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

<p>Difuzni krupnoćelijski B limfom je najče&scaron;ći limfom na svetu i obuhvata do 30% non Hodgkin limfoma u zapadnim zemljama i veći procenat u zemljama u razvoju i nerazvijenim zemljama. Obično nastaje de novo ali može nastati sekundarno kao rezultat progresije manje agresivnih limfoma. U većini slučajeva počinje u limfnim čvorovima. Histolo&scaron;ka slika nije uniformna kod svih podtipova DLBCL, zbog morfolo&scaron;kog diverziteta tumorskih ćelija i zbog pratećih neneoplastičnih procesa. Na&scaron;e istraživanje obuhvata 92 bolesnika koji su dijagnostikovani i lečeni na Institutu za onkologiju Vojvodine od 2003. do 2011. godine, od kojih je 66 imalo kompletna imunohistohemijska ispitivanja. Svi su lečeni standardnim protokolom koji uključuje rituksimab. Ispitivali smo morfolo&scaron;ki i imunohistohemijski podtip, ekspresiju imunohistohemijskih markera, pol, starost, stadijum, nodalno/ekstranodalno ishodi&scaron;te i preživljavanje. CD20 je bio pozitivan u svim dostupnim uzorcima. MUM1 u 62,07%, CD10 u 20,23%, BCL6 u 51,72%, CD30 u 10,81%, CD5 u 8,05%, Ki-67 u 30-92%, BCL2 u 39,33%, BAFFR u 46,15%, TACI u 43,94%, BCMA u 10,61%, VEGF u 27,7%, COX2 u 63,64%, CD43 u 18,52%, EBV LMP u 37,88%. Među obolelim ženama je statistički značajno veći procenat MUM1 pozitivnih i BAFFR pozitivnih bolesnika nego među obolelim mu&scaron;karcima. Kod starijih ispitanika je statistički signifikantno ređa ekspresija CD30, COX2, TACI i BCMA. Kod ekstranodalnih limfoma ekspresija TACI je če&scaron;ća nego kod nodalnih limfoma. Ispitanici sa pozitivnom ekspresijom COX2 i ispitanici sa pozitivnom ekspresijom TACI imaju značajno izraženiju ekspresiju Ki-67. Bolesnici sa negativnom ekspresijom BCL2 imaju statistički značajnu korelaciju sa negativnom ekspresijom CD5 i negativnom ekspresijom CD43. Bolesnici sa pozitivnom ekspresijom CD30 imaju statistički značajnu pozitivnu korelaciju sa ekspresijom BCMA. Ispitanici sa pozitivnom ekspresijom COX2 imaju statistički značajnu pozitivnu korelaciju sa ekpresijom TACI. Markeri aktivacije i diferencijacije limfocita nemaju statistički značajnu pozitivnu korelaciju sa boljom prognozom. Markeri signalnih puteva angiogeneze i inflamacije nemaju statistički značajnu pozitivnu korelaciju sa lo&scaron;ijom prognozom. Izgleda da treba biti racionalan i upotrebiti samo osnovna antitela za određivanje imunohistohemijskog podtipa i antitela potrebna za diferencijalnu dijagnozu. Čini se da je prognostički značaj imunohistohemijskih antitela (osim CD20) danas kada se u terapiji koristi Rituximab minimalan.</p> / <p>Diffuse large B-cell lymphoma is the most common lymphoma in the world and represents up to 30% of all NHL in western countries and more in developing and undeveloped countries. It is usually a primary disease but also it can develop secondary as a result of progression of low grade lymphomas. In most cases the disease begins in lymph nodes. Histological features are not uniform in all subtypes of DLBCL due to morphological diversity and following non-neoplastic processes. Our research includes 92 patients whom are diagnosed and treated at the Institute for Oncology of Vojvodina since 2003. to 2011. and 66 among them have complete immunohistochemical findings. All of them are treated with standard protocols including Rituximab. We have examined morphological and immunohistochemical subtype, expression of immunohistochemical markers, sex, age, stadium, nodal / extranodal origin and survival. CD20 was positive in all available samples. MUM1 in 62,07%, CD10 in 20,23%, BCL6 in 51,72%, CD30 in 10,81%, CD5 in 8,05%, Ki-67 in 30-92%, BCL2 in 39,33%, BAFFR in 46,15%, TACI in 43,94%, BCMA in 10,61%, VEGF in 27,7%, COX2 in 63,64%, CD43 in 18,52%, EBV LMP in 37,88%. Female patients have significantly more often MUM1 and BAFFR expression compared to male patients. Older patients have significantly less often CD30, COX2, TACI and BCMA expression. Extranodal lymphomas have more frequent TACI expression then nodal ones. Patients with COX2 expression and patients with TACI expression have significantly higher Ki-67 expression. Patients without BCL2 expression have a significant correlation with CD5 negative expression and CD43 negative expression. Patients with CD30 expression have significant correlation with BCMA expression. Patients with COX2 expression have significant correlation with TACI expression. Markers of activation and differentiation of lymphocytes do not have significant correlation with better prognosis. Markers of signaling pathways for angiogenesis and inflammation do not have a significant correlation with worst prognosis. It seems that we should be rational and use only basic antibodies for determination if immunohistochemical subtype and antibodies necessary for differential diagnosis. It seems that prognostic significance of immunohistochemical antibodies (except CD20) is minimal today in Rituximab era.</p>

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics. DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC. This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC.

O linfoma difuso de grandes células B (LDGCB) é o subtipo de linfoma mais comum em países em desenvolvimento. Entretanto, apesar de sua prevalência e importância, ainda existem poucas publicações com dados epidemiológicos para a população brasileira. Conhecer os fatores de prognóstico é imperativo para identificar os pacientes que responderão melhor ao tratamento, além de permitir sua individualização terapêutica. Alguns estudos demonstraram que pacientes com o mesmo Índice Internacional de Prognóstico (IPI) podem apresentar diferentes sobrevidas, justificando a necessidade de identificar novos marcadores biológicos de prognóstico. O objetivo deste estudo foi avaliar o impacto prognóstico da expressão dos genes BCL2, MDR1 e OCT1, de suas respectivas proteínas e da translocação t(14;18), nos desfechos de resposta completa (RC), sobrevida global (SG), sobrevida livre de doença (SLD) e sobrevida livre de progressão (SLP) em pacientes com LDGCB. Foram avaliados de forma retrospectiva 98 pacientes com LDGCB de novo tratados com R-CHOP. A expressão gênica foi avaliada por PCR em Tempo Real com RNA extraído de amostras parafinadas. A expressão proteica foi avaliada pelo método de imuno-histoquímica. A mediana de idade foi de 54,5 anos e 49 pacientes (50%) eram do sexo masculino. Sessenta e quatro pacientes (85,3%) obtiveram RC, com uma mediana de acompanhamento de 2,66 anos. A expressão mediana de BCL2, MDR1 e OCT1 foi 6,27; 0 e 24,49, respectivamente. Não encontramos impacto prognóstico da expressão do gene BCL2 na RC (p=0,277), SG (p=0,068) e SLD (p=0,860). Porém, a expressão de BCL2 >= à mediana associou-se à menor SLP (p=0,040). Encontramos associação entre expressão do gene OCT1 >= à mediana e pior prognóstico para SG (p=0,010) e SLP (p=0,016). Porém, não observamos impacto prognóstico da expressão de OCT1 para RC (p=0,464) e SLD (p=0,717). Não houve associação entre expressão do gene MDR1 e das proteínas BCL-2, Pg-p e OCT-1 com o prognóstico dos pacientes em relação à RC, SG, SLD e SLP. O número de sítios extralinfonodais (p=0,004 e p=0,005), estádio clínico (p < 0,001 para ambas), IPI (p < 0,001 para ambas) e nível de DHL (p=0,010 e p=0,008) apresentaram impacto prognóstico na SG e SLP, respectivamente. Quando os pacientes foram estratificados pelo estádio, IPI e idade, o grupo com expressão de OCT1 >= à mediana e IPI intermediário-alto ou alto risco apresentou pior SG (p=0,048) e o grupo com idade >= 60 anos e expressão de OCT1 >= à mediana apresentou pior prognóstico para SG e SLP (p=0,025 para ambas). Em conclusão, a expressão de MDR1 não apresentou impacto no prognóstico de portadores de LDGCB, porém, a expressão do gene BCL2 >= à mediana foi associada a menor SLP. Além disso, a hiperexpressão de OCT1 apresentou valor preditivo de prognóstico para a SG e SLP em pacientes com LDGCB tratados com R-CHOP / The diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype in developing countries. However, despite its prevalence and importance, there are still few publications showing the epidemiological data of the Brazilian population. Knowing the prognostic factors is imperative to identify patients supposed to better respond to treatment, as well as to allow their therapeutic individualization. Some studies have shown that patients with the same International Prognostic Index (IPI) may present different survival rates, thus justifying the need to identify new prognosis biomarkers. The aim of this study was to assess the prognostic impact of the genes BCL2, MDR1 and OCT1 and their respective proteins and t (14; 18) translocation on the complete response (CR), overall survival (OS), disease-free survival (DFS) and progression-free survival (PFS) of patients with DLBCL. We retrospectively