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Integrated sequencing strategies have provided a broader understanding of the genomic landscape and molecular classifications of multiple cancer types and have identified various therapeutic opportunities across cancer subsets. Despite pivotal advances in the characterization of genomic alterations in glioblastoma, targeted agents have shown minimal efficacy in clinical trials to date, and patient survival remains poor. In this review, we highlight potential reasons why targeting single alterations has yielded limited clinical efficacy in glioblastoma, focusing on issues of tumor heterogeneity and pharmacokinetic failure. We outline strategies to address these challenges in applying precision medicine to glioblastoma and the rationale for applying targeted combination therapy approaches that match genomic alterations with compounds accessible to the central nervous system.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor genePTPRJin 19% of cases. NoBRAFmutations were detected, but activatingRASmutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes.MDM2amplifications (24%) andTP53mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen inBRAFwild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.AUTHOR SUMMARYMelanoma, an aggressive cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, which most often occurs in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Clinical features of canine melanoma resemble those of human melanoma, particularly the less common sun-shielded human subtypes. However, whereas the genomic basis of diverse human melanoma subtypes is well understood, canine melanoma genomics remain poorly defined. Similarly, although diverse new treatments for human melanoma based on a biologic disease understanding have recently shown dramatic improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain universally poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving the lives of pet dogs while also helping to establish canine melanoma as a comparative model system for informing human melanoma biology and treatment. In order to better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such asRAS,MDM2, andTP53as well mutational patterns that share important similarities with human melanoma subtypes. We have also found a new putative cancer gene,PTPRJ, frequently mutated in canine melanoma. These data will guide additional biologic and therapeutic studies in canine melanoma while framing the utility of comparative studies of canine and human cancers more broadly.

A retrospective analysis of 130 patients was conducted in a Phase I oncology clinic to assess the effect of QTc formula selection on clinical trial eligibility. QTc values were calculated from screening electrocardiograms using 7 formulae (Bazett, Fridericia, Framingham, Hodges, Mayeda, Van de Water and Wohlfart). QTc values > 470 ms for females and > 450 ms for males were used to define prolongation. Concomitant medication potential for QTc prolongation was determined using a public database (AzCert). Ineligibility rates ranged from 3.1 % to 17.7 % (Framingham: 3.1 %, Van de Water: 3.1 %, Hodges: 3.1 %, Wohlfart: 3.1 %, Fridericia: 3.9 %, Bazett: 10.8 % and Mayeda: 17.7 %). A consistent ineligibility rate was achieved by using formulae-specific thresholds. Fifty one percent of patients were taking concomitant medications with QTc prolongation potential. The proportion of concomitant medications with the potential to prolong QTc was 11.57 % (96 of 830). Uniform criteria and guidelines for selection of QTc formulae need to be developed. Formulae-specific QTc thresholds also need to be specified.

Large-scale multiplexed identification of somatic alterations in cancer has become feasible with next generation sequencing (NGS). However, calibration of NGS somatic analysis tools has been hampered by a lack of tumor/normal reference standards. We thus performed paired PCR-free whole genome sequencing of a matched metastatic melanoma cell line (COLO829) and normal across three lineages and across separate institutions, with independent library preparations, sequencing, and analysis. We generated mean mapped coverages of 99X for COLO829 and 103X for the paired normal across three institutions. Results were combined with previously generated data allowing for comparison to a fourth lineage on earlier NGS technology. Aggregate variant detection led to the identification of consensus variants, including key events that represent hallmark mutation types including amplified BRAF V600E, a CDK2NA small deletion, a 12 kb PTEN deletion, and a dinucleotide TERT promoter substitution. Overall, common events include >35,000 point mutations, 446 small insertion/deletions, and >6,000 genes affected by copy number changes. We present this reference to the community as an initial standard for enabling quantitative evaluation of somatic mutation pipelines across institutions.

