Burkitt Lymphomagenesis Research Articles

Activation-induced deaminase expression defines mature B cell lymphoma in the mouse.

Germinal centers (GCs) are the sites of secondary antibody diversification and underlie the mechanism of action of many vaccination strategies. Activation-induced deaminase (AID) triggers secondary antibody diversification through the introduction of somatic changes in immunoglobulin genes which lead to the generation of antibodies of higher affinity and more specialized effector functions. However, AID can also target other genomic regions, giving rise to mutations and chromosome translocations with oncogenic potential. Many human lymphomas originate from mature B cells that have undergone the GC reaction, such as the diffuse large B cell lymphoma, the follicular lymphoma and Burkitt lymphoma, and carry chromosome translocations. Mature B cell lymphomagenesis has been modeled in the mouse by the genetic introduction of chromosome translocations. Here, we present an in-depth characterization of one such model, λ-MYC mice. We found that young pre-tumor stage mice had a prominent block in early B cell differentiation that resulted in the generation of very aggressive tumors lacking surface B cell receptor (BCR) expression, indicating that a large fraction of tumors in λ-MYC mice arise from B cell precursors rather than from mature B cells. Further, we assessed the contribution of AID to B cell lymphomagenesis in λ-MYC mice by using a genetic tracer of historical AID expression. Only a fraction of tumors contained cells of GC origin as defined by AID expression. AID-experienced tumors associated with longer survival and resembled mature B cell lymphomas. Thus, AID expression defines Burkitt lymphomagenesis in λ-MYC mice.

Molecular Detection of Epstein Barr Virus in Malaria Positive Patients in Khartoum State Hospitals, Sudan 2018

Background: Malaria and Epstein–Barr virus (EBV) infections are recognized cofactors in the genesis of endemic Burkitt lymphoma, the most common pediatric cancer in equatorial Africa. Objective: To detect Epstein - Barr virus in malaria positive patients in Khartoum state Materials and methods: A cross-sectional study was conducted in Khartoum State hospitals during the period from April 2018 to April 2019. Fifty specimens from malaria positive patients (16 males and 34 females) were collected and tested by conventional polymerase chain reaction (PCR) for detection of EBV. Results: Out of the 50 malaria positive patients samples tested by PCR, EBV was detected in 5 (10%) samples. Conclusion: The rate of EBV infection among Sudanese malaria patients was low (10 %, 5/50).

Increased Frequency of Mosaic Chromosomal Alterations in Peripheral Blood Samples of Sub-Saharan African Children with Endemic Burkitt Lymphoma

