Browsing by Author "Govender, Dhirendra"

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    Open Access
    An investigation of Epstein-Barr Virus (EBV) latency type and MYC gene aberrations in plasmablastic lymphoma diagnosed at Groote Schuur Hospital, Cape Town, South Africa
    (2020) Kriel, Raymond Frank; Ramburan, Amsha; Govender, Dhirendra
    Introduction: Plasmablastic lymphoma (PBL) is a rare, aggressive, AIDS-associated non-Hodgkin lymphoma. The pathogenesis of PBL is incompletely understood, however association with the Epstein-Barr virus (EBV) and the MYC gene, have been identified as important pathogenic mechanisms. Aims and objectives: To characterise the EBV latency in a cohort of patients diagnosed with PBL at Groote Schuur Hospital (GSH), by means of immunohistochemistry. To determine MYC gene aberrations using fluorescent in situ hybridisation (FISH). Materials and methods: The cohort comprised PBL cases diagnosed from 2005-2017. EBER ISH was used to confirm EBV infection. Manual immunohistochemistry using three monoclonal antibodies for EBV latent proteins, (EBNA1, EBNA2 and LMP1) was used to determine the latency type. Manual MYC FISH was performed on all PBL cases using a dual colour break apart rearrangement probe. Results: Forty-nine cases of PBL were included in this study. Forty-one cases were positive for EBER ISH. Thirty-seven (78.7%) cases showed HIV/EBV coinfection. Latency 0 was observed in 29 (70.7%) cases, latency 1 in 8 (19.5%) and latency 2 in 4 (9.8%) cases. MYC FISH was performed on all 49 PBL cases, of which 30 (61.2%) yielded a result. MYC was intact in 11 (36.7%), translocated in 8 (26.7%) and 11 (36.7 %) cases showed copy number variations. Conclusion: Our research demonstrated 37 (90.2%) of the EBV positive PBL cases showed a restricted latency pattern of 0 or 1. Furthermore we found that MYC gene aberrations consisting of translocations and copy number variations occurred in 19 cases (63.3%) , with copy number variations being higher than cited in current literature. Our study is also the first to investigate PBL EBV latency in SA. An uncommon finding was the existence of MYC gene aberrations in HIV positive, EBV negative PBL cases.
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    Open Access
    Differentiating follicular thyroid carcinoma from the follicular variant of papillary thyroid carcinoma
    (2022) Rikhotso, Tshikani Norman; Govender, Dhirendra
    Introduction: Differentiating follicular adenoma (FA), follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC) remain a diagnostic challenge in some cases. This is as a result of the assessment of nuclear features being highly subjective, and the threshold for confirming cytomorphological features for papillary thyroid carcinoma varying greatly among pathologists which results in poor interobserver agreement. Diagnostic challenges may be encountered with some encapsulated follicular-patterned neoplasms of the thyroid due to uncertainty about the presence of capsular or vascular invasion. Immunohistochemistry may provide a better alternative to distinguishing these entities. Aims and objectives: To study the expression of biomarkers HBME-1, CD15, CK-19 and BRAF V600E in follicular adenoma, follicular carcinoma and follicular variant of papillary thyroid carcinoma. To ascertain the usefulness of these markers in differentiating these follicular-patterned thyroid neoplasms. Materials and methods: This is a ten-year retrospective study in which seventy-nine cases consisting of follicular adenoma (n=26), follicular thyroid carcinoma (n=25) and follicular variant of papillary thyroid carcinoma (n=28) were retrieved and reviewed. Four immunohistochemical stains (CD15, CK-19, HBME-1 and BRAF V600E) were performed and scored in tumour tissue. Data were analysed to determine if there was any correlation between the expression of the immunomarkers and the three follicular patterned thyroid neoplasms. Results: The patients' ages ranged from 13 to 74 years. There was a female bias with a female-to-male ratio of 4:1. HBME-1 expression showing varying intensity and proportion was seen in 7 (28%) FA, 15 (65%) FTC and 22 (79%) FVPTC. CK-19 expression showing varying intensity and proportion was seen in 5 (19%) FA, 3 (13%) FTC and 18 (72%) FVPTC. BRAF V600E expression showing varying intensities and proportions was seen in 3 (11%) of FVPTC. FA and FTC cases were all negative for BRAF V600E. CD15 expression showing varying intensity and proportion was seen in 2 (8%) FA, 6 (24%) FTC and 9 (33%) FVPTC. Statistical analysis detected a significant association between group