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Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. As part of The Cancer Genome Atlas project, we report here an integrated analysis of 131 urothelial carcinomas to provide a comprehensive landscape of molecular alterations. There were statistically significant recurrent mutations in 32 genes, including multiple genes involved in cell-cycle regulation, chromatin regulation, and kinase signalling pathways, as well as 9 genes not previously reported as significantly mutated in any cancer. RNA sequencing revealed four expression subtypes, two of which (papillary-like and basal/squamous-like) were also evident in microRNA sequencing and protein data. Whole-genome and RNA sequencing identified recurrent in-frame activating FGFR3-TACC3 fusions and expression or integration of several viruses (including HPV16) that are associated with gene inactivation. Our analyses identified potential therapeutic targets in 69% of the tumours, including 42% with targets in the phosphatidylinositol-3-OH kinase/AKT/mTOR pathway and 45% with targets (including ERBB2) in the RTK/MAPK pathway. Chromatin regulatory genes were more frequently mutated in urothelial carcinoma than in any other common cancer studied so far, indicating the future possibility of targeted therapy for chromatin abnormalities.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

Chromosomal rearrangements leading to fusion transcripts represent a class of oncogenic aberrations that are of high interest for understanding cancer biology, treating cancer patients, and as targets for the development of new therapies. Transcriptome sequencing via RNA-Seq coupled with downstream bioinformatics software applications offers an effective method for identifying candidate fusion transcripts, and is more targeted and cost-effective than whole genome sequencing. Although many such fusion detection software tools have recently been made available, they often differ greatly in prediction accuracy, execution times, computational requirements, installation complexity, and in not being readily accessible to non-bioinformatician cancer researchers. Here we present the Trinity Cancer Transcriptome Analysis Toolkit (CTAT), a newly developed suite of RNA-Seq targeted fusion detection tools leveraging a combination of reference genome read-mapping and de novo transcriptome assembly, coupled to in silico fusion transcript validation, annotation, and visualization. We show that components of our Trinity CTAT fusion detection toolkit, including STAR-Fusion and FusionInspector, yield improved accuracy and run times as compared to alternative leading tools. As a demonstration of the utility of Trinity CTAT, we explore fusion transcript discovery in a large cohort of ∼300 chronic lymphocytic leukemia (CLL) patient samples. We identify several novel recurrent fusions that may represent drivers of CLL etiology and provide new avenues to therapies. In addition to fusion transcript identification, Trinity CTAT aims to provide cancer researchers with easy access to methods for analyzing cancer RNA-Seq, including transcript reconstruction, identification of mutations, expression analysis, and tumor heterogeneity. Trinity CTAT is freely available open source software and readily accessible to all cancer researchers via our public Galaxy web portal: https://galaxy.ncgas-trinity.indiana.edu/. Citation Format: Brian Haas, Timothy Tickle, Nathalie Pochet, Jing Sun, Peggy Hsu, Chip Stewart, Carrie Ganote, Ben Fulton, Catherine Wu, Aviv Regev. Fast and accurate fusion transcript detection using the Trinity Cancer Transcriptome Analysis Toolkit. [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 5266.

Nat. Genet. 47, 1326–1333 (2015); published online 12 October 2015; corrected after print 7 December 2015 In the version of this article initially published, two patients were stated on page 5 to have been excluded owing to insufficient follow-up data. These patients were included in the final analysis, but two additional patients were excluded owing to the presence of Noonan syndrome.

