Comprehensive Genomic Analysis of Adult Burkitt Lymphoma Identifies the B-Cell Receptor Signaling Pathway As a Potential Therapeutic Target

Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children. Although it accounts for only 1-5% of NHL in adults, approximately 60% of the BL cases diagnosed each year in western countries occur in patients >40 years of age. Although adult and pediatric BL cases are indistinguishable by molecular classification, pediatric patients have a significantly better outcome than adults. While translocation of MYC to the immunoglobulin heavy or light chain genes is characteristic of pediatric and adult BL, genetic differences may contribute to the superior clinical outcome of childhood cases.

Therefore, we aimed to identify the spectrum of additional genetic abnormalities that occur in adult and pediatric BL. Copy number analysis, gene expression profiling (GEP), and targeted sequencing of ~400 genes known to be mutated in NHLs were performed on a cohort of molecularly defined BL samples. Copy number abnormalities (CNAs) were identified by the Affymetrix 250k NspI SNP array in 73 BL tumors (28 adult, 45 pediatric), and sequencing was performed on 52 BLs (21 adult, 31 pediatric). Pediatric cases had fewer CNAs than adults. The most common focal abnormality identified was a gain on 13q31.3 encompassing MIR17HG. It was more frequent in adult compared to pediatric cases (35% vs 16%, p=0.085) and was associated with increased expression of miR-17~92 cluster members; and among adults, patients with this gain trended towards worse overall survival, though the number of cases with available information was small. Gain of 8q was found in ~20% of adult cases, but in no pediatric cases. Surprisingly, cases with 8q gain had significantly lower MYC mRNA expression (p< 0.001) and lower protein expression. In cases with MYC gain 0/4 cases were positive for MYC protein expression by immunohistochemistry; in contrast,6/10 cases with no MYC gain were positive for MYC expression. This suggests that gain of 8q is driven by another gene in the region. Additional genetic alterations included gains of genomic loci encompassing MCL1 and MDM4 (1q21-24) and losses encompassing RB1, p53 and CDKN2A/CDKN2B. Pathway analysis of genes differentially expressed by CN status showed an enrichment of genes involved in cell cycle regulation, the p53 signaling pathway, and the ubiquitin proteasome pathway.

The frequencies of mutations in commonly mutated genes including MYC, ID3, TP53, CCND3, DDX3X, ARID1A, and TCF3 were not significantly different in adult and pediatric BL. However, BCL2, (43%, p<0.001), ZFHX3 (24%, p<0.01), SPTBN5 (20%, p=0.02), RB1 (14%, p=0.06), BTG1 (14%, p=0.06), TCF4 (14%, p=0.06), and TNFRSF14 (14%, p=0.06), were exclusively mutated in adult BL. In contrast, mutations in CDH23 (29% vs 5%, p=0.04) and SMARCA4 (35% vs 19%, p=0.05) were more frequent in pediatric BL. When mutations were placed into oncogenic pathways, mutations in genes regulating the PI3K-AKT pathway did not shown significant differences between adult and pediatric cases. Mutations promoting tonic BCR signaling (TCF3 and ID3) by activation of the PI3K pathway had similar frequencies in the two age groups, however, BCR signaling effectors inducing chronic active NF-kB signaling (CD79A, SYK, MYD88, BCL10, CARD11) were significantly associated with adult BL (adult cases with any mutation: 19% vs 7%).

Gene expression studies suggested activated BCR signaling in BL cases with CN gain of miR-17~92. In vitro analysis of miR17~92 in BL cell lines (n=4) showed that functional loss of miR-17 ~ 92 expression using a miRNA sponge led to reduced proliferation. Treatment of BL cell lines with anti-IgM induced BCR activation in a time- and dosage-dependent manner, as estimated by increased phosphorylation of downstream mediators (SYK and BLNK). This activation was reduced upon loss of functional miR-17 ~ 92 expression in cell lines. Since miR-17~92 can directly inhibit proximal negative regulators of BCR signaling, treatment with the FDA-approved BTK inhibitor, Ibrutnib, further inhibited proliferation of BL cell lines carrying the miRNA sponge. The BCR signaling pathway is one example of how unique abnormalities in adult BL can provide possible targets for therapeutic intervention.