The Atlas of Blood Cancer Genomes (ABCG) Project: A Comprehensive Molecular Characterization of Leukemias and Lymphomas

Abstract Introduction Blood cancers are collectively common and strikingly heterogeneous diseases both clinically and molecularly. According to the WHO taxonomy, there are over 100 distinct myeloid and lymphoid neoplasms. Genomic profiling of blood cancers has been applied in a somewhat ad hoc fashion using diverse sequencing approaches including the use of targeted panels, whole exome sequencing, whole genome sequencing, RNA sequencing, etc. The lack of data uniformity has made it difficult to comprehensively understand the clinical and molecular spectrum within and across diseases. Systematic genomic approaches can address the central challenges in the diagnosis and treatment of blood cancers. For the diagnosis of blood cancers, the incorporation of genomics could greatly enhance the accuracy and speed of clinical diagnostics. Genomics could also inform their pathology classification. However, these applications must be preceded by a clear understanding of the particular genetic aberrations and expression profiles that unite and distinguish different leukemias and lymphomas. Therapeutic development can also be aided by genomic approaches through identification of new targets and establishing the relevance of existing targets and treatments. Targeted therapies including those directed at specific surface markers (e.g. CD19, CD30 and CD123) or molecular targets (e.g. BCR-ABL fusions, IDH1 mutations and EZH2 mutations) are rarely restricted to a single disease, with most occurring in multiple blood cancers. A systematic understanding of the presence or overlap of these targets within or across blood cancers would significantly expand the therapeutic possibilities and better enable the use of existing therapies in both common and rare cancers. However, such therapeutic possibilities need to be established through a rigorous, data-driven approach. We initiated the Atlas of Blood Cancers Genomes (ABCG) project to systematically elucidate the molecular basis of all leukemias and lymphomas by building upon advances in genomic technologies, our capabilities for data analysis and economies of scale. Using a uniform approach to systematically profile all blood cancers through DNA and RNA sequencing at the whole exome/whole transcriptome level, we aim to link genomic events with clinical outcomes, disease categories and subcategories, thereby providing a complete molecular blueprint of blood cancers. Methods/Results The ABCG project consists of collaborators from 25 institutions around the world who have collectively contributed samples from 10,481 patients comprising every type of blood cancer in the current WHO classification. The samples include thousands of myeloid leukemias and mature B cell lymphomas, hundreds of Hodgkin lymphoma and plasma cell myeloma, as well as every rare type of hematologic malignancy (along with case-matched normal tissue). All cases were de-identified and their associated pathology and detailed clinical information entered into a purpose-built web-based system that included disease-specific data templates. All cases were subjected to centralized pathology review and clinical data review by experienced hematopathologists and oncologists. All 10,481cases are being sequenced at the DNA and RNA level, and are being profiled to define the genetic alterations and expression changes that are characteristic of each disease. Analysis will include translocations, copy number alterations, and viral status. These molecular features will be examined in conjunction with genetic events, pathologic factors, and the clinical features. We have already generated results for ALK-negative anaplastic large B cell lymphoma and primary mediastinal B cell lymphomas (N=210). These data demonstrate novel subgroup and molecular discoveries that are enabled by integrative DNA and RNA sequencing analysis and the examination of molecular features across different diseases as well as within individual entities. In addition, other disease entities and the collective data will be presented in the meeting. Conclusion The ABCG project will comprehensively study the genetic and clinicopathological features of all blood cancers using systematic genomic approaches. We anticipate our data, approaches and results will serve as a lasting resource for the molecular classification and therapeutic development for leukemias and lymphomas. Disclosures McKinney: Novartis: Research Funding; Nordic Nanovector: Research Funding; Molecular Templates: Consultancy, Research Funding; Kite/Gilead: Honoraria, Speakers Bureau; Incyte: Research Funding; Genetech: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy; Celgene: Consultancy, Research Funding; BTG: Consultancy; Beigene: Research Funding; ADC Therapeutics: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Verastem: Consultancy. Behdad: Lilly: Speakers Bureau; Roche/Foundation Medicine: Speakers Bureau; Thermo Fisher: Speakers Bureau.

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Similarity Search Methods As an Alternative to Sub-Type Characterisation in Aggressive Lymphomas

Blood ◽

10.1182/blood.v128.22.3052.3052 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3052-3052

Author(s):

Chulin Sha ◽

Sharon Barrans ◽

Andrew Jack ◽

Cathy H Burton ◽

Alexandra Smith ◽

...

Keyword(s):

B Cell ◽

Similarity Search ◽

Research Funding ◽

Molecular Level ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Research Network ◽

