Abstract 3183: Deep-sequencing reveals distinct clonal evolution patterns of DLBCL relapse and identifies mutant MLL2 as potential early driver.
Authors: Jiang Yanwen , Redmond David , Nie Kui , Martin Peter , Melnick Ari , Tam Wayne , Elemento Olivier 
Published: August 2014

Abstract
Diffuse Large B Cell Lymphoma (DLBCL) is an aggressive lymphoma with 40% of the patients relapse to chemoresistant disease. Our current understanding of the molecular mechanisms associated with relapse is poor. Mainly, how clonal heterogeneity in primary tumors contributes to relapse is unclear. Therefore, we performed high-throughput sequencing of rearranged VDJ junctions in 20 pairs of matched diagnosis-relapse tumors. In DLBCL, VDJ junctions effectively tag clonal tumor populations. Moreover, somatic hypermutations (SHM) within rearranged VDJ provide additional information to study heterogeneity and track evolution. To achieve high coverage, we sequenced (2X150bp) across the 300-350bp VDJ sequences, generating 0.25-0.5 million VDJ junctions per tumor. We developed a custom bioinformatics pipeline to identify the VDJ and SHM patterns of each sequence.
In most pairs (n=19), diagnosis and relapse are clonally related, harboring the same major VDJ rearrangement. Phylogenetic analysis on the SHM profiles of the major VDJ arrangement uncovered two distinct scenarios of DLBCL relapse.
In scenario I (n=17), the major diagnosis and relapse clones clustered together, indicating a linear progression. We performed exome sequencing on 6 such pairs. As expected, diagnosis and relapse tumors harbored many common mutations, including mutations in CARD11, NOTCH1, and TP53. Furthermore, we observed that relapse tumors had acquired additional 70±22 mutations, among which 14±5 are in genes previously found mutated in DLBCLs, including EP300, EZH2, and MYC. Taken together, we hypothesize that in this scenario, a portion of the diagnosis tumor cells survived chemotherapy, acquired additional “facilitator” mutations, and developed into relapse tumors.
In scenario II (n=2), the major relapse clone clustered together with a highly divergent minor diagnosis clone forming a separate branch from the rest of the diagnosis clones on the phylogenetic tree. This particular pattern indicates that in this scenario although the relapse and diagnosis tumors came from the same B cell, they diverged early, acquired different mutations, and rose at different times. Indeed, exome sequencing on these pairs revealed that both diagnosis and relapse tumors had their distinctive DLBCL related mutations. Furthermore, there were several mutations common between diagnosis and relapse tumors, indicating that these mutations were acquired very early in the history of the tumors. Interestingly, MLL2 mutations were found in the common set of mutations in both pairs, strongly suggesting that MLL2 mutation could act as a “driver” or “facilitator” mutation in the earliest steps of DLBCL lymphomagenesis.
Taken together, we discovered two modes of DLBCL relapse by coupling deep-sequencing of the VDJ rearrangements and exomes, providing new knowledge about molecular pathogenesis of DLBCL relapse.
Citation Format: Yanwen Jiang, David Redmond, Kui Nie, Peter Martin, Ari Melnick, Wayne Tam, Olivier Elemento. Deep-sequencing reveals distinct clonal evolution patterns of DLBCL relapse and identifies mutant MLL2 as potential early driver. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2013-3183