3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item K167 immunohistochemistry quantification in breast carcinoma: a comparison of visual estimation, counting and immunoratio(2019) Joy Van Den Berg, EuniceItem Investigating known cancer suspectibility genes in black South African breast cancer individuals(2018-09-06) Pitere, ReabetsweBreast cancer is an increasingly common cause of morbidity and mortality in black South African women. Of all diagnosed cases of breast cancer in European populations, approximately 5-10% of these arise due to an inherited mutation in a cancer susceptibility gene. BRCA1 and BRCA2 gene mutations are the primary contributors to inherited breast cancer (IBC). However, mutations in other high, moderate and low susceptibility genes have also been identified. Previous studies indicate that approximately 10% of young black South Africans with breast cancer have a deleterious mutation in either BRCA1 or BRCA2 gene. It would, therefore, be pertinent to determine what is contributing to disease in the remainder of the young high-risk black South African breast cancer patients by investigating other genes which are known to confer cancer susceptibility. In addition to a breast cancer syndrome, biallelic BRCA2 mutations can also result in a Fanconi anaemia (FA) phenotype. Previous studies have identified overlapping BRCA2 mutations, c.582G>A and c.5771_5774delTTCA, in patients with either breast cancer or FA. Approximately 80% of black South African patients with FA are found to be homozygous for the c.637_643delTACCGCC mutation in the FANCG gene. The relationship between this FANCG mutation and breast cancer in the black South African population has not been previously determined. The main aim of the study was to increase current knowledge of the molecular basis of breast cancer in the black South African population. The study initially focussed on genotyping the two BRCA2 mutations (c.582G>A and c.5771_5774delTTCA) in a sample of black South African women with breast cancer to determine the frequencies of these proposed common mutations in this population. This was followed by the construction of haplotypes using the BRCA2 mutations to evaluate the presence of a founder effect for each mutation. The study also aimed to screen for the FANCG c.637_643delTACCGCC mutation in breast cancer patients to determine its role in breast cancer development.Item Investigation known cancer susceptibility genes in black SOUTH AFRICAN breast cancer individuals(2018) Pitere, ReabetsweBreast cancer is an increasingly common cause of morbidity and mortality in black South African women. Of all diagnosed cases of breast cancer in European populations, approximately 5-10% of these arise due to an inherited mutation in a cancer susceptibility gene. BRCA1 and BRCA2 gene mutations are the primary contributors to inherited breast cancer (IBC). However, mutations in other high, moderate and low susceptibility genes have also been identified. Previous studies indicate that approximately 10% of young black South Africans with breast cancer have a deleterious mutation in either BRCA1 or BRCA2 gene. It would, therefore, be pertinent to determine what is contributing to disease in the remainder of the young high-risk black South African breast cancer patients by investigating other genes which are known to confer cancer susceptibility. In addition to a breast cancer syndrome, biallelic BRCA2 mutations can also result in a Fanconi anaemia (FA) phenotype. Previous studies have identified overlapping BRCA2 mutations, c.582G>A and c.5771_5774delTTCA, in patients with either breast cancer or FA. Approximately 80% of black South African patients with FA are found to be homozygous for the c.637_643delTACCGCC mutation in the FANCG gene. The relationship between this FANCG mutation and breast cancer in the black South African population has not been previously determined. The main aim of the study was to increase current knowledge of the molecular basis of breast cancer in the black South African population. The study initially focussed on genotyping the two BRCA2 mutations (c.582G>A and c.5771_5774delTTCA) in a sample of black South African women with breast cancer to determine the frequencies of these proposed common mutations in this population. This was followed by the construction of haplotypes using the BRCA2 mutations to evaluate the presence of a founder effect for each mutation. The study also aimed to screen for the FANCG c.637_643delTACCGCC mutation in breast cancer patients to determine its role in breast cancer development.Item Prevalence of breast cancer in patients undergoing microdochetomy for a pathological nipple discharge(2016) Lesetedi, Chiapontroduction Although a pathological nipple discharge can be associated with breast cancer, most of the causes are benign. The current gold standard for diagnosis is