Molecular Features ◽

Unfavourable Prognosis ◽

Treatment Responses

Abstract Aggressive B-cell non-Hodgkin lymphomas are haematological malignancies that account for significant morbidity and mortality worldwide. They encompass a wide range of histological and clinical features: Diffuse large B-cell lymphoma (DLBCL) represents the most frequent subtype and Burkitt lymphoma (BL) occurs less frequently. Recent advances in molecular profiling have demonstrated considerable heterogeneity at the molecular level, and further classified DLBCL into germinal centre B-cell-like (GCB), activated B-cell-like (ABC), primary mediastinal B-cell lymphoma (PMBL) and type III subgroups, where ABC associates with the most unfavourable prognosis. Gene-expression profiling (GEP) also confirmed a subgroup with features intermediate between BL and DLBCL, and these cases particularly that have concurrent chromosomal rearrangements of MYC and BCL2are often associated with poor prognosis. Despite the progress made from GEP, the classification still has limited influence on clinical treatment decision-making. In fact, as more heterogeneity beyond the subtypes above has been discovered by recent next-generation sequencing studies the approach of dividing cases into limited subgroups makes less sense in clinical practice. It is clear that the subtypes overlap to an extent in the affected signalling and regulatory pathways, and that small groupings within subtypes exhibit clear mechanistic differences and treatment responses. We describe an alternative approach, where a large database of aggressive B-cell lymphomas is used in a similarity search to identify those cases most similar at a molecular level to a query case. The hypothesis is that the most similar cases provide the best guide to prognosis and treatment outcome in the query case, independent of any need to place the query case into a particular subtype. We used both large public datasets and data from our Haematological Malignancy Research Network (www.HMRN.org) to explore genes associated with pathogenic pathways and an unfavourable prognosis. We also defined similarity between cases according to their molecular features and treatment responses. We then trained the similarity search method, by employing a distance metric learning approach that has been successfully used in similar machine learning applications, to test our HMRN dataset which contains detailed clinical data with treatment and outcome information. The cross-validation result on the public dataset achieved nearly 90% accuracy in recognizing cases with similar overall survival, and initial test results on HMRN data also shows that over 80% cases can be correctly represented by similar cases. In summary, we present a similarity learning method as an alternative to the current sub-type classification method. This mathematical method is able to accurately recognise cases with similar molecular features and provides important information on predicted treatment response for any given query case. Disclosures Smith: Novartis: Research Funding; Celgene: Research Funding; Jansen Cilag: Research Funding; Amgen: Research Funding.

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Predictors of Relapse and Survival Following Autologous Stem Cell Transplant in Patients with Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2021-146734 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1832-1832

Author(s):

Marie Hu ◽

Marcus P Watkins ◽

Qing Cao ◽

Saba Raya ◽

David A. Russler-Germain ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Standard Of Care ◽

Disease Status ◽

Advisory Committees ◽

Factors Associated ◽

Molecular Features

Abstract Background: Although the majority of patients with diffuse large B-cell lymphoma (DLBCL) can be cured with intensive chemotherapy and rituximab, 30-40% of patients will be refractory to or relapse after first line treatment. For these patients, the current standard of care is salvage chemotherapy followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT). Prior studies have largely examined clinical risk factors associated with higher risk of relapse after ASCT; however, there is limited data integrating both pathologic and molecular features. Thus, we aimed to identify high-risk features associated with relapse and survival after ASCT using a combination of clinical, molecular, pathologic, and transplant characteristics. Methods: We retrospectively analyzed the medical records of all adult patients with DLBCL who underwent ASCT at our two institutions from 2010 to 2020. Patients with primary CNS lymphoma, primary mediastinal B-cell lymphoma, or Burkitt lymphoma were excluded. We analyzed demographics, clinical characteristics, cell of origin (COO) by immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH) testing, and treatment/transplant characteristics. The primary endpoints were 3-year progression-free survival (PFS) and overall survival (OS) from ASCT. The Kaplan-Meier method was used to estimate survival with univariate and multivariate Cox proportional hazards regression performed to identify factors associating with PFS and OS, summarized using hazard ratios (HR) with 95% confidence intervals (CI). Results: A total of 235 DLBCL patients underwent ASCT from 2010 to 2020. Median age at ASCT was 61 years (range: 25-75) and 63% were male. At DLBCL diagnosis, 80% had advanced stage disease, 74% had extranodal involvement, 13% had poor performance status, and 65% had elevated lactate dehydrogenase (LDH). 71 patients (30%) had a prior or concurrent indolent lymphoma diagnosis indicating transformed disease. Of the patients with available COO and molecular data, 115 (60%) had germinal center B-cell (GCB) phenotype by IHC, 10 (6%) had a single MYC rearrangement by FISH, and 35 (22%) had MYC plus BCL2 and/or BCL6 rearrangements (DHL/THL). After first-line treatment, 12% remained refractory and 62% later relapsed at a median of 13 months (range: 1-240). Patients received a median of 2 (range: 1-5) lines of treatment pre-ASCT. At time of ASCT, 66% were in complete response (CR) and 32% were in partial response (PR) by standard of care imaging and response criteria. With median follow-up of 5.2 years from time of ASCT, 98 patients (42%) relapsed and 78 (33%) died. 3-year PFS and OS were 58% (95% CI 51-64%) and 74% (95% CI 67-79%), respectively. In univariate analysis, factors associated with worse PFS and worse OS included 3 or more lines of treatment pre-ASCT (p<0.01 for both) and non-CR at ASCT (p<0.01 for both) (Figure 1A and B). Transformed disease was also associated with worse PFS (p=0.03). In multivariate analysis, non-CR at ASCT remained significant (HR 2.22, 95% 1.26-3.90, p<0.01) for worse OS, along with non-GCB COO (HR 1.81, 95% CI 1.03-3.18, p=0.04) and age >60 at ASCT (HR 1.92, 95% CI 1.06-3.47, p=0.03) (Figure 1C). Stratifying by COO and disease status at transplant, 3-year OS was best in the GCB/CR group (84%, 95% CI 73-90%), while worse but similar in the GCB/non-CR and non-GCB/CR groups (68%, 95% CI 51-80% and 71%, 95% CI 56-83%, respectively) (Figure 1D). The non-GCB/non-CR group had the worst 3-year OS (48%, 95% CI 27-67%). No individual factors beyond CR/non-CR at ASCT were associated with worse 3-year PFS. Notably, DHL/THL patients (77% of whom were in CR at time of ASCT) had similar PFS (p=0.08) and OS (p=0.30) to non-DHL/THL patients, suggesting that response to therapy may be more prognostic than high-risk molecular features alone. Conclusions: This analysis indicated that factors associated with OS after ASCT were disease status at time of transplant and COO, with non-GCB patients not in CR having the poorest outcomes. GCB patients not in CR were still able to be cured by ASCT at a high rate. Molecular rearrangements including DHL/THL were not prognostic, although interpretation may be limited by the modest number of DHL/THL patients. These findings may inform which patients should undergo ASCT, while the highest risk group may be better treated with alternatives including novel targeted agents or chimeric antigen receptor cell therapy. Figure 1 Figure 1. Disclosures Bachanova: FATE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; KaryoPharma: Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fehniger: Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; OrcaBio: Other; Kiadis: Other; HCW Biologics: Research Funding; Indapta: Other. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding.

