Doctoral Degrees (Anatomical Pathology)
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- ItemDevelopment and application of a pathology supported pharmacogenetic test for improved clinical management of South African patients with breast cancer and associated co-morbidities(Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Nicole; Kotze, Maritha J.; Pienaar, Fredrieka; Janse van Rensburg, Susan; Bezuidenhout, Juanita; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Anatomical Pathology.ENGLISH ABSTRACT: Three major challenges in the field of breast cancer have been identified as research priorities for this study. The first is the need to combine genetic testing of high-risk patients with familial breast cancer with pharmacogenetics to reduce recurrence risk in cancer survivors due to drug failure as a consequence of anti-cancer treatment that does not match the patient’s genotype. The second is the delineation of key pathways through which genes implicated in breast cancer and associated co-morbidities can serve as nutritional and drug targets across diagnostic boundaries. The third is the discovery of genetic alterations underlying familial breast cancer not attributed to mutations in the two major tumour suppressor genes, BRCA1 and BRCA2. The study population consisted of 164 breast cancer patients (60 Coloured/Mixed Ancestry and 104 Caucasian), of whom 88 patients were selected from a total of 813 individuals who provided informed consent for inclusion of their data in a genomics database resource generated at the interface between the laboratory and routine clinical practice. In addition, DNA samples of 101 cancer-free individuals above the age of 65 years were available for clinical validation of potentially causative variants in an extended female control group. In the first phase of this study, real-time polymerase chain reaction (PCR) TaqMan© technology was used to confirm the potential value of adding pharmacogenetic testing (CYP2D6 allele 4) to standard immunohistochemistry (IHC)-based breast tumour subtyping complemented by BRCA mutation screening and/or microarray gene profiling in eligible patients. In phase two of the study, common genetic risk factors for cardiovascular disease (CVD) were shown to be significantly associated with earlier age (10 years on average) of breast cancer onset/diagnosis (APOE E4 allele p=0.003; 95% CI: 4-15) and body mass index (BMI) (MTHFR 1298 A>C; p=0.01; 95% CI: 3-14) in patients stratified according to estrogen receptor (ER) status, after adjustment for potential confounders. Age at diagnosis/onset of breast cancer was significantly lower in patients with ER-negative versus ER-positive tumours, after adjustment for ethnicity (p=0.022), while BMI was significantly higher in patiens with ER-positive compared to ERnegative tumours after adjustment for age, ethnicity, and family history of cancer (p=0.035). These findings contributed to the development of an exome pre-screening algorithm (EPA) used in part 3 of this study to select three genetically uncharacterized breast cancer patients for whole exome sequencing (WES) performed in comparison with three ethnically concordant cancer-free controls. WES followed by variant calling using both the standard human genome reference sequence (hg19) and an ethnically concordant major allele reference genome (MARS) revealed a more than 20% discrepancy in the number of gene variants identified in the same samples. After exclusion of a large number of false-positives caused by minor alleles in hg19, two rare missense mutations (<1%) were identified in a family with ER-positive breast cancer: RAD50 R385C and MUC1 Q67E. Three different bioinformatics tools were used to predict functionality and both mutations were confirmed by Sanger sequencing and/or real-time PCR in the Pathology Research Facility (PRF) laboratory. Neither the RAD50 nor the MUC1 missense mutation were identified in the exomes of an unrelated breast cancer patient with triple-negative breast cancer or three population-matched control individuals. This study led to the development of a pathology-supported genetic testing framework for WES beyond the limitations of single-gene BRCA mutation screening in South African breast cancer patients. Our findings support previous WES results indicating that the majority of genetically uncharacterised familial breast cancer may be caused by a combination of low-moderate penetrance mutations exerting their effect in a high-risk environment reflected by high BMI. WES enables identification of genetic risk factors of relevance to both cancer development and tailored therapeutic intervention in a single genetic test.
- ItemAnalysis of the clinical utility of gene expression profiling in relation to conventional prognostic markers in South African patients with breast carcinoma(Stellenbosch : Stellenbosch University, 2014-12) Grant, Kathleen Ann; Kotze, Maritha J.; Wright, Colleen A.; Apffelstaedt, Justus P.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Anatomical Pathology.ENGLISH ABSTRACT: Breast cancer is a heterogeneous disease characterised by marked inter-individual variability in presentation, prognosis and clinical outcome. The recognition that morphological assessment has limited utility in stratifying patients into prognostic subgroups led to clinico-pathological classification of tumour biology, based on receptor expression using immunohistochemical (IHC) techniques. This standard is currently complemented by the development of gene expression profiling methodology that led to the identification of intrinsic molecular subtypes, reflecting tumour genetics as the true driver of biological activity in breast cancer. The study was based on the hypothesis that molecular classification of breast carcinomas integrated with established clinico-pathological risk factors will improve current diagnostic and risk management algorithms used in clinical decision-making. A pathology-supported genetic testing strategy was used to evaluate microarray-based gene profiling against diagnostic pathology techniques as the current standard. Clinico-pathological factors including age, number of positive axillary nodes, tumour size, grade, proliferation index and hormone receptor status was documented for 141 breast cancer patients (143 tumours) referred for microarray-based gene expression profiling between 2007 and 2014. Subsets of patients were selected from the database based on the inclusion criteria defined for three phases in which the study was performed, in order to determine 1) the percentage of patients stratified as having a low as opposed to high risk of distant recurrence using the 70-gene MammaPrint profile within the inclusion criteria, 2) correlation of HER2 status as determined by IHC and fluorescence in situ hybridisation (FISH) with microarray-based mRNA readout (TargetPrint), and 3) the relationship between hormone receptor determination as reported by standard IHC and molecular subtyping using the 80-gene BluePrint profile. Similar distribution patterns for MammaPrint low- and high-risk profiles were obtained irrespective of whether fresh tumour biopsies or formalin-fixed paraffin embedded (FFPE) tissue was used. During the first phase of the study, 60% of the 106 tumour specimens analysed with MammaPrint were classified as low-risk and 40% as high-risk using a newly-developed MammaPrint pre-screen algorithm (MPA) aimed at cost-saving. In the second phase of the study, performed in 102 breast tumours, discordant or equivocal HER2 results were found in four cases. Reflex testing confirmed the TargetPrint results in discordant cases, achieving 100% concordance regardless of whether fresh tumour or FFPE tissue was used for microarray analysis. For the third phase of the study 74 HER2-negative tumour samples were selected for comparative analysis. Statistically significant positive correlations were found between protein expression (IHC score) and mRNA (TargetPrint) levels for estrogen receptor (ER) (R=0.53, p<0.0001) as well as progesterone receptor (PR) (R=0.62, p<0.0001), while combined ER/PR tumour status was reported concordantly in 82.4% of these tumours. BluePrint was essential for interpretation of these results used in treatment decision-making. The MPA developed in South Africa in 2009 was validated in this study as an appropriate strategy to prevent chemotherapy overtreatment in patients with early-stage breast cancer. The use of microarray-based analysis proved to be a reliable ancillary method of assessing HER2 status in breast cancer patients. Risk reclassification based on the TargetPrint results helped to avoid unnecessary high treatment costs in false-positive cases, in addition to providing potentially life-saving treatment to those for whom it was indicated. While neither IHC nor TargetPrint estimation of intrinsic subtype correlated independently with the molecular subtype as indicated by BluePrint profiling, the ability to distinguish between basal-like and luminal tumours was enhanced when the combined protein and mRNA values was considered. Genomic profiling provided information over and above that obtained from routine clinico-pathological assessments. This finding supports the relevance of a pathology-supported genetic testing approach to breast cancer management, whereby advanced genomic testing is combined with existing clinico-pathological risk stratification methods for improved patient management.
- ItemThe development of malignancies in renal allograft recipients with special emphasis on Kaposi's sarcoma(Stellenbosch : Stellenbosch University, 2002-03) Moosa, M. R.; Du Toit, D. F.; Wranz, P. A. B.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Anatomical Pathology.ENGLISH ABSTRACT: Renal transplantation is undoubtedly the best treatment for patients with irreversible renal failure. As a prelude to establishing the nature of malignancies in renal transplant patients we sought to determine factors influencing the outcome of renal transplantation. The survival of renal allografts and of recipients is influenced by a number of demographic, clinical and therapeutic factors. Some of these factors have been better studied than others, and we sought to establish the influence of particular factors on our own patients and allografts. The total number and nature of malignancies developing in these patients subsequent to transplantation was also established. All patients transplanted in our unit between April 1, 1976 and March 31, 1999 were included in the study. In the study period, 542 patients received 623 renal allografts. Demographic details were analysed. Patient and graft outcomes were assessed using Kaplan-Meier survival analysis. The survival curves were compared using univariate analysis; results that were significant were subjected to multivariate analysis. The influence of a number of factors on graft and patient survival were assessed and compared. The impact of a variety of variables on the number and behaviour of malignancies was also established. Patient and graft survival were superior in recipients who were aged less than 40 years; cyclosporine improved graft survival but not patient survival. Early graft loss was associated with a high patient mortality rate. Contrary to the experience elsewhere, black and white patients had similar outcomes after renal transplantation. Of the 542 recipients 41(8.1%) developed malignancies with Kaposi's sarcoma occurring, in 21 patients and skin cancers in 13 patients. The relative risk for the Kaposi's sarcoma development was 235. Kaposi's sarcoma was the most common tumour in non-white patients (accounting for 79% of malignancies in this group) and occurred less than 2 years after transplantation. Kaposi's sarcoma was equally common in male and female recipients. Under cyclosporine the latent period to malignancies was reduced but the frequency remained unaffected. Kaposi's sarcoma skin lesions were present in all the affected patients, with the lower limbs the most common site of involvement. Kaposi's sarcoma responded to reduction of immunosuppression without the need for complete discontinuation, and with preservation of renal function. Extracutaneous involvement occurred in over one quarter of the patients and invariably proved fatal in all patients with visceral organ involvement. The histopathology of posttransplant Kaposi's sarcoma was the same as that described in the other epidemiological forms of the disease. White male recipients were at the greatest risk of developing skin cancers after renal transplantation. Squamous cell carcinomas were relatively more common and were found in sun-exposed areas. The lesions were treated only by local excision and none metastasized. Malignant lymphoma, breast cancer and lung cancer occurred in individual patients but the relative risk of all these lesions were close to unity. Patients with preexisting cancers did not develop recurrences following transplantation. SECTION 2 Both immunosuppression and immunostimulation are thought to play a role in the development of Kaposi's sarcoma after renal transplantation. We investigated the quantitative and qualitative aspects of the immune system of patients who had developed Kaposi's sarcoma. The lymphocyte phenotypes were established using flow cytometry while transformation studies were performed using mitogens. Pokeweed was used as the B-cell mitogen, and concanavalin A and phytahaemagglutinin were the T-cell mitogens. Cell mediated immunity was also tested using delayed type hypersensitivity skin tests and the serum immunoglobulin levels were estimated. Firstly, with regard to humoral immunity, 2/3 of the patients had normal serum immunoglobulin levels, although the B-cell count was reduced in all the patients on immunosuppression. B-cell transformation tests with pokeweed mitogen revealed that B-cell function was not impaired in patients with Kaposi's sarcoma. The patients with decreased immunoglobulin levels also appeared to be malnourished as evidenced by low albumin levels. Secondly, CD3 and CD4, but not CD8, cell counts were reduced in patients with Kaposi's sarcoma. The transformation analyses revealed significant differences compared to controls, with reduced responses in patients with Kaposi's sarcoma. Thirdly, natural killer (NK) cell numbers were also reduced in patients with Kaposi's sarcoma. There were no significant differences in delayed type hypersensitivity skin reactions that could not be accounted for by racial differences. Cellular immunity is impaired in patients with Kaposi's sarcoma with a reduction in the number of NK cells. Both of these components of the immune system are important in protection against malignant transformation. SECTION 3 Kaposi's sarcoma is an important complication of renal transplantation. If the human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma, the virus should be present in all Kaposi's sarcoma lesions and be drastically reduced or cleared from involved tissue on remission of the Kaposi's sarcoma. Fourteen renal transplant patients with cutaneous Kaposi's sarcoma, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of Kaposi's sarcoma, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. HHV-8 DNA was detected in all the cutaneous Kaposi's sarcoma and all the visceral Kaposi's sarcoma samples, as well as a number of Kaposi's sarcoma-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-Kaposi's sarcoma cases than that found in African endemic Kaposi's sarcoma cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low-grade viral replication that coincides with clinical remission of the Kaposi's sarcoma lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant Kaposi's sarcoma. SECTION 4 The recently discovered HHV-8 is an important factor in the aetiopathogenesis of Kaposi’s sarcoma. The reason for the exceptionally high prevalence of Kaposi's sarcoma in our area, as well as that of other developing countries, remains unexplained. We investigated the seroprevalence of the virus in the different healthy subjects as well as organ donor-recipient pairs. All recipients were tested at the time of transplantation, as were the paired donors. Control subjects tested were healthy blood donors, Renal Unit staff, and household contacts of patients with Kaposi's sarcoma. An enzyme-linked immunoassay (ELISA) to the whole virus was used for screening and all positives were confirmed using ELISA to the latent ORF 73 antigen. The prevalence of HHV-8 was similar in all groups and averaged less than 6%. After transplantation the seroprevalence increased to almost 20% but neither the transplanted kidney nor blood transfused perioperatively could account for the increase. Kaposi's sarcoma developed in 3 of the 116 patients transplanted. All patients with Kaposi's sarcoma were proven to be HHV-8 seropositive before the development of the disease. Two of the patients who developed Kaposi's sarcoma were seropositive before transplantation. No patient who received a graft from a seropositive donor developed Kaposi's sarcoma. We refute the notion that a high prevalence of HHV-8 in the general population is responsible for the high prevalence of Kaposi's sarcoma in our population or that the donor organ is a major source of infection in renal transplant recipients. Reactivation, rather than primary infection appears to be the source of the virus after renal transplantation.
- ItemCytokines and tuberculosis : an investigation of tuberculous lung tissue and a comparison with sarcoidosis(University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences., 2005-12) Bezuidenhout, Juanita; Walzl, Gerhard; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Anatomical Pathology.The formation of granulomas at the site of antigen presentation in both tuberculosis and sarcoidosis is an essential component of host immunity for controlling inflammation. Granuloma formation is a complex process that also requires recruitment and activation of lymphocytes and macrophages to the site of infection and arrangement into a granuloma. It is dependant on the activation of especially IFNγ secreting CD4+ T cells, resulting in a Th1 profile. However, it is suggested that a persistently high IFNγ is responsible for the damage caused by granulomatous disease and that moderating cytokines, resulting in a Th0 profile, are necessary to down-regulate the IFNγ response to more appropriate levels later in the disease process, after the antigen has been effectively contained. I propose that: “Cytokine profiles determine clinical and histopathological phenotypes of disease. This thesis tests the hypothesis that it will be reflected by cytokine expression profiles in granulomas in different forms of tuberculosis and in sarcoidosis.” To examine this, biopsy tissue was obtained from patients with pulmonary cavitary tuberculosis, pleural tuberculosis in HIV sero-negative and sero-positive patients, and sarcoidosis. The diagnosis of tuberculosis or sarcoidosis was confirmed, granulomas were characterised as necrotic or non-necrotic, sarcoidosis cases were graded histologically and in situ hybridisation was performed for IL-12-, IFNγ-, TNFα- and IL-4-mRNA. In all patients with pleural tuberculosis, a Th0 profile was noted, while necrotic granulomas were more evident in HIV positive than HIV negative patients. There was a clear association between TNFα and necrosis in tuberculous granulomas that may be ascribed to the increased apoptotic activity of TNFα. An increase in IFNγ correlated with an increase in necrosis, supporting the theory that high IFNγ levels later in disease is detrimental. This effect may be enhanced by a strong presence of TNFα positive cells. An increase in both Th1 and Th2 cytokine mRNA in HIV positive patients supports the theory that an overproduction of cytokines may be a mechanism to compensate for the failure of another immune effector mechanism. Findings in pulmonary tuberculosis were similar to those in pleural tuberculosis. In all sarcoidosis cases the presence of a very strong Th1 and TNFα, but no Th0 response was confirmed. None of the differences in either the histological grading, or the clinical outcome of patients were reflected in the cytokine profile. It is possible that this profile does not reflect the histological grade of disease or that it may reflect various stages of disease. These findings support the theory that a strong Th1 presence later in disease, in conjunction with TNFα may induce fibrosis, as most of these cases showed signs of at least focal fibrosis. Numerous aspects, including a T helper response are involved in granulomatous inflammation. The earlier dogma of good, beneficial (Th1) versus evil, detrimental (Th2), is an oversimplification of a very complex process. It is clear that the effect of a cytokine depends at least partially on the stage of disease. The balance between the various cytokines, and the levels of these cytokines contribute to their role in resolution or disease progression. An early, pure Th1 response may be beneficial if effectively clearing the granuloma-inducing antigen. At this stage, a Th2 presence will be harmful as clearing of the antigen will not be as effective. In chronic disease where failure to remove the antigen results in progression of granulomas with subsequent necrosis and/or fibrosis, a proinflammatory Th1 response may be detrimental and minimising of this effect is needed. An overly strong presence of the various cytokines may also be detrimental, while lower levels will be beneficial.