A recurrent clonally distinct Burkitt lymphoma case highlights genetic key events contributing to oncogenesis

Abstract Burkitt lymphoma (BL) is characterized by a translocation of the MYC oncogene that leads to the upregulation of MYC expression, cell growth and proliferation. It is well‐established that MYC translocation is not a sufficient genetic event to cause BL. Next‐generation sequencing has recently provided a comprehensive analysis of the landscape of additional genetic events that contribute to BL lymphomagenesis. Refractory BL or relapsing BL are almost always incurable as a result of the selection of a highly chemoresistant clonally related cell population. Conversely, a few BL recurrence cases arising from clonally distinct tumors have been reported and were associated with a favorable outcome similar to that reported for first‐line treatment. Here, we used an unusual case of recurrent but clonally distinct EBV+ BL to highlight the key genetic events that drive BL lymphomagenesis. By whole exome sequencing, we established that ID3 gene was targeted by distinct mutations in the two clonally unrelated diseases, highlighting the crucial role of this gene during lymphomagenesis. We also detected a heterozygous E1021K PIK3CD mutation, thus increasing the spectrum of somatic mutations altering the PI3K signaling pathway in BL. Interestingly, this mutation is known to be associated with activated phosphoinositide 3‐kinase delta syndrome (APDS). Finally, we also identified an inherited heterozygous truncating c.5791CT FANCM mutation that may contribute to the unusual recurrence of BL.


| INTRODUCTION
Burkitt lymphoma (BL) is characterized by a translocation of the MYC oncogene that leads to the upregulation of MYC expression, cell growth and proliferation. It is well-established that MYC translocation is not a sufficient genetic event to cause BL. [1][2][3][4] Nextgeneration sequencing (NGS) has recently provided a comprehensive analysis of the landscape of additional genetic events that contribute to BL lymphomagenesis. Additional recurrent mutations are observed in the genes encoding TCF3 and its negative regulator, ID3, with up to 70% of tumors bearing mutations in one or both of the genes, suggesting that TCF3/ID3 play a key role in BL lymphomagenesis. 2,4 Furthermore, Epstein-Barr virus (EBV), a ubiquitous oncogenic virus, is associated with B-cell lymphomas, including BL and Hodgkin lymphoma. 5 The role of EBV in BL is still unclear, and it has been suggested that EBV-positive (EBV+) and -negative cases might arise from different cells of origin. EBV+ BL may arise from late germinal center lymphoblast memory B-cells and EBV− BL from an earlier stage of differentiation. Despite its aggressiveness, BL treated in first-line by intensive combination chemotherapy with rituximab and central nervous system (CNS) preventative procedures has resulted in event-free survival rates of 80% to 90%. 6 By contrast, refractory BL or relapsing (R/R) BL are almost always incurable as a result of the selection of highly chemoresistant clonally related cell populations. Conversely, a few BL recurrences arising from clonally distinct tumors have been reported and have been associated with a favorable outcome similar to that reported of first line treatment. [7][8][9][10][11] Here, we used an unusual case of recurrent but clonally distinct EBV+ BL to highlight the key genetic events that drive BL lymphomagenesis. After lymph node biopsy, a diagnosis of BL with a typical immunophenotype was confirmed (BL1). Conventional cytogenetics showed that the tumor harbored the characteristic translocation t(8;22)(q24;q11) that juxtaposes the MYC and IGL loci with the loss of Y as an additional cytogenetic alteration (Supporting Information Figure S1A).

| CASE REPORT
Staging indicated stage IV disease without any involvement of the bone marrow or CNS (Supporting Information Figure S2A). The patient was treated with dose-dense chemotherapy and rituximab, followed by autologous stem cell transplantation (ASCT) conditioned by BEAM (carmustine, etoposide, cytarabine, and melphalan) 12 . A complete remission (CR) was obtained. Two years later, the patient presented with facial paralysis and abdominal pain. PET scan showed multiple enlarged lymph nodes and a massive infiltration of the kidneys (Supporting Information Figure S2B). Bone marrow biopsy displayed massive infiltration (around 30%) by typical BL cells, and the CNS was considered infiltrated. The diagnosis of Burkitt leukemia was confirmed (BL2). In this case, cytogenetics showed that the tumor harbored the characteristic translocation t(8;14)(q24;q32) that juxtaposes the MYC and IGH loci, with a short interstitial deletion of the long arm of chromosome 13 as an additional cytogenetic aberration (Supporting Information Figure S1B). The main immunological features of BL1 and BL2, both EBV+/BCL2 neg/BCL6+, are summarized in Supporting Information Table S1 and representative histopathologic pictures provided in Supporting Information Figure S3A/3B. The patient received two cycles of R-HyperCVAD (rituximab, cyclophosphamide, dexamethasone, methotrexate, doxorubicin, vincristine, cytarabine) with intrathecal methotrexate injection. CR was obtained, and the patient underwent an allogeneic transplantation from an HLA-identical sibling donor that was conditioned by a myeloablative regimen (cyclophosphamide and total-body irradiation). To date, the patient is considered in CR, with a follow-up of 18 months. No abnormal toxicities, including unusual prolonged pancytopenia or mucosal toxicity, were observed throughout the different therapeutic sequences.

