Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin Lymphoma, with ~4,400 Canadians diagnosed annually. DLBCL patients are treated with a standard frontline immunochemotherapy (R-CHOP) comprised of several chemotherapeutics and the monoclonal antibody rituximab, which binds to the B-cell surface marker CD20. While R-CHOP is generally effective for DLBCL, for patients where frontline treatment fails (relapsed-refractory DLBCL, rrDLBCL), prognosis is poor with a median survival time of 6 months. Although numerous rrDLBCL salvage therapies have been developed, their efficacies have been limited partially due to the heterogeneity of DLBCL and an incomplete understanding of the genomic features associated with relapse disease. We hypothesize that the genomic landscape of rrDLBCL will be distinct from diagnostic DLBCL and contain recurrent mutations contributing to both treatment failure and advanced disease. To this end, we performed ultra-deep targeted sequencing of 63 genes (CAPP-Seq) on 135 rrDLBCL liquid biopsies and found six genes significantly enriched for mutations at relapse. KMT2D and TP53 were mutated in half of all rrDLBCL samples, with TP53 enriched for dominant negative hotspot mutations. 8% of rrDLBCL cases harbour MS4A1 mutations, which encodes CD20. Mutations in MS4A1 were clonally selected following treatment and in vitro attenuated the binding of rituximab and other anti-CD20 antibodies, including those undergoing clinical trials. To expand upon these findings, we performed whole exome sequencing on 155 rrDLBCL samples and found mutations in TET2 and TMEM30A significantly depleted at relapse which, in conjunction with enrichment of mutations in KMT2D and CREBBP, suggest broad epigenetic changes in rrDLBCL. Using additional copy number information from 77 rrDLBCL liquid biopsies (n=222) we observed a high burden of copy number variants in rrDLBCL and novel recurrent deletions of RNA regulators HNRNPU and HNRNPD, the MHC Class I regulator IRF2, and recurrent gains involving the B-cell proliferation factors IKZF3 and TCF3, representing candidate therapeutic targets. 13 regions were significantly enriched for events in rrDLBCL, including these novel events and others regulating apoptosis (TP53, PTEN, BCL2) and proliferative signaling (MIR17HG, BCL6). Overall, we have further characterized the genetics of rrDLBCL and identified mechanisms of treatment resistance and possible therapeutic targets.
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Thesis advisor: Morin, Ryan
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