Next Generation Sequencing of B Cell Antigen Receptors Expressed in African Endemic Burkitt Lymphoma

The endemic form of Burkitt lymphoma (eBL), the most common pediatric cancer in sub-Saharan Africa, is the prototypic infection-related malignancy. Virtually 100% of eBL tumor cells carry Epstein-Barr virus (EBV) DNA, and eBL is closely ecologically associated with holoendemic P. falciparum malaria. The cell that undergoes transformation in eBL is an antigen-experienced B lymphocyte that has reached the germinal center and initiated the process of somatic hypermutation (SHM). The tumor cells in eBL express functional B-cell antigen receptors (BCRs), most commonly of the IgM isotype. The rearranged immunoglobulin heavy (IGH) and light (IGκ/λ) chain genes that encode Burkitt BCRs show evidence of SHM, demonstrating that eBL cells have uncoupled the processes of class switch recombination and SHM. Several lines of evidence suggest that BCR signaling pathways are active in eBL cells and that BCR signaling may contribute to the pathogenesis and maintenance of the disease. The uniquely rearranged BCR genes in eBL also represent a tumor-specific molecular signature that can be used to detect and quantitate eBL tumor cells in different tissue compartments. Therefore, Burkitt-associated BCRs have potential as both a disease biomarker and a therapeutic target.

We utilized next-generation sequencing (NGS) to identify BCR gene rearrangements in eBL tumor cells obtained at the time of diagnosis from 22 patients, ages 4 to 12 (median: 7 years), with histologically confirmed Burkitt lymphoma who presented to the Uganda Cancer Institute in Kampala, Uganda. Thirteen of the patients were male and 9 were female; 3 patients were HIV-positive. Genomic DNA was isolated from cryopreserved tumor biopsies, and NGS of the IGH and IGκ/λ loci was performed to identify dominant BCR rearrangements in the eBL tumor cells. Sequence reads spanned a 130-nucleotide interval from the middle of framework region 3 (FR3) in the V gene segment to the 5’ region of the J gene segment. The reads captured the entirety of the CDR3 region. A single dominant IGH V-D-J rearrangement comprising >35% of all sequence reads was identified in 16 of the 22 samples. A single dominant IGκ or IGλ rearrangement was likewise seen in most cases with a single dominant IGH rearrangement. Two dominant but independent light chain rearrangements were seen in three eBL cases with a single dominant IGH rearrangement; whether both productive light chains are expressed at the RNA level is under active investigation. The utilization of IGH V- and J-gene segments in Burkitt-associated BCRs from this cohort of 22 patients was highly non-uniform, with utilization of IGHJ04-01 observed in 10 of the 16 cases with a single dominant IGH rearrangement. Analysis of BCR SHM patterns has revealed enrichment in CDRs as compared to FRs, suggesting antigen selection in eBL tumor cells, as well as a higher than expected rate of somatic mutations that create potential N-linked glycosylation sites. Deep sequencing of DNA extracted from peripheral blood mononuclear cells (PBMC) obtained at the time of diagnosis from 13 of the 22 patients identified sequences that were identical to the dominant IGH sequences observed in each patient’s tumor in 9 cases (69%). 5 of the 9 patients had early stage (Ziegler A) disease, demonstrating that Burkitt lymphoma cells commonly circulate in the blood.

Current studies include sequencing of rearranged IGH genes present in PBMC after completion of primary therapy to determine if response is correlated with disappearance of the putative tumor-associated BCR sequences. Each eBL tumor sample is undergoing RNA analysis to confirm expression of the established dominant IGH and IGκ/λ rearrangements, as well as to determine the dominant BCR isotype. The complete light and heavy chain variable region sequences of the tumor-associated BCRs are being cloned by PCR with V gene leader peptide- and CDR3-specific primers to enable comprehensive assessment of SHM and BCR stereotypy. Identification of complete variable region sequences will also lay the foundation for synthesis of recombinant full-length membrane-associated and soluble forms of Burkitt BCRs. Reconstitution of Burkitt BCRs in vitro will allow evaluation of their antigenic specificity and signaling properties.