Immunoglobulin heavy variable (IgHV) gene mutation and micro-RNA expression in Burkitt\u2019s lymphoma at Moi Teaching and Referral Hospital in Western Kenya

Introduction: Burkitt\u2019s lymphoma (BL) is a virus associated childhood B-cell cancer common in Eastern Africa. Continued survival of B-cells in germinal centres depend on expression of high affinity immunoglobulins (Ig) to complementary antigens by somatic hypermutation of Ig genes. Cellular microRNAs, non-coding RNAs have been reported to play role in cell cycle regulation. Both viral antigen dependent mutation and micro-RNA expression maybe involved in BL pathogenesis. Objective: To describe immunoglobulin heavy variable (IgHV) rearrangement and micro-RNA expressions in BL tumours. Methods: Genomic DNA were extracted and purified from BL tissue blocks at Moi Teaching and Referral Hospital, before amplification using IgHV consensus primers and sequencing. The sequences were then aligned with germline alleles in IMGT/ V-QUEST\uae database. Total RNA extracted from tissue blocks and cell lines were used to determine relative expression of hsamiR-34a and hsa-miR-127. Results: In all tumours, allele alignment scores and number of mutations range were 89.2-93.2%, 15-24 respectively. The range of IgHV amino acid changes were higher in EBER-1+ (15-25) than EBER-1- (9-15). In MYC+ tumours, the relative expression were: hsa-miR-127(2.09);hsa-miR-34a (2.8) and MYC- hsa-miR-127 (1.2), hsa-miR-34a (1.0). Conclusion: B-cell in BL contained somatic mutated IgHV gene and upregulated cellular microRNAs with possible pathogenetic role(s)

Cytokines associated with Burkitt’s lymphoma in western Kenya

Abstract Objective Burkitt’s lymphoma (BL) is a common aggressive non-Hodgkin’s lymphoma in East and Central Africa among children. Persistent infections with Epstein Barr virus or Plasmodium falciparum are associated with immune hyperstimulation. It is hypothesised that inadvertent cytokine responses to infections indirectly or directly influence B cell neoplastic transformation through c-myelocytomatosis (c-myc) gene translocation. We sought to describe cytokines in children and adolescents with BL. Participants were recruited from western Kenya with parental consent, diagnosis confirmed using histology and consensus panel of immunohistochemistry antibodies. T helper1/2/17A and transforming growth factor-β1 (TGF-β1) cytokines were estimated using cytometric bead array in plasma. Complete blood counts (CBC) were determined by Beckman Coulter®. Results Out of 104 enrolled participants, 32% were confirmed BL and 68% grouped as non-BL. Mean (pg/ml) levels of cytokines in BL and non-BL were: interleukin (IL)-6 100.3 and 39.4 p = 0.152; IL-10 11.5 and 12.5 p = 0.363; IL-17A 17.8 and 64.9 p = 0.094 respectively. Expressions of interferon-γ, IL-2 and tumour necrosis factor-α were low and TGF-β1 undetectable in both groups. Mean CBC differed between the two groups before and after chemotherapy, WBC being significantly so. Interleukin-6, IL-17A and IL-10 responses to infections in the study area may be associated with pathogenesis and be potential therapeutic targets

Genetic diversity of ´Candidatus Phytoplasma palmicola‘ in Ghana and Mozambique

International audienc

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

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

(A) the dendrogram classifies the samples into EBV-positive and EBV-negative BL independently on the specific subtype with an accuracy of 96% (45/47). (B) GSEA C2 analysis on genes differentially expressed between EBV-positive and EBV-negative cases detects a significant enrichment for the <i>LMP-1</i> gene set signature. GSEA: gene set enrichment analysis.

<p>(A) the dendrogram classifies the samples into EBV-positive and EBV-negative BL independently on the specific subtype with an accuracy of 96% (45/47). (B) GSEA C2 analysis on genes differentially expressed between EBV-positive and EBV-negative cases detects a significant enrichment for the <i>LMP-1</i> gene set signature. GSEA: gene set enrichment analysis.</p

(A) Unsupervised hierarchical clustering of expressed EBV genes demonstrates a diversity of non-canonical latency-associated gene expression programs with a subset of viral episome initiating lytic reactivation as indicated by expression of genes corresponding to the lytic program. (B) LMP-2A is expressed by 40 to 50% of neoplastic cells. LMP-2A stain, O.M.: 40x; (C) LMP-2A expression is identified in a proportion of neoplastic cells ranging from 20 to 30%. LMP-2A stain, O.M.: 40x; (D) BZLF1/ZEBRA positivity is expressed by 5 to 10% of neoplastic cells. BZLF1/ZEBRA stain, O.M.: 40x; (E) BZLF1/ZEBRA expression is detected in few neoplastic cells. BZLF1/ZEBRA stain, O.M.: 40x; (F) BMRF-1/Ea-D expression is observed in 50% of neoplastic cells. BMRF-1/Ea-D stain, O.M.: 40x; (G) BMRF-1/Ea-D protein expression in 5% to 10% of neoplastic cells is shown. BMRF-1/Ea-D stain, O.M.: 40x; (H) BHRF-1/Ea-R staining is found in 60% of neoplastic cells. BHRF-1/Ea-R stain, O.M.: 40x; (I) BHRF-1/Ea-R is