and HBME-1 and CK-19 (H-score, proportion and intensity). The post-hoc comparison revealed that follicular adenoma was significantly more likely to have negative HBME-1 staining and a lower H-score when compared to follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. The follicular variant of papillary thyroid carcinoma was significantly more likely to have an HBME-1 intensity of three and a higher H-score compared to follicular adenoma. Post-hoc comparison revealed that follicular adenoma was significantly more likely to have a negative CK-19 and a lower H-score when compared to follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. Follicular variant of papillary thyroid carcinoma was significantly more likely to have a CK-19 intensity of three and a higher H-score compared to follicular thyroid carcinoma. HBME-1 had an overall specificity and sensitivity of 72% and 74% for distinguishing FA from FTC and FVPTC (benign from malignant). CK-19 had an overall specificity and sensitivity of 80.8% and 43.8% for distinguishing FA from FTC and FVPTC (benign from malignant). CK-19 had specificity of 87% and sensitivity of 72% for distinguishing FTC from FVPTC. Combining HBME-1 and CK-19 did not significantly increase the sensitivity and specificity of these markers. There was statistically no significant association between group and BRAF V600E and CD15 biomarkers. Conclusion: The study, within the small sample size power limitations, has shown that CK-19 may have a role in distinguishing follicular thyroid carcinoma from follicular variant of papillary thyroid carcinoma. In addition, HBME-1 and CK-19 may be used in differentiating benign follicular-patterned thyroid lesions from malignant follicular patterned thyroid lesions.
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    Open Access
    Human immunodeficiency virus (HIV) and Human papillomavirus (HPV) infection and cell cycle regulators in preinvasive lesions and invasive carcinomas of the anus
    (2017) De Jager, Louis Johann; Govender, Dhirendra
    Introduction: Anal cancer is a rare disease which accounts for 1.5% of gastrointestinal tract malignancies. The majority of these carcinomas are squamous cell carcinomas and are associated with high risk-HPV infection. HIV infection appears to interact synergistically with high risk-HPV in the development of squamous cell carcinoma at this site. Aims and objectives: To review the pathology of anal carcinomas and anal intraepithelial neoplasia (AIN) diagnosed between 2003 and 2012. To investigate the frequency of high risk-HPV infection and HIV infection in premalignant and malignant epithelial anal lesions using immunohistochemistry and to investigate the effect of these infections on Langerhans cell density. To investigate the role of cell cycle and WNT signalling pathway markers in the pathogenesis of these lesions. Materials and methods: This was a retrospective study and 51 cases of anal carcinoma and precursor lesions were identified during the study period. Where possible, blocks which contained normal and dysplastic tissue and invasive carcinoma were selected. Ten immunohistochemical stains (p24, p16, pRb, E-cadherin, CD1a, Langerin, Bcl-2, Ki-67, HPV L1 capsid protein and β-catenin) were performed and scored in normal, dysplastic and carcinomatous tissue. Data were analysed to determine if there were statistically significant differences in the expression of markers in different subtypes of carcinomas, grades of differentiation of carcinomas and in the range from normal to carcinoma. Results: The patients' ages ranged from 24 to 81 years. There were 26 females and 24 males; one patient did not have age or sex information available. Twenty-one cases did not have information available on HIV status. Eleven cases demonstrated squamous cell dysplasia only and 40 cases demonstrated invasive carcinoma, 36 of these being squamous cell carcinomas. p24 was positive in only two known HIVpositive cases. p16 demonstrated block positive staining in 35 out of 36 squamous cell carcinomas and 14 out of 18 high grade squamous intraepithelial lesions. There was a significant decrease in the proportion of pRb-positive cells from well to poorly differentiated squamous cell carcinomas (p=0.03). HIV status did not influence the expression of markers. The subtype of carcinoma did not have a significant effect on the proportion of pRb-positive cells. Differentiation of squamous cell carcinoma had a significant effect on the E-cadherin expression score (the more well differentiated a carcinoma, the higher the E-cadherin score; p=0.04). There was a significant