Cancer is principally considered a genetic disease, and numerous mutations are thought essential to drive its growth. However, the existence of genomically stable cancers and the emergence of mutations in genes that encode chromatin remodelers raise the possibility that perturbation of chromatin structure and epigenetic regulation are capable of driving cancer formation. Here we sequenced the exomes of 35 rhabdoid tumors, highly aggressive cancers of early childhood characterized by biallelic loss of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. We identified an extremely low rate of mutation, with loss of SMARCB1 being essentially the sole recurrent event. Indeed, in 2 of the cancers there were no other identified mutations. Our results demonstrate that high mutation rates are dispensable for the genesis of cancers driven by mutation of a chromatin remodeling complex. Consequently, cancer can be a remarkably genetically simple disease.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease characterized by multiple low-frequency alterations including somatic mutations, copy number alterations (CNAs) and chromosomal rearrangements. We sought to identify previously unrecognized low-frequency genetic events, integrate recurrent alterations into comprehensive signatures and associate these signatures with clinical parameters. For these reasons, our multi-institutional international group assembled a cohort of 304 primary DLBCLs from newly diagnosed patients, 87% of whom were uniformly treated with state-of-the-art therapy (rituximab-containing CHOP regimen) and had long term followup. Tumors were subjected to whole exome sequencing with an extended bait set that included custom probes designed to capture recurrent chromosomal rearrangements. In this cohort, 47% of samples had available transcriptional profiling and assignment to associated disease subtypes. Analytical pipelines developed at the Broad Institute were used to detect mutations (MuTect), CNAs (Recapseq+Allelic Capseq) and chromosomal rearrangements (dRanger+Breakpointer) and assess clonality (Absolute). To analyze formalin-fixed paraffin-embedded tumors without paired normals we developed a method which utilized 8334 unrelated normal samples to stringently filter recurrent germline events and artifacts. Significant mutational drivers were identified using the MutSig2CV algorithm and recurrent CNAs were assessed with GISTIC2.0. In addition, we utilized a recently developed algorithm, CLUMPS2, to prioritize somatic mutations which cluster in 3-dimensional protein structure. With this approach, we identified > 90 recurrently mutated genes, 34 focal amplifications and 41 focal deletions, 20 arm-level events and > 200 chromosomal rearrangements in the DLBCL series. Of note, 33% of the mutational drivers were also perturbed by chromosomal rearrangements or CNAs, underscoring the importance of a comprehensive genetic analysis. In the large DLBCL series, we identified several previously unrecognized but potentially targetable alterations including mutations in NOTCH2 (8%) and TET2 (5%). The majority of identified chromosomal rearrangements involved translocations of potent regulatory regions to intact gene coding sequences. The most frequently rearrangements involved Ig regulatory elements which were translocated to BCL2, MYC, BCL6 and several additional genes with known roles in germinal center B-cell biology. After identifying recurrent somatic mutations, CNAs and chromosomal rearrangements, we performed hierarchical clustering and identified subsets of DLBCLs with comprehensive signatures comprised of specific alterations. A large subset of tumors shared recurrent alterations previously associated with follicular lymphoma including mutations of chromatin modifiers such as CREBBP, MLL2, and EZH2 in association with alterations of TNFRSF14 and GNA13 and translocations of BCL2. This cluster was enriched in GCB-type DLBCLs and contained a subset with select genetic alterations associated with an unfavorable outcome. An additional cohort of tumors was characterized by alterations perturbing B-cell differentiation including recurrent BCL6 translocations or alterations of PRDM1. A subset of these DLBCLs had alterations of NOTCH2 and additional pathway components or mutations of MYD88 in association with TNFAIP3, CD70 and EBF1, a master regulator of B-cell differentiation. An additional group of DLBCLs exhibited frequent MYD88 mutations in association with alterations of CD79B, PIM1, TBL1XR1 and ETV6 and BCL2 copy gain; these tumors were highly enriched for ABC-type DLBCLs. This coordinate signature and additional alterations of p53 pathway components were associated with outcome. We explored bases for the identified genetic alterations in DLBCL by performing an in silico mutational signature analysis. The most frequent mutational signatures were those of spontaneous deamination (aging) and AID with rare cases of microsatellite instability. We also assessed the clonality of identified genetic features to define cancer cell fraction and establish the timing of specific genetic events. The comprehensive genetic signatures of clinically annotated DLBCLs provide new insights regarding approaches to targeted therapy. Disclosures Link: Kite Pharma: Research Funding; Genentech: Consultancy, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Pfreundschuh:Boehringer Ingelheim, Celegene, Roche, Spectrum: Other: Advisory board; Roche: Honoraria; Amgen, Roche, Spectrum: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Clonal evolution in response to therapy is a central feature of disease relapse. This raises a fundamental question in cancer biology: what enables the relapse clone to replace the pre-treatment clone? In other words, is the increased fitness of the relapse clone due to a lower death rate during therapy (less sensitivity to therapy) or a higher growth rate following therapy (superior ability to compete during repopulation)? We sought to address this question in chronic lymphocytic leukemia (CLL), as its relatively indolent disease kinetics enable the study of serially collected samples from the same patient over time. We recently reported the genetic characterization of 278 samples from patients enrolled in the German CLL Study Group CLL8 trial (Nature, in press). These samples were collected prior to first therapy with FC or FCR, and studied using whole-exome sequencing (WES). From this cohort, we further analyzed by WES 59 patients (FC [n = 28] or FCR [n = 31]) at time of relapse. We found that clonal evolution is the rule rather than the exception (57 / 59 CLLs), with TP53 alterations found in relapse in 15 cases. This series constitutes a unique opportunity to dissect the clonal dynamics of treated CLL. We therefore quantified clone-specific death and growth rates by targeted deep sequencing of serial peripheral blood samples, beginning at pre-treatment and ending at relapse. Given the expected minimal mutation detection sensitivity (0.1-1%) by targeted deep sequencing, we only selected samples with >1% CLL cells by flow cytometry. Such samples were available for 23 of 59 patients, with a median of 6 samples/patient (range 3-10). Based on the mutations identified by WES in the pre-treatment and relapse samples, we designed patient-specific multiplexed assays for targeted deep sequencing (median sequencing depth - 6561). A series of normal samples were sequenced together with patient samples to account for sequencing errors. The measurements of the CLL cell fraction in the sample, by sequencing and by flow cytometry, were highly correlated (r=0.89, p Clone-specific growth rates following therapy were calculated based on the measurements taken after therapy end, following exponential growth rate calculation. To calculate the clone-specific death rate during therapy, we applied two complementary approaches. First, measurements were taken after 3 cycles of therapy and the death rate per cycle was calculated. Second, clone-specific growth rates were back extrapolated to estimate the size of the population at the end of therapy, a method we have validated with an ultrasensitive emulsion droplet sequencing approach for targeted mutation detection. We discerned different mechanisms of relapse based on whether the relapse clone harbored mutated TP53 (TP53mut) or other mutations. In CLLs where the relapse clone contained TP53mut(n=10), the TP53mut clone showed lower death rate during therapy compared with the pre-treatment TP53 wildtype (TP53wt) clone (2.4 and 3.8 median log10 reduction, respectively; P = 0.02). On the other hand, the TP53mut clone showed only modestly higher growth rates during repopulation compared with the TP53wt clone (median growth rate of 0.8%/day vs. 0.56%/day, P = 0.13). Thus, differential sensitivityto therapy plays a primary role in TP53mut clonal evolution. In contrast, in the remaining cases whose relapse clone harbored mutations other than in TP53 (e.g., NOTCH1, ATM, SF3B1), we did not find differential sensitivity (median log10 clone reduction of 3.9 for the pre-treatment clone vs. 3.8 for the relapse clone, P=0.9). The primary engine leading to takeover by the relapse clone was a median of 1.5-fold higher growth rate during repopulation compared with the pretreatment clone. These data uncover evolutionary mechanisms in a personalized fashion directly from patient samples. Complementary efforts to apply these methods to define evolutionary mechanisms with targeted therapy are well under way. Thus, precise quantitation of clone-specific fitness in the context of therapy provides the required knowledge infrastructure to design the next generation of therapeutic algorithms, to maximize overall tumor elimination, instead of merely selecting one clone over another. Disclosures Tausch: Gilead: Other: Travel support. Fink:Roche: Honoraria, Other: travel grant. Hallek:Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding.