The longitudinal evolution of a myeloma genome from diagnosis to plasma cell leukemia has not previously been reported. We used whole-genome sequencing (WGS) on 4 purified tumor samples and patient germline DNA drawn over a 5-year period in a t(4;14) multiple myeloma patient. Tumor samples were acquired at diagnosis, first relapse, second relapse, and end-stage secondary plasma cell leukemia (sPCL). In addition to the t(4;14), all tumor time points also shared 10 common single-nucleotide variants (SNVs) on WGS comprising shared initiating events. Interestingly, we observed genomic sequence variants that waxed and waned with time in progressive tumors, suggesting the presence of multiple independent, yet related, clones at diagnosis that rose and fell in dominance. Five newly acquired SNVs, including truncating mutations of RB1 and ZKSCAN3, were observed only in the final sPCL sample suggesting leukemic transformation events. This longitudinal WGS characterization of the natural history of a high-risk myeloma patient demonstrated tumor heterogeneity at diagnosis with shifting dominance of tumor clones over time and has also identified potential mutations contributing to myelomagenesis as well as transformation from myeloma to overt extramedullary disease such as sPCL.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species., Author summary Melanoma, a cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, most often occurring in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Canine melanoma clinically resembles human melanoma, particularly sun-shielded subtypes. However, canine melanoma genomics remain poorly defined. Similarly, although new treatments for human melanoma based on genomic understanding have shown improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving lives of pet dogs and helping to define canine melanoma’s role in comparative studies that also inform human melanoma understanding. To better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such as RAS, MDM2, and TP53 alongside mutational patterns sharing important similarities with human melanoma subtypes. We have also found a new putative cancer gene, PTPRJ, frequently mutated in canine melanoma. These data will guide biologic and therapeutic studies in canine melanoma while broadly framing the utility of comparative studies of canine and human cancers.

Contains fulltext : 80661.pdf (Publisher’s version ) (Closed access) Age-related hearing impairment (ARHI), or presbycusis, is the most prevalent sensory impairment in the elderly. ARHI is a complex disease caused by an interaction between environmental and genetic factors. Here we describe the results of the first whole genome association study for ARHI. The study was performed using 846 cases and 846 controls selected from 3434 individuals collected by eight centers in six European countries. DNA pools for cases and controls were allelotyped on the Affymetrix 500K GeneChip for each center separately. The 252 top-ranked single nucleotide polymorphisms (SNPs) identified in a non-Finnish European sample group (1332 samples) and the 177 top-ranked SNPs from a Finnish sample group (360 samples) were confirmed using individual genotyping. Subsequently, the 23 most interesting SNPs were individually genotyped in an independent European replication group (138 samples). This resulted in the identification of a highly significant and replicated SNP located in GRM7, the gene encoding metabotropic glutamate receptor type 7. Also in the Finnish sample group, two GRM7 SNPs were significant, albeit in a different region of the gene. As the Finnish are genetically distinct from the rest of the European population, this may be due to allelic heterogeneity. We performed histochemical studies in human and mouse and showed that mGluR7 is expressed in hair cells and in spiral ganglion cells of the inner ear. Together these data indicate that common alleles of GRM7 contribute to an individual's risk of developing ARHI, possibly through a mechanism of altered susceptibility to glutamate excitotoxicity.

Individuals with type 2 diabetes have an increased risk of developing non-alcoholic fatty liver disease (NAFLD), and NAFLD patients are also at greater risk for developing type 2 diabetes. Although the relationship between type 2 diabetes and NAFLD is highly interconnected, the pathogenic mechanisms linking the two diseases are poorly understood. The goal of this study was to identify genetic determinants of hepatic lipid accumulation through association analysis using histological phenotypes in obese individuals. Using the Illumina HumanOmniExpress BeadChip assay, we genotyped 2,300 individuals on whom liver biopsy data were available. We analyzed total bilirubin levels, which are linked to fatty liver in severe obesity, and observed the strongest evidence for association with rs4148325 in UGT1A (P

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare and understudied cancer with a dismal prognosis. SCCOHT's infrequency has hindered empirical study of its biology and clinical management. However, we and others have recently identified inactivating mutations in the SWI/SNF chromatin remodeling gene SMARCA4 with concomitant loss of SMARCA4 protein in the majority of SCCOHT tumors.(1-4) Here we summarize these findings and report SMARCA4 status by targeted sequencing and/or immunohistochemistry (IHC) in an additional 12 SCCOHT tumors, 3 matched germlines, and the cell line SCCOHT-1. We also report the identification of a homozygous inactivating mutation in the gene SMARCB1 in one SCCOHT tumor with wild-type SMARCA4, suggesting that SMARCB1 inactivation may also play a role in the pathogenesis of SCCOHT. To date, SMARCA4 mutations and protein loss have been reported in the majority of 69 SCCOHT cases (including 2 cell lines). These data firmly establish SMARCA4 as a tumor suppressor whose loss promotes the development of SCCOHT, setting the stage for rapid advancement in the biological understanding, diagnosis, and treatment of this rare tumor type.