microdochectomy and this means that many women will undergo this invasive procedure for benign causes. Demographic data of patients, clinical characteristics, and preoperative radiological investigations which can select patients at risk of cancer may help to reduce the number of patients operated for benign causes but there is little data to confirm this, especially from sub-Saharan Africa. Aim This study aimed to determine the prevalence of cancer in patients who had microdochectomy for pathological nipple discharge in a population in South Africa and evaluate patients’ demographics and clinical characteristics as indicators of underlying cancer. Patients and methods Clinical, radiological and histological data from 153 patients who underwent a microdochectomy for a pathological nipple discharge at two South African breast clinics was collected. Results Invasive or in-situ cancer was found in 12 patients (7.84%) and in all patients, cancer was associated with a bloody nipple discharge. Bloody discharge had a sensitivity of 100% in indicating cancer, specificity of 55.32%, positive predictive value of 16%, and negative predictive value of 100%. Patients with breast cancer were also more likely to be above 50 years (p=0.04). Preoperative mammogram and ultrasound were poor in detecting cancer (0/12). Conclusion In our population, patients with an isolated bloody nipple discharge (no mass) should have microdochectomy done, while many other patients can be managed expectantly with surgery only offered in individualised cases. Thorough clinical examination to determine the true colour and nature of the discharge is vital in the initial assessment of these patients. Preoperative radiology is not helpful in determining the presence of cancer (in an isolated pathological nipple discharge) and microdochectomy still remains the gold standard in diagnosing cancer in these patients.Item 3-Dimensional reconstruction of the breast tumour microenvironment: mediation of tumour progression by T(REG) lymphocytes and NK cells(2015-04-21) Augustine, Tanya NadineBreast tumour progression involves complex interactions between malignant cells and the tumour microenvironment. It is increasingly apparent that immunity is a critical determinant for tumour progression. T regulatory (TREG) lymphocytes, which dominate tumour infiltrating lymphocyte populations, are implicated in facilitating tumour immunoediting processes and suppressing Natural Killer (NK) cell anti-tumour function. To investigate such cellular interaction, experimentation traditionally involves using reductionist 2-dimensional culture systems that do not recapitulate the spatial dimensions of the in vivo microenvironment. Three-dimensional (3D) culture systems, conversely, recreate these dimensions, allowing tumour cells to assume a phenotype more representative of the tumour microenvironment. Given that immunity is a critical factor in determining tumour progression, a novel 3D culture system was established to investigate the interactions between TREG lymphocytes, NK cells and hormone-dependent (MCF-7) or hormone-independent (MDA-MB-231) breast cancer cells. Lymphocyte subpopulations were magnetically isolated, with the efficacy of the sorting procedure verified using flow cytometry. To generate 3D cultures, cell populations were resuspended in growth factor-reduced Matrigel and cultured for 72 hours. This culture system proved effective for RNA extraction for downstream applications; for immunolocalisation of selected tumour biomarkers (ER-α, TGF-β, MUC-1 and EGFR) for qualitative analysis; and for acquisition of cytokine data (IL-1β, IL-2, IL-6, TNF-α, IFN-, CCL2, CCL4 and CXCL8) for quantitative multivariate statistical analysis. Immune mediation was shown to induce the disruption of cell-cell associations, altering the expression of biomarkers and secreted cytokine profiles. Collectively, these results reflect tumour cell subversion of NK cell and/or TREG lymphocyte function to promote tumour progression by generating an inflammatory microenvironment. While hormone-dependent and hormone-independent breast cancer cells differed in their specific response to immune mediation, the mechanisms by which they elicited responses resulted in similar outcomes – that of enhanced evasive and invasive capacity. It is necessary to further elucidate the relationship between the investigated cytokines, biomarkers and immune cells, to understand their interactions and potentially provide more information for therapeutic intervention, given that these factors may contribute to tumours not responding favourably to combined modalities of therapy.Item Occupations and breast cancer in women treated at a tertiary hospital in Johannesburg(2015-04-17) Abrahams, Odette NatashaIntroduction This is the first study in South Africa to look for an association between breast cancer and occupations in