IMMUNOHISTOCHEMICAL AND MOLECULAR CHARACTERIZATION OF BURKITT’S LYMPHOMA IN CHILDREN FROM P. FALCIPARUM, HIV AND EBV PREVALENT SETTING AT MTRH IN WESTERN KENYA

Abstract

Burkitt’s lymphoma (BL) is a B cell non-Hodgkin’s lymphoma (NHL) that affects children in equatorial belt of Africa. The region overlaps geographic areas with high prevalences of infectious diseases. Differential diagnosis of BL with tumours presenting similar clinical and morphological feature presents a challenge in the region. Many Kenyan public health institutions use clininal features and morphology by haematoxylin and eosin (H&E) to diagnose BL. This study carried out molecular characterisation of BL by immunohistochemistry (IHC), c-myc gene translocation, IgVH mutation and cellular microRNA expression at Moi Teaching and Referral Hospital (MTRH) in high prevalence of EBV, HIV-1/2 and Plasmodium falciparum setting in Western Kenya. A prospective comparative study of 104 children with clinical and histological diagnosis of B cell NHL was undertaken. Formalin fixed paraffin embedded (FFPE) tissue sections were stained by H&E, followed by IHC for CD10, CD20, CD38, CD44, BCL-2, MYC protein and Ki-67, c-myc gene t(8;14) translocation by fluorescence in-situ hybridization (FISH), IgVH mutations, miRNA expression, HIV-1/2 and EBV ELISA, pfHRP, HIV-1 RNA and haematogical and cytokine analyses were done at MTRH histopathology, AMPATH Reference Laboratory and the Department of Human Pathology and Oncology, University of Siena. Of the recruited NHL participants, BL accounted for 23.9%. Boys were 78.8% of cases, aged 3-16, with a mean of 8.8 ±3.7 years. Ethnic distribution of cases were; Luhya (54.6%), Kalenjin (21.2%), Luo and Kisii (9.1%) and mainly came from poorer socioeconomic backgrounds. The presentations sites were; abdomen (46%), jaw (33%) and others (21%). All BL tumours showed moderate to strong expression of CD10, CD20, CD38, high Ki-67 proliferative index (100%), MYC+ or MYC- expression. A MYC+ status was associated with an unfavourable outcome. Incidence of BL was 1.4 times greater in EBER+ participants (OR: 1.39, 95% CI: 0.16–12.19) and 1.6 times greater in HIV+ (OR: 1.58, 95% CI: 0.35–7.18), regardless of age and gender. There were variable P.falciparum and WBC values in various study groups. The number of IgVH gene mutations ranged from 15-25 in BL EBER+/- cases and showed elevated expressions of hsa-miR-34a and hsa-miR-127 compared to control cell lines. The Th2, Th17, IL-6 and IL-10 cytokines were elevated, while Th1 cytokines IFN-γ, IL-2 and TNF-α were decreased in BL cases compared to non-BL cases. Other non lymphoid paediatric tumours occurred at MTRH. Expression of CD10, CD20, CD38, Ki-67 and MYC+/- can permit a more accurate BL diagnosis in addition to mutated IgVH and upregulated IL-6, IL-10; hsa-mR-127 and-34a. Immunostaining of MYC protein can serve as a screening tool for which FISH test may be necessary. Pathogenesis mechanisms associated with various immune modulating infectious agents and microRNA’s appeared to exist. Other molecular, immunoregulatory determinants and apparent changing anatomic site o