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Burkitt Lymphoma Genome Sequencing Project (BLGSP): Introduction

Blood ◽

10.1182/blood.v128.22.1760.1760 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1760-1760 ◽

Cited By ~ 1

Author(s):

Daniela S Gerhard ◽

Bruno Grande ◽

Nicholas Griner ◽

Corey Casper ◽

Sarah E. Gerdts ◽

...

Keyword(s):

Rna Sequencing ◽

Molecular Characterization ◽

Genome Sequencing ◽

Burkitt Lymphoma ◽

Research Funding ◽

Somatic Mutations ◽

Rna Seq ◽

Dna And Rna ◽

Pathology Review

Abstract Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.

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Genomic and Transcriptional Characterization of Primary Mediastinal Large B Cell Lymphoma

Blood ◽

10.1182/blood-2021-149745 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2398-2398

Author(s):

Rebecca J Leeman-Neill ◽

Devang Thakkar ◽

Sarah L. Ondrejka ◽

Eric D. Hsi ◽

Amy Chadburn ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Research Funding ◽

Immune Escape ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Dna And Rna ◽

Large B Cell Lymphoma ◽

Recurrent Mutations ◽

Large B Cell

Abstract Introduction: Primary mediastinal large B-cell lymphoma (PMBL) is a rare non-Hodgkin lymphoma subtype that occurs predominantly in young adults, with an overall favorable prognosis. The cell of origin is presumed to be thymic medullary B-cells and the gene expression profile of PMBL is similar to classic Hodgkin lymphoma. Recent studies have begun unravelling the genomic alterations underlying PMBL. Frequent, recurrent mutations (e.g. B2M, TNFAIP3, SOCS1, STAT6, GNA13) have been reported, but most of the studies have analyzed a small number of cases. To gain further insights into disease biology, we recruited 63 cases of PMBL as part of the Atlas of Blood Cancer Genomes (ABC-G) initiative, a consortium consisting of 25 institutions. Methods: Formalin-fixed paraffin-embedded (FFPE) biopsies and clinical data were collected. All cases were subjected to centralized review by an experienced panel of hematopathologists to ensure accurate diagnosis. Whole-exome DNA and RNA sequencing was performed using the Illumina platform and the DNA and RNA reads aligned to the GRCh38 genome and transcriptome respectively. Exonic variants were filtered using a set of paired normal samples and population-based databases to identify putative driver mutations, which were then aggregated at the gene level. Mutational analysis was performed on 56 samples that passed quality filtering and expression analysis on 45 samples. RNAseq data was normalized using DESeq2. Results: The cohort included samples from 16 males and 24 females, with a median age of 33 years (range 16 - 72) at the time of diagnosis. The majority of patients were treated with R-CHOP (47%) or R-EPOCH (43%), with 93% of patients surviving through the end of follow-up (median follow-up: 60.1 months). Besides the known recurrent mutations involving the JAK-STAT (STAT6 -21%, SOCS1 - 26%), NFKB (TNFAIP3 - 27%, NFKB1A - 7%), immune escape (B2M - 20%, LTB - 11%, IRF8 - 9%, IRF4 -9%), and chromatin modification (ZNF217 - 16%, CREBBP - 11%, KMT2D -11%) pathways , we discovered recurrent somatic variants in novel candidate driver genes in this disease, including NOTCH4 (7%), DICER1 (11%), MCL1 (7%), amongst others. EZH2, EP300, and XPO1 mutations were not detected. CIITA mutations and fusions were observed in 14% and 11% of cases, respectively, with novel partner genes (IGHA2, IGHG1, CDC6) detected in 67% of the fusion positive cases. Copy number alterations included gains at 2p16.1 (REL - 20%) and 9p24.2 (JAK2/PDL1/PDL2 - 24%), as well as loci not previously implicated in PMBL, 8q24.3 and 9q34.3 (each in 20%). Of note, CIITA alterations and 9p24 gains were virtually mutually exclusive, highlighting diverse mechanisms of immune escape in this entity. The transcriptomes of cases harboring CIITA alterations demonstrated differential enrichment of genes involved in protein glycosylation. The PMBLs in our series showed significant enrichment of the reported PMBL genetic classifier score, compared to nodal diffuse large B cell lymphoma (DLBCL) (p=0.0003). Finally, the gene expression profile of thymic B cells was more similar to that of PMBL than nodal DLBCL (p=0.0144). Conclusions: Our study, representing one of the largest comprehensive genomic and transcriptomic analyses of PMBL, expands the mutational landscape of PMBL, provides evidence for biologically distinct disease subsets and suggests an origin of PMBLs from thymic B-cells. Disclosures Hsi: AbbVie: Research Funding; Eli Lilly: Research Funding; Cytomx: Honoraria; Seattle Genetics: Honoraria. McKinney: BTG: Consultancy; Celgene: Consultancy, Research Funding; Epizyme: Consultancy; Genetech: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria, Speakers Bureau; Molecular Templates: Consultancy, Research Funding; Nordic Nanovector: Research Funding; Novartis: Research Funding; Pharmacyclics: Consultancy; Verastem: Consultancy; Beigene: Research Funding; ADC Therapeutics: Consultancy, Speakers Bureau. Jaye: Stemline Therapeutics: Honoraria. Cohen: Genentech, Takeda, BMS/Celgene, BioInvent, LAM, Astra Zeneca, Novartis, Loxo/Lilly: Research Funding; Janssen, Adaptive, Aptitude Health, BeiGene, Cellectar, Adicet, Loxo/Lilly, AStra ZenecaKite/Gilead: Consultancy. Behdad: Lilly: Speakers Bureau; Roche/Foundation Medicine: Speakers Bureau; Thermo Fisher: Speakers Bureau. Dave: Data Driven Bioscience: Current equity holder in publicly-traded company.