Introduction: Burkitt lymphoma (BL) is an aggressive B-cell lymphoma with reciprocal IG∷MYC translocations and accounts for 50-75% of childhood cancers in sub-Saharan Africa. Mosaic chromosomal alterations (mCAs) are large, somatically acquired structural chromosomal alterations that are clonal and observed in detectable fractions of circulating mononuclear cells. mCAs have been linked to increased hematologic cancer risk, but their association with BL, a mononuclear B cell tumor, is unknown. We hypothesized that the frequency and distribution of mCAs in peripheral leukocytes would be elevated in children with BL, potentially serving as a biomarker of chromosomal instability relevant to BL risk. Methods: We investigated peripheral leukocyte DNA extracted from children enrolled in the Epidemiology of Burkitt Lymphoma in East African Children and Minors (EMBLEM) study (2010-2016) and the Infections and Childhood Cancer case-control study conducted in Malawi (2005-2010). We used genome-wide single nucleotide polymorphism (SNP) array data to investigate the frequency, type, and distribution of mCAs >2Mb in size detected in peripheral leukocytes of 931 pediatric BL cases with 3,645 healthy children in Uganda, Tanzania, and Kenya, and with 177 non-BL pediatric cancers in Malawi. Eagle2 and SHAPEIT4 software were used for phasing to infer haplotypes for SNP array data and WGS data. MoChA (v2014-05-14) was used to detect mCAs. Parallel analysis using similar methods was performed on whole genome sequences (WGS) from paired tumor-normal blood samples from two Ugandan BL patients from the BL Genome Sequencing Project (BLGSP) and from paired BL- and lymphoblastoid-derived cell lines. Results: Most samples (92%) were from the EMBLEM study, whereas 8% were from Malawi. The mean age of the BL cases and controls was similar (~7.4 years in both, P=0.60). Table 1 shows detected autosomal mCAs for BL cases and controls by country. Overall, a total of 250 autosomal mCAs were detected in 78 (8.4%) of the 931 BL cases. By comparison, a total of 188 autosomal mCAs were detected in 131 (3.4%) of the 3822 controls. Autosomal mCAs were detected in cell fractions ranging from 0.5% to 99.2% (Figure S1). The average number of autosomal mCAs per person (i.e., total of mCAs detected/number of individuals with mCAs detected) was higher in the BL cases compared to controls (3.21 [SD 3.24] versus 1.44 [SD 2.31], P-value=4.50×10-5. The higher frequency of mCAs in blood samples of children with BL compared to controls was independent of age, sex, P falciparum status, and country (OR=2.8, 95% CI=2.06-3.81, P-value=5.70×10-11, Table 1. mCAs on the X chromosome in girls were also associated with BL (5% versus 1.7%, OR=3.12, 95% CI=1.47-6.29, P-value=1.65×10-3). We did not observe an association for mCAs on the Y chromosome in boys. Analysis of paired tumor-normal samples confirmed the presence of the IG∷MYC translocation in tumor, but the same pipelines did not detect the translocation in the corresponding normal blood sample. In informative tumor-normal cases, some mCAs were only detected in tumor, others were detected in both tumor and normal blood, whereas others were detected mCAs in normal only suggesting some mCAs in blood could arise from pre-BL clones. mCAs only observed in BL cases were predominantly gains on chromosome 1q and 8 (Figure 1) which contain major drivers of Burkitt lymphomagenesis including PLEKHO1, MCL1, PSMB4, ILF2, HAX1, ATP8B2 and CKS1B.Conclusion: We report a high frequency of mCAs in healthy sub-Saharan African children and even higher frequency in pediatric BL cases. The mCAs in children with BL clustered on chromosomes 1q and 8 in regions that overlap with several significantly mutated BL genes, suggesting that mCAs may be potential biomarkers important in the natural history of BL. Using paired tumor-normal sample WGS data, we report both similar and unique mCAs in the paired samples suggesting pre-BL clones in blood. These methods are scalable to large cohorts with SNP array data to investigate health effects associated with mCAs in Africa. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Omipalisib (GSK458), a Dual an-PI3K/mTOR Inhibitor, Exhibits in Vitro and In Vivo activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Background: Reports of recurrent genomic alterations in Burkitt lymphoma (BL) have identified multiple recurrent alterations that result in activation of PI3K highlighting the importance of the PI3K/AKT/mTOR pathway in Burkitt lymphomagenesis. In a cell line model of resistant BL, we have previously identified an increase of PI3K/AKT/mTOR pathway activation suggesting a role in therapy resistance. While inhibition of PI3K-delta using the isoform specific inhibitor idelalisib has demonstrated clinical activity in indolent lymphomas, limited single agent activity has been observed in more aggressive variants. Pre-clinical investigation of idelalisib in BL indicated similar somewhat limited in vitro activity with synergistic activity in combination with chemotherapy. Broader inhibition of both upstream PI3K and downstream mTOR may exhibit more significant anti-lymphoma activity.Objectives: Investigate the in vitro and in vivo activity of the dual pan-PI3K/mTOR inhibitor omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.Methods: Experiments were conducted in Raji, Raji 4RH (chemotherapy-rituximab resistant), Ramos, and Daudi BL cells. Cell viability following exposure to omipalisib +/- chemotherapy was analyzed using Cell-Titer Glo and Alamar blu assays. Induction of apoptosis was assessed by flow cytometry for Annexin V (AV)-propidium iodide (PI) staining. Downstream effects of omipalisib on PI3K/Akt/mTOR signaling were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using PI staining. Synergy of combination exposures was determined by calculation of the combination index (CI) using CalcuSyn software. In vivo activity was evaluated using disseminated Raji and subcutaneous Ramos SCID mouse xenograft models. The survival end point was hind limb paralysis in the disseminated model and tumor diameter >2cm in the subcutaneous model. Mice were treated with vehicle or omipalisib daily by oral gavage. Median survival was compared by Kaplan-Meier analysis.Results: Exposure of BL cells to omipalisib for 24-72 hours resulted in a dose- and time-dependent decrease in viable cells at nM concentrations (48h IC50 values: Raji=1.2uM, Raji 4RH=0.02uM, Ramos=0.01uM, Daudi=0.01uM) (Figure 1A). Marked induction of apoptosis occurred following 72h exposure to omipalisib primarily in chemosensitive cells with half-maximal effect noted at approximately 200nM, but requiring significantly higher concentrations to induce apoptosis in therapy resistant Raji 4RH cells (%AV positive at 200nM: Raji=40.7%, Raji 4RH=4.4%, Ramos=59.4% and Daudi=46.9%). Downstream of PI3K/Akt/mTOR, S6 and GSK3β showed reduced phosphorylation after 30 minute omipalisib exposure. G1 cell cycle arrest occurred in all cell lines following exposure to omipalisib for 72 hours; however, chemotherapy-resistant Raji 4RH cells arrested in G2/M at higher concentrations. BL cells exposed to omipalisib in combination with either doxorubicin or dexamethasone, exhibited synergistic anti-tumor activity (CI<0.9) with synergistic induction of apoptosis in therapy sensitive cells exposed to omipalisib and chemotherapy. NOD-SCID mice injected via tail vein with Raji-luc (provided by Dr. Mitchell Cairo) and treated with omipalisib demonstrated decreased luciferase signal compared to controls (Figure 1C) while mice with established subcutaneous Ramos xenografted tumors treated with omipalisib exhibited slower tumor progression compared to controls (Figure 1B), though with only modest prolongation of survival (median 28 vs 34 days, n=15/group, p<0.05).Conclusion: Dual PI3K-mTOR inhibitor omipalisib suppresses the PI3K/Akt/mTOR pathway leading to induction of apoptosis, impaired BL cell proliferation in vitro and in vivo and exhibits synergistic in vitro activity when combined with cytotoxic chemotherapy highlighting the relevance of PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-clinical activity of targeting the PI3K/Akt/mTOR pathway in Burkitt lymphoma.

