Browsing by Author "Naidoo, Richard"
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- ItemOpen AccessBiomarker identification in HIV and non-HIV related lymphomas(2016) Magangane, Pumza Samantha; Naidoo, Richard; Govender, DhirenDLBCL is the most common lymphoma subtype occurring in older populations as well as in younger HIV infected patients. The current treatment options for DLBCL are effective for most patients yet the relapse rate is high. While many biomarkers for DLBCL exist, they are not in clinical use due to low sensitivity and specificity. In addition, these biomarkers have not been studied in the HIV context. Therefore, the identification of new biomarkers for HIV negative and HIV positive DLBCL, may lead to a better understanding of the disease pathology and better therapeutic design. Initially differences in the clinicopathological features between HIV negative and HIV positive DLBCL patients were determined by conducting a retrospective study of patients treated at GSH. Subsequent to this, potential protein biomarkers for DLBCL were determined using MALDI imaging mass spectrometry (IMS) and characterised using LCMS. The expression of one of the biomarkers, heat shock protein (Hsp) 70, was confirmed on a separate cohort of samples using immunohistochemistry. Our results indicate that the clinicopathological features for HIV negative and HIV positive DLBCL are similar except for median age, and frequency of elevated LDH levels. Several clinicopathological factors were prognostic for all DLBCL cases including age, gender, stage and bone marrow involvement. In addition, tumour extranodal site was also a prognostic indicator for the HIV negative cohort. The biomarkers identified in the study consisted of four protein clusters including glycolytic enzymes, ribosomal proteins, histones and collagen. These proteins could differentiate between control and tumour tissue, and the DLBCL subtypes in both cohorts. The majority (41/52) of samples in the confirmation cohort were negative for Hsp70 expression. The HIV positive DLBCL cases had a higher percentage of cases expressing Hsp70 than their HIV negative counterparts. The non-GC subtype also frequently overexpressed Hsp70, confirming MALDI IMS data. Expression of Hsp70 correlated with poor outcome in the HIV negative cohort. In conclusion, this study identified potential biomarkers for HIV negative and HIV positive DLBCL from both clinical and molecular sources. These may be used as diagnostic and prognostic markers complementary to current clinical management for DLBCL.
- ItemOpen AccessExpression levels of miRNA-127 in a cohort of HIV-positive and HIV-negative Diffuse Large B-Cell Lymphoma.(2018) Olivier, Chera; Naidoo, Richard; Govender, DhirenDiffuse Large B Cell Lymphoma is one of the most common Non-Hodgkin’s Lymphomas. It is prevalent in older age patients but as of late there has been a rise in the younger population in South Africa due to the rise of HIV. DLBCL is quite an aggressive cancer but can be treated, however the relapse rate is high. There are prognostic indicators which can be seen as factors which can be indicative of a poor outcome for patients. Micro-RNA(miRNA) are small non-coding RNA which can remain stable to be tested. There are several miRNAs which may be linked to prognosis, including miRNA-21 whose upregulated expression has been associated with bad prognosis. However, this is not specific to DLBCL and some studies done have indicated that there may be other miRNAs which are better suited to be biomarkers for DLBCL. Studies have pointed to the direction of miRNA127 as a more reliable microRNA in its association with prognosis in breast, cervical and gastric cancer as well as DLBCL. Objective: The primary aim of this study was to determine the association between miRNA127 and prognostic markers including immunohistochemical stains, survival status and the IPI factor to determine its significance as a prognostic indicator. An additional aim was to determine the correlation between HIV status and expression level of miRNA-127. Design: A total of 42 DLBCL cases were collected from the archive of Division of Anatomical Pathology, University of Cape Town/NHLS Groote Schuur. The H&E slides were assessed before RNA was extracted from FFPE tissue and converted to cDNA. Real time quantitative RT-qPCR was used to assess the expression of the microRNA. Normal tissue as well as reactive lymph node tissue were used as controls. The expression patterns were also correlated to the clinical information to determine if there was any relationship. Results: Out of the 42 cases used, 10 cases were silenced, and 31 cases had high miRNA-127 expression. The expression levels were correlated with the IPI factors and the other clinicopathologic features however no significant conclusion were determined. Conclusion: We found high expression of miRNA-127 cases in the majority of the DLBCL cases. There was no correlation between HIV status and the expression of miRNA-127, nor between the expression and any of the clinicopathological feature. For future studies it is advised that more equally distribution of samples (both HIV status and gender) are obtained, this will allow for a better comparative study.