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Whole Genome Sequencing of B-Cell Neoplasms with t(14;19)(q32;q13) Reveals Different Entities

Blood ◽

10.1182/blood-2021-150891 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3709-3709

Author(s):

Anna Carbó-Meix ◽

Francesca Guijarro ◽

Luojun Wang ◽

Romina Royo ◽

Isabel Granada ◽

...

Keyword(s):

B Cell ◽

Base Pair ◽

Research Funding ◽

Differential Expression Analysis ◽

Lymphocytic Leukemia ◽

Whole Genome ◽

Rna Seq ◽

Molecular Features ◽

Genomic Complexity ◽

Mutational Status

Abstract Introduction: The t(14;19)(q32;q13) is a rare cytogenetic abnormality found in <0.1% of all B-cell neoplasms. The molecular features of this translocation are not well characterized. IGH-BCL3 rearrangement has been found in some tumors identified as "atypical" chronic lymphocytic leukemia (CLL) with aggressive clinical evolution. This translocation has also been observed in other B-cell neoplasms without clear evidence of the target gene. The mechanisms generating this translocation, the genomic profile of alterations of these cases, and whether different molecular features may be associated with specific entities are not known. Aim: To elucidate the genomic features of B-cell neoplasms carrying the t(14;19) and their relationship to pathological characteristics of the tumors. Materials and methods: We sequenced the whole-genome (WGS) of 13 cases in which the t(14;19) had been identified by conventional cytogenetics and/or FISH using a BCL3 break-apart probe. In six of these cases we performed RNA-seq. Pathological and clinical revision was conducted in all cases, 8 of them with tissue biopsies. Results: The breakpoints of the t(14;19) were characterized at base-pair resolution using WGS. All breakpoints in chr14 were found within any of the class switch recombination (CSR) regions suggesting an aberrant CSR as the mechanism causing this alteration. The breakpoints on chr19 were found upstream (13 kb) the 5' untranslated region (UTR) of BCL3 in 8/13 (61.5%) cases. One additional case had the breakpoint further upstream (49 kb) of BCL3 truncating CEACAM16. The four remaining cases had breakpoints downstream of BCL3; two cases within CBLC, one in BCAM, and one after NECTIN2. Of note, the further upstream BCL3 case and the downstream BCL3 cases had mutated IGHV, while all upstream BCL3 cases had unmutated IGHV. Based on RNA-seq data, all upstream BCL3 cases (n=5) showed an upregulation of BCL3, while one downstream case with RNA-seq available showed upregulation of NECTIN2 and low levels of BCL3. The pathology review identified the four downstream BCL3 cases as marginal zone lymphomas whereas the cases with breakpoints upstream BCL3 (n=3 with tissue available) and the case further upstream BCL3 were classified as "atypical" CLL. We next characterized the genomic landscape of these tumors based on the breakpoint on chr19 (upstream and downstream BCL3). The analysis of the WGS showed a lower number of mutations, copy number alterations (CNA), and structural variants (SV) in the upstream BCL3 group compared to the downstream BCL3 cases (mean of 2429.5 vs 6271.7 somatic mutations, 3.1 vs 11.7 CNA, and 4.4 vs 18 SV, respectively). In terms of specific driver mutations, the downstream BCL3 group carried mutations in genes previously described in MZL, such as KMT2D, NOTCH2, or KLF2 found in two cases. All but one case with the breakpoint upstream BCL3 carried trisomy 12 (tri12), which was absent in all cases with a downstream breakpoint. Finally, we performed a differential expression analysis between 5 atypical CLL cases with BCL3 rearrangements vs 4 CLL without t(14;19) [all unmutated IGHV]. This analysis showed 578 genes upregulated and 720 genes downregulated in the BCL3-rearranged cases (q <0.05), including remarkable differences in the expression of previously described CLL hallmark genes, such as upregulation of EBF1 and downregulation of LEF1, FMOD, ADTRP, CLNK, IGSF3, TCF4. An analysis of the RNA-seq data of 294 CLL cases lacking the t(14;19) (Puente et al., Nature 2015) indicated that this transcriptional program was not related to IGHV mutational status nor to the presence of tri12. Nonetheless, we identified a small set of tri12 mutated IGHV CLL lacking the t(14;19) with a similar modulation of the expression of the above hallmark genes. Conclusions: We have characterized the breakpoints of the t(14;19) at base-pair resolution and evidenced marked molecular and pathological differences of the tumors according to the location of the breakpoint. Tumors carrying the breakpoint downstream BCL3 exhibit a higher genomic complexity, driver alterations, and pathological features corresponding to MZL. Contrarily, tumors with the breakpoint upstream of BCL3 upregulate BCL3 and display lower genomic complexity as well as CLL-like features. Nonetheless, these cases have a different gene expression profile compared to conventional CLL characterized by LEF1 downregulation and EBF1 overexpression. Disclosures Navarro: Nocartis: Honoraria; Roche: Honoraria; EUSA: Consultancy, Research Funding; Pharma: Consultancy; GILEAD: Research Funding; Pharma: Research Funding.

