• High-throughput sequencing of primary African Burkitt lymphoma tumors suggests disrupted immunoglobulin rearrangements in BL progenitors.• Extensive mutation of expressed and nonexpressed IGH rearrangements suggests multiple active mutational processes in BL tumors. suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.
We thank Ralf Küppers for his interest in our article 1 and for his thoughtful commentary. 2 We address each of the points he raises below. First, Küppers claims that, in contrast to what has been demonstrated in mice,3 not all normal human B-cell progenitors undergo concurrent biallelic D H J H rearrangement during B-cell development, and he cites an analysis performed with Southern blot techniques of 26 B-cell lines derived from 3 individuals to support this claim. 4 We analyzed publicly available IGH high-throughput sequencing (HTS) data derived from analysis of B-cell genomic DNA from 60 healthy individuals (available at https://clients.adaptivebiotech. com/immuneaccess), and we conclude that the data strongly suggest that a very high fraction of normal human B cells do indeed undergo biallelic D H J H rearrangement. These IGH HTS data were acquired using the same sequencing platform used for our studies. 5 Second, Küppers points out that it is physiological for human B cells, and not an abnormal feature of Burkitt lymphoma (BL) cells, that nonproductive V H D H J H rearrangements undergo somatic hypermutation (SHM) as efficiently as productive V H D H J H rearrangements. He reports that this observation is attributable to the fact that nonproductive rearrangements are transcribed and that transcription is a prerequisite for being targeted by somatic hypermutation. In our study, however, we observed evidence of comparable hypermutation of incomplete D H J H rearrangements, which are not transcribed. Hypermutation on incomplete D H J H rearrangements has not been reported previously, to our knowledge. We interpret this novel finding as evidence of an aberrant mutational process that is broadly active across the immunoglobulin loci in BL tumors.Third, while one early report suggested that MYC translocation into the IGH locus may occur preferentially within the J H gene segment in endemic BL and preferentially within the S m switch region in sporadic BL, 6 subsequent studies have demonstrated that the translocation breakpoints in both endemic and sporadic BL regularly occur in the switch and J H regions.7-10 Therefore, the possible breakpoint locations on chromosome 14 span a genomic interval of ;280 kb, from the J H region through the switch region. For the V H D H J H or D H J H rearrangement carried on the IGH allele into which MYC is translocated to be undetectable by our HTS platform, the translocation breakpoint would have to occur within the ,200-bp interval that is amplified and sequenced by our sequencing strategy. If the breakpoint occurs outside this extremely narrow ,200-bp window, our sequencing strategy would still detect the IGH rearrangement on this allele, even if it is adjacent to MYC. While disruption of the polymerase chain reaction (PCR) target region by the MYC; IGH translocation is theoretically 11 JULY 2017 x VOLUME 1, NUMBER 16 1261 possible, it is a highly unlikely event, and it is therefore improbable that it could explain the lack of detection of a second rearranged IGH allele in 85...
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. Disclosures No relevant conflicts of interest to declare.
Introduction: Burkitt lymphoma (BL) is an aggressive B-lineage non-Hodgkin lymphoma that has traditionally been classified into 3 subtypes: "endemic", "sporadic", and HIV-associated. The highest incidence of BL - prompting the "endemic" classification - is observed in children in sub-Saharan Africa, where the distribution of the disease is closely associated with that of P. falciparum malaria. The tumor cells in most cases of BL from sub-Saharan Africa are infected with Epstein Barr virus (EBV). In contrast, a minority of BL tumors from Europe and North America - which are commonly described as "sporadic" - are EBV positive. Although excellent outcomes have been observed for patients with BL treated with multi-agent chemotherapy in Europe and North America, the outcomes of BL patients in sub-Saharan Africa are suboptimal. We hypothesized that improved understanding of the molecular heterogeneity that exists within BL, particularly the molecular features that distinguish cases from sub-Saharan Africa from those from Europe/North America, will enable more effective treatment strategies that can readily be implemented in low-resources settings. Methods: Tumor biopsies were collected from 19 pediatric patients who presented to the Uganda Cancer Institute with maxillofacial tumors histologically confirmed to be BL. Total RNA was extracted and polyA-selected sequencing libraries were prepared. Paired-end, 50-base pair sequencing was performed on the Illumina HiSeq 2500 platform at a depth of 100 million reads per sample. A publicly available RNA sequencing dataset from 28 BL cases from Europe and North America that were previously analyzed by both microarray (NEJM 2006; 354[23]: 2431-2442) and RNA sequencing (Nature 2012; 490[7418]: 116-120) was analyzed in parallel for comparison. RNA sequencing on the 28 sporadic BL tumors was performed on polyA-selected sequencing libraries with paired-end, 108-base pair sequence reads generated on the Illumina HiSeq 2000 platform. The reads from all 47 BL tumors were aligned to the human and EBV (GenBank ID KC207813.1) genomes using the STAR aligner. Tumors were deemed EBV positive if the ratio of mapped viral reads to human reads exceeded 0.001%, since the EBV genome is 0.005% the size of the human genome. Normalization and differential expression analysis were performed on aligned reads using the DESeq2 R package. Somatic variants were identified by the Genome Analysis Tool Kit. Analysis of alterative splicing was performed with the SGSeq R package. Results: All of the Uganda BL cases but only 70% of the previously published European/North American cases were obtained from children less than 18 years; 61% and 87% were from male patients, respectively. One half of patients in both groups presented with Ann Arbor stage I or II disease, while the remainder presented with higher stage disease. The median survival in the 19 Ugandan patients was less than one year. Sequence reads from 15 (79%) of the Ugandan tumors and 4 (15%) of the European/North American tumors aligned to the EBV genome. Although BL is reported to express only latency type I EBV genes, expression of latency type I, II, and III genes was detected. Among the 4 tumors from Uganda that did not contain EBV-derived reads, 1 was from a child who was HIV-seropositive, while the others were from HIV-seronegative patients. Unsupervised hierarchical clustering and principal component analysis of all annotated genes failed to separate EBV positive and EBV negative tumors. Cluster analysis of the Ugandan tumors revealed no association with clinical variables. Ongoing analyses are being performed to identify associations between differentially expressed genes and somatic mutations and alternative splicing that may distinguish the tumor subtypes. Conclusions: While the terms "endemic" and "sporadic" have traditionally been used to classify BL, this nomenclature is archaic and does not accurately capture the underlying biology of the tumor subtypes. We performed a comparative whole transcriptome analysis of BL tumors from patients in Uganda to a dataset of BL tumors from patients in Europe/North America to identify features that distinguish the two cohorts. While cluster analysis failed to clearly separate tumors by their EBV or clinical status, ongoing analyses are being performed to identify alterations that may contribute to their distinct gene expression profiles. Disclosures No relevant conflicts of interest to declare.
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