Though outcomes for pediatric Burkitt lymphoma (BL) have improved significantly in recent decades with intensive multi-agent chemotherapy and the addition of rituximab, chemotherapy resistance remains a significant impediment to cure following relapse. Activation of the PI3K/AKT pathway has been implicated in Burkitt lymphomagenesis and increased PI3K/AKT activation has been associated with worse outcomes in adults with aggressive B-cell non-Hodgkin lymphoma (B-NHL). Inhibitors of the PI3K/AKT pathway have been approved for the treatment of refractory indolent B-NHL and continue to be investigated for treatment of aggressive B-NHLs. We investigated the activation of the PI3K/AKT pathway in a cell line model of resistant BL and the ability to target this pathway with small molecule inhibitors in BL cell lines. We found that cell lines resistant to rituximab and chemotherapy exhibited increased activation of PI3K/AKT and that inhibition of AKT or PI3K results in in vitro anti-lymphoma activity. To investigate the role of PI3K/AKT activation on the efficacy of cytotoxic chemotherapy, we exposed cells to inhibitors in combination with chemotherapy and noted a synergistic increase in response to chemotherapy. Overall these findings highlight the role of PI3K/AKT in chemotherapy resistance in BL cells and may represent a tractable therapeutic target.

EBV-Encoded Latent Genes.

Epstein-Barr virus (EBV) is one of the most widespread human pathogens. EBV infection is usually asymptomatic, and it establishes life-long latent infection. EBV latent infection sometimes causes various tumorigenic diseases, such as EBV-related lymphoproliferative diseases, Burkitt lymphomas, Hodgkin lymphomas, NK/T-cell lymphomas, and epithelial carcinomas. EBV-encoded latent genes are set of viral genes that are expressed in latently infected cells. They include virally encoded proteins, noncoding RNAs, and microRNAs. Different latent gene expression patterns are noticed in different types of EBV-infected cells. Viral latent gene products contribute to EBV-mediated B cell transformation and likely contribute to lymphomagenesis and epithelial carcinogenesis as well. Many biological functions of viral latent gene products have been reported, making difficult to understand a whole view of EBV latency. In this review, we will focus on latent gene functions that have been verified by genetic experiments using EBV mutants. We will also summarize how viral latent genes contribute to EBV-mediated B cell transformation, Burkitt lymphomagenesis, and epithelial carcinogenesis.

Relevance of ID3-TCF3-CCND3 pathway mutations in pediatric aggressive B-cell lymphoma treated according to the non-Hodgkin Lymphoma Berlin-Frankfurt-Münster protocols.

Mature B-cell non-Hodgkin lymphoma is the most common subtype of non-Hodgkin lymphoma in childhood and adolescence. B-cell non-Hodgkin lymphomas are further classified into histological subtypes, with Burkitt lymphoma and Diffuse large B-cell lymphoma being the most common subgroups in pediatric patients. Translocations involving the MYC oncogene are known as relevant but not sufficient for Burkitt lymphoma pathogenesis. Recently published large-scale next-generation sequencing studies unveiled sets of additional recurrently mutated genes in samples of pediatric and adult B-cell non-Hodgkin lymphoma patients. ID3, TCF3 and CCND3 are potential drivers of Burkitt lymphomagenesis. In the study herein, frequency and clinical relevance of mutations in ID3, TCF3 and CCND3 were analyzed within a well-defined cohort of 84 uniformly diagnosed and treated pediatric B-cell non-Hodgkin lymphoma patients of the Berlin-Frankfurt-Münster group. Mutation frequency was 78% (ID3), 13% (TCF3) and 36% (CCND3) in Burkitt lymphoma (including Burkitt leukemia). ID3 and CCND3 mutations were associated with more advanced stages of the disease in MYC rearrangement positive Burkitt lymphoma. In conclusion, ID3-TCF3-CCND3 pathway genes are mutated in more than 88% of MYC-rearranged pediatric B-cell non-Hodgkin lymphoma and the pathway may represent a highly relevant second hit of Burkitt lymphoma pathogenesis, especially in children and adolescents.

Targeting BET Bromodomains in Pre-Clinical Models of Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway.Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values.Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI<0.9) at multiple dose combinations while other combinations exhibited primarily additive effects.Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. DisclosuresNo relevant conflicts of interest to declare.

Virus-encoded microRNA contributes to the molecular profile of EBV-positive Burkitt lymphomas.