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RNA Sequencing of Hodgkin Lymphoma Reed-Sternberg Cells Uncovers a Plasma Cell Signature and Escape from NK Cell Recognition

Blood ◽

10.1182/blood-2019-131163 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 549-549 ◽

Cited By ~ 1

Author(s):

Ethel Cesarman ◽

Mikhail Roshal ◽

Jonathan Reichel ◽

Wagner Florian ◽

Bhavneet Binder ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Rna Sequencing ◽

Cell Lines ◽

Plasma Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Provision Of Services ◽

Slam Family

Introduction: Previous gene expression profiling studies of classical Hodgkin lymphoma (cHL) have been confined to cell lines and microdissected HRS cells from tissue biopsies given the difficulty of isolating sparse Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. We previously used flow sorting to separate HRS cells from fresh or viably frozen cHL biopsies, and performed the first full exome sequencing of HRS cells. Here we report use of the same cell separation approach to examine the HRS cell transcriptome using RNA sequencing. Methods: We used flow cytometric cell sorting and low-input RNA sequencing to generate full transcriptome data from viable primary HRS cells, along with intratumor B cells. Nine primary cases of cHL and four cell lines were assessed for RNA expression, expressed mutations, cell type of origin, signaling pathways, gene fusions and pathogen identification. We used immunohistochemistry to evaluate expression of PDIA6 and CD48 in the 9 cases sequenced and a tissue microarray containing 16 additional cases of cHL. Flow cytometry for CD48 was performed in two cell lines and 5 primary cases. Results: Clustering show that primary HRS cells have a transcriptional profile that is unique, and different from that of intratumoral B cells, as well as cHL cell lines. Comparison of HRS cells with normal cellular subsets indicated plasma cell differentiation, suggesting that the cell of origin is a B cell on its way to becoming a plasma cell. Clustering with B cells showed much lower similarity. Consistent with plasma cell differentiation, we uncovered an unfolded protein response UPR) signature, shared with plasma cell neoplasms and, to a lesser extent, activated B cell (ABC) diffuse large B cell lymphoma, but not other B cell lymphoma types, including primary mediastinal B cell lymphoma (PMBCL). Among other UPR response genes, PDIA6 showed strong downregulation at the RNA level (2.4 logFC, p=9.4E-17). This finding was validated by immunohistochemistry for PDIA6, which showed strong positivity in the HRS cells of all 25 cases examined, confirming that this is a common feature of cHL, including nodular sclerosis and mixed cellularity subtypes. Top upregulated genes included those involved in oncogenesis (HGF/MET, NFkB/apoptosis inhibition), stem cell differentiation (homeobox genes MEIS1 and PBX1), and mitotic checkpoints, mitotic spindle formation and DNA repair, possibly explaining the unique nuclear morphology of HRS cells. Downregulation of MHC-1 and MHC-2 driven antigen processing and presentation was confirmed, and so was overexpression of PDL1 (CD274). Importantly, we detected loss of SLAM family receptors, which serve as activation signals for NK cells providing an additional mechanism for tumor immune evasion. One of these is CD48 (-2.63 logFC, p=1.56E-05), which was confirmed to be strongly downregulated. This finding was confirmed by immunohistochemistry (25 cases) and flow cytometry (2 cell lines and 5 primary cases) on the expanded sample set. Given that only some cHL cases are associated with EBV infection, it has been speculated that other viruses are involved in negative cases. However, our analysis did not reveal additional viruses in the HRS cells. Conclusions: Our data indicate that cHL more closely resembles plasma cells than B cells, and plasma cell malignancies than other lymphomas. The salient feature of plasmacytic differentiation is a UPR, which is seen in HRS cells and multiple myeloma. In contrast, UPR is not a feature of primary mediastinal B cell lymphoma, which is thought to be the DLBCL most similar to cHL clinically, immunophenotypically and in terms of gene expression patterns. We also provide an integrated view of potential immune evasion mechanisms by HRS cells that potentially explain lack of anti-tumor T or innate response. These include lack of antigen presentation due to B2M mutations, overexpression of PDL1 and PDL2, immunosuppressive cytokine secretion and, for the first time, a demonstration of lack of NK activating receptors of the SLAM family. Lack of SLAM family receptors may explain lack of NK cells clearance of HRS cells in the face of MHC-I downregulation. It has long been suspected that cHL is a tumor where there likely exists a previously undiscovered virus in addition to EBV, but RNA sequencing failed to reveal additional infectious transcripts in the HRS cells. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Brody:Kite Pharma: Research Funding; Celldex Therapeutics: Research Funding; Genentech: Research Funding; Acerta Pharma: Research Funding; Oncovir, Inc.: Research Funding; BMS: Research Funding; Merck: Research Funding. Dave:Data Driven Bioscience: Equity Ownership.