The discovery and reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue--such as the inability to biopsy or test tissue from the central nervous system directly. RNAs originating from hard to access tissues, such as neurons within the brain and spinal cord, have the potential to get to the periphery where they can be detected non-invasively. The formation and extracellular release of microvesicles and RNA binding proteins have been found to carry RNA from cells of the central nervous system to the periphery and protect the RNA from degradation. Extracellular miRNAs detectable in peripheral circulation can provide information about cellular changes associated with human health and disease. In order to associate miRNA signals present in cell-free peripheral biofluids with neurodegenerative disease status of patients with Alzheimer's and Parkinson's diseases, we assessed the miRNA content in cerebrospinal fluid and serum from postmortem subjects with full neuropathology evaluations. We profiled the miRNA content from 69 patients with Alzheimer's disease, 67 with Parkinson's disease and 78 neurologically normal controls using next generation small RNA sequencing (NGS). We report the average abundance of each detected miRNA in cerebrospinal fluid and in serum and describe 13 novel miRNAs that were identified. We correlated changes in miRNA expression with aspects of disease severity such as Braak stage, dementia status, plaque and tangle densities, and the presence and severity of Lewy body pathology. Many of the differentially expressed miRNAs detected in peripheral cell-free cerebrospinal fluid and serum were previously reported in the literature to be deregulated in brain tissue from patients with neurodegenerative disease. These data indicate that extracellular miRNAs detectable in the cerebrospinal fluid and serum are reflective of cell-based changes in pathology and can be used to assess disease progression and therapeutic efficacy.

The development of accurate clinical biomarkers has been challenging in part due to the diversity between patients and diseases. One approach to account for the diversity is to use multiple markers to classify patients, based on the concept that each individual marker contributes information from its respective subclass of patients. Here we present a new strategy for developing biomarker panels that accounts for completely distinct patient subclasses. Marker State Space (MSS) defines ‘‘marker states’’ based on all possible patterns of high and low values among a panel of markers. Each marker state is defined as either a case state or a control state, and a sample is classified as case or control based on the state it occupies. MSS was used to define multi-marker panels that were robust in cross validation and training-set/test-set analyses and that yielded similar classification accuracy to several other classification algorithms. A three-marker panel for discriminating pancreatic cancer patients from control subjects revealed subclasses of patients based on distinct marker states. MSS provides a straightforward approach for modeling highly divergent subclasses of patients, which may be adaptable for diverse applications.

In the U.S., the incidence rates of breast cancer (BC) among Caucasian (CA) women are lower than those of African American (AA), however, AA women have a significantly higher mortality rate. AA women often present at a younger age, with later stage and higher grade tumors. In fact, AA women are 3x more likely to be diagnosed with the more aggressive molecular subtype, triple-negative breast cancer (TNBC), than CA women. Socioeconomic and lifestyle factors may be contributory; however, it is imperative that we investigate the underlying molecular biology that may be the cause of health disparities between AA and CA with TNBC. In this study, gene expression profiling, using the Almac BC DSA Research Tool, was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage (Node 0) TNBC. Unsupervised hierarchical clustering revealed a pattern of differential gene expression in the AA cohort compared to CA. Using the TNBC type: A Subtyping Tool for TNBC, we found a distinct distribution pattern of TNBC molecular subtypes in the AA cohort, which was very different than the CA cohort; Basal-like (14%), Immunomodulatory (43%) and Mesenchymal (43%). Gene expression analyses, comparing the AA and CA cohort (fold change > 2.0, p-value Citation Format: Julie E. Getz, Davyd B. Teoh, Sara Nasser, Christophe R. Legendre, Waibhav Tembe, Venkata Yellapantula, Mary Ellen Ahearn, Carmen R. Gomez, Merce Jorda, Shuk Mei Wong, Mark D. Pegram, John D. Carpten, Lisa L. Baumbach-Reardon. High caveolin-1 expression in a cohort of African American women with triple-negative breast cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C54.

Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.

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 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.