- ItemOpen AccessHuman Papillomavirus DNA extraction and genotype analysis by multiplex real time polymerase chain reaction from formalin fixed paraffin wax-embedded cervical carcinoma specimens(2019) Price, Brendon; Govender, Dhirendra; Naidoo, RichardIntroduction: Cervical squamous cell carcinoma is most commonly caused by persistent infection by high risk human papillomavirus (hrHPV) genotypes. The exact type of hrHPV varies geographically and is the basis for HPV–based vaccination for cervical squamous cell carcinoma prevention. Little is known regarding local hrHPV genotypes within the Western Cape population of South Africa. Aims and objectives: This was a pilot study aiming to extract of high quality genomic DNA from archival FFPE cervical squamous cell carcinoma cases and identify hrHPV genotypes by multiplex real time PCR (RT-PCR). Materials and methods: A retrospective search identified a total of 57 cases of cervical squamous cell carcinoma for the period 2004-2014. This was reduced to a final number of 23 that exhibited sufficient tumour burden for DNA extraction. The most common age group was 40-49 years. HIV status was as follows: two HIV-positive, 14 HIV-negative and 7 HIV unknown. DNA was extracted from archival FFPE cervical squamous cell carcinoma samples using QIAGEN QIAamp® DNA FFPE Tissue kit. Housekeeping genes were detected by endpoint PCR using standard primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences to determine the quality and integrity of extracted DNA for downstream PCR amplification experiments. HrHPV DNA amplification was optimised using a touchdown PCR technique with L1 consensus gene GP5+/GP6+ primers. HrHPV genotypes were detected using a four colour multiplex hrHPV genotyping kit. Samples showing positive results in overlapping probe filter detection spectra were subjected to DNA Sanger sequencing for final confirmation of specific hrHPV genotype. Results: Standard xylene DNA extraction methods using QIAamp® system yielded adequate amounts of DNA with average final concentration of 463.2 ng/l and A260/A280 ratio of 1.86. Housekeeping genes were successfully detected in all samples, confirming that no significant DNA degradation of target sequences occurred within the archival time range of 2004-2014. HPV L1 detection via GP5+/GP6+ primers with endpoint PCR was not achieved via standard cycling conditions and required the use of a touchdown technique with gradually decreasing annealing temperatures. This method successfully identified HPV L1 sequences in 22 out of 23 cases. Multiplex RT-PCR with four colour hydrolysis probes identified hrHPV genotypes in 22 of 23 cases with relative frequencies of HPV genotypes: 16>>18=39=45>33. Most cases showed infection with a single hrHPV genotype (HPV 16 and one case with HPV 33) with four cases demonstrating two genotypes (two with HPV 16&18, one with 16&33 and one with 39&45) and one case with three genotypes (HPV 16, 39, 45). Interestingly, none of the HIV-positive cases showed multiple hrHPV genotype infection. Four hrHPV cases with overlapping spectra for HPV 18/31 and 45/59 were subjected to Sanger sequencing for confirmation of genotype. Three of four cases showed 100% match for genotypes 18 and 45 with the final case demonstrating only co-infective HPV 16.Conclusion: Commercial DNA extraction kits yield adequate amounts of intact, amplifiable DNA in archival FFPE cervical carcinoma specimens. Touchdown PCR is necessary for HPV detection in extracted FFPE DNA cases using GP5+/GP6+ L1 primers. RT-PCR using multicolour hydrolysis probes is a rapid, sensitive technique for hrHPV genotype screening of cervical squamous cell carcinoma specimens. A three colour detection system rather than four colour kit is recommended for future studies in order to avoid extra cost in DNA sequencing cases with overlapping spectra. This pilot study demonstrates hrHPV genotype prevalence similar to that in other populations and suggests that vaccination with currently available formulations would provide a sufficiently wide coverage of HPV genotypes. Future studies will include application of the FFPE DNA extraction, endpoint PCR and RT-PCR techniques to the remainder of the cases in the original cohort.