Background The evolution of gene expression is a challenging problem in evolutionary biology, for which accurate, well-calibrated measurements and methods are crucial. Results We quantified gene expression with whole-transcriptome sequencing in four diploid, prototrophic strains of Saccharomyces species grown under the same condition to investigate the evolution of gene expression. We found that variation in expression is gene-dependent with large variations in each gene's expression between replicates of the same species. This confounds the identification of genes differentially expressed across species. To address this, we developed a statistical approach to establish significance bounds for inter-species differential expression in RNA-Seq data based on the variance measured across biological replicates. This metric estimates the combined effects of technical and environmental variance, as well as Poisson sampling noise by isolating each component. Despite a paucity of large expression changes, we found a strong correlation between the variance of gene expression change and species divergence (R2 = 0.90). Conclusion We provide an improved methodology for measuring gene expression changes in evolutionary diverged species using RNA Seq, where experimental artifacts can mimic evolutionary effects. GEO Accession Number: GSE32679

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

As a consequence of the accumulation of insertion events over evolutionary time, mobile elements now comprise nearly half of the human genome. The Alu, L1, and SVA mobile element families are still duplicating, generating variation between individual genomes. Mobile element insertions (MEI) have been identified as causes for genetic diseases, including hemophilia, neurofibromatosis, and various cancers. Here we present a comprehensive map of 7,380 MEI polymorphisms from the 1000 Genomes Project whole-genome sequencing data of 185 samples in three major populations detected with two detection methods. This catalog enables us to systematically study mutation rates, population segregation, genomic distribution, and functional properties of MEI polymorphisms and to compare MEI to SNP variation from the same individuals. Population allele frequencies of MEI and SNPs are described, broadly, by the same neutral ancestral processes despite vastly different mutation mechanisms and rates, except in coding regions where MEI are virtually absent, presumably due to strong negative selection. A direct comparison of MEI and SNP diversity levels suggests a differential mobile element insertion rate among populations., Author Summary We embarked on this study to explore the 1000 Genomes Project (1000GP) pilot dataset as a substrate for Mobile Element Insertion (MEI) discovery and analysis. MEI is already well known as a significant component of genetic variation in the human population. However the full extent and effects of MEI can only be assessed by accurate detection in large whole-genome sequencing efforts such as the 1000GP. In this study we identified 7,380 distinct genomic locations of variant MEI and carried out rigorous validation experiments that confirmed the high accuracy of the detected events. We were able to measure the frequency of each variant in three continental population groups and found that inherited MEI variants propagate through populations in much the same way as single nucleotide polymorphisms, except that MEI are more strongly suppressed in protein coding parts of the genome. We also found evidence that the MEI mutation rate has not been constant over human population history, rather that different populations appear to have different characteristic MEI mutation rates.