<p>(A) Unsupervised hierarchical clustering of expressed EBV genes demonstrates a diversity of non-canonical latency-associated gene expression programs with a subset of viral episome initiating lytic reactivation as indicated by expression of genes corresponding to the lytic program. (B) LMP-2A is expressed by 40 to 50% of neoplastic cells. LMP-2A stain, O.M.: 40x; (C) LMP-2A expression is identified in a proportion of neoplastic cells ranging from 20 to 30%. LMP-2A stain, O.M.: 40x; (D) BZLF1/ZEBRA positivity is expressed by 5 to 10% of neoplastic cells. BZLF1/ZEBRA stain, O.M.: 40x; (E) BZLF1/ZEBRA expression is detected in few neoplastic cells. BZLF1/ZEBRA stain, O.M.: 40x; (F) BMRF-1/Ea-D expression is observed in 50% of neoplastic cells. BMRF-1/Ea-D stain, O.M.: 40x; (G) BMRF-1/Ea-D protein expression in 5% to 10% of neoplastic cells is shown. BMRF-1/Ea-D stain, O.M.: 40x; (H) BHRF-1/Ea-R staining is found in 60% of neoplastic cells. BHRF-1/Ea-R stain, O.M.: 40x; (I) BHRF-1/Ea-R is expressed in 10% of neoplastic cells. BHRF-1/Ea-R stain, O.M.: 40x.</p

(A) RNA-Seq technology reveals the presence of EBV and of other viruses. In particular, 5/20 cases contain human herpesvirus 5 (CMV), 4/20 human herpesvirus 8 (KSHV), and 1/20 human T-lymphotropic virus 1 (HTLV-1). (B) Immunohistochemical evaluation demonstrates the presence of CMV in the stromal cells in the adjacent reactive lymphoid tissue. CMV stain, Original Magnification (O.M.): 40x. (C) KSHV positivity is shown, respectively in few neoplastic cells and in the endothelial cells within the neoplastic proliferation. LANA-1 (LN53 antibody), O.M.: 40x; (D) LANA-1 (AT4C11 antibody) O.M.: 40x.

<p>(A) RNA-Seq technology reveals the presence of EBV and of other viruses. In particular, 5/20 cases contain human herpesvirus 5 (CMV), 4/20 human herpesvirus 8 (KSHV), and 1/20 human T-lymphotropic virus 1 (HTLV-1). (B) Immunohistochemical evaluation demonstrates the presence of CMV in the stromal cells in the adjacent reactive lymphoid tissue. CMV stain, Original Magnification (O.M.): 40x. (C) KSHV positivity is shown, respectively in few neoplastic cells and in the endothelial cells within the neoplastic proliferation. LANA-1 (LN53 antibody), O.M.: 40x; (D) LANA-1 (AT4C11 antibody) O.M.: 40x.</p

(A) The presence of mutations in genes previously described in BL is reported, including <i>MYC</i> (50%), <i>DDX3X</i> (35%), <i>ID3</i> (30%), <i>ARID1A</i> (25%), <i>RHOA</i> (20%), <i>TCF3</i> and <i>TP53</i> (15%), and <i>CCND3</i> 1/20 (5%). In addition, a new mutation is shown, involving <i>CCNF</i> and detected in 20% of the cases. (B) Bar plot showing the frequency comparison of virus presence and driver mutations between endemic and sporadic BL. For each comparison we report the p-value associated with rejecting the null hypothesis of equal eBL and sBL prevalences. (C) Distribution of mutations in 5 driver genes. Red points indicate endemic BL, while blue points the sporadic ones.

<p>(A) The presence of mutations in genes previously described in BL is reported, including <i>MYC</i> (50%), <i>DDX3X</i> (35%), <i>ID3</i> (30%), <i>ARID1A</i> (25%), <i>RHOA</i> (20%), <i>TCF3</i> and <i>TP53</i> (15%), and <i>CCND3</i> 1/20 (5%). In addition, a new mutation is shown, involving <i>CCNF</i> and detected in 20% of the cases. (B) Bar plot showing the frequency comparison of virus presence and driver mutations between endemic and sporadic BL. For each comparison we report the p-value associated with rejecting the null hypothesis of equal eBL and sBL prevalences. (C) Distribution of mutations in 5 driver genes. Red points indicate endemic BL, while blue points the sporadic ones.</p