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

Tumor suppressor CD99 is downregulated in plasma cell neoplasms lacking CCND1 translocation and distinguishes neoplastic from normal plasma cells and B-cell lymphomas with plasmacytic differentiation from primary plasma cell neoplasms

CD99(MIC2) is a widely expressed cell surface glycoprotein and functions as a tumor suppressor involved in downregulation of SRC family of tyrosine kinase. CD99 expression is tightly regulated through B-cell development. The principal aims of this study were to investigate the clinical utility of CD99 expression (i) in distinguishing normal plasma cells from primary plasma cell neoplasms; (ii) in detection of minimal residual disease in primary plasma cell neoplasms; and (iii) in distinguishing plasma cell component of B-cell lymphomas from primary plasma cell neoplasms. We analyzed expression of CD99 by flow cytometry and immunohistochemistry in lymph nodes, peripheral blood, and bone marrow samples. CD99 showed stage-specific expression with highest expression seen in precursor B and plasma cells. In contrast to the uniform bright expression on normal plasma cells, CD99 expression on neoplastic plasma cells was lost in 39 out of 56 (69.6%) cases. Furthermore, 8 out of 56 samples (14%) showed visibly (>10-fold) reduced CD99 expression. Overall, CD99 expression was informative (absent or visibly dimmer than normal) in 84% of primary plasma cell neoplasm. In the context of minimal residual disease detection, CD99 showed superior utility in separating normal and abnormal plasma cells over currently established antigens CD117, CD81, and CD27 by principal component analysis. Preservation of CD99 expression was strongly associated with cyclin D1 translocation in myeloma (p < 0.05). B-cell lymphomas with plasma cell component could be distinguished from myeloma by CD99 expression. In summary, we established that tumor suppressor CD99 is markedly downregulated in multiple myeloma. The loss is highly specific for identification of abnormal cells in primary plasma cell neoplasms, and can be exploited for diagnostic purposes. The role of CD99 in myeloma pathogenesis requires further investigation

Revealing the Impact of Recurrent and Rare Structural Variations in Multiple Myeloma

Whole genome sequencing (WGS) studies have started to reveal the critical role of structural variants (SVs) in multiple myeloma (MM) pathogenesis and evolution. We have recently revealed the existence of three main classes of complex events in 30 MM patients: chromothripsis, chromoplexy and templated insertions (Maura F et al, Nat Comm, 2019). Here, drawing on a large cohort of 768 MM patients enrolled in the MMRF CoMMpass study (NCT01454297), we comprehensively characterized the landscape of SVs and their functional implications. Low coverage long-insert WGS (median 4-8X) was available from all patients, of whom 591 also had RNAseq data. Overall, we identified a median of 15 total SVs (range 1-253). Fifty-one percent of SVs (n = 8766) were defined as part of complex events, with a median of one per patient (range 0-14). Chromothripsis, chromoplexy and templated insertions involving >2 chromosomes were observed in 21%, 11% and 21 %, respectively. Chromothripsis was the only SV class with clear prognostic implications after adjustment for molecular and clinical features, resulting in adverse PFS (adjusted HR = 1.57; 95% CI 1.13-2.22; p = 0.008) and OS (adjusted HR = 2.4; 95% CI 1.5-3.83; p < 0.001). Templated insertions emerged as the cause of CCND1-IGH and MYC translocations in 34 % and 73 % of cases, respectively. This is particularly important given the capability of templated insertions to connect and amplify multiple regions of the genome, involving several oncogenes and regulatory regions (e.g. super enhancers). Twenty-four patients (3.1 %) had translocation between an immunoglobulin locus and a non-canonical driver gene (e.g. PAX5, CD40 and MAP3K14), showing outlier expression by RNAseq where available. SV hotspot analysis was carried out using the Piecewise Constant Fitting algorithm, comparing the local SV breakpoint density to an empirical background model (Glodzik et al, Nat Genet, 2017). To identify functionally important hotspots, we integrated: 1) local cumulative copy number data, 2) amplification and deletion peaks identified by GISTIC v2 (q < 0.1), 3) gene fusion data and 4) differential expression analysis with adjustment for main molecular subgroups (limma; Bonferroni-Holm adjusted p-values < 0.01). Ninety-eight hotspots were identified (Figure 1), of which 71 (72%) have not previously been reported. Among these novel hotspots, 23 (33 %) contained a known or suspected driver gene, including TNFRSF17 (encoding CAR-T target BCMA), SYK (BCR signal transduction) and KLF2 (key myeloma transcription factor and germline predisposition locus). Active enhancer regions were present in 29 of the novel hotspots (41 %), including 65 % of those with a concurrent putative driver gene involved. For 34 hotspots (48 %) no clear target gene or regulatory region emerged. SV hotspots and GISTIC peaks covered 13 % of the genome. Overall 38 % of simple and complex SVs had at least one breakpoint falling within a recurrently involved region. The majority of chromoplexy, chromothripsis and templated insertions involved recurrent regions (64, 76 and 86 %, respectively). Simple events were most commonly rare, ranging from 74 % of deletions to 45 % for translocations. Quantifying the global functional impact of the remaining 72 % of non-recurrent or rare SVs, we observed that genes involved by a rare SV were significantly enriched for outlier expression (z-score +/- 2) compared to a permutation background model. Rare deletions and duplications exerted their effects within 10 Kb of the gene body. Translocations and templated insertions were associated with overexpression up to 1 Mb from the gene, but had no effect when involving the gene body, consistent with a major enhancer hijacking mechanism. Finally, we sought to understand the role of recurrent and rare SVs in evolutionary dynamics, analyzing 27 patients that progressed with branching evolution. Seventy-two acquired SVs involved a hotspot region (42 driver and/or enhancer; 48 unknown), while 328 were rare. In conclusion, the SV landscape in multiple myeloma is characterized by multiple recurrently involved genes and regulatory regions. These regions account for the majority of complex SVs, indicating strong positive selection of these events. Nonetheless, the majority of SVs remain unaccounted for. Rare SVs were associated with outlier gene expression and may contribute to the tumor evolutionary trajectory of individual patients. Disclosures Papaemmanuil: Celgene: Research Funding. Landgren:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees