GenomeVIP: A cloud platform for genomic variant discovery and interpretation

Identifying genomic variants is a fundamental first step toward the understanding of the role of inherited and acquired variation in disease. The accelerating growth in the corpus of sequencing data that underpins such analysis is making the data-download bottleneck more evident, placing substantial burdens on the research community to keep pace. As a result, the search for alternative approaches to the traditional “download and analyze” paradigm on local computing resources has led to a rapidly growing demand for cloud-computing solutions for genomics analysis. Here, we introduce the Genome Variant Investigation Platform (GenomeVIP), an open-source framework for performing genomics variant discovery and annotation using cloud- or local high-performance computing infrastructure. GenomeVIP orchestrates the analysis of whole-genome and exome sequence data using a set of robust and popular task-specific tools, including VarScan, GATK, Pindel, BreakDancer, Strelka, and Genome STRiP, through a web interface. GenomeVIP has been used for genomic analysis in large-data projects such as the TCGA PanCanAtlas and in other projects, such as the ICGC Pilots, CPTAC, ICGC-TCGA DREAM Challenges, and the 1000 Genomes SV Project. Here, we demonstrate GenomeVIP's ability to provide high-confidence annotated somatic, germline, and de novo variants of potential biological significance using publicly available data sets.</jats:p

Proteogenomics connects somatic mutations to signalling in breast cancer

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. We describe quantitative mass spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers of which 77 provided high-quality data. Integrated analyses allowed insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. The 5q trans effects were interrogated against the Library of Integrated Network-based Cellular Signatures, thereby connecting CETN3 and SKP1 loss to elevated expression of EGFR, and SKP1 loss also to increased SRC. Global proteomic data confirmed a stromal-enriched group in addition to basal and luminal clusters and pathway analysis of the phosphoproteome identified a G Protein-coupled receptor cluster that was not readily identified at the mRNA level. Besides ERBB2, other amplicon-associated, highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets

Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients

Comprehensive and Integrated Genomic Characterization of Adult Soft Tissue Sarcomas

Sarcomas are a broad family of mesenchymal malignancies exhibiting remarkable histologic diversity. We describe the multi-platform molecular landscape of 206 adult soft tissue sarcomas representing 6 major types. Along with novel insights into the biology of individual sarcoma types, we report three overarching findings: (1) unlike most epithelial malignancies, these sarcomas (excepting synovial sarcoma) are characterized predominantly by copy-number changes, with low mutational loads and only a few genes (, , ) highly recurrently mutated across sarcoma types; (2) within sarcoma types, genomic and regulomic diversity of driver pathways defines molecular subtypes associated with patient outcome; and (3) the immune microenvironment, inferred from DNA methylation and mRNA profiles, associates with outcome and may inform clinical trials of immune checkpoint inhibitors. Overall, this large-scale analysis reveals previously unappreciated sarcoma-type-specific changes in copy number, methylation, RNA, and protein, providing insights into refining sarcoma therapy and relationships to other cancer types

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Abstract A74: Differential Wnt signaling in African American and Caucasian women with triple-negative breast cancer

