Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

Abstract

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.

Fig. 1.

Significantly mutated genes in 49 patients with DLBCL. (A) The rate of synonymous and nonsynonymous mutations is displayed as mutations per megabase, with individual DLBCL samples ranked by total number of mutations. (B) The heat map represents individual mutations in 49 patient samples, color-coded by type of mutation. Only one mutation per gene is shown if multiple mutations were found in a sample. Left: Histogram shows the number of mutations in each gene. Percentages represent the fraction of tumors with at least one mutation in the specified gene. Right: The 15 genes with the lowest q₁-value, ranked by level of significance. (C) Base substitution distribution of individual samples, ranked in the same order as in A.

Fig. 2.

Somatic mutations in DLBCL affect genes of various classes. Sites of somatic mutations in significantly mutated genes called by analysis pipeline and passing manual review. A diagram of the relative positions of somatic mutations is shown for MLL2, TNFRSF14, BTG1, MEF2B, ACTB, and P2RY8. The type of the mutation is indicated in the key (Bottom). The overall validation rate of mutation calls was 97.9% [of 47 selected mutations tested, only one (ACTB G20A) failed to validate; see Table S5].

Fig. 3.

Selection for functionally inconsequential mutations in BCL2 and sites of somatic mutations in BCL2. Nonsynonymous mutations in BCL2 are preferentially located outside of BH-domains.