Burkitt lymphoma (BL) is an aggressive neoplasm characterized by consistent morphology and phenotype, typical clinical behavior and distinctive molecular profile. The latter is mostly driven by the MYC over-expression associated with the characteristic translocation (8;14) (q24; q32) or with variant lesions. Additional genetic events can contribute to Burkitt Lymphoma pathobiology and retain clinical significance. A pathogenetic role for Epstein-Barr virus infection in Burkitt lymphomagenesis has been suggested; however, the exact function of the virus is largely unknown.In this study, we investigated the molecular profiles (genes and microRNAs) of Epstein-Barr virus-positive and -negative BL, to identify specific patterns relying on the differential expression and role of Epstein-Barr virus-encoded microRNAs.First, we found significant differences in the expression of viral microRNAs and in selected target genes. Among others, we identified LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, and the tyrosine kinases encoding STK10/STK33, all provided with potential pathogenetic significance. GCET2, also validated by immunohistochemistry, appeared to be a useful marker for distinguishing EBV-positive and EBV-negative cases. Further, we provided solid evidences that the EBV-encoded microRNAs (e.g. BART6) significantly mold the transcriptional landscape of Burkitt Lymphoma clones.In conclusion, our data indicated significant differences in the transcriptional profiles of EBV-positive and EBV-negative BL and highlight the role of virus encoded miRNA.

Deregulation of the PI3K/Akt Signal Transduction Pathway Is Associated with the Development of Chemotherapy Resistance and Can be Effectively Targeted to Improve Chemoresponsiveness in Burkitt Lymphoma Pre-Clinical Models

Abstract Multi-agent chemotherapy regimens have significantly improved long-term survival of pediatric Burkitt Lymphoma (BL) to over 80%. However, patients with refractory or relapsed disease have a dismal prognosis, stressing the need to identify mechanisms of therapeutic resistance and develop novel therapeutic approaches. Recent data demonstrated tonic B-cell receptor (BCR) activation of PI3K in BL (Srinivisan et al, Cell 2009); and implicated PI3K, in coordination with MYC, in Burkitt lymphomagenesis (Sander et al, Cancer Cell 2012). In order to investigate mechanisms of resistance, our laboratory generated several resistant BL cell lines. We exposed Raji cells to escalating doses of rituximab +/- human serum and generated several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or therapy (rituximab-chemotherapy) resistant (Raji 2R and Raji 4RH) (TRCL) cell lines. We then screened for aberrant activation of signal transduction pathways using western blot, phospho-flow cytometry and phosphoproteomics to define pathways associated with the development of resistance. While total Akt expression was similar between all cell lines tested, Raji 2R and Raji 4RH cells had increased phosphorylation of Akt at Ser473 and Thr308, indicating Akt activation, when compared to Raji, Raji 7R and Raji 8RH by western blot and phospho-flow cytometry. Phosphoproteomic analysis comparing Raji and Raji 4RH identified an increase of at least 2 fold in phosphorylation of 315 proteins in Raji 4RH cells, including multiple direct targets of AKT (e.g. GSK3B, WEE1, FOXO1 and PRAS40). Altered phosphorylation of proteins downstream of Akt (e.g. BAD, 4EBP1, GSK3B and ERK) was also detected by western blot in Raji 2R and Raji 4RH compared to Raji, Raji 7R and Raji 8RH. These findings suggest that activation of PI3K/Akt may play a role in the development of chemoresistance noted in our cell line model. KEGG analysis of phosphoproteome and gene expression comparison of Raji to Raji 4RH cells identified the BCR pathway as one of the top pathways altered. Additionally, the microRNA (miR) 17-92 has been noted to increase Akt activation through its inhibitory effects on PTEN, a key regulator of Akt activation. A recent copy number analysis indicated that a recurrent gain in 13q, containing the MIR17HG locus, was also associated with higher mir17 RNA levels and early relapse in pediatric BL patients (Schiffman et al, Br J Haematol 2011). An analysis of miR expression in Raji 2R and Raji 4RH compared to Raji noted a 1.93 and 3.77 fold increase in miR17 respectively. These findings suggest possible mechanisms for increased PI3K/Akt activation in our chemoresistant cell lines. In vitro exposure to MK-2206, a pan-Akt inhibitor, resulted in dose- and time-dependent decreases in viability of TRCLs (IC50 at 72 hours: Raji 2R = 2.6µM; Raji 4RH = 3.2µM) and to a slightly lesser degree Raji and RRCL (Raji= 4.4µM; Raji 7R = 4.0µM; Raji 8RH = 5.2µM) (TRCL vs. Raji/RRCL, p&lt;0.05), by alamarBlu assay. Cell cycle analysis following exposure to MK2206 identified G1 cell cycle arrest in Raji/RRCL, but G2/M cell cycle arrest was observed in TRCL, suggesting differing mechanisms of anti-proliferative effect between sensitive and resistant cells. The combination of MK2206 and either doxorubicin or dexamethasone resulted in synergistic decreases in cell viability at a variety of dose combinations in both sensitive and resistant cells, as well as in BL Ramos and Daudi cells, using the Chou-Talalay method to measure synergy. In Raji and Ramos, the combination of MK2206 and doxorubicin also resulted in a higher degree of PARP cleavage compared to either agent alone, though no PARP cleavage was noted in Raji 2R and Raji 4RH cells likely related to impaired apoptotic potential previously noted in these cell lines. An siRNA knockdown of Akt in Raji cells resulted in a decrease in viable cells compared to controls following exposure to doxorubicin. Similar effects on cell proliferation, cell cycle and synergy were seen with upstream inhibition of PI3K by the PI3Kδ inhibitor idelalisib. Our data suggests that constitutive activation of the PI3K/Akt pathway, through activation of the BCR pathway or over-expression of miR17, is associated with the development of resistance in BL cell lines, and inhibition of PI3K/Akt can sensitize BL cells to the effects of some chemotherapeutic agents. Targeting the PI3K/Akt signaling pathway may be a clinically relevant approach in pediatric BL. Disclosures Cairo: Celgene: Research Funding.