Intra-tumoral genetic heterogeneity and clonal evolution play a central role in disease relapse, and constitute one of the foremost obstacles to cancer cure. To measure intra-tumoral genetic heterogeneity, we recently developed an analytic framework which utilizes whole-exome sequencing (WES) to calculate the proportion of cancer cells within a sample harboring each mutation. Applying this approach to a clinically heterogeneous cohort of chronic lymphocytic leukemia (CLL), we previously discovered that the intra-tumoral genetic heterogeneity in primary patient samples prior to treatment initiation anticipates clonal evolution and is linked to adverse clinical outcome with therapy (Landau et al., Cell 2013). To definitively evaluate the value of intra-tumoral heterogeneity in predicting clinical outcome in CLL, we performed an analogous analysis of pre-treatment samples collected from study subjects enrolled on the CLL8 trial of the GCLLSG (Hallek et al., Lancet 2010). In this multicenter phase III study, 817 patients were randomized to receive as first-line therapy fludarabine and cyclophosphamide either alone or in combination with rituximab. Paired pre-treatment tumor DNA from CD19+ selected peripheral blood mononuclear cells (PBMC), and non-tumor DNA from CD19- PBMC were available from 309 subjects (61% IGHV unmutated, 26% del(11q), 4.5% del(17p)). From these 309 sample pairs, we performed SNP array analysis and WES. Somatic mutations were identified as single nucleotide variants (SNVs), indels and copy number alterations (CNAs), detected in the tumor DNA but not in matched germline DNA. Subsequently, we utilized the algorithm ABSOLUTE to calculate the purity and ploidy of each sample, and classify every mutation as either clonal (i.e., present in all leukemic cells in the sample) vs. subclonal (i.e., present in a subset of leukemic cells). Progression free survival (PFS) was defined as the primary outcome measure. Preliminary sequencing results for 275 of 309 CLL samples are currently available. Across these samples, WES identified 7290 somatic SNVs and indels. Overall mutation rate for these samples was 0.8/Mb, similar to prior large-scale reports of DNA sequencing of CLL. By applying the algorithm MutSig, which identifies putative driver genes recurrently mutated more than expected by chance, we detected 16 of 24 previously described CLL drivers (e.g., TP53, ATM, SF3B1 and NOTCH1) to significantly affect this cohort. In addition, due to the large sample size we have identified 9 novel putative drivers in CLL including MGA, IKZF3 and ARID1A. CNA analysis identified the previously reported recurrent CNAs including (del(13q), del(11q), del(17p), trisomy 12 and del(8p)). At this time, subclonal analysis by ABSOLUTE is available for 216 of 309 CLLs. Across the 216 samples, we identified 2041 clonal and 2106 subclonal mutations. A putative driver gene mutation was identified as clonal in 102 (47.2%) patients, and as subclonal in 81 (37.5%) patients. The presence of a clonal driver was not significantly associated with differences inPFS (logrank P= 0.236). In contrast, the presence of a subclonal driver was associated with a significantly shortened PFS (logrank P= 0.006, Figure 1A-B). A Cox regression model that included the presence of a subclonal driver as well as the treatment arm assignment (FC vs. FCR), retained the presence of a subclonal driver as statistically significant in association with shorter PFS (HR 1.56 [95%CI 1.12-2.19], P=0.009). The presence of a subclonal driver was furthermore associated with shorter overall survival (logrank P=0.02, Fig 1C-D). This preliminary analysis demonstrates that pre-treatment characterization of intra-tumoral genetic heterogeneity can identify patients at risk for inferior outcome with first-line fludarabine-based therapy in CLL. With completion of this analysis for the entire cohort of 309 patients, we plan to perform a multivariable analysis to evaluate the clinical impact of clonal and subclonal driver mutations in CLL. Further ongoing studies include a longitudinal genetic evaluation of the subset of patients with subsequent disease relapse in order to unravel the evolutionary dynamics underlying disease progression. Figure 1 Figure 1. Disclosures Fischer: Roche: Travel grants Other. Hallek:Janssen, Pharmacyclics: Consultancy, Research Funding.

Introduction. Primary testicular lymphoma (PTL) and primary central nervous system lymphoma (PCNSL) are large B-cell lymphomas (LBCL) that occur in immune privileged (IP) sites and share certain clinical and molecular features. To date, the treatment of these IP lymphomas is largely empiric and more effective targeted therapies are needed. Methods. To define actionable genetic features of IP lymphomas, we performed comprehensive genomic analyses of 21 PCNSLs and 7 PTLs and validated specific alterations in an independent cohort of 43 additional PTLs. Recurrent copy number alterations (CNAs) were detected using high-density single nucleotide polymorphism (SNP) arrays and the GISTIC algorithm and integrated with transcriptional profiles to identify candidate driver genes. Recurrent somatic mutations were identified using a combination of whole exome sequencing (WES) of paired tumor/normal samples and whole transcriptome sequencing (RNA-Seq) of the additional tumors without paired normal samples. Results. In systemic diffuse large B-cell lymphomas (DLBCLs), multiple low-frequency CNAs and associated target genes decrease p53 activity and perturb cell cycle regulation; infrequent somatic mutations of TP53 also deregulate these pathways (Cancer Cell, 2012; 22:359-372). In contrast, PCNSLs and PTLs primarily exhibit bi-allelic deletion of the upstream regulator of p53 activity and cell cycle, CDKN2A (~70% PCNSLs and ~80% of PTLs) and rarely have copy loss or somatic mutations of TP53 or CNAs of additional pathway components. The most commonly mutated genes in PCNSL and PTL, CD79B and MYD88, are also perturbed in a subset of systemic DLBCLs. However, mutations of these two genes are much more frequent in IP lymphomas (70% MYD88 and 61% CD79B of analyzed PCNSLs and PTLs) and these alterations are commonly found in the same cases (57% of cases in this series). These data indicate that concurrent oncogenic activation of the B-cell receptor (BCR) and the Toll-like receptor (TLR) signaling pathways is a characteristic feature of IP lymphomas with implications for targeted therapies. Among the IP lymphomas, ~20% of PCNSLs and ~40% PTLs exhibit 3q12.3/NFKBIZ copy gain and increased expression of the NFKBIZ protein product, IκB-ζ, an atypical IκB family member induced by TLR signaling. In our PTL series, MYD88 wild-type tumors had the highest 3q12.3/NFKBIZ copy gains, and ~90% of all analyzed PTLs had structural bases for NFκB activation via the TLR pathway. Lentiviral-mediated IκB-ζ knockdown decreased expression of the IκB-ζ target genes, IκB-α and BCL-xL, and induced apoptosis of LBCL cell lines with MYD88 L265P mutations, NFKBIZ gain or both alterations. In addition, enforced expression of NFKBIZ enhanced the growth of LBCLs with normal NFKBIZ copy numbers. Taken together, these data suggest that many IP lymphomas depend upon oncogenic MYD88/NFKBIZ signaling. Although the majority of CNAs and somatic mutations were shared by PCNSLs and PTLs, certain alterations were primarily observed in PTL. In both the initial and independent validation series, > 40% of PTLs exhibited copy gain of chromosome 9p24.1/CD274 (PD-L1) / PDCD1LG2 (PD-L2) and associated overexpression of the PD-1 ligands. These observations were of particular interest because 9p24.1 copy gain is a characteristic abnormality in two additional lymphoid malignancies, primary mediastinal LBCL and classical Hodgkin lymphoma, PD-1 signaling promotes tumor immune evasion and the PD-1 pathway is targetable. We also identified one PTL in which a novel translocation juxtaposed the regulatory elements of TBL1XR1 (chromosome 3) to the start codon-bearing exon 2 of PDCD1LG2 (PD-L2) (chromosome 9). This translocation, which was detected by RNA-Seq and confirmed by 5’ RACE and a newly developed split-apart FISH assay, resulted in dramatic overexpression of the PD-L2 protein. These data suggest that PTLs utilize several genetic mechanisms to deregulate the PD-1 ligands and limit anti-tumor immunity. Conclusions. Integrative and comparative genomic studies define PCNSL and PTL as related but unique lymphoid malignancies with targetable genetic alterations, and associated p53 deficiency and cell cycle deregulation, concurrent oncogenic BCR and TLR signaling and PD-1 dependent immune evasion that warrant further clinical investigation. Note: B.C. and M.G.M.R have made equal contributions to this research. Disclosures Feuerhake: Roche Pharma Research and Early Development (pRED) from 2008-2012: Employment. Freeman:Merck: on the PD-1 pathway Patents & Royalties; EMD-Serrono: on the PD-1 pathway Patents & Royalties; Boehringer-Ingelheim: on the PD-1 pathway Patents & Royalties; Amplimmune: on the PD-1 pathway Patents & Royalties; Roche: on the PD-1 pathway Patents & Royalties; Bristol-Myers-Squibb: on the PD-1 pathway Patents & Royalties; Novatis: on the PD-1 pathway, on the PD-1 pathway Patents & Royalties. Shipp:Sanofi: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Membership on an entity's Board of Directors or advisory committees.

