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.

Oesophageal squamous cell carcinoma (OSCC) is a highly malignant tumour that has a poor prognosis and shows marked regional variation in its incidence, implicating environmental factors. South Africa is one of several countries that has areas of high incidence. The exact aetiopathogenesis of OSCC is not well understood. Current environmental risk factors include alcohol, tobacco, human papillomavirus (HPV) infection and nutritional factors including; low intake of Vitamins A, C and riboflavin, lack of fruit and vegetables, ingestion of fungal contaminated foods and consumption of extremely hot beverages. This study was a retrospective immunohistochemical study done on paraffin embedded tissues. The histopathology, grading and staging of all resected squamous cell carcinomas over a twenty one year period from 1982 to 2002, were reviewed. Sixty eight patients were identified; all had an oesophagectomy for OSCC at Groote Schuur Hospital, a tertiary referral centre. Clinical details regarding gender, race, age, smoking or alcohol usage and survival data were collected. Survival data was updated to 23 June 2003. Two paraffin blocks representing OSCC and normal mucosa for each patient were retrieved from the archives in the Division of Anatomical Pathology. In addition, 16 cases of reflux oesophagitis were included for comparison. Initial immunohistochemical staining for HPV (Dako- clone KlH8) was undertaken but the negative results necessitated a shift in the focus of this study to that of cell cycle regulatory proteins. The tissues were evaluated for p53 (Dako - clone D0-7), p2l (Novocastro - clone 4Dl0), cyclin DI (Dako - clone DCS-6) and cyclin E (Novocastro - clone 13A3). Expression was interpreted as positive if 10% or more of the tumour cell population stained. Expression was also stratified into three levels (1, 2 and 3) depending on the percentage positive staining. Normal mucosa did not stain for any of the cell cycle regulators. OSCC stained as follows: 61.8% for p53, 27.9% for p21, 22.1 % for cyclin E and 44.1% for cyclin Dl. Reflux oesophagitis stained as follows: 31.2% for cyclin DI, 12.5% for p21 and 0% for both p53 and cyclin E. Subsequent statistical analysis failed to reveal any prognostic significance to the expression of cell cycle regulators, nor could expression or level of expression be associated with stage, grade, age, gender or alcohol use. There was however a significant relationship between cyclin DI and smoking. In addition, expression of p53 discriminated between malignant and reactive oesophageal lesions. Advancing age proved to be associated with an increased risk of mortality. Lastly, histopathological staging proved to be the most significant prognostic factor in this study.

Includes bibliographical references (leaves 49-55). / Beta-catenin is normally complexed with adenomatous polyposis coli (APC) protein and E-cadherin adhesion molecule, and localized on the cell membrane. If APC/beta-catenin is disrupted, beta-catenin transfers into the nucleus, where it functions as a transcriptional activator, causing unregulated cell proliferation. The localisation of beta-catenin in H NPCC adenomas has not been studied but a shift in beta-catenin to the nucleus has been previously demonstrated in a range of col 0 recta I cancers, including those in HNPCC. The aim of the first part of the study was to determine whether there is a beta-catenin shift occurring as an early event in HNPCC tumours. Coded sections of tumours were immunohistochemically stained with antibody against beta-catenin and counterstained in haematoxylin. 14 HNPCC adenomas, 13 HNPCC carcinomas, 10 FAP adenomas, 10 FAP carcinomas, 10 sporadic adenomas and carcinomas and 10 juvenile polyps -three with dysplasia and seven without- were studied. A score was given for loss of membrane staining (0-1), presence of cytoplasmic staining (0-2) or nuclear staining (0- 2) and a total out of five obtained. An shift in beta-catenin was demonstrated at the adenoma phase in HNPCC. HNPCC, tumours were compared with sporadic tumours and a statistically significant similarity in prevalence of beta-catenin shift found in adenomas and carcinomas. The early shift in beta-catenin in HNPCC led to the second part of the study evaluating the "down-stream" effects of this shift in HNPCC tumours. TheHNPCC sections were immunohistochemically stained with E-cadherin, cmyc and cyclin 01. The results showed a positive correlation between Ecadherin loss, increased cyclin 01 and a shift in beta-catenin. No significant change in c-myc or correlation between c-myc and a shift of beta-catenin was found. In conclusion the study indicates that disruption of the APC/beta-catenin pathway plays a similar role in HNPCC tumours to that in sporadic tumours. A notable exeption is the effect on c-myc and further study is needed in this regard.

Includes bibliographical references.

Breast cancer management continues to be a challenge due to the heterogeneity of the disease and the fact that individuals with the same stage and pathological diagnosis may respond differently to treatment as a result of differences in gene expression. Several components of the Her-2/PI3K/Akt pathway were evaluated for their relevance as potential prognostic markers or indicators for treatment. A secondary objective was to evaluate the CISH technique for its suitability for analysis of Her-2 gene amplification on archived, Papanicolaou-stained fine needle aspiration (FNA) samples. Tissue blocks from a retrospective series of 93 primary breast carcinoma cases were selected, based on their Her-2 status. Twenty six of these cases received trastuzumab treatment while 67 did not. Expression of Her-2, ER, PI3K, PTEN, p-Akt, BCL2, NFκB, MDM2 and p53 were analysed and compared with various clinicopathological features. The CISH technique was evaluated for its suitability for analysis of Her-2 gene amplification on archived, Papanicolaou-stained FNA samples.

Includes abstract. / Includes bibliographical references. / Breast carcinoma is a common cancer in South African women. In the Republic of South Africa, 41 new cases of breast cancer are diagnosed per 100 000 population and the mortality rate is 21 per 100 000 population. Many risk factors have been implicated in the carcinogenesis of this disease; including smoking, family history and hormones, however, this only accounts for about 1/3 of the cases diagnosed. Some studies have implicated Human papillomavirus (HPV) as a possible aetiologic agent in the pathogenesis of breast carcinoma however the results have been inconsistent and sometimes controversial. This study was designed to determine the presence of HPV in breast cancer in a South African cohort and to investigate its influence on the cell cycle. A retrospective and prospective cell block study was undertaken.

Includes bibliographical references (leaves 40-44). / The aims of this study were to determine the age at presentation and the racial distribution of prostatic adenocarcinoma in the Western Cape region and correlate this with histological grade; to correlate the expression of androgen receptor, bcl-2, p53 and Cox-2 with the Gleason grade of disease and patient demographic data and to establish a method to determine androgen receptor (AR) gene amplification in formalin fixed prostatic carcinoma tissue.

Includes abstract. Includes bibliographical references.

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.

Background: Castleman disease (CD) is a lymphoproliferative disorder with four subtypes, some of which are aetiologically linked to Human Herpes virus 8 (HHV-8) which is known to cause diseases preferentially occurring in HIV-infected individuals. There has been a notable increase in the number of patients with HIV/HHV-8 associated CD diagnosed in the Groote Schuur hospital complex. Aims: The aim of the study was to determine the role of DC-SIGN, DC-SIGNR, p24 and HHV-8 (LANA-1) in Castleman disease. Our objectives were to identify the presence of DC-SIGN and DC-SIGNR in HHV-8 infected cells, determine whether HHV-8 and p24 (HIV) co-infection occurs in the same cells and to determine whether HHV-8 infects B and/or T cells. This study not only represents the largest and first immunophenotypic investigative evaluation of CD but also signifies the first double staining immunohistochemical analysis of CD diagnosed at Groote Schuur hospital. Methods: This was both a retrospective descriptive as well as an analytic cross-sectional immunohistochemistry study. Fifty cases of CD diagnosed at the Division of Anatomical Pathology, National Health Laboratory Service, Groote Schuur hospital over a ten and half year period were included in the study. Double immunohistochemistry was used to characterise HHV-8 infected cells using LANA-1 antibody, in conjunction with DC-SIGN, DC-SIGNR, p24, CD20 and CD3. Immunophenotypic analysis was then performed to assess 1) the number of infected HHV-8 cells and 2) number and distribution of cells co-expressing HHV-8 and DC-SIGN, DC-SIGNR, p24, CD20 and CD3. The immunophenotypic profiles were then compared to the CD morphologic subtypes. Results: The study cohort included 26 male and 24 female patients (M: F = 1.08:1), mean age 37.7 years. There were 16 hyaline vascular CD (HV-CD), 16 plasmablastic CD (Pb-CD). Nine plasma cell CD and 9 mixed-CD subtypes. There was a statistically significant association between HIV (n=45) and HHV-8 (n=40) positivity (p < 0.0002). CD4 counts and HAART enrolment were not predictive of CD development (p = 0.6120). Concurrent Kaposi sarcoma was seen in 16% (n=8) of the cohort. When comparing Pb-CD and HV-CD, there were statistically significant differences in density of LANA-1 infected cells (p<0.0002), LANA-1/DC-SIGN co-expressing cells (p <0.0072) and LANA-1/p24 co-expressing cells (p<0.0001). Conclusions: The findings of this study suggest that DC-SIGN may have a role in HHV-8 entry into cells. Furthermore, there is evidence that HIV and HHV-8 co-infection may function synergistically in CD. It is possible that DC-SIGN and DC-SIGNR facilitate dual viral entry into cells and influence viral replication and persistent infection.