Breast cancer detection has not resulted in the expected decrease in mortality suggesting that current screening strategies are not finding metastasis-prone lesions before metastasis occurs and instead are detecting many lesions that will not progress to metastatic disease. Although several improvements in the management of breast cancer have been made, an estimated 90% of breast cancer related deaths are due to the development of distant metastasis to organs such as lung, bone, liver, and brain. Metastatic breast cancer affects 10-15% of breast cancer patients, is often refractory to therapy and associated with poor outcomes. However, a blood test indicating a high-risk population at the time of breast cancer diagnosis might shift the current strategy and improve outcomes. Efforts for identifying predictive markers of breast cancer metastasis have been impeded by a lack of data defining the genetic and epigenetic determinants of the disease. Numerous studies suggest that DNA methylation could be a useful biomarker for improving the clinical management. Recently circulating tumor (ct)DNA has attracted attention for clinical use in the context of risk prediction, prognostication and prediction in human cancer. Various types of DNA alterations have been reported in ctDNA but methylated DNAs are chemically stable and are among the most sensitive and specific ctDNA cancer markers. The objective of our study was to identify and validate a blood-based DNA methylation biomarker for breast cancer patients at high risk of distant metastasis. Using the HumanMethylation27K bead array, we recently identified RUNX3 hypermethylation involving 3 CpG probes in 32 breast brain metastasis tumor tissue compared to a series of 50 unmatched early stage breast cancer samples. We calculated the area under the curve (AUC) to exceed 0.8, with a higher AUC achieved in Her2-positive samples. In an independent, first of its kind analysis, we performed whole genome bisulfite sequencing (WGBS) of ctDNA from plasma samples representing three pools of 40 women with distant metastatic breast cancer to various organs, 40 women with metastasis-free breast cancer, and 40 non-breast cancer controls. In this unbiased analysis, these data demonstrated RUNX3 hypermethylation in the plasma of patients with metastatic breast cancer in the same region corresponding to the CpG probes of the methylation array. We are now performing bisulfite pyrosequencing of each individual plasma sample, and in an additional cohort of 50 archival primary/brain metastasis tumor pairs with matching serum and clinical outcomes data. To determine regional specific differences we have also selected two additional regions of RUNX3 not differentially methylated for validation by bisulfite pyrosequencing. Determining the extent to which RUNX3 hypermethylation occur in the context of brain metastasis versus other distant sites are ongoing studies. Together these studies point towards RUNX3-regional specific methylation as a potential blood-based biomarker of breast cancer metastasis. Such a biomarker will help establish surveillance methods and eventually preventative regimens that will increase the likelihood of long-term progression-free survival for breast cancer patients at risk of distant relapse. Citation Format: Bodour Salhia, Gerald C Gooden, Kyle N Johnson, Waibhav Tembe, Garrick Wallstrom, Winnie Liang, Manmeet Ahluwalia, Steven N Kalkanis, Tom Mikkelsen, Steven A Toms. RUNX3 hypermethylation in circulating tumor DNA is a biomarker of breast cancer distant metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-09.

Oncogenic fusion genes underlie the mechanism of several common cancers and also constitute or encode important diagnostic and therapeutic targets. Studies using next-generation sequencing technologies, particularly RNA sequencing (RNA-seq), have revealed a growing number of recurrent fusions in a variety of cancers. As such, establishing analytic parameters, including the limit of detection, sensitivity, and specificity of detecting fusions in tumor RNA specimens is important. We have developed reference materials useful in assessing the ability to detect fusion events in RNA or DNA samples. These consist of a set of nine synthetic poly-adenylated RNA transcripts that correspond to common cancer fusion gene sequences. These synthetic fusion RNAs can be spiked at known concentrations into total RNA in the first stage of the mRNA sample prep and used as in-line controls to evaluate the sensitivity and specificity of the detection of these mutations. As a demonstration of the utility of these controls, we performed a series of spike-in experiments to investigate performance characteristics of three commonly used fusion-detection software algorithms. Our results show all three algorithms to be capable of detecting fusions in a dose-dependent manner. The results also show differences in performance among the algorithms for detecting specific fusions. Citation Format: Winnie Liang, Stephanie JK Pond, Waibhav D. Tembe, Han-Yu Chuang, Christophe Legendre, Nancy Kim, Valerie Montel, Shukmei Wong, Timothy K. McDaniel, David Craig, John Carpten. mRNA spike-in control materials for cancer fusion gene detection assays. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3571. doi:10.1158/1538-7445.AM2014-3571