Unbiased high-throughput massively parallel sequencing methods have transformed the process of discovery of novel putative driver gene mutations in cancer. In chronic lymphocytic leukemia (CLL), these methods have yielded several unexpected findings, including the driver genes SF3B1, NOTCH1 and POT1. Recent analysis, utilizing down-sampling of existing datasets, has shown that the discovery process of putative drivers is far from complete across cancer. In CLL, while driver gene mutations affecting >10% of patients were efficiently discovered with previously published CLL cohorts of up to 160 samples subjected to whole exome sequencing (WES), this sample size has only 0.78 power to detect drivers affecting 5% of patients, and only 0.12 power for drivers affecting 2% of patients. These calculations emphasize the need to apply unbiased WES to larger patient cohorts. To this end, we performed a combined analysis of CLL WES data joining together our previously published cohort of 159 CLLs with data from 103 CLLs collected by the International Cancer Genome Consortium (ICGC). The raw sequencing reads from these 262 primary tumor samples (102 CLL with unmutated IGHV, 147 with mutated IGHV, 13 with unknown IGHV status) were processed together and aligned to the hg19 reference genome. Somatic single nucleotide variations (sSNVs) and indels were detected using MuTect. Subsequently, inference of recurrently mutated genes was performed using the MutSig algorithm. This method combined several characteristics such as the overall mutation rate per sample, the gene specific background mutation rate, non-synonymous/synonymous ratio and mutation clustering to detect genes that are affected by mutations more than expected by chance. This analysis identified 40 recurrently mutated genes in this cohort. This included 22 of 25 previously identified recurrently mutated genes in CLL. In addition, 18 novel candidate CLL drivers were identified, mostly affecting 1-2% of patients. The novel candidates included two histone proteins HIST1H1D and HIST1H1C, in addition to the previously identified HIST1H1E. Another was IKZF3, affected by a recurrent sSNV resulting in a p.L162R change in its DNA binding domain, in close proximity to a region recently identified as critical for lenalidomide resistance in multiple myeloma (MM). An additional recurrently mutated gene was nuclear RNA export factor 1 (NXF1), which along with previously known recurrently mutated genes (SF3B1, XPO1, DDX3X), highlights the importance of RNA processing to CLL biology. Finally, this search for putative CLL driver genes also identified ASXL1 and TRAF3, already characterized as drivers in acute myeloid leukemia and MM, respectively. Of the 59 of 262 samples for which RNA-seq data were available, 76% of the identified driver mutations were detected and thereby validated. Validation using RNAseq detection of driver mutations and targeted sequencing within the entire cohort are ongoing. The larger size of our cohort enabled the separate application of the somatic mutation discovery process to samples with mutated or unmutated IGHV. Among the 147 samples with mutated IGHV, only 5 driver genes (TP53, SF3B1, MYD88, CHD2, RANBP2) retained significance. In contrast, analysis of the 102 IGHV unmutated samples revealed a distinct and more diverse pattern of recurrently mutated genes (lacking MYD88 and CHD2, and including NOTCH1, RPS15, POT1, NRAS, EGR2, BRAF, MED12, XPO1, BCOR, IKZF3, MAP2K1, FBXW7 and KRAS). This extended cohort also allowed for better resolution of the clinical impact of those genetic variants with greater than 4% prevalence in the cohort. For example, samples with POT1 mutations were found to be associated with shorter time from sample to therapy compared with those with wild-type POT1 (P= 0.02). Our study demonstrates that with larger cohort size, we can effectively detect putative driver genes with lower prevalence, but which may nonetheless have important biological and clinical impact. Moreover, our interrogation shows that subset analysis can reveal distinct driver patterns in different disease subsets. In particular, the marked clinical difference between CLLs with mutated and unmutated IGHV may reflect the higher likelihood of the latter group to harbor a broader spectrum of driver mutations with a more complex pattern of co-occurrence. Disclosures Brown: Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy.