assessed 98 patients with de novo DLBCL treated with R-CHOP. The gene expression was assessed through real-time PCR using RNA extracted from paraffin samples. The protein expression was assessed through the immunohistochemistry method. The median age was 54.5 years; 49 patients (50%) were men. Sixty-four patients (85.3%) had CR and median follow-up 2.66 years. The median expression of BCL2, MDR1 and OCT1 was 6.27; 0 and 24.49, respectively. We did not find the prognostic impact of the BCL2 gene expression on CR (p = 0.277), OS (p = 0.068) and on DFS (p = 0.860). However, the expression of BCL2 >= the median was associated with the lower PFS (p = 0.040). We found association between OCT1 gene expression >= the median and worse prognosis for OS (p = 0.010) and PFS (p = 0.016). However, we did not find the prognostic impact of OCT1 expression on CR (p = 0.464) and DFS (p = 0.717). There was no association between the MDR1 gene expression and the BCL-2, Pg-p and OCT-1 proteins, and the patients\' prognosis regarding CR, OS, DFS and PFS. The number of extranodal sites (p = 0.004 and p = 0.005), the clinical status (p <0.001 for both), the IPI (p < 0.001 for both) and DHL levels (p = 0.010 and p = 0.008) presented PFS, respectively. When the patients were stratified by stage, IPI and age, the group with OCT1 expression at the median and intermediate-to-high or high-risk IPI had worse OS results (p = 0.048) and the patients in the age group >= 60 years and expression of OCT1 >= the median presented worse prognosis for OS and PFS (p = 0.025 for both). Therefore, the MDR1 expression had no impact on the prognosis of DLBCL carriers. However, the expression of the BCL2 gene >= the median was associated with lower PFS. In addition, the OCT1 hyperexpression presented a predictive prognosis value for OS and PFS in patients with DLBCL treated with R-CHOP

O linfoma difuso de grandes células B (LDGCB) é o subtipo de linfoma mais comum em países em desenvolvimento. Entretanto, apesar de sua prevalência e importância, ainda existem poucas publicações com dados epidemiológicos para a população brasileira. Conhecer os fatores de prognóstico é imperativo para identificar os pacientes que responderão melhor ao tratamento, além de permitir sua individualização terapêutica. Alguns estudos demonstraram que pacientes com o mesmo Índice Internacional de Prognóstico (IPI) podem apresentar diferentes sobrevidas, justificando a necessidade de identificar novos marcadores biológicos de prognóstico. O objetivo deste estudo foi avaliar o impacto prognóstico da expressão dos genes BCL2, MDR1 e OCT1, de suas respectivas proteínas e da translocação t(14;18), nos desfechos de resposta completa (RC), sobrevida global (SG), sobrevida livre de doença (SLD) e sobrevida livre de progressão (SLP) em pacientes com LDGCB. Foram avaliados de forma retrospectiva 98 pacientes com LDGCB de novo tratados com R-CHOP. A expressão gênica foi avaliada por PCR em Tempo Real com RNA extraído de amostras parafinadas. A expressão proteica foi avaliada pelo método de imuno-histoquímica. A mediana de idade foi de 54,5 anos e 49 pacientes (50%) eram do sexo masculino. Sessenta e quatro pacientes (85,3%) obtiveram RC, com uma mediana de acompanhamento de 2,66 anos. A expressão mediana de BCL2, MDR1 e OCT1 foi 6,27; 0 e 24,49, respectivamente. Não encontramos impacto prognóstico da expressão do gene BCL2 na RC (p=0,277), SG (p=0,068) e SLD (p=0,860). Porém, a expressão de BCL2 >= à mediana associou-se à menor SLP (p=0,040). Encontramos associação entre expressão do gene OCT1 >= à mediana e pior prognóstico para SG (p=0,010) e SLP (p=0,016). Porém, não observamos impacto prognóstico da expressão de OCT1 para RC (p=0,464) e SLD (p=0,717). Não