected in our sample set. Pathway analysis revealed K-RAS regulation and apoptosis and survival pathways in these tumor samples. Analysis of paired-end data from these three tumors did not reveal the presence of translocations at the recently discovered TCF7L2 locus (Bass et al, Nature Genetics, 2011). The concomitant mutations in MAPK (KRAS) and PI3K (PIK3CA, INPPL1, INPP4B) pathways suggest that combination therapies might be required for effective treatment of these tumors. By harnessing the power of Next-Gen Sequencing, our study has revealed a series of therapeutically actionable events in mCRC that might provide insights into treating these difficult to treat advanced tumors. 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 5072. doi:1538-7445.AM2012-5072

Considered independently, lung cancer in never-smokers would rank among the ten most common causes of cancer mortality. Driver pathways and potential therapeutics must be identified for this clinically relevant subpopulation. We hypothesize that novel mutations and pathways identified by whole genome sequencing (WGS), whole transcriptome sequencing (WTS), and methylation profiling drive tumorigenesis in adenocarcinomas (AC) of never-smoker (NS) patients, and represent potential therapeutic targets. We have completed WGS, WTS, and methylation profiling on two lung ACs from female, never-smokers, one early-stage and one stage IV, and one female smoker (S) patient with early-stage lung AC. Approximately 100 short nucleotide variants (SNV) were discerned from the early-stage and stage IV NS lung ACs. Of interest, these NS patients lacked alterations in common genes associated with lung cancer such as EGFR and KRAS. In comparison, the lung AC from a smoker contained 78 SNVs, including a well-characterized KRAS mutation. Mutations in MAGEC1, a tumor marker in melanoma, were observed in common between the early-stage NS tumor and the smoker lung AC. The early-stage NS tumor contained a mutation in PIK3C3 and CSNK1E, a casein kinase involved Wnt signaling. The stage IV NS tumor demonstrated mutations in tumor suppressor genes such as p53, LATS2, and ATM. With relatively few mutations discerned from NS lung ACs, WTS and methylation profiling were performed. WTS showed 1,083 genes differentially regulated in the early-stage NS and Ingenuity Pathway Analysis implicated G-protein coupled receptor signaling. 2,000 genes were differentially expressed in the stage IV NS lung AC with sonic hedgehog implicated as a significantly regulated pathway. Besides gene expression, methylation profiling revealed ∼3,100 differentially methylated genes in the early-stage NS lung AC compared to normal lung DNA, with 65% hypo-methylated, including genes involved in lung carcinogenesis such as RET and BCL2. In the stage IV NS lung AC, ∼35,000 genes were differentially methylated compared to normal lung DNA (10X the early-stage NS) with ∼62% hypo-methylated. Thus, while an early-stage smoker lung AC displayed a classic driver mutation in KRAS, lung ACs from never-smokers had relatively quiet genomes. Despite few mutations, thousands of genes were differentially regulated at the mRNA level or by methylation. We continue to validate the genes implicated in NS lung AC by mutation, differential expression, and differential methylation, as well as integrating the data across platforms to discern significantly regulated pathways. We believe that the genes and pathways implicated by WGS, WTS, and methylation profiling will lead to a better understanding of NS lung AC and identify possible therapeutic interventions. 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 5068. doi:1538-7445.AM2012-5068