Patterns of oncogene co-expression at single-cell resolution influence clinical outcome in Diffuse Large B-Cell Lymphoma.

Intratumor heterogeneity (ITH) in oncogene expression is common in cancer, but it is not known if oncogenes exert combinatorial effects at the single cell level to influence clinical outcome. We address this question using quantitative spectral imaging to simultaneously measure the cellular co-expression of the oncogenes MYC, BCL2 and BCL6 in clinically annotated cohorts of Diffuse Large B-Cell Lymphoma (DLBCL). Unlike in non-malignant lymphoid tissue, where the co-expression of these oncogenes is spatially constrained, DLBCL samples show multiple permutations of oncogenic co-expression at the single cell level. Oncogene co-expression follows clustered and non-random spatial distribution, and is typically stable across different regions of a tumour. Interestingly, we noted that the extent of cells with the unique oncogene combination MYC+BCL2+BCL6- (M+2+6-) associates consistently with patient survival across three independent DLBCL cohorts. We then show that the fraction of M+2+6- co-expressing cells can be inferred from quantitative single oncogene data, as the overall frequencies of co-expression of these oncogenes are stochastic. Predicted values of M+2+6- cellular co-expression frequency from immunohistochemistry of MYC/BCL2/BCL6 (n=316) and multiple independent gene expression datasets (n=2522; 8 cohorts) consistently correlate with survival after R-CHOP chemotherapy, offering a novel strategy for identification of high-risk DLBCL. Finally, we use comparative RNAseq analysis of patient-derived M+2+6+ and M+2+6- B-cells to identify cyclin D2 as a potential BCL6-repressed mediator of the aggressive behavior of M+2+6- population. Overall, our work demonstrates that patterns of oncogene co-expression analyzed at single-cell resolution are clinically relevant, with implications for both diagnostics and target discovery in other cancer types. Primary germinal centre B-cells derived from fresh tonsil of independent donors were isolated (Caeser et al. 2019 Nat Commun) and either MYC and BCL2 or MYC, BCL2 and BCL6 were overexpressed. Four samples of each condition were assessed for gene-expression by RNA sequencing.