Shawn Yost

Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin

BackgroundMucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale.MethodsWe sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases.ResultsOur study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGFβ pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm.ConclusionsMNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.

BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma

BackgroundMalignant mesothelioma (MM) arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces. The majority of MMs are pleural and have been associated with asbestos exposure. Previously, pleural MMs have been genetically characterized by the loss of BAP1 (40-60%) as well as loss of NF2 (75%) and CDKN2A (60%). The rare peritoneal form of MM occurs in ~10% cases. With only ~300 cases diagnosed in the US per year, its link to asbestos exposure is not clear and its mutational landscape unknown.MethodsWe analyzed the somatic mutational landscape of 12 peritoneal MM of epitheloid subtype using copy number analysis (N = 9), whole exome sequencing (N = 7) and targeted sequencing (N = 12).ResultsPeritoneal MM display few copy number alterations, with most samples having less than 10 Mbp total changes, mostly through deletions and no high copy number amplification. Chromosome band 3p21 encoding BAP1 is the most recurrently deleted region (5/9), while, in contrast to pleural MM, NF2 and CDKN2A are not affected. We further identified 87 non-silent mutations across 7 sequenced tumors, with a median of 8 mutated genes per tumor, resulting in a very low mutation rate (median 1.3 10−6). BAP1 was the only recurrently mutated gene (N = 3/7). In one additional case, loss of the entire chromosome 3 leaves a non-functional copy of BAP1 carrying a rare nonsense germline variant, thus suggesting a potential genetic predisposition in this patient. Finally, with targeted sequencing of BAP1 in 3 additional cases, we conclude that BAP1 is frequently altered through copy number losses (N = 3/12), mutations (N = 3/12) or both (N = 2/12) sometimes at a sub-clonal level.ConclusionOur findings suggest a major role for BAP1 in peritoneal MM susceptibility and oncogenesis and indicate important molecular differences to pleural MM as well as potential strategies for therapy and prevention.

Mutascope: sensitive detection of somatic mutations from deep amplicon sequencing

Summary: We present Mutascope, a sequencing analysis pipeline specifically developed for the identification of somatic variants present at low-allelic fraction from high-throughput sequencing of amplicons from matched tumor-normal specimen. Using datasets reproducing tumor genetic heterogeneity, we demonstrate that Mutascope has a higher sensitivity and generates fewer false-positive calls than tools designed for shotgun sequencing or diploid genomes. Availability: Freely available on the web at http://sourceforge.net/projects/mutascope/. Contact: oharismendy@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.

High-Resolution Mutational Profiling Suggests the Genetic Validity of Glioblastoma Patient-Derived Pre-Clinical Models

Recent advances in the ability to efficiently characterize tumor genomes is enabling targeted drug development, which requires rigorous biomarker-based patient selection to increase effectiveness. Consequently, representative DNA biomarkers become equally important in pre-clinical studies. However, it is still unclear how well these markers are maintained between the primary tumor and the patient-derived tumor models. Here, we report the comprehensive identification of somatic coding mutations and copy number aberrations in four glioblastoma (GBM) primary tumors and their matched pre-clinical models: serum-free neurospheres, adherent cell cultures, and mouse xenografts. We developed innovative methods to improve the data quality and allow a strict comparison of matched tumor samples. Our analysis identifies known GBM mutations altering PTEN and TP53 genes, and new actionable mutations such as the loss of PIK3R1, and reveals clear patient-to-patient differences. In contrast, for each patient, we do not observe any significant remodeling of the mutational profile between primary to model tumors and the few discrepancies can be attributed to stochastic errors or differences in sample purity. Similarly, we observe ∼96% primary-to-model concordance in copy number calls in the high-cellularity samples. In contrast to previous reports based on gene expression profiles, we do not observe significant differences at the DNA level between in vitro compared to in vivo models. This study suggests, at a remarkable resolution, the genome-wide conservation of a patient’s tumor genetics in various pre-clinical models, and therefore supports their use for the development and testing of personalized targeted therapies.

Evaluation of ultra-deep targeted sequencing for personalized breast cancer care

IntroductionThe increasing number of targeted therapies, together with a deeper understanding of cancer genetics and drug response, have prompted major healthcare centers to implement personalized treatment approaches relying on high-throughput tumor DNA sequencing. However, the optimal way to implement this transformative methodology is not yet clear. Current assays may miss important clinical information such as the mutation allelic fraction, the presence of sub-clones or chromosomal rearrangements, or the distinction between inherited variants and somatic mutations. Here, we present the evaluation of ultra-deep targeted sequencing (UDT-Seq) to generate and interpret the molecular profile of 38 breast cancer patients from two academic medical centers.MethodsWe sequenced 47 genes in matched germline and tumor DNA samples from 38 breast cancer patients. The selected genes, or the pathways they belong to, can be targeted by drugs or are important in familial cancer risk or drug metabolism.ResultsRelying on the added value of sequencing matched tumor and germline DNA and using a dedicated analysis, UDT-Seq has a high sensitivity to identify mutations in tumors with low malignant cell content. Applying UDT-Seq to matched tumor and germline specimens from the 38 patients resulted in a proposal for at least one targeted therapy for 22 patients, the identification of tumor sub-clones in 3 patients, the suggestion of potential adverse drug effects in 3 patients and a recommendation for genetic counseling for 2 patients.ConclusionOverall our study highlights the additional benefits of a sequencing strategy, which includes germline DNA and is optimized for heterogeneous tumor tissues.

Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors

Background Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal) pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations). Methods Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas. Results We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48) and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value <0.01), across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% (p-value < 10–15), supporting clonality. In the second pair, no common mutations of 184 variants were validated (p-value >0.99), supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair. Conclusion We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.

Correction: Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin

No abstract.

Abstract 5191: Genome-wide mutational analysis reveals core signaling pathways in mucinous neoplasms of the appendix

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mucinous Neoplasm of the Appendix (MNA) is a rare malignancy often diagnosed after metastasis to the peritoneal surfaces resulting in Mucinous Carcinomatosis Peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer (CRC) and not based on molecular rationale. We sequenced the whole exomes of 10 MCPs to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases using deep sequencing and microdroplet PCR. These studies demonstrate that MNA has a different molecular makeup than CRC. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (21/29) and are characterized by cAMP-PKA pathway activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with CRC including gain of 1q (5/10), Wnt and TGFβ pathway alteration. In contrast, mutations in TP53 (1/10) and APC (0/10), common in CRC, are rare in MNA. Coincident activation of the Ras-Raf-MEK-ERK and cAMP-PKA signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm. Thus, MNA genome-wide mutational analysis reveals genetic alterations distinct from CRC, in support its unique pathophysiology and suggests MEK and PKA inhibition as new therapeutic opportunities. Citation Format: Hakan Alakus, Michele Babicky, Pradipta Ghosh, Shawn Yost, Kristen Jepsen, Yang Dai, Angelo Arias, Michael Samuels, Evangeline Mose, Michael Peterson, Andrew Lowy, Kelly Frazer, Olivier Harismendy. Genome-wide mutational analysis reveals core signaling pathways in mucinous neoplasms of the appendix. [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 5191. doi:10.1158/1538-7445.AM2014-5191

Abstract 219: Splicing inhibitors reduce human AML CD34+ cell survival and self-renewal during MDS/AML evolution in a leukemia stem cell supportive niche assay.

Introduction. Myelodysplastic syndromes (MDS) are relatively common neoplasms of hematopoietic stem cells, which commonly evolve to acute myeloid leukemia (AML). Recent results suggest that MDS evolution is controlled by mutations in splicing related genes and epigenetic modifiers of gene expression. Alternative splicing driven by these mutations has been implicated in the evolution of MDS to AML. However, little is known about the cell type and context specific functional effects of these mutations on leukemia stem cells (LSC) that promote AML therapeutic resistance. Therefore, we investigated 1) the effect of splicing inhibitors on LSC survival and self-renewal during the MSD/AML progression in a bone-marrow stromal co-cultures that recapitulates key aspects of the human LSC niche and 2) the genomic mutations in LSC and non-LSC populations during disease progression. Methods. Whole exome sequencing on CD34+ and CD34- cells before and after MDS/AML progression . Mouse bone marrow cell lines transfected to produce human SCF, IL3 and G-CSF, were used as a stromal monolayer. Human CD34+ cells were selected from MDS and AML primary samples (n=6). As a normal control, cord blood CD34+ cells (CB, n=3) were utilized. Two SF3B1-targeted splicing inhibitors: FD 895 and a FD-analog were added at the initiation of co-culture at concentrations ranging from 0.1 to10 uM. Results. Whole exome DNA sequencing analysis revealed that a loss of chr21 was observed at different frequencies in CD34- and CD34+ cells. We identified a RUNX1 missense mutation with increasing prevalence in CD34+ during progression. Notably, loss of heterozygosity and a missense mutation in the histone methyltransferase EZH2 gene, implicated in MDS progression, was detected only in CD34+ post-progression to AML. After 2 weeks of stromal co-culture, survival of the cells was investigated by colony forming assay in methylcellulose. While the splicing inhibitors demonstrated no cytotoxicity towards normal CB, MDS and AML samples showed a dose-dependent inhibition of colony formation. To analyze the effect of splicing inhibitors on LSC self-renewal, replating assays were performed. While compounds at high doses mediated only a slight decrease in colony formation in CB samples, MDS and AML samples exhibited a dose dependent inhibition of LSC survival (38.2+/-8.1% p Citation Format: Larisa Balaian, Michael Burkart, Shawn Yost, Sophie Rozenhak, Edward D. Ball, Kelly Frazer, Olivier Harismendy, Catriona Jamieson. Splicing inhibitors reduce human AML CD34+ cell survival and self-renewal during MDS/AML evolution in a leukemia stem cell supportive niche assay. [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 219. doi:10.1158/1538-7445.AM2013-219