- ItemOpen AccessInvestigating the relationship between miRNA expression and epithelial mesenchymal transition in colorectal cancer(2016) Jaca, Anelisa; Naidoo, Richard; Locketz, Michael LIntroduction: Epithelial-mesenchymal transition (EMT) is characterized by the loss of an epithelial phenotype and gain of a mesenchymal phenotype, i.e., migratory and metastatic properties. The EMT process is therefore characterized by a low expression of E-cadherin and high expression of mesenchymal markers (e.g., N-cadherin, snail and vimentin). It is stated that cells which have undergone EMT also gain stem cell features. Therefore, both EMT and stem cell phenotypes have been implicated in carcinogenesis and metastasis of tumour cells. Furthermore, EMT is regulated by small non-coding molecules (miRNAs) that either function as tumour suppressors or oncogenes (oncomirs). Tumour suppressor miRNAs reverse EMT while oncomirs activate it. Therefore, investigating the relationship between miRNAs and EMT is important in addressing metastasis of colorectal cancers (CRC). Aims and Objectives: The aim of the study was to determine the association between miRNA (miRNA-21 and miRNA-34a) expression levels and EMT in CRC. In addition, this investigation aimed to correlate miRNA and EMT data with clinicopathologic features of the study cohort. Methodology: A total of 100 CRC (including 8 known HNPCC cases) Formalin Fixed Paraffin Embedded (FFPE) tissue blocks and their corresponding H&E slides were collected from the archives of the Division of Anatomical Pathology at the University of Cape Town. Subsequently, the FFPE tissue blocks were sectioned at 3μm and IHC analysis of 4 EMT markers (E-cadherin, N-cadherin, snail-1 and vimentin) and 1 stem cell marker (CD44V6) was performed. The stains were then evaluated and scored by a pathologist. The IHC data were then correlated with clinicopathologic features. Furthermore, 59 cases (FFPE tissues and corresponding H&E slides) which included the 8 HNPCCs were randomly selected for miRNA analysis. The H&Es were examined by a pathologist to demarcate normal and tumour regions. RNA was then extracted from 59 tumours and 12 normal tissues using a High Pure FFPET Isolation Kit (Roche). Subsequently, cDNA was synthesized and qRT-PCR was performed to determine the expression levels of miRNA-21 and miRNA-34a. MiRNA-21 and miRNA-34a expression levels were ascertained using the relative quantification method. Moreover, the clinical significance of the two miRNAs was evaluated in relation to MSI status. Therefore, IHC analysis of MLH1, MSH2 and MSH6 mismatch repair proteins was performed on the Ventana platform. Statistical analysis was performed using Fisher's and Pearson's Chi Square tests in Stata 12 to correlate EMT and clinicopathologic data. Additionally, the Mann-Whitney non-parametric test in GraphPad prism 6 was used to determine miRNA-21 and miRNA-34a expression in relation to EMT and MSI data. Results: Our results showed low expression of E-cadherin in 77% of cases. In addition, there was decreased expression of N-cadherin and vimentin in 98% whilst snail-1 expression was decreased in 65% of the cases. Low expression of CD44v6 was also seen in 78% of the cases. There was no correlation between EMT/stem cell markers and clinicopathologic data. Furthermore, increased miRNA-21 expression was significantly associated with grade, lymph node metastasis and age of patients. There was a significant correlation between high miRNA- 21 expression and down-regulated snail-1 and N-cadherin expression. MiRNA-34a expression was not associated with any of the clinicopathologic features. In addition, high miRNA-34a expression was linked with low expression of snail-1 and CD44v6. Increased miRNA-21 expression was related with MSS tumours, whereas there was no relationship between miRNA- 34a and MSI status. Conclusion: Our investigation shows that there is an inverse association between miRNA (miRNA-21 and miRNA-34a) expression and two EMT (N-cadherin and snail-1) markers in our colorectal cancer cohort. Our data also show that both miRNA-21 and miRNA-34a cannot be used as biomarkers to determine progression of the cancer. Contrary to previous studies, our findings indicate that miRNA-21 does not activate EMT in this CRC cohort. However, similar to other studies our results confirm that miRNA-34a may be repressing snail-1 expression, thereby inhibiting EMT in the cancer.
- ItemOpen AccessInvestigation of microRNA expression in thyroid carcinoma among South Africa patients(2017) Mokhesi, Neo; Naidoo, Richard; Govender, Dhiren; Ross, Ian L; Dandara, ColletObjective: Thyroid cancer affects approximately 298 million people worldwide and the major challenge is reliably distinguishing patients who present with poor prognosis from those who do not. There are genetic markers that have been shown to be associated with poor clinical outcome in thyroid cancer, which include mutations in the BRAF and RAS genes. In addition to genetic variation, recent studies have reported on the effects of micro-ribonucleic acids' (miRNAs) differential expression observed in tumour and normal tissue as another possible marker of thyroid cancer prognosis. Therefore, miRNA expression signatures in thyroid cancer could be used as biomarkers for prognosis and diagnosis. This study compared the expression of miRNAs in papillary thyroid cancer and follicular thyroid cancer. Methods: As part of a preliminary study, 66 differentiated thyroid cancer samples were obtained from patients attending Groote Schuur Hospital and used in the study. MiRNA miScript polymerase chain reaction (PCR) Array (Qiagen) was used to determine the differential miRNA expression profiles between follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC). Real time PCR was employed to confirm the expression levels of miRNA- 21 and miRNA-122. Results: 17 miRNAs were upregulated in PTC and 14 in FTC. There were significant differences in the miRNA expression between FTC and PTC. For example, miRNA-21 was the most upregulated miRNA in PTC and miRNA-122 in FTC. We found no correlation of the expression of these miRNAs to clinicopathological features. We observed an association of BRAF mutation positivity to advanced tumour stage and advanced age of presentation however, no correlation was seen to miRNA-21 or miRNA-122 expression. Conclusion: Although we did not observe correlations between miRNAs and any of the clinicopathological features, microRNA expression profile signatures were able to differentiate between PTC and FTC and could potentially be further validated as diagnostic markers.
- ItemOpen AccessInvestigation of protein biomarkers in HIV positive and HIV negative associated DLBCL(2020) Hlatshwayo, Lerato; Naidoo, RichardIntroduction: Diffused large B-cell lymphomas (DLBCL) is the most common aggressive nonHodgkin lymphoma (NHL) worldwide, constituting up to 40% of all cases globally. The incidence of HIV-associated lymphoma has decreased since the introduction of combination antiretroviral therapy (cART) in the mid-1990s. However, NHL, especially DLBCL remains the most common cause of morbidity and mortality among people living with HIV/AIDS, especially in sub-Saharan Africa where 70% of the global HIV/AIDS population reside. Gene expression profiles (GEP) identified based on the cell of origin (COO) two distinct DLBCL subtypes; germinal-centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. These subtypes differ in their genetic abnormalities and response to treatment regimens. Aim: We aimed to investigate in detail, protein distribution profile from FFPE tissue in HIV and non-HIV related DLBCL subtypes. Methods: FFPE DLBCL lymph node tissue samples from HIV and non-HIV related DLBCL were subjected to MALDI-imaging, in order to get the spatial distribution of proteins in DLBCL tissue. Proteins were extracted from tissue samples and subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify proteins present in FFPE DLBCL tissue. The protein profiles from the above-mentioned samples were compared and characterized by cancer pathways. Results: This study had 12 DLBCL cases and 2 human tonsil controls diagnosed from 2009- 2011. These cases were retrieved using the NHLS database. The overall age of DLBCL patients by the time they were diagnosed ranged from 18 to 73 years, with a median age of 48 years. MALDI-IMS peak detection function identified 1466 different m/z values from both the HIV negative and HIV positive DLBCL cases. There were only 50 exclusive m/z values that distinguished the DLBCL subtypes, Using LC-MS/MS we identified a total of 88 proteins, by comparing these proteins, we observed 6 differentially expressed among the DLBCL subtypes and controls Fructose-bisphosphate aldolase C was the only significantly differentially expressed proteins between HIV negative ABC DLBCL and HIV positive ABC DLBCL subtype (p value=1,47738). 10 Conclusion: Using proteomic techniques, we identified and visualized differentially expressed protein in DLBCL subtypes and controls. The majority of these proteins belonged to glycolysis, ATP synthesis, and cellular movement.
