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.

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.

Includes bibliographical references. / Hepatoxicity, especially liver fibrosis. is the major concern with long-term, low-dose oral methotrexate (MTX) therapy for psoriasis. The histological features are nonspecific and may resemble those of nonalcoholic steatohepatitis (NASH). Moreover most of the risk factors of MTX induced liver injury are also associated with NASH. In this study serial liver biopsies of 24 psoriatic patients on lOng-term MTX were investigated to determine whether they had risk factors for NASH and the histological features of a NASH-like pattern of liver injury that could have been contributing to the prevalence and progression of liver injury that occurred while on MTX.

Includes bibliographical references (leaves 66-75). / The aim of the first study was to examine clinical, histopathological and immunohistochemical features of ampullary carcinomas and to determine whether any of these features had significant prognostiC value. The immunohistochemical panel was selected after a literature review and included p53, Ki-67, MUC1, MUC1core, MUC2 and CA 19.9. The data was analyzed by multivariate analysis.

Malignant rhabdoid tumours of the kidney are rare childhood neoplasms. Extra-renal rhabdoid tumours are known to have a distinctive biological behaviour and do not always occur in the paediatric age group. As the histogenesis of rhabdoid tumours, and their apparent relationship to nephroblastoma is still unclear, careful assessment of new cases is required. This investigation illustrates diverse ultrastructural, light microscopic and immunohistochemical findings. These features are related to each other and to the biological behaviour of renal rhabdoid tumours, and six extra-renal lesions with rhabdoid features obtained from the Pathology Archives of the Red Cross Children's Hospital and Groote Schuur Hospital. In this series primitive epithelial elements are a dominant feature, but ultrastructural features of one renal rumour suggest diverse differentiation. The extra-renal lesions investigated include three undifferentiated rhabdoid lesions, a primitive neuro-ectodermal tumour, a malignant epithelioid Schwannoma and a possible undifferentiated hepatocellular carcinoma; all showing areas of extensive rhabdoid differentiation. Pseudo-rhabdoid cells in an additional two cases were also examined. These particular tumours were a nephroblastoma and a fibro-lamellar carcinoma of the liver. These rhabdoid tumour mimics were ultrastructurally different from true rhabdoid cells. Strong immunohistochemical co-expression of Vimentin and cytokeratin in rhabdoid tumour cell inclusions has been noted by previous investigators. (Vogel, 1984) (Gansler, 1991), (Berry, 1992). We speculate that the predominant line of differentiation in renal rhabdoid tumours is epithelial although, as in nephroblastoma multiple lines of differentiation may occur. The extra-renal lesions appear to represent more than one entity, but once again epithelial or neuro-epithelial differentiation appears to be present. Ultrastructural examination is a more useful investigation than immunohistochemistry because of inherent non-specific uptake of antibodies by the filamentous cytoplasmic inclusions.

Includes bibliographical references.

Includes bibliographical references. / Families with hereditary non-polyposis colorectal carcinoma (HNPCC) are not uncommon along the West-Coast of South Africa. These patients present with early onset carcinomas mostly colorectal, predominantly in the right colon. They may develop tumours of other organs, including uterus, breast, stomach and skin. To evaluate and compare the microscopic characteristics of three groups of colorectal carcinomas (HNPCC, early onset colorectal carcinomas and sporadic colorectal carcinomas). 2. To determine the features most characteristic of the group.

DLBCL 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.

Introduction: 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.

INTRODUCTION: Constitutional Mismatch Repair Deficiency (CMMR-D) syndrome is a rare tumour predisposition and polyposis syndrome that presents in childhood. It is caused by mutations in mismatch repair (MMR) genes that result in a tumour spectrum including colorectal cancers, high-grade gliomas, non-Hodgkin T-cell lymphomas and leukaemias. It is characterized by biallelic germline mutation of one of four possible MMR genes resulting in loss of protein expression that can be identified by applying immunohistochemistry to formalin fixed paraffin embedded tissue sections. Use of MMR immunohistochemistry is established in the setting of Lynch syndrome (LS); however, the pattern of loss of staining in the background, non-tumour tissue is unique to CMMR-D syndrome. CMMR-D syndrome is seen in LS families and occurs as a result of consanguinity or founder effect. The South African population has LS families concentrated in the Western Cape and Northern Cape Provinces and the mixed ancestry population shows a unique MLH1 c1528C>T mutation which may have implications on the incidence, penetrance and severity of CMMR-D syndrome seen in our population. The diagnosis of CMMR-D syndrome includes clinical findings outlined in the European Consortium's Care of CMMRD document and confirmation of the biallelic mutation in one of the MMR genes. MMR immunohistochemistry can be used in the diagnosis of CMMR-D syndrome by identifying cases for targeted molecular genetic tests. However, MMR immunohistochemical staining patterns are not usually described in detail, particularly the loss of staining of the affected gene in the background, non-tumour tissue, the key feature of CMMR-D syndrome. METHODS: We performed a retrospective analysis of archival formalin fixed paraffin embedded tissue of children attending Red Cross Children's Hospital with tumours that form part of the CMMR-D spectrum, outlined by the Care for CMMRD criteria. We used the criteria of high-grade gliomas (WHO Grade III or IV) occurring before 25 years of age, cutaneous lesions suggestive of CMMR-D syndrome and patients with a first or second degree relative diagnosed with LS. MMR immunohistochemistry was applied, and the staining pattern was documented in terms of proportion of tumour staining and intensity of staining using a modified Allred Scoring system. Specific attention was given to the characterization of the staining pattern of the background normal tissue. RESULTS: 21 samples taken from 18 patients were evaluated. 16 samples represented brain tumours, predominantly high-grade gliomas. Three samples were excluded due to suboptimal staining despite positive external controls. 12 samples showed intact staining of all four MMR stains. Two samples showed staining of unknown significance. Four samples from 3 different patients showed staining patterns compatible with MMR deficiency. This included two patients, each with a biopsy showing high-grade glioma and two samples of the same patient taken at a 1-year interval of a Burkitt lymphoma. Of these four samples, three samples showed loss of staining in background non-tumour tissue with positive external control, the unique staining pattern for CMMR-D syndrome. These cases will be referred for confirmatory testing by molecular genetic techniques. CONCLUSION: MMR immunohistochemistry can be used in the evaluation of CMMR-D syndrome, but care is needed in evaluating adequacy of staining, the pattern and scoring of staining of both the tumour and the background non-tumour tissue. Endothelial cells are easy to identify and evaluate as background tissue which is useful in extra-intestinal tumours. Neurons and choroid plexus can also be evaluated as background tissue in brain tumour samples. Selection bias in this study resulted in the underrepresentation of lymphomas and colorectal carcinomas. Improved characterization and search for Non-Hodgkin T-cell lymphomas and inclusion of samples of colorectal carcinomas of adolescents and adults would be needed to include these tumours. Use of MMR immunohistochemistry in postmortem tissue samples is not recommended because of suboptimal staining, even with a short post-mortem interval of 1 day. The diagnosis of CMMR-D syndrome depends on clinical application of Care for CMMRD criteria, MMR immunohistochemistry in conjunction with molecular genetic testing. It is important to identify cases of CMMR-D syndrome and offer cancer screening to prevent development of other cancers in the index patient. It also provides an opportunity for genetic counselling and testing of the parents and at-risk siblings.