Abstract In the U.S., breast cancer (BC) incidences among African American (AA) and Caucasian (CA) women are similar, however, AA have a significantly higher mortality rate (20%). In addition, AA women often present with tumors at a younger age, with a higher grade/later stage, and are more likely to be diagnosed with the highly aggressive triple-negative breast cancer (TNBC) subtype. Although multiple factors may contribute to the observed health disparities in TNBC, it is essential that we identify the molecular characteristics and underlying biological differences between CA and AA TNBC. We initially conducted a gene expression study using archived formalin-fixed, paraffin-embedded (FFPE) samples obtained from CA and AA women diagnosed with Node 0 TNBC. Briefly; total RNA was isolated from 10 μm scrolls from each FFPE block, cDNA synthesis, and each sample was hybridized to a breast-enriched gene expression array (Affymetrix, BC DSA Research Tool). Expression analysis was conducted using GeneSpring 12.1 analytical software. PCA analysis revealed that the samples clustered well with respect to ethnicity and unsupervised hierarchical clustering analysis resulted in distinct subgroups based on ethnicity. Differentially expressed genes (DEG) from each cohort were selected using ANOVA analysis (fold change > 2.0, p value <.05) followed by the Benjamin/Hochberg for multiple-testing correction. Finally, the DEG was imported into GeneGo MetaCore, which revealed that the majority of functionally enriched pathways were associated with the Wnt/β-catenin signaling pathway in the AA-TNBC cohort. Additionally, TNC, CAV1, TCF4 and FOX03A, genes associated with this pathway, were significantly upregulated in the AA-TNBC cohort. We have thus far validated these findings for CAV1 and FOX03A by qRTPCR, using an independent cohort of Node 0 TNBC FFPE samples from AA and CA women. These observations are being investigated in-vitro, using a multi-ethnic TNBC cell line panel, and siRNA-mediated knockdown of the Wnt-associated genes. The effects will be evaluated through a series of functional assays including proliferation, migration, invasion, apoptosis, and epithelial-mesenchymal transition (EMT). We believe that our observations suggest that the Wnt/β-catenin pathway may contribute to the more aggressive phenotype observed in AA women diagnosed with TNBC. These studies have important implications for further understanding of TNBC and the associated health disparities that exist in AA women diagnosed with this disease; ultimately leading to the development of targeted therapies for treating this aggressive subtype of BC. Citation Format: Julie E. Getz, Davyd B. Teoh, Sara Nasser, Tembe Waibhav, Legendre R. Christophe, Venkata Yellapantula, Mary Ellen Ahearn, Carmen R. Gomez, Merce Jorda, Mark D. Pegram, John D. Carpten, Lisa L. Baumbach. Differential Wnt signaling in African American and Caucasian women with triple-negative breast cancer. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A74

Abstract C54: High caveolin-1 expression in a cohort of African American women with triple-negative breast cancer

Abstract 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 <.05), resulted in 190 differentially expressed genes (DEG). Pathway enrichment analysis conducted in MetaCore GeneGo, revealed that the DEGs were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC cohort. Furthermore, several genes in the Wnt/β-catenin pathways were over-expressed in the top 10-enrichment pathways. We validated our results using RT-qPCR and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort (p-value 1.22 x 10-05). An independent cohort of FFPE samples, from AA and CA women with early stage TNBC, was used to create a tissue microarray (TMA). Immunohistochemistry results showed no difference in localization of Cav1 between the AA and CA cohorts, however, the AA cohort had significantly higher levels of Cav1 staining (p-value 0.04). Additionally, using RT-qPCR, we demonstrated that CAV1 mRNA was significantly higher in the AA TNBC Cohort (p-value 0.48). Furthermore, endogenous Cav1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, using siRNA, we demonstrated that CAV1 silencing resulted in a significant decrease in cell proliferation, for each of the TNBC cell lines while it showed no effect on the luminal ER+ cell lines. Our combined study results suggest that CAV1 over-expression may be a biological contributor to the observed health disparity between AA women and CA diagnosed with early stage TNBC. 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

V(D)J Sequence Capture for DNA-Based Minimal Residual Disease Detection in Multiple Myeloma

Abstract Introduction Minimal residual disease (MRD) negativity after initial therapy is a strong predictor of survival in multiple myeloma. Tracking of clonal immunoglobulin V(D)J rearrangements by next generation sequencing is highly sensitive for MRD and does not require immediate analysis of fresh samples. However, previous studies have found variable rates of baseline V(D)J sequence capture, which could limit tracking. In this study, we aimed to define the sample-related and disease-related factors that influence V(D)J capture. Methods We included 177 patients with plasma cell myeloma who had available stored mononuclear cells from a baseline bone marrow aspirate. Each sample was sequenced by two assays: The LymphoTrack® VDJ assays from Invivoscribe, and our in-house myeloma panel myTYPE, as a molecular control for detectable tumor derived DNA in the samples. MyTYPE positivity was defined by one or more single nucleotide variant, insertion, deletion, translocation or copy number variation that is known to occur in myeloma. Results and discussion The V(D)J capture rate in our whole cohort was 81 %, as compared with 95 % in the myTYPE positive samples, demonstrating the importance of tumor cell content for V(D)J capture. This was confirmed in multivariate logistic regression (Figure 1), where myTYPE positivity was a strong independent predictor of V(D)J capture success, with an odds ratio (OR) of 6.61 (95 % CI 2.22-24.81, p = 0.002). Plasma cell content estimated from bone marrow aspirate smears also contributed to the multivariate model, with an OR of 1.3 for each 10 % increase in plasma cell content (95 % CI 0.96-1.84, p=0.109), but this did not reach statistical significance after accounting for the strong effect of myTYPE. Finally, having lambda light chain restricted plasma cells was a strong predictor of V(D)J capture success (OR 6.91, 95 % CI 2.4-25.32, p = 0.001). Higher V(D)J capture rate in lambda-restricted myeloma as compared with kappa-restricted was mostly driven by a difference in immunoglobulin kappa gene (IGK) rearrangement capture (73 vs. 44 %, p < 0.001). As a potential explanation, we found up to 4 unique IGK rearrangements that are amenable to capture in lambda-restricted cases, as well as dramatically lower somatic hypermutation (SHM) of the IGK variable region in clonal rearrangements, as compared with kappa-restricted cases. SHM has previously been shown to cause V(D)J capture failure by interfering with PCR primer annealing. Both of these factors can be attributed to IGK inactivation by rearrangements involving the "kappa deleting element" region, affecting both IGK alleles in lambda-restricted plasma cells (Perfetti et al, Immunology, 2004). As an explanation for low plasma cell content in the samples used in this study, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: from the initial pull used for aspirate smear, to the final pull that is commonly used for research. Supporting the important role of hemodilution, we found V(D)J capture rates of 97 % in clinical samples (early pull aspirates) from our institution that were analyzed with the same NGS assays, as long as the bone marrow plasma cell infiltration was above 5 %. V(D)J capture probability appears to be determined by two factors: The abundance of clonal cells (i.e. tumor cell content), and the degree to which clonal sequences can be amplified by the assay (which is negatively affected by SHM). Thus, increasing the tumor cell content in samples as much as practically possible (i.e., optimal bone marrow aspirates and enrichment of CD138+ plasma cells) may compensate for SHM and improve V(D)J capture rates beyond 95 %. Conclusion V(D)J capture rates of at least 95 % are feasible in multiple myeloma using LymphoTrack® NGS assays, when the sample quality is good. The most important reason for V(D)J capture failure is low tumor cell content due to bone marrow aspirate hemodilution. Optimal performance depends on the use of early pull aspirates and/or subsequent tumor cell enrichment. Figure 1: Predicting V(D)J capture. Regression lines and individual data points are colored according to myTYPE status (red = positive; blue = negative); V(D)J capture probability on the y-axis (capture yes/no on the second y-axis) and bone marrow plasma cell percentage by aspirate smear on the x-axis; split into panels according to light chain restriction (right = lambda; left = kappa). Disclosures Ho: Invivoscribe, Inc.: Honoraria. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Jacobsen:Invivoscribe, Inc.: Employment. Huang:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership. Landgren:Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding

Baseline identification of clonal V(D)J sequences for DNA-based minimal residual disease detection in multiple myeloma.

Tracking of clonal immunoglobulin V(D)J rearrangement sequences by next generation sequencing is highly sensitive for minimal residual disease in multiple myeloma. However, previous studies have found variable rates of V(D)J sequence identification at baseline, which could limit tracking. Here, we aimed to define the factors influencing the identification of clonal V(D)J sequences. Bone marrow mononuclear cells from 177 myeloma patients underwent V(D)J sequencing by the LymphoTrack assays (Invivoscribe). As a molecular control for tumor cell content, we sequenced the samples using our in-house myeloma panel myTYPE. V(D)J sequence clonality was identified in 81% of samples overall, as compared with 95% in samples where tumor-derived DNA was detectable by myTYPE. Clonality was detected more frequently in patients with lambda-restricted disease, mainly because of increased detection of kappa gene rearrangements. Finally, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: From the initial pull used for aspirate smear, to the final pull that is commonly used for research. In conclusion, baseline clonality detection rates of 95% or higher are feasible in multiple myeloma. Optimal performance depends on the use of good quality aspirates and/or subsequent tumor cell enrichment