black women. Breast cancer is one of the commonest forms of malignancy experienced by women in South Africa and its incidence is increasing (1). Approximately six million women work in South Africa, some of these women are likely to be exposed to carcinogenic agents during their daily working lives. Many occupational carcinogens to the breast have been described and there is some evidence suggesting that many more synthetic chemicals used in different industries may also have carcinogenic properties that have not fully been explored as yet (2). This study plans to identify occupations that place black women at risk of breast cancer in the South African context. Thereafter, it will assess if there is an association between shift work (a known carcinogen to the breast) and breast cancer in black women in South Africa. The findings of the study could be of particular significance to the local context, given that women are entering the workforce in increasing numbers possibly putting more women at risk. Aims The aims of the study are to identify occupations that possibly increase the risk of breast cancer in South Africa, and to specifically assess if there is an association between shift work and breast cancer in black South African women. Objectives To determine whether there are associations between different occupations and breast cancer in black women by calculating the odds ratios (ORs) for breast cancer in different occupations in South Africa. To examine the association between shift work and breast cancer in black women adjusting for variables that may confound the association. Methods The study is an unmatched case-control study using secondary data from the existing Johannesburg Cancer Case Control Study (JCCS) database. The JCCS study is a large ongoing study that recruits black male and female cancer patients with all types of cancers receiving treatment at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), a tertiary hospital in Johannesburg. All women patients recruited from 1 January 2001 to 31 December 2009 were included in the analysis. This included 1 903 cases and 3 990 controls. An expert group was set up to estimate the likelihood of occupational engagement in shift work for the existing occupational groupings present in the JCCS database. These occupations were classified into: a) high probability of shift work; b) possibility of shift work and c) unlikely to do shift work. ORs were estimated using logistic regression. Those who had never worked were the reference category. Bivariate analysis was then carried out to estimate ORs between individual occupations and breast cancer and later to estimate ORs between the likelihood of involvement in shift work and breast cancer. Multivariate logistic regression followed a forward stepwise approach and all the possible confounders present in the JCCS database were considered. These included age, smoking, drinking alcohol, age at first menarche, parity, age at menopause, use of oral contraception and retroviral status. Results With regards to occupation, the bivariate analyses showed significant ORs for breast cancer in the occupational categories of health, education, social services, retail, hospitality, construction, food, textile and manufacturing, with the highest OR in education (OR 2.33, 95% CI: 1.44 – 3.78) and social service (OR 2.39, 95% CI: 1.24 – 4.58) followed by office workers (OR 2.17, 95% CI: 1.47-3.20) and health workers (OR 2.01, 95% CI: 1.33-3.04). Agriculture (OR 0.55, 95% CI: 0.32 – 0.94) and domestic workers (OR 0.96, 95% CI: 0.75 – 1.22) had ORs under one. Following the adjustments for possible confounders, no statistically significant ORs were found between specific occupations and breast cancer. However, manufacturing had the highest OR (OR 1.44, 95% CI: 0.42- 4.94), followed by office workers (OR 1.44 95% CI: 0.31- 5.94) and health workers (OR 1.31, 95% CI: 0.36-4.76) as compared to the never worked group. In the bivariate analyses there was an association between a possibility of doing shift workers and breast cancer (OR 1.66, 95% CI: 1.41-1.97). Similarly, women who had a high likelihood of participating in shift work had an elevated odds ratio for the disease (OR 1.76, 95% CI: 1.44-2.15). After adjusting for confounders in the multivariate model the ORs for both shift work groups were increased with women who possibly did shift work having an OR of 2.18 (95% CI: 1.34- 3.56) and those who had a high likelihood of carrying out shift work having an OR of 2.13 (95% CI: 1.26- 3.61). Conclusions The bivariate analyses identified elevated ORs for breast cancer in some occupations but in multivariate analyses no statistically significant OR were found. Nevertheless ORs>1 were found for the occupational categories of manufacturing, office and health workers. This study reports a strong association of breast cancer and shift work, which is supported by the literature. However shift work still remains necessary for the functioning of many industries. Many gaps still exist, however, and this study has tried to address one of the neglected areas of occupational risks for breast cancer development.Item Inherited breast and ovarian cancer: a review of the available genetic counselling and testing services in Johannesburg(2013) Jefferies, MarianneFive to ten percent of both breast and ovarian cancer cases are attributable to dominantly inherited mutations in genes that predispose to cancer, with a large proportion caused by mutations in the breast and ovarian cancer predisposing genes BRCA1 and BRCA2. Testing for these inherited cancers is indicated for individuals identified as being at high risk, or moderate to high risk, of having a cancer syndrome based on their family history of breast and/or ovarian cancer. Screening for high-risk individuals through services such as genetic counselling, has the potential to improve outcomes for these individuals and lower mortality rates. This study focused on individuals who attended genetic counselling for breast and/or ovarian cancer at the Genetic Counselling Clinics of the Division of Human Genetics, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg from 2001 to 2010. The study was divided into a file review on 218 counsellees and a telephonic interview of 50 counsellees. Focusing on breast and/or ovarian cancer, the study aimed to review who attends genetic counselling and why; who is offered genetic testing; what testing is offered and performed and; who pays for the testing, as well as gain a better understanding of how the service is received by counsellees. The study found that the majority of counsellees are white females, at a high risk of inherited breast and/or ovarian cancer, attend the genetic counselling session alone and are self-referred. There is an under representation of the black and coloured populations and an over representation of the Ashkenazi Jewish population in the cohort. The study‟s findings showed that a main motivator for individuals attending genetic counselling was for BRCA mutation testing, with the majority of testing offered being nationally based testing. The study also demonstrated that the service is generally well received and counsellees reported having a positive experience. Overall, the study pointed to the general lack of understanding and public awareness of genetic counselling, with suggestions to market to both the general population and to other medical professionals in order to reach more high risk individuals. On a practical level, a follow up service was suggested to ensure counsellees adhered to screening measures, informing counsellees on changes to testing protocols and identifying family members who may be at an increased risk of inherited breast and/or ovarian cancer.Item A comparison between oestrogen and HER-2 immunohistochemical staining of core and excision biopsy specimens in breast cancer(2014-02-06) Vermaak, Jacobus StephanusImmunohistochemical staining for oestrogen and human epidermal growth factor receptor (HER)-2 is essential in the management of breast cancer patients. Two histological specimens are usually obtained during a breast cancer patient’s treatment: the core biopsy and the final excision specimen.Item Chromosomal radiosensitivity in South African breast cancer patients before and after radiotherapy(2013-03-19) Muller, XantheneIntroduction: Radiosensitivity (RS) of South African women with breast cancer was investigated as it has been studied in European women, but to date this has not been studied in South African women. The micronucleus assay was used to determine the amount of DNA damage on lymphocytes of breast cancer patients. Materials and Methods: The first component to this study involved the collection of blood samples from breast cancer patients and healthy individuals. For the second component, blood samples from breast cancer patients were collected before and after the completion of radiotherapy (RT). A centromeric micronucleus assay using the Fluorescent in situ Hybridisation (FISH) pancentromeric probe was used to investigate the origin of the micronuclei (MN) and to distinguish between radiation-induced [centromere negative (CM-)] and spontaneous [centromere positive (CM+)] MN. Results: Micronucleus frequencies were slightly higher in breast cancer patients than those observed in lymphocytes of healthy donors. This was noted for the different radiation doses and indicated a trend towards an enhanced chromosomal radiosensitivity in this cancer population. Results were compared before and after radiotherapy. The micronucleus scores for the 0 Gy (sham irradiated samples) were significantly higher (p < 0.05) post radiotherapy. This is an expected result as ionising radiation causes more damage. However, blood samples from post-therapy patients, were shown to have fewer MN after subsequent in vitro 2 Gy and 4 Gy irradiation respectively. When assessing the centromeric micronucleus assay results, a significantly (p < 0.05) higher number of CM- MN was observed than CM+ MN after RT, thereby indicating that ionising radiation causes more breaks in the chromosomes (clastogenic damage). Discussion and Conclusion: This study demonstrates that a group of South African breast cancer patients have slightly higher micronucleus frequencies compared to a population of healthy women, indicating a trend towards a higher sensitivity to radiation.Item Oestrogen receptor mutations and their influence on breast cancer growth(2012-03-12) Amoils, Karin DagmarOestrogen receptor (ER) mutations have been identified for both ERα and ERβ in previous studies. The effects of the deletion variants due to splice mutations on clinical parameters, prognosis and treatment were examined in 61 breast carcinoma patients and 13 control samples from elective reduction mammoplasty procedures, respectively. RNA extracted from fine needle aspirates (FNAs) of breast tissue was reverse transcribed and using nested PCR and sequence analysis the presence of these variants elucidated. Using Χ2 and Fisher’s exact tests their significance with respect to clinical parameters such as tumour size, nodal involvement, stage, presence or absence of metastases, menstrual status and hormone responsiveness was examined. Kaplan-Meier survival analysis was also determined. The T-47D breast cancer cell line was cloned with two clones being selected for further analysis, namely TCA3 (hormone sensitive) and TCC1 (hormone resistant). These clones were treated for ten passages with oestrogen metabolites, 17-β- oestradiol and oestriol; oestrogen precursors, androstenedione and cholesterol; an anti-oestrogen, 4-hydroxy-tamoxifen; and the aromatase inhibitor aminoglutethimide, respectively. RNA was extracted from the cells initially and after the tenth passage and the ERα and ERβ exon profiles were examined using RT-PCR and sequence analysis. After the tenth passage hormone response tests were performed every 24 hours (up to 96 hours) with cell number being determined using the MTT assay. The results indicate that ERα and ERβ variants do not have any affect with respect to menstrual status and nodal involvement (N). Expression of ERα2 and ERα4 are required by the mouse monoclonal antibody (DAKO ® Clone 1D5) in the immunocytochemical assay used for the recognition of the protein in order to assess ER status and therefore show significance. ERαΔ2 and, contrary to previous investigations, the variant ERαΔ3 were not found to play a role in tumourigenesis. ERαΔ5 was observed to be more prevalent in ERα-positive patients and was usually co-expressed with the complete ERα5 indicating heterodimerization. ERαΔ5 showed no significance with respect to progression of disease or response to hormone treatment. An increase in the ratio of ERαΔ4: wild-type ERα4 indicated an increase in metastatic potential of diseased tissue. ERα4 and ERαΔ4 heterodimers were present in both T-47D clones and after 10 passages the TCA3 clone grown in 10-8M aminoglutethimide indicated a complete loss of ERα4 without altering hormone responsiveness. These results suggest that ERαΔ4 may play a role in progression of disease but not in the acquisition of tamoxifen resistance. ERαΔ6 was observed in 15% of patients but not in the T-47D clones or the control samples. An increase in the expression of ERαΔ6 among patient samples significantly increased their metastatic potential (p=0.018). ERαΔ6 was also observed as significant with respect to stage of disease (p=0.023) indicating the possible relevance of ERαΔ6 in progression of the disease. ERαΔ7 was the most frequently observed variant and did not show any significance with regard to any of the clinical parameters examined. The presence of ERαΔ7 did not show significance with regard to hormone response in vivo but in vitro the presence of this variant, expressed as a heterodimer with the wild-type ERα7, conferred greater sensitivity to tamoxifen in the tamoxifen resistant clone TCC1. Multiple exon deletions of ERα were also observed. The two more significant multiple deletion variants were those involving ERαΔ4, namely, ERαΔ2-ERαΔ6 and ERαΔ4-ERαΔ6. The multiple variant ERαΔ4-ERαΔ6 may be involved in tumour progression. ERβ variants were not examined in as much detail as ERα variants due to insufficient material available for analysis. The two domains, the DNA binding domain and the ligand binding domain, of ERβ were analyzed in a few of the patients and in the T-47D clones. They were not found to be significant with respect to the clinical parameters investigated and the ERβ profiles of the TCA3 and TCC1 clones remained unchanged after 10 passages under varying growth conditions.