AID expression in peripheral blood of children living in a malaria holoendemic region is associated with changes in B cell subsets and Epstein-Barr virus.

The development of endemic Burkitt's lymphoma (eBL) is closely associated with Epstein-Barr virus (EBV) infection and holoendemic malaria infections. The role of EBV in the development of malignancy has been studied in depth, but there is still little known about the mechanisms by which malaria affects Burkitt's lymphomagenesis. Activation induced cytidine deaminase (AID) expression is necessary for the introduction of c-myc translocations that are characteristic of BL, but a link between AID and EBV or malaria is unclear. To determine whether frequency of malaria exposure leads to increased AID expression in peripheral blood mononuclear cells (PBMC) we examined two cohorts of children in western Kenya with endemic and sporadic malaria transmission dynamics. High frequency of malaria exposure led to increased expression of AID, which coincided with decreases in the IgM(+) memory B cells. In the children from the malaria endemic region, the presence of a detectible EBV viral load was associated with higher AID expression compared to children with undetectable EBV, but this effect was not seen in children with sporadic exposure to malaria. This study demonstrates that intensity of malaria transmission correlates with AID expression levels in the presence of EBV suggesting that malaria and EBV infection have a synergistic effect on the development of c-myc translocations and BL.

Recurrent RHOA mutations in pediatric Burkitt lymphoma treated according to the NHL-BFM protocols.

Burkitt lymphoma (BL) is the most frequent B-cell lymphoma in childhood. Genetically, it is characterized by the presence of an IG-MYC translocation which is supposed to be an initiating but not sufficient event in Burkitt lymphomagenesis. In a recent whole-genome sequencing study of four cases, we showed that the gene encoding the ras homolog family member A (RHOA) is recurrently mutated in pediatric BL. Here, we analyzed RHOA by Sanger sequencing in a cohort of 101 pediatric B-cell lymphoma patients treated according to Non-Hodgkin's Lymphoma Berlin-Frankfurt-Münster (NHL-BFM) study protocols. Among the 78 BLs in this series, an additional five had RHOA mutations resulting in a total incidence of 7/82 (8.5%) with c.14G>A (p.R5Q) being present in three cases. Modeling the mutational effect suggests that most of them inactivate the RHOA protein. Thus, deregulation of RHOA by mutation is a recurrent event in Burkitt lymphomagenesis in children.

