A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in the branch of Anatomical Pathology. 21 July 2017. / Aims and objectives The study design aimed to assess and validate various laboratory techniques in the detection of EBV in HIV positive patients with diffuse large B cell lymphoma. The sensitivity and specificity of each technique was determined, as was the presence of an asymptomatic (latent) or lytic phase infection and the viral strain. DLBL samples occurring in HIV seropositive patients were used as a vehicle for these laboratory procedures which included chromogenic in situ hybridisation (EBER), immunohistochemistry (EBNA 2, LMP 1), real time PCR, (EBNA 1, LMP 2 and BZLF 1) and nested PCR (EBNA 2). Materials and Methods 46 cases of previously diagnosed DLBL from HIV positive individuals were identified and retrieved from the archives of the Department of Anatomical Pathology of the University of Witwatersrand and NHLS. All in-situ hybridisation, immunohistochemical and PCR laboratory procedures were carried out in accordance with the Standard Operating Procedures of the Anatomical Pathology Molecular Laboratory, using appropriate negative and positive controls throughout. Ethical clearance was obtained (M140273). Results/Conclusion A 20% frequency of EBV in HIV positive DLBL cases was established. All EBV infections were found to be in the lytic phase, with an almost equal distribution of latency patterns II and III and an equal distribution of EBV strains 1 and 2. EBER in situ hybridisation was confirmed to be the most sensitive and reliable method of viral detection, and the presence of the BZLF 1 gene determined by real time PCR was found to be a reliable indicator of a lytic infection. / LG2018

Introduction: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS) is subdivided according to the cell-of-origin (COO) classification into germinal centre B-cell (GCB) and activated B-cell (ABC) subtypes, each with different molecular profiles and clinical behaviour. This study aims to describe the pattern of the COO subtypes, the proportion of Epstein-Barr virus (EBV) co-infection, and their influence on survival outcomes in a setting of high HIV prevalence. Materials and Methods: This retrospective cohort study included patients diagnosed with de novo DLBCL NOS at our tertiary academic centre in Cape Town, South Africa over a 14-year period. Immunohistochemical stains were performed for COO classification, according to the Hans algorithm. Tumour EBV co-infection was established by EBV-encoded ribonucleic acid in situ hybridisation (EBER-ISH) staining. The effect of the COO subtypes and EBV co-infection on overall survival were described by means of univariate, bivariate and multivariate analyses. Results: A total of 181 patients with DLBCL NOS were included, which comprised 131 HIV-uninfected and 50 HIV-infected patients. There was an equal distribution of GCB and ABC subtypes in the HIV-infected and HIV-uninfected groups. EBV co-infection was detected in 16% of the HIV-infected cases and in 7% of the HIV-uninfected cases (p=0.09). There was no significant difference in the incidence of EBV co-infection between the GCB and ABC subtypes (p=0.67). HIV-infected patients with CD4≥150 cells/mm3 had similar survival to HIV-uninfected patients (p=0.005). Multivariate regression analysis showed that in the HIVinfected group with marked immunosuppression (CD4 <150 cells/mm3), there was significantly poorer overall survival compared to the HIV-uninfected group (HR 2.4, 95% CI 1.3–4.1). There were no statistically significant differences in overall survival by DLBCL COO subtype. Conclusions: There was no difference in the proportion of DLBCL COO subtypes, regardless of HIV status. EBV co-infection was more common in the HIV-infected group, but less than described in the literature. Unexpectedly, there were no significant differences in survival outcomes between the GCB and ABC subtypes. Higher CD4 counts in the HIV-infected group had good survival outcomes, while lower CD4 counts predicted adverse survival outcomes. Further research is needed to explore the genetic mutational landscape of HIVassociated DLBCL.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12871号 / 論医博第2087号 / 新制||医||1006(附属図書館) / 31589 / 北海道大学大学院医学研究科臨床医学コース / (主査)教授 三森 経世, 教授 長澤 丘司, 教授 河野 憲二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The chicken bursa of Fabricius is a primary lymphoid tissue important for B-cell development. Our long-term goal is to understand the role of bursal microenvironment in an early B-cell differentiation event initiating repertoire development through immunoglobulin gene-conversion in the chick embryo. We hypothesize that early bursal B-cell differentiation is guided by signals through cytokine receptors. Our theory is based on previous evidence for expression of the receptor tyrosine kinase superfamily members and interleukin receptors in unseparated populations of bursal B-cells and bursal tissue. Knowledge of the expressed genes that are responsible for B-cell differentiation is a prerequisite for understanding the bursal microenvironment’s function. This project uses transcriptomic analysis to examine gene expression across an early B-cell differentiation event. RNA-seq was performed with total RNA isolated from developing B-cells at embryonic day (ED) 16 and ED 19 (n=3). Approximately 90 million high quality clean reads where obtained from the cDNA libraries. The analysis revealed differentially expressed genes involved in Wnt signaling pathway, Jak-STAT pathway, metabolic pathways, tyrosine metabolism, Toll-like receptor signaling pathway, MAPK signaling pathway, and cellhesion molecules. The transcripts for surface receptors, signal transduction and transcription factors identified in this study represent gene candidates for controlling B-cell differentiation in response to bursal microenvironmental factors.

The growth of B-cell lymphoma, LSCC-RP9, in culture was inhibited by spleen cells from bursa-immunized agammaglobulinemic (A-gamma) chickens. This inhibition was mediated by suppressor T-cells. The growth of transplantable LSCT-RP6 B-cell lymphoma was suppressed in A-gamma chickens, while that of the control SPCT-RP11 T-cell tumor was not affected. Furthermore, the incidence and growth of the LSCT-RP6 tumor in normal recipients were decreased when it was co-transplanted with spleen cells from bursa immunized A-gamma chickens. The results suggest that suppressor T-cells inhibit the growth of B-cell lymphoma.

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