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae both from extension of the tumors and from therapeutic interventions which damage the optic chiasm, the pituitary stalk, and the hypothalamic area. Using whole exome sequencing we identified mutations in beta-catenin (CTNNB1) in nearly all adamantinomatous craniopharyngiomas (11/12; 92%) and recurrent mutations in BRAF (V600E) in all papillary craniopharyngiomas (3/3; 100%). Targeted genotyping revealed BRAF V600E in 95% of papillary craniopharyngiomas (36 of 39 tumors) and CTNNB1 mutation in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype and no other recurrent mutations or genomic aberrations were detected in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

The sequencing of aggressive pediatric solid tumors is revealing remarkably stable genomes. In the cases of malignant rhabdoid and retinoblastoma, there is a paucity of recurrently mutated genes, and oncogenesis appears to be driven, at least in part, by epigenetic deregulation. It has been suggested that pediatric tumors characterized by oncogenic fusions will exhibit relatively few additional somatic driver aberrancies. Ewing sarcoma, the second most common pediatric bone tumor, is characterized by rearrangements of the EWS gene and ETS-family transcription factor genes, most commonly FLI and ERG. In experimental models, Ewing sarcoma demonstrates dependency on the expression of the resulting chimeric fusion products. As such, Ewing sarcoma represents a paradigm for studying the genomic landscape of fusion-driven cancers. To this end, we performed whole-exome sequencing of 96 Ewing sarcoma tumors and 11 Ewing sarcoma cell lines, as well as whole-genome sequencing, transcriptome sequencing, and copy-number analysis of a subset of these samples. We found that Ewing sarcoma is one of the most genetically normal cancers sequenced to date, but that treatment, which generally employs genotoxic chemotherapy and radiation, is associated with an increase in mutation rate and single nucleotide substitutions associated with DNA damage. There was a marked absence of recurrent mutations in immediately druggable targets, such as tyrosine kinases, calling into question the feasibility of utilizing tumor sequencing to nominate targeted therapies for patients with Ewing sarcoma. Rather, these results highlight the importance of directly targeting the EWS/ETS fusion events or identifying synthetic lethal dependencies. To this end, we clarified a number of outstanding questions regarding the EWS/ETS fusions. We found that reciprocal ETS/EWS fusions are not expressed in Ewing sarcoma and therefore unlikely to play a role in Ewing pathogenesis as is seen with reciprocal fusions of PML-RARα in acute promyelocytic leukemia. We also found that wild-type FLI and wild-type ERG are not expressed in Ewing sarcoma tumors. However, there appears to be a role for ETS gene deregulation in this disease beyond the expression of EWS/ETS fusion proteins because we found recurrent somatic events in ERF and ETS1. We also identified a small number of other recurrently mutated genes that likely collaborate with EWS/ETS fusions in a minority of cases and confirmed that loss of STAG2 occurs in approximately 15% of Ewing sarcoma tumors. Thus, massively parallel sequencing of a large collection of Ewing sarcoma tumors supports the notion that fusion-driven pediatric malignancies bear quiet genomes, underscores the importance of identifying new treatment approaches targeting EWS/ETS fusions, and also identifies new genetic abnormalities that warrant further biological validation. Citation Format: Brian Crompton, Chip Stewart, Amaro Taylor-Weiner, Gabriela Alexa, Kyle Kurek, Monica Calicchio, Adam Kiezun, Scott Carter, Sachet Shukla, Swapnil Mehta, Aaron Thorner, Carmen de Torres, Cinzia Lavarino, Mariona Sunol, Aaron McKenna, Andrey Sivachenko, Kristian Cibulskis, Michael Lawrence, Lauren Ambrogio, Daniel Auclair, Ivan Rosshandler, Angela Schwarz-Cruz y Celis, Miguel Rivera, Carlos Rodriguez-Galindo, Mark Fleming, Todd Golub, Gad Getz, Jaume Mora, Kimberly Stegmaier. The genomic landscape of pediatric Ewing sarcoma. [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 999. doi:10.1158/1538-7445.AM2014-999

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae both from extension of the tumors and from therapeutic interventions which damage the optic chiasm, the pituitary stalk, and the hypothalamic area. Using whole exome sequencing we identified mutations in beta-catenin (CTNNB1) in nearly all adamantinomatous craniopharyngiomas (11/12; 92%) and recurrent mutations in BRAF (V600E) in all papillary craniopharyngiomas (3/3; 100%). Targeted genotyping revealed BRAF V600E in 95% of papillary craniopharyngiomas (36 of 39 tumors) and CTNNB1 mutation in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype and no other recurrent mutations or genomic aberrations were detected in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms. Citation Format: Amaro N. Taylor-Weiner, Priscilla K. Brastianos, Peter E. Manley, Robert T. Jones, Dora Dias-Sangata, Aaron Thorner, Fausto Rodriguez, Lindsay Bernardo, Laura Schubert, Chip Stewart, Mark Keiran, William C. Hahn, Adam Resnick, David Louis, Sandro Sangata, Gad Getz. Exome sequencing reveals BRAF mutations in papillary craniopharyngiomas. [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 935. doi:10.1158/1538-7445.AM2014-935

The availability of large numbers of formalin-fixed, paraffin-embedded (FFPE) tumor DNA samples, together with accurate and specific clinical annotations, can be a great resource for in-depth analysis of correlations of somatic and germline DNA alterations with clinical outcome. However, the effects of this tissue preservation technique on next-generation DNA sequencing technologies and downstream analyses are still being investigated. Furthermore, usually the archives of such FFPE samples do not include matched normal tissue, which complicates the process of identifying somatic alterations. We are in the process of analyzing lung adenocarcinoma, colorectal and prostate patient datasets, which contain both an FFPE and a fresh-frozen tumor together with a matched blood normal sample. The availability of both a frozen and an FFPE tumor sample enables us to compare the detected somatic point mutations and indels, taking into account the fact that these pairs are produced from different aliquots of DNA. This analysis includes calculating the power to detect variants in the FFPE sample given that they have been observed in the fresh frozen tumor, as well as the validation rate of clonal SNVs between the two samples. We are also developing a method for classification of SNVs as germline or somatic without a paired normal sample by taking advantage of the fact that most tumor samples contain a substantial fraction of normal cells. Because stromal contamination has a different effect on the allelic fraction of somatic vs. germline SNVs, we can apply algorithms to estimate tumor purity and absolute somatic copy numbers in order to distinguish the two types of events. We present preliminary results from these analyses. Citation Format: Petar Stojanov, Scott L. Carter, Ivan Imaz Rosshandler, Andrey Sivachenko, Alberto Salido Guadarrama, Karla Vazquez, Sandra Romero Cordoba, Kristian Cibulskis, Carrie Sougnez, Douglas Voet, Gordon Saksena, Lee Lichtenstein, Lihua Zou, Scott Frazer, Chip Stewart, Rameen Beroukhim, Matthew Meyerson, Michael S. Lawrence, Gad Getz. Analysis of formalin-fixed paraffin-embedded (FFPE) samples. [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 5135. doi:10.1158/1538-7445.AM2013-5135

Small cell lung carcinoma (SCLC) is a highly lethal tumor for which few targetable genetic alterations have been identified. SCLC is tightly associated with an extended history of tobacco use, and recent DNA sequencing studies have revealed highly mutated SCLC cancer genomes. Interestingly, two studies analyzing the human SCLC genome report distinct sets of putative driver mutations, highlighting the challenge of identifying functional events in highly mutated cancers. In the absence of environmental mutagens, a previously established genetically engineered mouse model of SCLC initiated by combined loss of Trp53 and Rb1 recapitulates the salient clinical features of human SCLC including tumor histologic progression and frequent distant metastases. This model has been shown to acquire DNA copy number alterations shared with human SCLC, suggesting that cross-species cancer genomics may identify a subset of driver events in human cancers. In order to characterize the spectrum of acquired events in murine SCLC and identify evolutionarily conserved drivers of SCLC progression, we define the somatic genome and transcriptome of a large panel of murine SCLC at single nucleotide resolution using exome, genome and RNA sequencing. We uncover complex subclonality in primary murine SCLC tumors and detect evidence for clonal selection during metastatic spread. As expected in the absence of environmental mutagens, we demonstrate a low somatic mutation frequency in murine SCLC. Using integrative genomic analyses we uncover alterations in chromatin remodeling enzymes and identify Pten as a critical tumor suppressor in this model. Engineered Pten deletion in murine SCLC dramatically accelerates tumorigenesis and fundamentally alters the genomic evolution of these tumors. This work represents the first large-scale comprehensive genomic characterization of a genetically engineered mouse cancer model at single nucleotide resolution, has implications for the nature of tumor evolution in mouse cancer models and identifies a potential therapeutic target in a subset of human SCLC. Citation Format: David G. McFadden, Thales Papagiannakopoulos, Kristian Cibulskis, Chip Stewart, Scott Carter, Amaro Taylor-Weiner, Arjun Bhutkar, Carrie Sougnez, Alison Dooley, Erica Shefler, Eric Lander, Stacey Gabriel, Gad Getz, Tyler Jacks. Comprehensive genomic analysis of murine small cell lung carcinoma uncovers recurrent Pten alterations that drive tumor progression and alter tumor genome evolution. [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 1084. doi:10.1158/1538-7445.AM2013-1084

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community., National Human Genome Research Institute (U.S.) (HG03067-05)

