MYC, BCL2 and BCL6 co-localisation patterns at single-cell resolution underlie their prognostic significance in diffuse large B-cell lymphoma

BackgroundCancers often overexpress multiple clinically relevant oncogenes. However, it is not known if multiple oncogenes within a cancer combine uniquely in specific cellular sub-populations to influence clinical outcome. We studied this phenomenon using the prognostically relevant oncogenes MYC, BCL2 and BCL6 in Diffuse Large B-Cell Lymphoma (DLBCL). MethodsQuantitative multispectral imaging simultaneously measured oncogene co-expression at single-cell resolution in reactive lymphoid tissue (n=12) and four independent cohorts (n=409) of DLBCL. Mathematically derived co-expression phenotypes were evaluated in DLBCLs with immunohistochemistry (n=316) and eight DLBCL cohorts with gene expression data (n=4186). Bulk and single-cell RNA sequencing was performed on patient-derived B-cells with induced co-expression of MYC, BCL2 and BCL6. ResultsUnlike in non-malignant lymphoid tissue where the co-expression of MYC, BCL2 and BCL6 in a B-cell is limited, DLBCLs show multiple permutations of oncogenic co-expression in malignant B-cells. The percentage of cells with a unique combination MYC+BCL2+BCL6-(M+2+6-) consistently predicts survival in contrast to that of other combinations (including M+2+6+). An estimated percentage of M+2+6-cells can be derived from any quantitative measurement of the component individual oncogenes, and correlates with survival in immunohistochemistry and gene expression datasets. Comparative transcriptomic analysis of DLBCLs and transformed patient-derived B-cells identifies cyclin D2 (CCND2) as a potential BCL6-repressed regulator of proliferation in the M+2+6-population. ConclusionsUnique patterns of oncogene co-expression at single-cell resolution affect clinical outcomes in DLBCL. Similar analyses evaluating oncogenic combinations at the cellular level may impact diagnostics and target discovery in other cancers.