difference in E-cadherin expression between normal tissue and squamous cell carcinoma, and dysplastic tissue and squamous cell carcinoma (p=0.002 and p=0.004, respectively). Differentiation, subtype of squamous cell carcinoma and HIV status did not influence the density of CD1a/Langerin-positive Langerhans cells. No significant difference in the density of CD1a/Langerin-positive cells was demonstrated amongst normal, dysplastic and squamous cell carcinoma tissue, regardless of HIV status. The differentiation, subtype of squamous cell carcinoma and HIV status, did not have a significant effect on the Bcl-2 expression. There was a significant difference in Bcl-2 expression among normal, dysplastic and cancerous tissue (p=0.02). There was no significant difference in the Ki-67 proliferation index amongst the different subtypes of squamous cell carcinoma and the degrees of differentiation. HPV L1 capsid IHC only stained two squamous cell carcinomas and nine cases with dysplastic squamous epithelium (AIN I and AIN II). There was no case which showed abnormal localisation of β-catenin. Conclusion: Less than 20% of HIV-positive cases showed positive p24 staining. p24 does not appear to be a useful stain to determine HIV status in non-lymphoid tissues. p16 is known to be a surrogate marker for high risk-HPV infection, and the fact that 35 out of 36 squamous cell carcinomas showed block positive staining suggests that the majority of squamous cell carcinomas in this study were associated with high risk-HPV infection. The mean density of CD1a- and Langerin-positive cells was increased in HIVpositive patients. HPV L1 capsid IHC showed a low sensitivity of detecting AIN and invasive SCC of the anus. Including vaccinations against high risk-HPV in the South African Expanded Programme on Immunisation may reduce the burden of anal dysplastic lesions and invasive squamous cell carcinoma in future.
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    Open Access
    Human 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, Richard
    Introduction: 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.
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    Open Access
    Neuroendocrine neoplasms of the digestive tract: retrospective classification according to the 2019 WHO grading system, and evaluation of the immunohistochemical marker INSM1
    (2020) Aldera, Alessandro Pietro; Locketz, Michael Louis; Govender, Dhirendra
    Neuroendocrine Neoplasms (NENs) of the Gastrointestinal Tract (GIT) are a heterogeneous group of tumours with varied biologic potential and clinical outcomes. They are classified as well differentiated neuroendocrine tumours (WD NET) or poorly differentiated neuroendocrine carcinomas (PD NECs) based on morphology. WD NETs are further subtyped (grade 1, 2 or 3) by evaluating the mitotic rate and Ki-67 proliferative index. The most recent grading system was published in 2019 by the World Health Organisation (WHO). Insulinoma-associated protein 1 (INSM1) is a transcription factor that is expressed in neuroendocrine cells, and recent studies have shown that it is a sensitive and specific marker for neuroendocrine differentiation. The aims of this study were to grade NENs of the GIT according to the 2019 WHO grading system, and to evaluate the expression of INSM1 in order to assess its sensitivity and specificity as a marker of neuroendocrine differentiation compared to chromogranin A (CgA) and synaptophysin (SYN). Sixty-nine GIT NENs diagnosed between 2003 and 2017 at Groote Schuur Hospital were included in this study. The mitotic rate and Ki-67 proliferation index were evaluated for each case. We identified 38 grade 1 NETs, 16 grade 2 NETs, 1 grade 3 NET, 13 small cell type NECs and 1 large cell type NEC. INSM1 immunohistochemical staining was performed on all cases. To assess specificity, we evaluated the expression of INSM1 in other GIT primary tumours (adenocarcinoma, gastrointestinal stromal tumour, lymphoma, leiomyoma and Kaposi sarcoma). Eighty percent of our NEN cases stained with INSM1. We found the sensitivity of INSM1 to be higher than CgA (68%), but lower than SYN (87%) and the combined use of CgA-SYN (94%) when considering all NENs. When evaluating only the PD NEC cases, INSM1 had a higher sensitivity than CgA (50%) and SYN (64%), and an equal sensitivity to the combined use of CgA-SYN (79%). We conclude that the high sensitivity and specificity of INSM1 make it a valuable standalone marker for assessing neuroendocrine differentiation. The nuclear reactivity and high sensitivity of INSM1 make it the preferred neuroendocrine marker for PD NEC.
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    Open Access
    The role of stem cells and WNT signalling pathway in renal cell carcinoma
    (2020) Madlala, Siphelele Clifford; Govender, Dhirendra; Roberts, Riyaadh
    Introduction: Renal cell carcinoma (RCC) accounts for 87% of all kidney cancers. Despite advances in diagnostic techniques and management, renal cell carcinoma remains a lethal tumour accounting for substantial mortality and morbidity. The poor prognosis arises from metastasis, chemoradiation resistance and disease relapse. Cancer stem cells, a subpopulation of tumour cells with capacity to self-renew and reconstitute tumour heterogeneity have been implicated as the root cause of poor prognosis. Therefore, a better understanding of biomarkers of cancer stem cells will be useful for risk stratification, prognostication and may lead to novel targeted therapies that will ultimately alter the management of many patients. Aims and objectives: To review the morphological subtypes of renal cell carcinomas diagnosed in the Division of Anatomical Pathology, National Health Laboratory Service, Groote Schuur Hospital over a 10-year period. To identify cancer stem cells in various histopathological subtypes of renal cell carcinoma using immunohistochemical markers (CD133 and CD105). To review the WNT signalling pathway in renal cell carcinomas using selected protein expression by immunohistochemistry (β-Catenin).Materials and methods: Ten-year retrospective study in which sixty-four cases of renal cell carcinoma were retrieved and reviewed. Four immunohistochemical stains (β-catenin, HIF-1α, CD133 and CD105) were performed and scored in tumour tissue. Data were analysed to determine if there was any correlation between expression of the biomarkers and the histopathological subtypes of renal cell carcinoma. Results: The mean age of the patients was 56-years (range, 35 to 81 years). Females constituted just over half (52%, n = 33) of the study patients. All 64 cases were confirmed as renal cell carcinomas, with 29 (45%) clear cell renal cell carcinomas, 14 (22%) papillary renal cell carcinomas, 9(14%) chromophobe renal cell carcinomas, 9 (14%) multicystic renal cell carcinomas and 3 (5%) sarcomatoid renal cell carcinomas. Ten (16%) cases showed abnormal β-Catenin cytoplasmic localisation. The majority of cases (n=6, 60%) showing abnormal β-Catenin localisation were clear cell renal cell carcinomas. However, there was no significant correlation between abnormal and normal β-Catenin localisation and RCC histopathological subtype (p = 0.766). CD133 immunohistochemical studies showed low expression in 52 (81 %) cases and high expression in 12 (19 %) cases. There was no correlation between low and high CD133 expression and histopathological RCC subtype (P = 0.800). CD105 immunostaining showed tumour cell immunopositivity in one case of clear cell renal cell carcinoma whilst the rest of the cases were negative. The low, moderate and high microvascular density categories had 24, 10 and 32 cases respectively. There was no significant correlation between low, moderate, and high microvascular densities and the histopathological RCC subtype (P = 0.320). HIF-1α immunohistochemical studies showed low expression in 39 (61 %) cases and high expression in 25 (39 %) cases. There was no significant correlation between levels of HIF-1α expression and the histopathological RCC subtype (P =0.972).Conclusion: Within the power limitations of this small study,β-catenin abnormal expression, microvascular densities and levels cytoplasmic CD133 and HIF-1α were not associated with any histopathological subtype of renal cell carcinoma.
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    Open Access
    The role of tissue inflammation in Kenyan women with breast cancer
    (2022) Abdulrehman, Shahin Sayed; Govender, Dhirendra; Naidoo, Richard
    Background 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.
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    Open Access
    TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis
    (BioMed Central Ltd, 2015) Francisco, Ngiambudulu; Hsu, Nai-Jen; Keeton, Roanne; Randall, Philippa; Sebesho, Boipelo; Allie, Nasiema; Govender, Dhirendra; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam
    BACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF / ) mice and compared outcomes of disease against TNF f/f control and global TNF / mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF / mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF / mice were resistant to infection and presented with a phenotype similar to that in TNF f/f control mice. Impaired immunity in TNF / mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB.