Subtyping Burkitt Lymphoma by DNA Methylation

Burkitt lymphoma (BL) is an aggressive germinal center B-cell-derived malignancy. Historically, sporadic, endemic, and immunodeficiency-associated variants were distinguished, which differ in the frequency of Epstein–Barr virus (EBV) positivity. Aiming to identify subgroups based on DNA methylation patterns, we here profiled 96 BL cases, 17 BL cell lines, and six EBV-transformed lymphoblastoid cell lines using Illumina BeadChip arrays. DNA methylation analyses clustered the cases into four subgroups: two containing mostly EBV-positive cases (BL-mC1, BL-mC2) and two containing mostly EBV-negative cases (BL-mC3, BL-mC4). The subgroups BL-mC1/2, enriched for EBV-positive cases, showed increased DNA methylation, epigenetic age, and, in part, proliferation history compared to BL-mC3/4. CpGs hypermethylated in EBV-positive BLs were enriched for polycomb repressive complex 2 marks, while the CpGs hypomethylated in EBV-negative BLs were linked to, for example, B-cell receptor signaling. EBV-associated hypermethylation affected regulatory regions of genes frequently mutated in BL (e.g., CCND3, TP53) and impacted superenhancers. This finding suggests that hypermethylation may compensate for the lower mutational burden of pathogenic drivers in EBV-positive BLs. Though minor, significant differences were also observed between EBV-positive endemic and sporadic cases (e.g., at the SOX11 and RUNX1 loci). Our findings suggest that EBV status, rather than epidemiological variants, drives the DNA methylation-based subgrouping of BL.