Doctoral Degrees (Anatomical Pathology)

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Browsing Doctoral Degrees (Anatomical Pathology) by browse.metadata.advisor "Kotze, Maritha J."

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    Analysis 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.
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    Development 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.
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    Identification of clinically-informative biomarkers within the spectrum of gastro-oesophageal reflux disease in the South African population
    (Stellenbosch : University of Stellenbosch, 2006-03) Van Rensburg, C. J.; Kotze, Maritha J.; Wright, C.; De Jong, G.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Anatomical Pathology.
    Patients with chronic gastro-oesophageal reflux disease are predisposed to Barrett’s metaplasia and oesophageal adenocarcinoma. The availability of molecular markers that can objectively identify patients with Barrett’s oesophagus at increased risk of carcinoma is highly desirable. A literature search was conducted to identify potentially useful biomarkers for genotype-phenotype correlation studies in South African patients with Barrett’s oesophagus. The COX-2, c-myb and c-myc genes selected for mRNA expression analysis were analysed in 26 patients with Barrett’s metaplasia (BM) without dysplasia; 14 with Barrett’s oesophagus and dysplasia (BD); 2 patients with Barrett’s adenocarcinoma (BAC); 19 with erosive oesophagitis (ERD); 25 with non-erosive oesophagitis (NERD) and 19 control individuals with a normal gastroscopy and no gastro-oesophageal reflux disease (GORD) symptoms. In the BD/BAC group, 69% (11/16) showed increased c-myb mRNA expression compared with 35% (9/26) in the BM group (p = 0.03). A statistically significant difference (p = 0.002) in c-myb expression was also observed between Barrett’s patients (20/42, 48%) and the control groups (9/63, 14%). In the BD patients, 21% (3/14) had increased c-myc mRNA expression compared with none in those with BM (p < 0.05) and BAC. No significant differences in mRNA expression levels were observed between ethnic groups for the genes analysed. In an attempt to determine whether the low expression level of c-myc in the study cohort may be related to possible gene-gene interaction, DNA samples of 199 individuals were subjected to genotyping of the functional GT-repeat polymorphism in the promoter region of the NRAMP1/SLC11A1 gene. Both these genes are involved in iron metabolism and c-myc is known to repress NRAMP1/SLC11A1. Genotype and allele frequencies were similar in all the groups studied with the 3/3 genotype being the most common. However, none of the three above-mentioned BD patients with increased c-myc mRNA expression had the 3/3 genotype. Therefore, although small in number, c-myc-NRAMP1/SLC11A1 interaction may be of adverse significance in patients with allele 2. TP53 mutation analysis was performed on 68 Barrett’s patients, and TP53 immuno-staining on oesophageal biopsy specimens of 55 subjects. Sporadic TP53 mutations were not identified in any of the patients with BM or dysplasia without BAC. Immuno-histochemistry staining of 2+ and 3+ intensity was similar in patients with metaplasia and dysplasia (58%). The low mutation frequency and relative non-specificity of TP53 immunostaining observed in Barrett’s patients seem to preclude its widespread use as a screening tool. TP53 mutation detection may however be useful for risk stratification once dysplasia has been diagnosed, as mutations G245R and D281Y were identified in two patients with BAC. Of the genes studied in the South African population, c-myb represents the most useful marker for early detection of an increased cancer risk in Barrett’s patients. In future, patients with Barrett’s oesophagus may benefit from genetic assessment to complement existing cancer surveillance and treatment strategies.