Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

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Sporadic Burkitt lymphoma (sBL) can be delineated from diffuse large B-cell lymphoma (DLBCL) by a very homogeneous mRNA expression signature. However, it remained unclear whether all three BL variantsFsBL, endemic BL (eBL) and human immunodeficiency virus-associated BL (HIV-BL)F represent a uniform biological entity despite their differences in geographical occurrence, association with immunodeficiency and/or incidence of Epstein-Barr virus (EBV) infection. To address this issue, we generated micro RNA (miRNA) profiles from 18 eBL, 31 sBL and 15 HIV-BL cases. In addition, we analyzed the miRNA expression of 86 DLBCL to determine whether miRNA profiles recapitulate the molecular differences between BL and DLBCL evidenced by mRNA profiling. A signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was obtained that differentiated BL from DLBCL. The miRNA profiles of sBL and eBL displayed only six differentially expressed miRNAs, whereas HIV and EBV infection had no impact on the miRNA profile of BL. In conclusion, miRNA profiling confirms that BL and DLBCL represent distinct lymphoma categories and demonstrates that the three BL variants are representatives of the same biological entity with only marginal miRNA expression differences between eBL and sBL.

CDK9 is a member of the CDC2-like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B- and T-cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B- and T-cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T-lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma also showed strong nuclear staining. Diffuse large B-cell, Burkitt's lymphomas, and peripheral T-cell lymphomas, among T-cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B-cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkin's lymphomas, Burkitt's lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated.

The Epstein–Barr virus (EBV) is linked to various B-cell lymphomas, including Burkitt lymphoma (BL), classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) at frequencies ranging, by routine techniques, from 5 to 10% of cases in DLBCL to >95% in endemic BL. Using higher-sensitivity methods, we recently detected EBV traces in a few EBV-negative BL cases, possibly suggesting a “hit-and-run” mechanism. Here, we used routine and higher-sensitivity methods (qPCR and ddPCR for conserved EBV genomic regions and miRNAs on microdissected tumor cells; EBNA1 mRNA In situ detection by RNAscope) to assess EBV infection in a larger lymphoma cohort [19 BL, 34 DLBCL, 44 cHL, 50 follicular lymphomas (FL), 10 T-lymphoblastic lymphomas (T-LL), 20 hairy cell leukemias (HCL), 10 mantle cell lymphomas (MCL)], as well as in several lymphoma cell lines (9 cHL and 6 BL). qPCR, ddPCR, and RNAscope consistently documented the presence of multiple EBV nucleic acids in rare tumor cells of several cases EBV-negative by conventional methods that all belonged to lymphoma entities clearly related to EBV (BL, 6/9 cases; cHL, 16/32 cases; DLBCL, 11/30 cases), in contrast to fewer cases (3/47 cases) of FL (where the role of EBV is more elusive) and no cases (0/40) of control lymphomas unrelated to EBV (HCL, T-LL, MCL). Similarly, we revealed traces of EBV infection in 4/5 BL and 6/7 HL cell lines otherwise conventionally classified as EBV negative. Interestingly, additional EBV-positive cases (1 DLBCL, 2 cHL) relapsed as EBV-negative by routine methods while showing EBNA1 expression in rare tumor cells by RNAscope. The relapse specimens were clonally identical to their onset biopsies, indicating that the lymphoma clone can largely loose the EBV genome over time but traces of EBV infection are still detectable by high-sensitivity methods. We suggest EBV may contribute to lymphoma pathogenesis more widely than currently acknowledged.

Objectives: Recent studies using next-generation sequencing (NGS) analysis disclosed the importance of the intrinsic activation of the B-cell receptor (BCR) pathway in the pathogenesis of sporadic Burkitt lymphoma (sBL) due to mutations of TCF3/ID3 genes. Since no definitive data are available on the genetic landscape of endemic Burkitt (eBL), we first assessed the mutation frequency of TCF3/ID3 in eBL compared with sBL and subsequently the somatic hypermutation status of the BCR to answer whether an extrinsic activation of BCR signaling could also be demonstrated in Burkitt lymphoma.Methods: We assessed the mutations of TCF3/ID3 by RNAseq and the BCR status by NGS analysis of the immunoglobulin genes (IGs).Results: We detected mutations of TCF3/ID3 in about 30% of the eBL cases. This rate is significantly lower than that detected in sBL (64%). The NGS analysis of IGs revealed intraclonal diversity, suggesting an active targeted somatic hypermutation process in eBL compared with sBL.Conclusions: These findings support the view that the antigenic pressure plays a key role in the pathogenetic pathways of eBL, which may be partially distinct from those driving sBL development.

BackgroundWe previously reported ongoing mutational and isotype switch events in the immunoglobulin (Ig) heavy chain (H) locus in hairy cell leukemia. Those analyses raised questions on the incidence and type of selective influences occurring on the tumor B-cell receptor of hairy cell leukemia.

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