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Higher Stability of Mutant mRNA As Compared to Wild-Type mRNA in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2019-128516 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1499-1499

Author(s):

Maher Albitar ◽

Zijun Yidan Xu-Monette ◽

Wanlong Ma ◽

Yingjun Wang ◽

Deng Manman ◽

...

Keyword(s):

Board Of Directors ◽

Rna Sequencing ◽

Allele Frequency ◽

Research Funding ◽

B Cell Lymphoma ◽

Variant Allele ◽

Mrna Levels ◽

Variant Allele Frequency ◽

Advisory Committees ◽

Dna And Rna

Cellular RNA levels are tightly regulated by very complex nuclear and cytoplasmic processes. The regulation of mutant mRNA in cancer cells is rarely studied. We explored the effects of mutations on mRNA levels in patients with diffuse large B-cell lymphoma (DLBCL). Using next generation sequencing (NGS) and variant allele frequency (VAF) of mutant RNA, we compared relative mutant mRNA or variant allele frequency (RNA-VAF) with variant allele frequency of mutant DNA (DNA-VAF) in the same samples from patients with DLBCL. Methods: RNA and DNA were extracted from 427 FFPE samples from patients with DLBCL. We sequenced the DNA using 177 gene panel and the RNA using 1408 gene panel. The DNA sequencing is based on Single Primer Extension (SPE) library preparation with unique molecular identifier (UMI) (Qiagen, Germantown, MD). The RNA sequencing is based on hybrid capture. Sequencing data of DNA is analyzed using the DRAGEN Platform. Sequence duplicates were removed before calculating VAF. The RNA sequencing data is analyzed using Illumina basespace. RNA VAF is calculated also after removing duplicates using Isaac variant caller. Only mutations detected by both DNA and RNA variant callers are compared. Results: A total of 1770 mutations were detected using the DNA panel and 2207 mutations were detected using the larger RNA sequencing panel. We focused on the most commonly mutated genes that included in both DNA and RNA panels and compared the VAF of the same mutations between DNA and RNA. The selected genes are: KMT2D, NOTCH2, CARD11, MYC, MYD88, EZH2, TP53, CD79B, BCL2, and TET2. The overall VAF in the RNA was significantly higher (P<0.00001) (median:43.9%, minimum: 6%, maximum: 100%) as compared with that of the DNA (median: 28.8%, minimum: 3.5%, maximum: 95%). When each gene is considered individual, all genes showed significantly higher VAF in RNA as compared with DNA. As expected some mutations were detected in DNA, but not in in RNA and vice versa. However, the number of mutations detected in these 10 genes using DNA sequencing was significantly (P= 0.0001) higher (#658) as compared with mutations detected in RNA (#471). Most of the missed mutations by RNA were termination mutations. The most striking RNA-missed mutations were in NOTCH2. The DNA testing showed 81 mutations, while the RNA testing listed only 19 mutations. Almost all NOTCH2 mutations missed by RNA sequencing wer Pro6ArgfsTer27, which leads to early termination of mRNA (loss of function). When we looked at overall NOTCH2 mRNA levels, NOTCH2 mRNA was significantly higher (P=0.002, Kruskal-Wallis ANOVA) in samples with NOTCH2 mutation detected in both DNA and RNA as compared with mutations detected in DNA only. The NOTCH2 mRNA levels were also lower in samples with mutations detected in DNA only (P=0.046) as compared with wild-type NOTCH2. Conclusion: This data suggests that stability of mutant mRNA is significantly higher for most mutations and most genes. However, there are exceptions, especially when the mutations are termination at early amino acid. NOTCH2 pro6ArgfsTer27 mutation is an example of early termination of transcription, which leads to significant instability and reduction in NOTCH2 mRNA levels acting as a tumor suppressor, while other mutations in the gene lead to over expression and more oncogenenic function. This data suggests that molecular profiling of cancer should include evaluating RNA mutations and expression levels and not all mutations detected in a gene are the same. Furthermore, increased stability of most mutant mRNA may have some implication on methods used for detected minimal residual disease. Figure Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Jazz: Consultancy; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2. Piris:Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees. Kantarjian:Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; Novartis: Research Funding; Jazz Pharma: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Research Funding; Astex: Research Funding; Takeda: Honoraria; BMS: Research Funding; Cyclacel: Research Funding; Ariad: Research Funding.

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Advancing precision medicine in clinical oncology: Whole exome paired tumor-normal DNA and RNA sequencing at a single-institution cancer center.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e14006 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e14006-e14006

Author(s):

Kevin McDonnell ◽

Amit Kulkarni ◽

Melissa Woodhouse ◽

Sidney A Smith ◽

Christine Hong ◽

...

Keyword(s):

Clinical Trial ◽

Precision Medicine ◽

Rna Sequencing ◽

Clinical Oncology ◽

Tumor Board ◽

Cancer Center ◽

Rna Seq ◽

Dna And Rna ◽

Whole Exome ◽

Board Review

e14006 Background: Next generation sequencing (NGS) allows for reliable, comprehensive and cost-effective identification of clinically actionable genetic and genomic alterations. The increasing adoption of NGS in clinical oncology has increased our ability to identify germline alterations predisposing to cancer development as well as somatic changes enabling prescription of individualized cancer treatment and enhanced clinical trial participation. Here we summarize implementation of an NGS-based precision medicine initiative involving oncology patients from a single institution cancer center. Methods: IRB-approved NGS matched whole exome (WES) germline and solid tumor somatic tumor sequencing together with somatic tumor RNA sequencing (RNA-seq) were performed using germline DNA extracted from peripheral blood lymphocytes and nucleic acids for tumor DNA and RNA sequencing obtained from formalin-fixed, paraffin-embedded tumor specimens. Results of sequencing and analyses were presented to a multi-disciplinary tumor board to establish recommendations for management of germline pathogenic variation, therapeutic drug matching, clinical trials eligibility and molecularly informed patient prognosis. Results: A total of 1,005 patients completed sequencing. Germline and somatic WES exceeded 100X and 250X mean target coverage, respectively; somatic RNA-seq exceeded 200 million mean reads. Patients ranged in age from 17 to 90 years. The study cohort comprised comparable numbers of female (51%) and male (49%) patients. Ethnicities and races were broadly represented with 22% of participants identifying as Hispanic, 14% as Asian, 4% as Black, 55% as Non-Hispanic White and 5% as other. The most common solid tumor histological classification was colorectal (18%), followed by breast (16%), prostate (7%), head and neck (7%), sarcoma (7%), ovarian (5%), melanoma (4%) and lung (3%). Bioinformatic analyses and precision medicine tumor board review established that 12% of patients harbored a germline pathogenic variant and 43% carried clinically actionable genetic/genomic alterations; a majority of patients met molecular requirements for participation in a clinical trial. Conclusions: This study confirms the feasibility and utility of clinical NGS and precision medicine tumor board review in clinical oncology to identify germline genetic pathology, deliver personalized cancer therapeutics, increase clinical trial enrollment and clarify diagnosis and prognosis.

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Asymptomatic Multiple Myeloma - Background of Progression, Evolution, and Prognosis

Blood ◽

10.1182/blood.v128.22.235.235 ◽

2016 ◽

Vol 128 (22) ◽

pp. 235-235 ◽

Cited By ~ 3

Author(s):

Anja Seckinger ◽

Anna Jauch ◽

Martina Emde ◽

Susanne Beck ◽

Marcel Mohr ◽

...

Keyword(s):

Rna Sequencing ◽

Plasma Cell ◽

Research Funding ◽

De Novo ◽

Accumulation Rate ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Paired Samples ◽

Whole Exome ◽

Cell Accumulation

Abstract BACKGROUND. Asymptomatic multiple myeloma (AMM) evolves from monoclonal gammopathy of unknown significance (MGUS) and progresses to symptomatic myeloma characterized by end-organ damage. Aim of our study was to address the determinants of evolution and progression of AMM, their molecular background, and whether they are present upfront or evolve de novoin a multistep process on the background of an ongoing genetic instability. METHODS . CD138-purified plasma cell samples of 2369 consecutive patients with MGUS, asymptomatic, and symptomatic myeloma were investigated by fluorescence-in-situ-hybridization (n=304/432/1633), 951 (n=62/259/630) by gene expression profiling. Sixty-five paired samples at AMM and disease progression were assessed by iFISH, 28 of these were further assessed by array-comparative-genomic-hybridization, as well as whole exome- (WES), and RNA-sequencing. Serum/urine samples (n=8398) allowed modelling of plasma cell accumulation in AMM and MGUS, respectively (n=322/196). RESULTS . Up-front tumor mass, plasma cell accumulation rate and molecular characteristics, including alterations in gene expression and presence of progression-associated chromosomal aberrations, i.e. t(4;14), deletions of 13q14, 17p13, 8p21, gains of 1q21, as well as hyperdiploidy, drive and predict evolution and progression of AMM. But for hyperdiploidy, the same factors drive progression from symptomatic to relapsed myeloma and also in AMM rather their number than the specific single aberration impact on time to progression. This means that the mechanisms driving progression to symptomatic myeloma are (at least in part) the same driving progression under treatment. Molecularly, all chromosomal aberrations, most transcriptomic changes, and most frequent mutations detected in symptomatic myeloma including NRAS, KRAS, DIS3, HIST1H1E are already present in MGUS or AMM. In paired AMM/MM samples, 22/27 (81%) show a stable clonal pattern, 5/27 (19%) the de novo appearance of expressed clones, including KRAS or FAM46C. No significant transcriptomic differences are found by RNA-sequencing. (Sub-)Clonal complexity with 4-5 discernable clusters of 103-363 single nucleotide variants with an allele frequency of ≥10% remains fairly constant during disease progression with most being detectable in both AMM and MM, incompatible with clonal outgrowth to any reason in these patients. In CONCLUSION, evolution and progression of AMM are driven and can be well predicted by factors being present upfront, i.e. tumor mass, plasma cell accumulation rate, and the set of molecular alterations. Progression is, contrary to current thinking, in the vast majority of patients not driven by de novo acquired expressed clonal alterations. This is proven in our set of paired samples on the level of chromosomal numeric or structural alterations (as per iFISH and aCGH), expressed clonal single nucleotide variants (as per whole exome- and RNA-sequencing), and remaining subclonal complexity. This in turn disproves other de novo alterations (e.g. methylation), as the subclone harboring these would then need to become clonal. Disclosures Hillengass: Sanofi: Research Funding; Amgen: Consultancy, Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hose:Takeda: Other: Travel grant; EngMab: Research Funding; Sanofi: Research Funding.

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RPS15 and TP53 Co-Mutation Drives B Cell Malignancy through Altered Translation and MYC Activation in a Murine Model

Blood ◽

10.1182/blood-2020-138900 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Catherine Gutierrez ◽

Tamara Ouspenskaia ◽

Doris Fu ◽

Peyton Waddicor ◽

Anat Biran ◽

...

Keyword(s):

B Cell ◽

Rna Sequencing ◽

Research Funding ◽

Target Genes ◽

Mrna Translation ◽

Gene Set Enrichment Analysis ◽

Mutant Mice ◽

Enrichment Score ◽

Publicly Traded ◽

The Impact

Amongst the novel putative drivers identified by large-scale sequencing studies of chronic lymphocytic leukemia (CLL) is the ribosomal protein RPS15. Mutated in 5.3% of CLL, it co-occurs with heterozygous TP53 alterations in 36% of RPS15-mutated samples. Mutation of this mediator of ribosome maturation and translation is associated with poor disease prognosis and enriched in cohorts with del(17p) and relapsed CLL, suggesting a role in disease progression and therapeutic resistance. However, the impact of RPS15 mutation on B cell function and CLL development, in the presence or absence of TP53 mutation, has yet to be characterized. To this end, we developed overexpression HG3 CLL cell lines modeling four common RPS15 mutations (G134R, H137Y, S138F, and S139F) and a conditional knock-in mouse model of the S138F mutation with and without heterozygous Trp53 deletion (generated by crossing Rps15 and Trp53 mutant mice with Cd19-Cre mice). To characterize the impact of RPS15 mutation on transcription, we performed RNA-sequencing on splenic B cells from 3-month-old Rps15WT, Rps15Het and Rps15Hom mice (3 per cohort). We identified 255 and 670 upregulated and 596 and 777 downregulated genes in the Rps15MT vs Rps15WT mice (Rps15Het and Rps15Hom, respectively; log2FC>0.5, p<0.05). Gene set enrichment analysis (GSEA) revealed strong enrichment for MYC target genes that was also evident upon RNA-sequencing of the HG3 RPS15-S138F MT vs WT overexpression lines, and of 3 primary untreated CLLs with heterozygous RPS15 mutation (compared to 3 RPS15WT CLLs of similar genetic background). Pathway analysis of differentially expressed signatures across murine, cell line and primary CLL models revealed a common enrichment in translational machinery, such as mRNA splicing/processing, rRNA processing, and snRNP assembly (normalized enrichment score>1, nominal p-value<0.05). To evaluate whether RPS15 mutant proteins incorporate into ribosomes, we performed polysome profiling of the HG3 lines. All overexpressed RPS15-WT and MT proteins were observed to integrate into the small ribosomal subunit and mature ribosomes, potentially impacting translation. Next, ribosome profiling of HG3 RPS15-WT and S138F cells revealed 2,334 genes with differential translation efficiency (TE) between RPS15-S138F vs WT cells and 2,425 genes between RPS15-S138F vs WT in TP53 knock-out cells (log2FC>0.5, p<0.05). GSEA of differentially translated genes in RPS15 MT- vs WT cells revealed a strong enrichment for TP53-related genes, consistent with the activation of stress pathways by RPS15 mutant expression. RPS15 MT- vs WT cells with TP53-deletion, however, exhibited a strong increase in TE of MYC target genes and components of the ribosomal machinery. This finding suggests that loss of TP53 surveillance allows RPS15 MT cells to induce MYC-mediated changes in mRNA processing and translation - potentially setting the stage for oncogenesis. To determine whether Rps15 mutation can drive CLL-like disease, we engineered 6 novel mouse lines with B cell restricted expression of alterations through crossing with CD19-Cre mice: Rps15WT, Rps15Het, and Rps15Hom mutant mice alone or co-expressing Trp53 deletion. We detected circulating CLL-like (B220+CD5+) cells in 5 of 30 (17%) Rps15Het mice by 20 months of age, but not in 30 age-matched Rps15WT mice. We also detected CLL-like cells in 6 of 30 (20%) Trp53+/- mice by 17 months, indicating that Trp53 deletion alone can induce CLL-like disease. Interestingly, we found CLL-like cells in 2 of 30 Rps15Het/Trp53+/- mice as early as 15 months of age. The cohorts of Rps15Hom and Rps15Hom/Trp53+/- mice, however, have been monitored for 18 months of age with no disease occurrences, indicating that a double dosage of Rps15 mutation may be detrimental to disease formation. Altogether, Rps15 heterozygous mutation can drive CLL development in mice, and our early data hint that co-mutation with Trp53 may shorten the latency of CLL-like disease. Overall, RPS15 mutant protein can incorporate into the ribosome and induce changes in mRNA translation, resulting in MYC activation predominantly in the context of TP53 loss. Our mouse studies indicate that mut-Rps15 drives CLL development, with a more aggressive disease course when combined with Trp53 deletion. Our results collectively suggest that RPS15 and TP53 co-mutation drives CLL development through translational dysregulation and MYC-mediated signaling. Disclosures Neuberg: Pharmacyclics: Research Funding; Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company. Getz:Broad Institute: Patents & Royalties: MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, POLYSOLVER and TensorQTL; Pharmacyclics: Research Funding; IBM: Research Funding; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

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