To compare the sensitivity of somatostatin receptor scintigraphy done using [111In-DTPA-DPhe1]octreotide with that of other imaging methods in the localization of gastrinomas in patients with the Zollinger-Ellison syndrome.Prospective study.Referral-based clinical research center.80 consecutive patients with the Zollinger-Ellison syndrome.Conventional tumor localization studies (ultrasonography, computed tomography [CT], magnetic resonance imaging [MRI], selective angiography, and bone scanning) and somatostatin receptor scintigraphy done using [111In-DTPA-DPhe1]octreotide with single-photon emission CT imaging at 4 and 24 hours. Patients with possible liver metastases had biopsies done for confirmation, and 15 patients had exploratory laparotomies done to assess primary tumor localization.Extrahepatic gastrinomas or liver metastases were identified by ultrasonography in 19% of patients, by CT in 38% of patients, by MRI in 45% of patients, by angiography in 40% of patients, and by somatostatin receptor scintigraphy in 70% of patients. Somatostatin receptor scintigraphy was as sensitive as the other tests combined (59%), and when the results of all other tests were added to the somatostatin receptor scintigraphy results, tumors were localized in 75% of patients. Among patients with a possible primary tumor, the results of ultrasonography were positive in 9%, the results of CT were positive in 31%, the results of MRI were positive in 30%, the results of angiography were positive in 28%, and the results of somatostatin receptor scintigraphy were positive in 58%. Somatostatin receptor scintigraphy was as sensitive as all of the other imaging studies combined; when the results of scintigraphy were added to the results of the other studies, possible primary tumors were identified in 68% of patients. In 24 patients who had histologically proven metastatic liver disease, sensitivities for the detection of any metastatic liver lesions were 46% for ultrasonography, 42% for CT, 71% for MRI, 62% for angiography, and 92% for somatostatin receptor scintigraphy. Somatostatin receptor scintigraphy was significantly better than all of the conventional imaging methods in the identification of gastrinomas later found at surgery (P = 0.004), but it still missed 20% of gastrinomas.Somatostatin receptor scintigraphy is the single most sensitive method for imaging either primary or metastatic liver lesions in patients with the Zollinger-Ellison syndrome. Because of its sensitivity, simplicity, and cost-effectiveness, it should be the first imaging method used in these patients. For patients with negative results on somatostatin receptor scintigraphy, guidelines about the use of other imaging studies are proposed.

Background DNA capture technologies combined with high-throughput sequencing now enable cost-effective, deep-coverage, targeted sequencing of complete exomes. This is well suited for SNP discovery and genotyping. However there has been little attention devoted to Copy Number Variation (CNV) detection from exome capture datasets despite the potentially high impact of CNVs in exonic regions on protein function. Results As members of the 1000 Genomes Project analysis effort, we investigated 697 samples in which 931 genes were targeted and sampled with 454 or Illumina paired-end sequencing. We developed a rigorous Bayesian method to detect CNVs in the genes, based on read depth within target regions. Despite substantial variability in read coverage across samples and targeted exons, we were able to identify 107 heterozygous deletions in the dataset. The experimentally determined false discovery rate (FDR) of the cleanest dataset from the Wellcome Trust Sanger Institute is 12.5%. We were able to substantially improve the FDR in a subset of gene deletion candidates that were adjacent to another gene deletion call (17 calls). The estimated sensitivity of our call-set was 45%. Conclusions This study demonstrates that exonic sequencing datasets, collected both in population based and medical sequencing projects, will be a useful substrate for detecting genic CNV events, particularly deletions. Based on the number of events we found and the sensitivity of the methods in the present dataset, we estimate on average 16 genic heterozygous deletions per individual genome. Our power analysis informs ongoing and future projects about sequencing depth and uniformity of read coverage required for efficient detection.

Retrotransposons comprise over 40% of the human genome and represent an important class of genetic variation. Though most elements are stationary ancient relics, it has recently been shown that some 100 elements remain highly active, copying and pasting themselves throughout the genome with each generation. Retrotransposon insertions can disrupt gene function, modulate gene expression, and lead to genomic rearrangement. These events have previously been implicated in cancer, including an account of insertional mutagenesis in APC as an early event in colon cancer progression. Due to the inherent difficulty in localizing repeat elements, however, an extensive investigation of retrotransposons’ role in cancer has not yet been performed. We developed RetroSeq, a computational framework to identify novel insertions of retrotransposons from paired-end sequencing data. In simulated data, RetroSeq identifies retrotransposon insertions with 99% sensitivity and 99% specificity. We ran RetroSeq on whole-genome sequencing data from a panel of tumors (including 9 colorectal, 20 non-small cell lung, 24 prostate and 22 breast tumors) and matched normal samples to determine the extent of somatic retrotransposon activity. We find a broad range of novel retrotransposon insertion events specific to each tumor, with some insertions falling in exonic as well as intronic and intergenic regions. These events were validated experimentally and integrated with expression and methylation data to provide a global view of retrotransposon activity in these samples. In sum, using RetroSeq we are able to localize novel retrotransposon insertions in paired-end sequencing data and provide evidence for the reactivation of retrotransposons in cancer. 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 5060. doi:1538-7445.AM2012-5060

Summary Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other carcinomas) and extended the set of known PTC driver alterations to include EIF1AX , PPM1D , and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF -mutant tumors, and multidimensional analyses highlighted a potential involvement of oncomiRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the disease.

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

Background Rare coding variants constitute an important class of human genetic variation, but are underrepresented in current databases that are based on small population samples. Recent studies show that variants altering amino acid sequence and protein function are enriched at low variant allele frequency, 2 to 5%, but because of insufficient sample size it is not clear if the same trend holds for rare variants below 1% allele frequency. Results The 1000 Genomes Exon Pilot Project has collected deep-coverage exon-capture data in roughly 1,000 human genes, for nearly 700 samples. Although medical whole-exome projects are currently afoot, this is still the deepest reported sampling of a large number of human genes with next-generation technologies. According to the goals of the 1000 Genomes Project, we created effective informatics pipelines to process and analyze the data, and discovered 12,758 exonic SNPs, 70% of them novel, and 74% below 1% allele frequency in the seven population samples we examined. Our analysis confirms that coding variants below 1% allele frequency show increased population-specificity and are enriched for functional variants. Conclusions This study represents a large step toward detecting and interpreting low frequency coding variation, clearly lays out technical steps for effective analysis of DNA capture data, and articulates functional and population properties of this important class of genetic variation.