Predictive biomarkers in muscle-invasive bladder cancer

Organ-confined muscle-invasive bladder cancer (MIBC) is treated with either radical cystectomy (RC) or radiotherapy (RT) and carries survival rates of only 40-60%. There is no clear survival advantage between the two therapies and therefore biomarkers that could aid in the choice of treatment would be of use in this disease. Both the abundance of the DNA repair protein, MRE11, and the genotype of a single nucleotide polymorphism in the MRE11 gene, have been found to have potential as predictive biomarkers of RT outcome in MIBC.
The aim of this thesis was to investigate biomarkers relevant to MIBC cancer-specific survival (CSS). To achieve this aim, an automated MRE11 IHC biomarker was investigated, with the goal of obtaining the technical validation required to progress this potential biomarker toward a prospective study. Following on from this the specificity of MRE11 IHC as a biomarker for MIBC was tested by performing MRE11 IHC on a cohort of anal cancer samples. Next the rs1805363 G&GT;A germline SNP in MRE11 was investigated, with the aim of validating the previously reported association between the presence of the rs1805363 A minor allele and increased MRE11 isoform 2 expression. The role of MRE11 isoform 2 in the repair of radiation-induced DNA damage was then examined. Finally, the crowdsourcing of IHC scoring, through a smartphone gaming app, was investigated as a tool to reliably score potential IHC biomarkers with increased efficiency.
The re-optimisation of an automated MRE11 IHC protocol was found to increase the reliability of IHC scoring and significantly alter the distribution of scores for patients within a MIBC TMA tissue cohort when compared to an insufficiently optimised assay.
However, this study did not have the statistical power to validate MRE11 as a predictive biomarker of RT response in MIBC. Additionally MRE11 IHC was not found to be predictive of patient relapse-free survival in anal cancer.
In the analysis of the rs1805363 G&GT;A germline MRE11 SNP, the carriage of the rs1805363 A minor allele was found to correlate with a significant increase in the expression of MRE11 isoform 2 in patient tumour biopsies. Cells which expressed an MRE11 isoform 2 construct were also found to have a significant reduction in survival following irradiation, increased levels of endogenous DNA damage and slower kinetics of DSB repair compared to cells which expressed the MRE11 isoform 1 construct.
In the investigation into crowdsourcing as a method of scoring IHC, the crowdsourced scores for six of the eleven IHC stains analysed were found to have a strong correlation with scores produced by trained researchers. The crowdsourced scores were also found to be of sufficient accuracy to identify MRE11 and CK20 as potential predictive and prognostic biomarkers in MIBC. However, as implemented by this study, the use of crowdsourcing did not result in an increase in the speed of data analysis.
In conclusion, this thesis provided more evidence for MRE11 as a key target for investigations into MIBC biomarkers. The finding that MRE11 isoform 2 expression results in a DNA repair defect is an important consideration for future studies of this protein as a biomarker. Crowdsourcing was identified as a potentially useful tool for screening biomarkers. However, to reach its full potential further investigation into the optimal application of this methodology is needed.

Advisor: Kiltie, A
Nicholson, J