Despite remarkable therapeutic advances in malignant melanoma, an incomplete understanding of melanoma's extraordinary genetic diversity, complex disease course and ability to resist treatment continues to impede rational, curative clinical intervention. This is particularly true for the understudied “dark matter” of the melanoma genome, i.e. cases that lack mutations in the three most commonly-mutated melanoma genes (BRAF, NRAS, NF1) and that display extensive mutational heterogeneity. Clearly a pressing need exists for transformative approaches to melanoma medicine. We have therefore broadened our traditional focus on melanoma in human patients to include canine melanoma as a model of human disease. Given canine melanoma's clinicopathological resemblance to human non-cutaneous triple-negative subtypes, study of this disease in each species may inform the other. Further, the accelerated clinical course of canine melanoma and flexibility in veterinary clinical trial design may allow for evaluation of novel treatment designs that would not be possible in human cancer trials. Thus, we have undertaken a first-in-dog genomic characterization study of canine melanomas from a cohort of 37 dogs to further explore the landscape of this comparative model. This analysis combines comprehensive genomics (whole genome, whole genome long-insert, RNA sequencing, and array comparative genomic hybridization) in 7 matched canine melanoma/normal tissues with targeted analysis in an additional 27 tumors and 3 cell lines. These canine tumors display striking patterns of mutation and subtype heterogeneity resembling that of their non-cutaneous human counterparts with the majority being BRAF, NRAS, and NF1 wild type and lacking recurrent single nucleotide variants. Despite heterogeneity at the single nucleotide level, shared patterns of structural variation and gene expression exist and these patterns suggest core pathways that may define therapeutic vulnerabilities in these disease subtypes. These data provide the genomic framework for further clinical pursuit of melanoma in the canine model as a paradigm for drug development. Citation Format: William P. Hendricks, Victoria Zismann, Berry Button, Christophe Legendre, Waibhav Tembe, Holly Yin, Michael Bittner, Donald Berry, Matthew Huentelman, Chand Khanna, Patricia LoRusso, Matthew Breen, Aleksandar Sekulic, Jeffrey Trent. The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2014-3417

The brain is a common site of metastatic disease in patients with breast cancer, which has few therapeutic options and dismal outcomes. The purpose of our study was to identify common and rare events that underlie breast cancer brain metastasis. We performed deep genomic profiling, which integrated gene copy number, gene expression and DNA methylation datasets on a collection of breast brain metastases. We identified frequent large chromosomal gains in 1q, 5p, 8q, 11q, and 20q and frequent broad-level deletions involving 8p, 17p, 21p and Xq. Frequently amplified and overexpressed genes included ATAD2, BRAF, DERL1, DNMTRB and NEK2A. The ATM, CRYAB and HSPB2 genes were commonly deleted and underexpressed. Knowledge mining revealed enrichment in cell cycle and G2/M transition pathways, which contained AURKA, AURKB and FOXM1. Using the PAM50 breast cancer intrinsic classifier, Luminal B, Her2+/ER negative, and basal-like tumors were identified as the most commonly represented breast cancer subtypes in our brain metastasis cohort. While overall methylation levels were increased in breast cancer brain metastasis, basal-like brain metastases were associated with significantly lower levels of methylation. Integrating DNA methylation data with gene expression revealed defects in cell migration and adhesion due to hypermethylation and downregulation of PENK, EDN3, and ITGAM. Hypomethylation and upregulation of KRT8 likely affects adhesion and permeability. Genomic and epigenomic profiling of breast brain metastasis has provided insight into the somatic events underlying this disease, which have potential in forming the basis of future therapeutic strategies.

Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq appeared more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.