The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma.

BackgroundBurkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression by its own encoded microRNAs.MethodsUsing microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNAs. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases.ResultsWe showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Rα and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression.ConclusionsOur preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.

Epigenetics, c-Myc and Aggressive B-cell Lymphomas

Epigenetics, c-Myc and Aggressive B-cell Lymphomas

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

Synergy between PI3K Signaling and MYC in Burkitt Lymphomagenesis

In Burkitt lymphoma (BL), a germinal center B-cell-derived tumor, the pro-apoptotic properties of c-MYC must be counterbalanced. Predicting that survival signals would be delivered by phosphoinositide-3-kinase (PI3K), a major survival determinant in mature B cells, we indeed found that combining constitutive c-MYC expression and PI3K activity in germinal center B cells of the mouse led to BL-like tumors, which fully phenocopy human BL with regard to histology, surface and other markers, and gene expression profile. The tumors also accumulate tertiary mutational events, some of which are recurrent in the human disease. These results and our finding of recurrent PI3K pathway activation in human BL indicate that deregulated c-MYC and PI3K activity cooperate in BL pathogenesis.

EBV Reactivation and Chromosomal Polysomies: Euphorbia tirucalli as a Possible Cofactor in Endemic Burkitt Lymphoma.

Burkitt lymphoma is endemic in the Equatorial Belt of Africa, its molecular hallmark is an activated, MYC gene mostly due to a chromosomal translocation. Especially in its endemic clinical variant, Burkitt lymphoma is associated with the oncogenic Epstein-Barr virus (EBV), and holoendemic malaria acts as an amplifier. Environmental factors may also cooperate in Burkitt lymphomagenesis in the endemic regions, such as plants used as traditional herbal remedies. Euphorbia tirucalli, a plant known to possess EBV-activating substances, has a similar geographical distribution to endemic Burkitt's Lymphoma and is used as a hedge, herbal remedy and toy in the Lymphoma BeltI. In this study we aimed at determining if exposure to Euphorbia tirucalli could contribute to lymphomagenesis, and at which extent. Lymphoblastoid and cord blood-derived cell lines were treated with plant extracts, and the expression of EBV-coded proteins was checked, to assess EBV reactivation. The occurrence of chromosomal translocations was then investigated by FISH. Our preliminary results suggest that E. tirucalli is able to reactivate EBV and determine chromosomal alterations, which leads to c-MYC altered expression. The existence of genomic alterations might determine the accumulation of further genetic alteration, which could eventually lead to a transformed phenotype.

Primary Bilateral Burkitt Lymphoma of the Lactating Breast

Burkitt lymphoma (BL) is a highly aggressive neoplasm, which frequently affects the ileocecal region in the sporadic form and the jaw in the endemic form; however, the breast is a rare primary site of this tumor. Here we describe a case of primary bilateral breast BL presenting during lactation in a 23-year-old woman. Excisional biopsy of breast masses demonstrated a B-cell lymphoma with a characteristic 'starry sky' pattern highly suggestive of BL. The neoplastic cells strongly expressed CD20 and CD10, and showed proliferative activity as measured by Ki-67. An IGH-MYC gene fusion indicating the presence of a typical Burkitt translocation t(8;14)(q24;q32) in the tumor tissue was detected by fluorescent in situ hybridization. The present case, along with a comprehensive review of the literature, demonstrates that BL of the breast should be considered in the differential diagnosis of lesions of the breast during lactation. Whether hormonal or antigenic factors trigger Burkitt lymphomagenesis in the lactating breast warrants further investigation.

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.

An Epstein-Barr Virus Anti-Apoptotic Protein Constitutively Expressed in Transformed Cells and Implicated in Burkitt Lymphomagenesis: The Wp/BHRF1 Link

Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth–transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc–driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro–transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo.