Signatures of biological heterogeneity associated with treatment outcome in diffuse large B-cell lymphoma.

7063 Background: Genetic subtypes of diffuse large B-cell lymphoma (DLBCL) capture biological differences between tumors that influence the response to immunochemotherapy (Schmitz et al., NEJM 2018). However, nearly 40% of DLBCL cases remain unclassified. The addition of gene expression signatures can accelerate classification and inform therapeutic intervention. Methods: We generated single-cell RNA sequencing of 103 DLBCL patient biopsies from Weill Cornell Medicine, New York Presbyterian Hospital, and the National Institutes of Health. We also utilized bulk genomic data from a discovery cohort (n=311, Ennishi et al., J Clin Oncol 2019) and a validation cohort (n=574, Schmitz et al., NEJM 2018). Results: Single cell sequencing allowed us to isolate the malignant B cells and develop genetic subtype signatures. The MCD signature was associated with poor overall survival (p<10 -6 ), as was the BN2 signature within ABC tumors (p<10 -3 ). When applied to unclassified tumors, the subtype signatures identified characteristic genetic alterations including SLC1A5 mutations in MCD (p<10 -5 ), UBE2A mutations in BN2 (p<10 -13 ), C10orf12 truncations and copy number loss in EZB (p<10 -6 ), and SGK1 mutations in ST2 (p<10 -7 ). Moreover, we discovered that most DLBCL tumors (80%) contained two or more genetic subclones (median 2, range 1-5) based on DNA copy number differences. The genetic subclones had distinct phenotypes based on expression of six recurrent gene expression meta-signatures, herein termed themes. The germinal center (GC) B cell, memory B cell, plasma cell, and pan-B cell themes reflect B cell differentiation whereas two other themes – cell cycle and cell growth – reflect proliferative and metabolic states that are independent of the differentiation states. Surprisingly, 23% of DLBCL (24/103) harbored genetic subclones expressing B cell differentiation themes that distinguished them from other malignant cells in the same tumor. The GC B cell theme was associated with a favorable response to R-CHOP chemotherapy (p<0.02), as expected, while the cell growth theme (but not the cell cycle theme) was associated with adverse survival (p<0.02). Conclusions: Our study revealed that genetic subtypes have distinct gene expression signatures. We further demonstrated a role for tumor subclones in generating intra-tumoral biological diversity. We developed signatures of inter and intra-tumoral heterogeneity that are associated with overall survival.