What drives pediatric Burkitt lymphoma? Timing events in the evolution of cancer using single‐cell whole genome sequencing

Introduction: Burkitt lymphoma (BL) is a rare but highly aggressive B-cell non-Hodgkin lymphoma. BL exhibits a characteristic immunophenotype that is positive for pan-B cell markers and CD10. The genetic hallmark of BL is the translocation of the MYC oncogene under the regulation of an immunoglobulin (IG) heavy or light chain enhancer, resulting in MYC protein overexpression. Notably, MYC translocation by itself is not sufficient for BL oncogenesis and a variety of cancer genes are recurrently mutated. BL can be divided epidemiologically but also, more recently, genetically based on driver mutations including DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Despite high survival rates, pediatric BL patients suffer from long-term side effects and relapses are usually fatal. In order to develop more targeted and less toxic therapies, a better understanding is needed of the ethology of the disease. Therefore, our aim is to characterise the cell-of-origin (COO) and dissect the life history of BL subtypes, by pinpointing when during tumorigenesis somatic mutations play a role and by identifying the rate-limiting steps of malignant transformation. Method: We collected single-cell suspensions of BL patient samples, including lymph node biopsies, bone marrow aspirates, as well as ascites and pleural fluid. To perform flow cytometry, single cells were stained with DAPI and a panel of antibodies: CD10/CD45/CD19 or CD10/CD3/CD20. Live B cells were separated with the DAPI-CD45+ CD19+ or DAPI-CD3-CD20+ phenotype. Subsequently, CD10+ B cells and CD10- B cells were sorted in 96-well plates (Figure 1). To confirm the presence or absence of the IG::MYC translocation a PCR was perfomed using patient-specific primers that flank the translocation locus. Whole genome amplification was carried out on the DNA of single cells using the Primary Template-directed Amplification (PTA) technique that was subsequently whole genome sequenced. Results: From a bone marrow sample of a 4-year-old female BL patient we found that 1% of immune cells were CD10+ B cells, while 17% of B cells were negative for the CD10 marker (Figure 2A). In the lymph node sample from the same patient, we found that 87% of B cells were CD10+ and 9% were CD10- (Figure 2B). PCR confirmed the MYC translocation in 2/6 CD10+ and 0/6 CD10- bone marrow-derived B cells (Figure 3). Keywords: Genomics, Epigenomics, and Other -Omics, Non-Hodgkin (Pediatric, Adolescent, and Young Adult), Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.