Adrenocortical carcinoma (ACC) is a lethal tumor of the adrenal cortex with limited treatment options. The only possibility for cure is complete surgical resection, but 50-70% of patients present with metastatic disease, precluding curative surgery. The outcome for patients with ACC has remained unchanged in the past 25 years, with the overall 5-year survival of patients undergoing surgical resection being ∼40%. Patients that present with advanced disease fare considerably worse with 5-year survival rates of 10-20%. The standard chemotherapy regimen of mitotane (the only approved agent), etoposide, doxorubicin, and cisplatin, has a response rate of 23.2%. A better understanding of pathobiology is needed to improve the treatment results for this disease. Previous genomic studies combined with expression microarray studies have implicated lack of H19 expression coupled with IGF2 over-expression, beta-catenin activation, and loss of p53 pathway function as important events in ACC, but no single alteration has been seen in all ACC. We used a combination of whole genome sequencing, exome sequencing, and RNA-sequencing on three tumor/normal pairs from patients. Using data from spectral karyotyping of the two ACC cell lines, SW-13 and NCI-H295R, we mined the sequencing data for structural rearrangements in regions of common breakpoints. We identified a novel recurrent translocation, t(4;8)(p16.2;p23.1) in all three tumors that was not present in the normal sequence. Using fluorescent in situ hybridization (FISH) we validated the translocation in the three tumors and both SW-13 and H295R. An additional five of five ACC scored positive for the translocation, as did three malignant non-ACC tumors (malignant pheochromocytoma, pancreatic neuroendocrine tumor, and carcinoid) suggesting that this finding has implications beyond ACC. The recurrent translocation t(4;8) (p16.2; p23.1) involves two genes of unknown function, BC042823 (uc003gho.2, IMAGE:5275587) on chromosome 4 and BC030294 (ucoo3wrb.1, IMAGE:5396854) on chromosome 8. RNA- sequencing data and RT-PCR with primers designed to both derivative chromosomes revealed transcription from both derivative chromosomes. The cDNA products were sequenced by Sanger sequencing and show partial homology to a non-coding RNA, LOC100506990, at chr8:12,338,893-12,452,929. RNA-sequencing data revealed this locus is highly transcribed in normal adrenal gland but reduced in expression in the three ACCs. In contrast to other recurrent translocations previously found in other cancers, this translocation is only present in 18-30% of the tumor nuclei scored by FISH, suggesting that it is a necessary alteration in malignant progression, but underscoring the question of how it might cooperate with events in other sub-clones to initiate tumor formation and progression. Citation Format: Michael J. Demeure, Kaiti Schwartz, Aditi Bapat, Claire Linnehan, Hollie Benson, Rebecca Reiman, Lori Phillips, Christophe Legendre, Raghu Metpally, Ahmet Kurdoglu, David Craig, John Carpten, Winnie S. Liang, Waibhav Tembe, Kimberly J. Bussey. Whole-genome and RNA sequencing identify a novel recurrent translocation in adrenocortical carcinoma. [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 1781. doi:10.1158/1538-7445.AM2013-1781

Abstract 2920 Human myeloma cell lines (HMCL) provide both a discovery and validation platform to improve our understanding of the molecular pathogenesis of multiple myeloma. We have completed a project to characterize the underlying genetics of all commercially available HMCL with a primary goal of identifying appropriate model systems for findings from large scale patient studies like the multiple myeloma genomics initiative (MMGI). We first purchased all 33 commercially available HMCL from DSMZ, JCRB, ECACC, and ATCC. Subsequently each HMCL was thawed and cultured under strict parameters, which yielded cells for analysis, by Agilent 400k CGH, whole exome sequencing (Agilent 70Mb Exon+UTR), and mRNA sequencing. The combination of these three assays provides a detailed map of the genetic complexity underlying this deadly disease. For variant discovery, alignment was done using BWA followed by indel realignment, quality recalibration and duplicate removal. High quality calls were identified from the intersection of variants called by both Samtools and GATK. This identified a median of 32691 high confidence variants per sample with upper and lower quartile values of 34307.75 and 32241.25, respectively. To identify likely somatic mutations, we removed variants found in the 1000 genomes project and the NHLBI Exome Sequencing project. In addition, we removed variants present in dbSNP unless these mutations were also present in the COSMIC database. After these filtering steps a median of 702 potential mutations remained. From these lists we identified a median of 209.5 non-synonymous variants per sample and in genes which are typically expressed in the cohort, a median of 91 variants were found. Overall, these steps identified 2678 variants in 1978 genes. The primary goal was to identify appropriate models for novel findings from studies like the MMGI. For instance we identified HMCL with mutations in FAM46C and DIS3 among others. Secondarily, we focused on attempting to identify potential oncogenes and tumor suppressors through the integration of our three data types and data from published studies (Chapman et al. and Walker et al.). To identify potential oncogenes we focused on mutations that occurred at the same position in the genome or altered the same amino acid at a minimum. This identified 23 genes; including expected genes like KRAS (n=11) and, NRAS (n=7) but it also identified potentially activating mutations in IKBKB, SOX2, KDM4C, CD81, OSBP, NOTCH2, WDR92 and UBR2. To identify potential tumor suppressors we focused on genes that are typically expressed, which showed bi-allelic inactivation in two or more samples by either a homozygous deletion event, a deletion plus mutation, or two independent mutations. This identified 116 genes; including expected genes like TP53, CDKN2C, RB1, BIRC2/3, TRAF3, KDM6A, CDKN1B, FAM46C, and DIS3. Outside of the expected genes we identified recurrent inactivation in ANKRD11, ATP6AP1, ATXN1, BCL2L11, CDK8, RNF7, STS, TSPAN7, and TBL1XR1. These studies have highlighted the value in studying HMCL as most novel genes reported from recent studies were independently identified in this small cohort of samples. This is in large part because HMCL provide an unlimited DNA and RNA resource that allowed for multiple independent assays to be performed on each sample. Ultimately this study will provide the myeloma community with a detailed resource from which they can acquire appropriate model systems for their research goals from the various cell line repositories around the world. Disclosures: Keats: Tgen: Employment.

Background: Approximately 50% of all non-small cell lung carcinoma (NSCLC) cases in the United States are adenocarcinoma histology. While definitive lung cancer resection offers a chance for cure in stage I and II NSCLC, up to 57% of patients will have disease relapse or metastatic disease within 5 years. A majority of the relapsed cases will take place within 2 years of definitive surgical resection. Thus, pathologic staging alone does not stratify patients for prognosis sufficiently. Identifying patients with aggressive versus indolent tumors would have critical implications for directing treatment and designing future clinical trials. We propose using microRNA (miRNA) for discovery and validation of prognostic biomarkers for stage I and II lung adenocarcinoma. Methods: For the Discovery Cohort, we identified treatment-naïve AJCC 7th edition stage I and II lung adenocarcinoma patient samples from 26 disease-free survivors (DFS) and overall survival (OS) at least 47 months and 14 patients with DFS and overall survival less than 23 months. Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples. Differential miRNA array expression profiling between these two groups was assessed in the Discovery cohort on an 8x15 Agilent microarray printed with 886 miRNAs. Target miRNAs were selected based on minimum of 2-fold change in expression and Welch t-test p-value cut-off of 47 months and N=12 with DFS and OS47 months and N=4 DFS/OS 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 4148. doi:1538-7445.AM2012-4148

Background: Linkage studies have consistently identified the 17q21-22 chromosomal region to be important in hereditary prostate cancer, although no known causal variant has yet been identified. Three genes in this region are potential candidates based on known function as well as location in our region of interest. Mitogen-activated protein kinase kinase kinase 14 (MAP3k14) encodes a serine-threonine protein kinase that stimulates NF-κβ activity. The ARHGAP27 gene codes for a Rho-like small GTPase-activating protein and RND2 is part of the Rho GTPase family implicated in endosomal trafficking. Methods: All exons within the 17q21-22 chromosomal region were interrogated in 94 probands from hereditary prostate cancer families and linkage to the 17q21-22 region. Participants were from the University of Michigan Prostate Cancer Genetics Project and Johns Hopkins University. Enrichment was performed using the RainDance Technology following an emulsion PCR protocol. Sequencing was performed on the ABI SOLiD platform. Confirmation of mutation status among family members (both affected and unaffected) was performed using Sanger sequencing. Results: No deleterious truncating mutations were detected in the three candidate genes. Although missense variants were not observed in RND2, five missense variants were identified in the MAP3k14 and ARHGAP27 genes. Four of the five variants are novel (not described in dbSNP) and four out of the five variants are predicted to be damaging on protein function by SIFT and/or PolyPhen. Several of these variants demonstrated evidence of cosegregation within families based on sequencing of family members. Conclusions: We have identified five missense variants in MAP3k14 and ARHGAP27 in men with hereditary prostate cancer. Further research is ongoing in the laboratory to determine the frequency of these variants in a large prostate cancer case-control study. 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 1642. doi:1538-7445.AM2012-1642

Adrenocortical carcinoma (ACC) is an aggressive endocrine malignancy with a 5-year survival rate of 20–30%. There are few treatment options for patients in part because the rarity of the tumor makes its study difficult. Previous work in our lab and by others has highlighted the importance of IGF2-IGF1R signaling, beta-catenin signaling, dysregulation of p53, and the G2/M transition in ACC pathogenesis. In order to advance knowledge of ACC biology and facilitate drug target discovery, we performed whole genome sequencing on an ACC tumor and matched peripheral blood. An ACC sample was obtained during surgery on a 51 year old female who had locally recurrent disease that progressed despite chemotherapy. DNA extracted from both tumor and blood was processed independently, and libraries were paired-end sequenced on the Illumina HiSeq 2000 platform. Sequencing data was aligned to the reference genome, build hg18. Variations were predicted in the tumor and blood separately using two different SNP calling algorithms: samtools and SolSNP. After SNP prediction, results were combined to identify variations called by both algorithms, submitted to PolyPhen2 to assess predicted functional consequence, and filtered to yield those SNPs most likely to represent deleterious, non-synonymous changes in coding regions of the genome in the tumor specifically, as well as those shared by the tumor and blood (i.e. constitutional changes).Our preliminary analysis identified a total of 1,724,133 single nucleotide changes in peripheral blood and 2,820,476 in the tumor prior to filtering. The transition to transversion ratio was 2.2 for blood and 2.0 for tumor. We did not observe mutations in known tumor suppressor or oncogenes. There were no coding mutations observed in any of the genes in the canonical pathways for IGF2 or beta-catenin signaling; p53 was wild-type. Inspection of constitutional changes indicates that the patient harbors a constitutive putative mutation in E2F2. In mice, knock-outs of E2F transcription factors result in developmental problems within the adrenal cortex. In our preliminary analysis, we identified a network that may possibly explain the aberrant p53 function that characterizes ACC. The tumor harbors a predicted mutation in SETD8. SETD8 encodes Set8, a lysine methyltransferase that has been implicated in gene silencing via histone 4 methylation, and is a negative regulator of p53 through methylation of p53 at lysine 382. Loss of p53 activity leads to increased activity of c-myc and SP1, both of which drive the expression of E2F2. Further work is required to characterize whether and how the mutations in SETD8 and E2F2 alter protein function and play a role in ACC pathogenesis. Finally, putative mutations in genes involved homologous repair such as ATM, FANCM, and BRCA2 suggests DNA cross-linking agents like mitomycin C may be useful in treating this patient's cancer. Data analysis is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-264. doi:10.1158/1538-7445.AM2011-LB-264

Background: In order to understand the genetic events associated with disease progression and the development of drug resistance in multiple myeloma, we studied four longitudinally collected samples from a single, high risk, t(4;14) multiple myeloma patient. Samples over a four-year period were available from diagnosis, at time of first progression on lenalidomide, second progression while on bortezomib, at time of relapsed refractory disease and with end stage plasma cell leukemia. To better understand these genetic events, we utilized DNA extracted from germline tissue and CD138 purified bone marrow tumor cells to conduct whole genome sequencing (WGS) utilizing the Life Technologies SOLiD™ platform. Results: We have sequenced over 100 billion bases per sample with whole genome fragment library sequencing providing an average of 30X aligned coverage at all four time points studied. Each sample was aligned to the reference genome (NCBI36/hg18) with BFAST and post-alignment removal of duplicates and recalibration performed with Picard and Genome Analysis Toolkit (GATK) respectively. We then called point mutations with SolSNP and compared the germline sample to each tumor sample utilizing the Paired Mutation Walker. To date, we have identified 224 nonsynonymous point mutations within 217 genes. We then utilized the Sorting Intolerant from Tolerant (SIFT) algorithm and identified 109 potentially damaging point mutations which include 108 novel mutations in coding exons. Of the 224 somatic mutations observed, only 20 of these mutations are common to all tumor time points. Interestingly, the diagnostic and bortezomib refractory (second progression) samples had 11 mutations in common while the lenalidomide refractory (first progression) sample shared only 4 mutations in common with the bortezomib refractory sample. This suggests the presence of multiple clones at diagnosis that, due to treatment selection pressure, may emerge or regress as the dominant clone. Preliminary pathway analysis reveals the presence of mutations in genes regulating NFKB and Akt, whose pathways are often dysregulated in cancer. Conclusions: Whole genome sequencing has revealed evidence of mutational differences in exons between tumor and germline DNA indicative of the presence of multiple clones at diagnosis and evolution of these clones over time. This data supports the use of multiple drug therapies that target multiple clones and challenges the long held assumption that a patient who becomes refractory to a particular treatment will always be refractory to that treatment. Additional evaluation in a larger population is needed to confirm these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4838. doi:10.1158/1538-7445.AM2011-4838