houve associação entre expressão do gene MDR1 e das proteínas BCL-2, Pg-p e OCT-1 com o prognóstico dos pacientes em relação à RC, SG, SLD e SLP. O número de sítios extralinfonodais (p=0,004 e p=0,005), estádio clínico (p < 0,001 para ambas), IPI (p < 0,001 para ambas) e nível de DHL (p=0,010 e p=0,008) apresentaram impacto prognóstico na SG e SLP, respectivamente. Quando os pacientes foram estratificados pelo estádio, IPI e idade, o grupo com expressão de OCT1 >= à mediana e IPI intermediário-alto ou alto risco apresentou pior SG (p=0,048) e o grupo com idade >= 60 anos e expressão de OCT1 >= à mediana apresentou pior prognóstico para SG e SLP (p=0,025 para ambas). Em conclusão, a expressão de MDR1 não apresentou impacto no prognóstico de portadores de LDGCB, porém, a expressão do gene BCL2 >= à mediana foi associada a menor SLP. Além disso, a hiperexpressão de OCT1 apresentou valor preditivo de prognóstico para a SG e SLP em pacientes com LDGCB tratados com R-CHOP / The diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype in developing countries. However, despite its prevalence and importance, there are still few publications showing the epidemiological data of the Brazilian population. Knowing the prognostic factors is imperative to identify patients supposed to better respond to treatment, as well as to allow their therapeutic individualization. Some studies have shown that patients with the same International Prognostic Index (IPI) may present different survival rates, thus justifying the need to identify new prognosis biomarkers. The aim of this study was to assess the prognostic impact of the genes BCL2, MDR1 and OCT1 and their respective proteins and t (14; 18) translocation on the complete response (CR), overall survival (OS), disease-free survival (DFS) and progression-free survival (PFS) of patients with DLBCL. We retrospectively assessed 98 patients with de novo DLBCL treated with R-CHOP. The gene expression was assessed through real-time PCR using RNA extracted from paraffin samples. The protein expression was assessed through the immunohistochemistry method. The median age was 54.5 years; 49 patients (50%) were men. Sixty-four patients (85.3%) had CR and median follow-up 2.66 years. The median expression of BCL2, MDR1 and OCT1 was 6.27; 0 and 24.49, respectively. We did not find the prognostic impact of the BCL2 gene expression on CR (p = 0.277), OS (p = 0.068) and on DFS (p = 0.860). However, the expression of BCL2 >= the median was associated with the lower PFS (p = 0.040). We found association between OCT1 gene expression >= the median and worse prognosis for OS (p = 0.010) and PFS (p = 0.016). However, we did not find the prognostic impact of OCT1 expression on CR (p = 0.464) and DFS (p = 0.717). There was no association between the MDR1 gene expression and the BCL-2, Pg-p and OCT-1 proteins, and the patients\' prognosis regarding CR, OS, DFS and PFS. The number of extranodal sites (p = 0.004 and p = 0.005), the clinical status (p <0.001 for both), the IPI (p < 0.001 for both) and DHL levels (p = 0.010 and p = 0.008) presented PFS, respectively. When the patients were stratified by stage, IPI and age, the group with OCT1 expression at the median and intermediate-to-high or high-risk IPI had worse OS results (p = 0.048) and the patients in the age group >= 60 years and expression of OCT1 >= the median presented worse prognosis for OS and PFS (p = 0.025 for both). Therefore, the MDR1 expression had no impact on the prognosis of DLBCL carriers. However, the expression of the BCL2 gene >= the median was associated with lower PFS. In addition, the OCT1 hyperexpression presented a predictive prognosis value for OS and PFS in patients with DLBCL treated with R-CHOP