Deep Sequencing Reveals Clonal Evolution Patterns and Mutation Events Associated With Relapse In B Cell Lymphomas

Abstract

Roughly 30% of Diffuse Large B-cell Lymphoma (DLBCL) patients do not respond to standard treatment or relapse after initial therapy. Relapsed DLBCL treatments are limited and less effective, highlighting the need for new therapies. Unfortunately, our current understanding of the molecular mechanisms of DLBCL relapse is poor. We hypothesize that clonal heterogeneity may contribute to disease progression. Here, we sought to explore patterns of clonal heterogeneity and evolution during DLBCL relapse by exploiting unique features of B-cell lymphomas: VDJ recombination and somatic hypermutation (SHM); and to identify genetic events underling DLBCL relapse. We used high-throughput sequencing of PCR amplified IGH VDJ sequences in 17 diagnosis(D)-relapse(R) DLBCL pairs using Illumina MiSeq pair-end 2x150bp approach, which retrieved sequence information of almost the entire VDJ amplicon. Upon aligning reads to germline sequences we identified 0.46±0.16 million VDJs per tumor. The D and R tumors of all but one pair harbored the same major VDJ rearrangement, confirming they were clonally related regardless of the time to relapse (0.5-10 yr). We first compared VDJ clonal heterogeneity of each D and R tumor pair. Although the number of unique rearrangements per sample varied, there was no significant difference between D and R samples (8.0±9.2 vs 7.6±8.9 per 10K mapped reads, p=0.84, paired t-test). In addition to rearrangement, DLBCL VDJ sequences contain various degrees of SHM. Using SHM position information on each VDJ sequence, we delineated tumor subclonal VDJ population, and uncovered two distinct modes of DLBCL relapse by performing phylogenetic analysis of the subclonal VDJ population between the D and R samples of each pair. In scenario I (n=6), the major R subclones clustered in a separate branch from the D subclones (Divergent, Figure 1A), indicating that although R and D tumors were derived from the same B-cell (same VDJ recombination), their precursors diverged early, acquired different SHMs, and expanded separately. In the more frequent scenario II (n=10), the dominant D and R subclones clustered together, and shared the majority of the same SHMs (Linear, Figure 1B), suggesting the R tumor arose directly from the major D subclone following a linear fashion. Moreover, by taking into consideration of the subclone number, frequency and SHM degree, we compared the diversity of D tumor subclone structure (empirical entropy) between the divergent and linear groups, and observed that overall divergent D tumors had a more diverse pattern of subclones than that in linear D tumors (p=3e-06, t-test), indicating that subclonal heterogeneity of the D tumor could be used to predict relapse modes.Figure 1Figure 1. To further investigate these evolutionary patterns, we performed exome-seq on 8 pairs for which we had sufficient tissue (5 linear, 3 divergent). By comparing R to matched D sample, we found that linear mode R tumors gained roughly 4 times more coding-region single nucleotide variations (SNVs) than they lost (5.2±1.9 fold), while the divergent mode R tumors gained and lost similar number of SNVs (1.4±0.7 fold). These results are consistent with our VDJ phylogenetic analysis that tumors evolve divergently undergo evolution of their genomes in parallel and acquire different sets of mutations independently; whereas tumors evolve linearly only acquire additional mutations at relapse. Finally, exome-seq revealed potential molecular mechanisms of lymphomagenesis and relapse. First, 5 R samples had genetic lesions of CD58 or B2M, two genes involving in immune surveillance escape, suggesting that escaping immune surveillance via genetic alteration may be a common relapse strategy. Moreover, in all three divergent pairs, there were histone modifier mutations shared between D and R tumors, including mutations in MLL2, EP300, and SETDB1, suggesting that these mutations could act as early “drivers” or “facilitators” to establish aberrant epigenetic landscape in tumor initiating cells favoring malignant transformation. Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor mutations in relapse. Disclosures: Martin: Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau.

Oncogenic events rather than antigen selection pressure may be the main driving forces for relapse in diffuse large B‐cell lymphomas

“…Our analysis relied on a combined approach to evaluate intra‐tumoral diversity, using Ig‐HTS, cytogenetics and targeted sequencing of a panel of 34 genes relevant for lymphomagenesis. Using Ig‐HTS, we first confirmed the previous findings that DLBCL relapse can emerge according to two scenarios: an early divergent scenario, where the relapsing clone independently evolves from a common progenitor, and a late divergent scenario, where the relapsing clone usually derives from the diagnostic clone. Combining targeted HTS and cytogenetics to Ig‐HTS we showed oncogenetic divergent evolution in the IGHV early divergent group but, interestingly, we also found such evolution in 2/7 IGHV late divergent cases.…”

“…2). As previously described , we observed no difference in the prevalence of the two relapsing modes according to COO subtype, a trend that should be confirmed in larger series. Considering the statistical limitations due the size of our series, the type of divergence did not significantly influence the elapsed time between diagnosis and relapse either (respectively 55.0 ± 42.7 and 23.9 ± 19.8 months for early and late divergence, Mann–Whitney U test: P value = 0.20) (Fig.…”

“…Interestingly, we found here that discrepancies could occur in IGHV late divergent group, highlighting that relapses appeared to be the consequence of selective pressure following lymphoma treatment. Jiang et al also exploited early divergent pairs and ultradeep sequencing to demonstrate that epigenetic alterations might represent one of the earliest genetic events promoting DLBCLs. The sequencing depth used in this study did not allow enough sensitivity to clearly highlight the precocity of such genetic alterations.…”

“…The two modes of divergence at relapse had previously been demonstrated in B‐cell Non Hodgkin Lymphomas (B‐NHLs) harboring a BCL2‐IgH translocation by Ruminy et al and other authors in FLs, using either analysis of Sµ mutations patterns, V(D)J Sanger sequencing , Ig‐HTS , BCL2 mutations or whole exome sequencing . Similarly to what had been shown by Jiang et al , our results demonstrated that evolution of Ig SHM, SNVs and CNVs, explored by HTS, as well as cytogenetic abnormalities, was parallel in early divergent relapsed DLBCLs. Interestingly, we found here that discrepancies could occur in IGHV late divergent group, highlighting that relapses appeared to be the consequence of selective pressure following lymphoma treatment.…”

“…Indeed, FLs could relapse either according a direct and rapid evolution from the dominant clone or following an indirect evolution arising from earlier subclones, usually after years of remission . Very recently, a similar scenario has been demonstrated in DLBLCs . Using Ig high throughput sequencing (Ig‐HTS), the authors clearly showed that DLBCL relapse could also arise either from a distantly‐related clone independently evolved from a common precursor (early divergence), or from a clone more closely related to that of initial diagnosis (late divergence).…”