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Patterns of genetic alterations in B-NHL
Introduction
Non-Hodgkin lymphomas (NHL) are a heterogeneous group of lymphoid malignancies that predominantly arise from mature B cells (B-NHL). Although mature B-cell neo- plasms encompass 38 unique diagnostic subtypes, over 85% of cases fall within only seven histologies.1,2 Recent next-generation sequencing studies have shed light onto the key driver mutations in many of these B-NHL subtypes; for example, large studies of diffuse large B-cell lymphoma (DLBCL) have led to proposed genomic subtypes that have unique etiologies.3-5 However, many less common NHL subtypes such as mantle cell lymphoma (MCL) have not been as extensively characterized.6,7 Furthermore, until recently3,4 genetic alterations have been considered in a binary fashion as either driver events, which directly pro- mote disease genesis or progression, or passenger events, which have little or no impact on disease biology. In con- trast to this principle, most B-NHL do not result from a sin- gle dominant driver but instead result from the serial acqui- sition of genetic alterations that cooperate in lymphomage- nesis.8 The genetic context of each mutation likely deter- mines its oncogenic potential, and groups of mutations should therefore be considered collectively rather than as singular events. For example, the ‘C5’ and ‘MCD’ clusters identified in DLBCL by Chapuy et al. and Schmitz et al., respectively, are characterized by the co-occurrence of CD79B and MYD88 mutations.3,4 In animal models, the Myd88 L252P mutation (equivalent to human L265P) was found to promote downregulation of surface IgM and a phenotype resembling B-cell anergy.9 However, this effect could be rescued by the Cd79b mutation, showing that these co-occurring mutations cooperate.9 The characteriza- tion of other significantly co-occurring genetic alterations are therefore likely to reveal additional important coopera- tive relationships. We approached this challenge by per- forming genomic profiling of 685 B-NHL across different subtypes. Through this cross-sectional analysis we charac- terized genomic hallmarks of B-NHL and sets of significant- ly co-associated events that likely represent subtype-specif- ic cooperating genetic alterations. This study therefore pro- vides new insight into how co-occurring clusters of genetic alterations may contribute to molecularly and phenotypi- cally distinct subtypes of B-NHL.
Methods
An overview of our approach is shown in Online Supplementary Figure S1. For detailed methods, please refer to the Online Supplementary Information.
Tumor DNA samples
We collected DNA from 685 B-NHL tumors, including 199 FL, 196 MCL, 148 DLBCL, 107 BL, 21 high-grade B-cell lymphoma not otherwise specified (HGBL-NOS), and 14 high-grade B-cell lymphoma with MYC, BCL2 and/or BCL6 rearrangement (DHL) (Online Supplementary Table S1). All samples were archival and de- identified. The study was approved by the institutional review board of the University of Nebraska Medical Center (203-15-EP) and performed in accordance with the Declaration of Helsinki. A total of 462 samples were obtained from the University of Nebraska Medical Center, and were prioritized for inclusion in this study if they had previously undergone pathology review and been interrogated by Affymetrix U133 Plus 2.0 gene expression
microarrays10-12 (n=284). An additional series of 223 formalin-fixed paraffin-embedded tumors were provided by other centers. Samples were de-identified and accompanied by the patients’ diagnosis from the medical records, plus overall survival time and status when available. Medical record diagnosis was used in all cases except for those with fluorescence in situ hybridization (FISH) showing translocations in MYC and BCL2 and/or BCL6, which were amended to DHL. Sequencing results for a subset of 52 BL tumors were described previously.13 All MCL samples were either positive for CCND1 translocation by FISH or positive for CCND1 protein expression by immunohistochemistry, depending on the diagnostic practices of the contributing institution.
Next-generation sequencing
A total of 500-1000 ng of genomic DNA was sonicated using a Covaris S2 Ultrasonicator, and libraries prepared using KAPA Hyper Prep Kits (Roche) with TruSeq Adapters (Bioo Scientific) and a maximum of eight cycles of polymerase chain reaction (average of 4 cycles). Libraries were qualified by TapeStation 4200, quantified by Qubit and 10- to 12-plexed for hybrid capture. Each multiplexed library was enriched using our custom LymphoSeq panel encompassing the full coding sequences of 380 genes that were determined to be somatically mutated in B-cell lymphoma (Online Supplementary Table S2, Online Supplementary Methods), as well as tiling recurrent translocation breakpoints. Enrichments were amplified with four to eight cycles of polymerase chain reaction and sequenced on a single lane of an Illumina HiSeq 4000 with 100PE reads in high-output mode at the Hudson Alpha Institute for Biotechnology or the MD Anderson Sequencing and Microarray Facility. Variants were called using our previously validated ensemble approach,13,14 germline poly- morphisms were filtered using dbSNP annotation and the EXAC dataset containing 60,706 healthy individuals,15 and significantly mutated genes were defined by MutSig2CV.16 Copy number alterations (CNA) identified by CopyWriteR,17 which was vali- dated using three FL tumors with matched Affymetrix 250K SNP array (Online Supplementary Figure S2), and significant DNA CNA were determined by GISTIC2.18 Translocations were called using FACTERA,19 which we previously validated against MYC translocation status determined by FISH.20 Mutation and CNA data are publicly viewable through cBioPortal: https://www.cbio- portal.org/study/summary?id=mbn_mdacc_2013. Matched gene expression microarray data are available through the Gene Expression Omnibus, accession GSE132929. For further details, refer to the Online Supplementary Methods.
Results
Identification of significantly mutated genes and structural alterations
We used a 380-gene custom targeted sequencing approach, LymphoSeq, to interrogate the genomes of 685 mature B-NHL, sequencing to an average depth of 578X (minimum, 101X; maximum, 1785X) (Online Supplementary Table S1) for a total yield of 1.81 Tbp. Somatic nucleotide variants and small insertions/deletions were identified using an ensemble approach that we have previously vali- dated14 (Online Supplementary Table S3) and significantly mutated genes were identified using MutSig2CV (Online Supplementary Table S4). Matched germline DNA was avail- able from purified T cells of 20 tumors (11 FL and 9 MCL) and sequenced to validate the filtering of germline variants; 0/632 variants called within these tumors were identified in the matched germline samples, which indicates that the fil-
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