| Routine procedures
Conventional cytogenetics, EBV expression analysis and immunohistochemistry were performed using routine procedures. CDR3 sequence and VDJ analyses were performed using routine BIOMED2 procedures.
3.2 | Whole exome sequencing and targeted sequencing WES was performed on the DNA of BL1 and BL2 tumor tissues and compared to germline DNA from PBMCs obtained at the time of initial diagnosis. Data analysis was conducted as fully described in a previous work. 13 Somatic variant calling was performed by VarScan, using germline DNA as a reference. Variant annotation was performed by GenerateReports software, as previously described. 14 To confirm some genetic variants, BL1 and BL2 tumor DNA was also analyzed using our in-house dedicated lymphopanel, as previously reported. 15 Copy number analysis was done using the copynumber Biocon- To investigate functionally the Fanconi anemia (FA) pathway integrity, hypersensitivity to mitomycin C (MMC) and the profile of FancD2 monoubiquitination were analyzed in primary fibroblast cells obtained from skin biopsy. 16   signaling (PI3KR1, EIF4B, and FGFR2). 3 We identified 64 acquired somatic variants in BL1 and 26 acquired variants in BL2. BL1 and BL2 harbored several mutations previously described that target the MYC, TP53, SMARCA4, RHOA and ID3 genes (see Table 1  To the best of our knowledge, we report here for the first time a heterozygous acquired mutation E1021K in PIK3CD, detected in BL1 (Table 1), thus increasing the spectrum of somatic mutations altering the PI3K signaling pathway in BL. Interestingly, this gainof-function (GOF) mutation is known to be associated with activated phosphoinositide 3-kinase delta syndrome (APDS). 17 This inherited disorder, resulting from GOF mutations in PIK3CD, the gene encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3KCδ), includes recurrent pulmonary infections (98%), nonneoplastic lymphoproliferations (75%), herpesvirus infections (49%) and autoinflammatory diseases (34%). 17

| Clinical and potential therapeutic relevance
Our case report confirms the crucial role of the PI3K/Akt/mTOR pathway in BL and the spectrum of mutations that contributes to its deregulation. Importantly, the PI3KCD inhibitor idelalisib has been investigated in a panel of BL cell lines, including cell lines that exhibit a high degree of resistance to both chemotherapy and anti-CD20 immunotherapy, and demonstrate preclinical activity. 18

| Constitutional genetic background that may contribute to BL emergence
Because BL1 and BL2 were both EBV+ and EBV infection is considered as a risk factor for developing BL, we tried to detect some alterations in target genes that are involved in EBV immune response and favor EBV-associated lymphoproliferative disorders (LPDs).
Among these alterations are SH2D1A (SAP), XIAP, ITK, MAGT1, CD27, CD70, CTPS1, RASGRP1, and CORO1A deficiencies. 21 None of these genes were found to be altered in BL1, BL2 or germline DNA, suggesting that an EBV immune response deficiency involving these genes cannot be considered responsible for this unusual phenotype.
We then sought more specifically to identify alterations in genes involved in DNA repair. We identified a heterozygous stop-gain mutation (c.5791C>T; p.Arg1931*) in the FANCM gene. This mutation was also detected in the patient's sister, demonstrating (Continues) that the mutation is inherited (see pedigree in Supporting Information Figure S4). At the resolution level of a WES approach we did not detect a FANCM copy loss. FANCM was identified in 2005 as a member of the FA core complex. Its product FANCM plays an important role in the FA pathway involved in DNA damage responses and repair. 22 Interestingly, it has been demonstrated that the FANCM c.5791C>T nonsense mutation induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor. 23,24 The mutation causes an out-of-frame deletion of exon 22 due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. 24 To verify the functional consequences of the c.5791C>T mutation, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) with patient PBMC RNA. We confirmed that this mutation was also related to aberrant splicing (Supporting Information Figure S4) with the expression of the Δ22 allele. A qRT-PCR assay indicated that the Δ22 and wild-type alleles were equally expressed in the BL tumor cells (Supporting Information Figure S4). Of note, in an additional cohort of 29 BL cases with available tumor DNA, we did not find the FANCM c.5791C>T nonsense mutation (Supporting Information Table S3).
However we cannot rule out other mutations targeting this gene.
Biallelic inactivating mutations in FANCM favor early-onset cancer, were also significantly associated with familial breast cancer risk, with an overall odds ratio (OR) of 2.05. 27 Despite a quantitative difference, the pattern of somatic mutations observed in BL1 and BL2 did not differ significantly, suggesting that BL1 and BL2 were sustained by a similar mutational process rather than arising as a consequence of a treatment side effect (Supporting Information Figure S5). To conclude, this unusual observation highlights the key events that lead to the emergence of genetically distinct BL types (Figure 1). The role of the inherited heterozygous truncating c.5791C>T FANCM mutation is uncertain and could be purely coincidental. ID3 mutations are shared by the two clonally distinct diseases and represent a key secondary genetic event following MYC translocation. The PI3KCD mutation expands the spectrum of mutations targeting the PI3K pathway and offers potential therapeutic opportunities in BL.