Methylation of glutathione S-transferase pi 1 as an epigenetic biomarker for prostate cancer detection in black South African men

Abstract

African ancestry is a major risk factor for prostate cancer (PCa), contributing to a 1.6-fold increased incidence and a 2.5-fold increased mortality in African Americans compared to European Americans. These are further exacerbated in Black men from South Africa, who experience double the disease burden compared to their African American counterparts, as addressed in Chapter 2. Hypermethylation of the Glutathione S-transferase pi 1 (GSTP1) gene, the focus of this thesis, has been shown to be a valuable diagnostic and prognostic biomarker for PCa with higher accuracies than the widely used and ‘golden standard’ prostate-specific antigen (PSA) screening test. Epigenetic biomarkers, like differential GSTP1 methylation, have the potential to improve disease outcomes by allowing for early, accurate diagnosis and prompt treatment of aggressive PCa. However, the lack of validation studies in Black South Africans makes it difficult to know whether this biomarker is applicable within this high-risk population. Reviewing in Chapter 2 different methylation-based methodologies, the aim of this dissertation was to determine whether GSTP1 could be used as an epigenetic biomarker for PCa detection in Black South African men. In Chapter 3, GSTP1 methylation-specific primers and probes were assessed to determine their suitability to detect the methylation status in prostate tissue derived from men of African ethnicity, specifically Black South Africans. Identifying low-frequency African-specific single nucleotide polymorphisms (SNPs) within published primer/probes, I designed a novel African-specific assay. Ultimately, I tested and standardised the new African-relevant GSTP1 methylation primer/probe sets for their feasibility using my selected method, digital PCR (dPCR), discussing the advantages, limitations and cost-effectiveness, to be used within a routine South African relevant diagnostic/prognostic setting. In Chapter 4, I tested the feasibility of the optimised African-specific GSTP1 dPCR methylation assay developed in Chapter 3, to distinguish PCa from commonly occurring non-cancerous benign prostatic hyperplasia (BPH) in 100 South African men. My cohort consisted of 66 men with a clinicopathological diagnosis of PCa and 34 with a diagnosis of no PCa with BPH. While methylation status, along with age (a known PCa risk factor) and PSA, were not significant predictors of PCa risk in this study, I observed significant differential hypermethylation in prostate tissue derived from men with PCa over men with BPH (P < 0.001). The designed GSTP1 dPCR methylation assay was able to distinguish between PCa and BPH in Black South African men, with an area under the receiver operating characteristic curve (AUC) of 0.907, which was further enhanced when combined with PSA (AUC = 0.957). Here I show the significant potential of GSTP1 hypermethylation as a PCa biomarker in the South African setting. While other studies have mainly focused on GSTP1 methylation in Europeans, in this dissertation I provided a unique, African perspective on GSTP1 methylation in aggressive PCa. As such, I highlighted important African variants that need to be considered when designing GSTP1 differential methylation diagnostic/prognostic assays, I discussed the different methodologies available when considering cost- effective testing, while I tested the applicability of one of the most recent technical approaches, namely dPCR. Data presented provided substantial evidence that GSTP1 is a suitable target for PCa screening in tissue biopsies from Black South Africans. Furthermore, I showed that this methylation-based biomarker has the potential to complement PSA screening for improved diagnosis of clinically significant PCa, specifically differentiating PCa from age-related BPH, with significant potential to not only provide early diagnosis, but importantly reduce associated mortality rates. With further improvements, as discussed in Chapter 5, translation into liquid biopsies, and large-scale validation across different ethnic groups in diverse South Africa, GSTP1 can be a cornerstone for better management of aggressive PCa and could help combat the health disparity.