- ItemOpen AccessA morphologic and immunohistochemical analysis of gastric adenocarcinoma with regard to the presence of E-cadherin and localisation of β-catenin staining(2014) Roberts, Riyaadh; Naidoo, Richard; Locketz, MichaelIncludes abstract. Includes bibliographical references.
- ItemOpen AccessSequence analysis of the E-cadherin (CDH1) gene in a cohort of gastric cancers seen in the Western Cape(2013) Jaca, Anelisa; Naidoo, RichardGastric cancer is commonly seen in the Western Cape. There are numerous factors that contribute to the development of this cancer and these include both environmental and genetic factors. Amongst the genetic factors, the E-cadherin (CDH1) gene is said to play a major role in the development of gastric cancer. Both germline and somatic mutations have been identified in the CDH1 gene. The germline mutations span the entire exon of this gene and are seen in approximately 29% to 56% of familial gastric cancers. On the other hand, CDH1 somatic mutations are seen in approximately 31% of sporadic cancers. These mutations have been identified in exons 7 to 10, with exons 8 and 9 being the commonly mutated regions. The primary objective of this study was to determine the prevalence of CDH1 genetic mutations in gastric cancer in the Western Cape and to correlate these findings with the demographic/clinicopathologic data.
- ItemOpen AccessThe role of tissue inflammation in Kenyan women with breast cancer(2022) Abdulrehman, Shahin Sayed; Govender, Dhirendra; Naidoo, RichardBackground The immune landscape of breast cancer (BC) molecular subtypes from Sub Saharan Africa is understudied and findings are mainly extrapolated from studies in Caucasians. The objective of this study was to describe the cellular and signalling milieu of BC tumour microenvironment and its associations with BCrisk factors, tumour characteristics and molecular subtypes in Kenyan women with breast cancer. Methods: Molecular subtyping of 838 cases of BC was performed based on Immunohistochemistry (Figure 1). Risk factor data and tumour characteristics were retrieved from an existing database. Visual quantification of overall Tumour Infiltrating Lymphocytes (TILs) was performed on Haematoxylin and Eosin-stained whole slide sections of 226 BC cases based on the guidelines of the International TIL working group. Tissue Microarrays (TMAs) of tumour were constructed, stained with immunohistochemistry for CD3, CD4, CD8, CD68, CD20 and Fox P3, scanned and analysed on the Leica Aperio platform. Cytokine profiles of ER positive and Triple Negative BC were performed using microarray gene arrays and results validated with the ELISA platform. Multivariable polytomous logistic regression models were used to determine associations between BC risk factors and tumour molecular subtypes. Linear and logistic regression models were used to assess the associations between risk factors and tumour features with TILs and TIL subtypes. Results: Distribution of molecular subtypes for luminal A, luminal B, HER2-enriched, and triple negative (TN) breast cancers was 34.8%, 35.8%, 10.7%, and 18.6%, respectively. Higher TILs were associated with high KI67, high grade and HER2 status, luminal B subtype, and smaller tumour size. The TILs were predominantly composed of CD3, CD8 and CD68 with a much smaller contribution of CD4 and CD20 and FOxP3. Differential expression of inflammatory related cytokines (GM-CSF, IL8, TGF β1 and MDC), at both the gene and the protein expression level were observed between Triple negative and ER positive BC. Conclusions: Our findings with regards to enrichment of TILs in more aggressive breast cancers are like what has been previously published, however the differential expression of inflammatory cytokines in breast cancer subtypes some of which has also been described in Caucasian and Asian populations warrants further studies as potential targets for therapeutic approaches and biomarkers of diagnosis and prognosis.