Abstract B04: RAD51 expression as a biomarker of homologous recombination deficiency in ovarian cancer
Authors: Hoppe Michal M. , Tan David SP , Lim Diana GZ , Karnezis Anthony , Huntsman David , Steel Jennifer , Liu Xinxue , Paul James , Lewsley Liz-Anne , Siddiqui Nadeem , Brown Robert , Jeyasekharan Anand D. 
Published: December 2017

RAD51 is a critical component of the homologous recombination pathway, forming a nucleoprotein filament that enables strand exchange and templated error-free DNA repair. The tumor suppressors BRCA1 and BRCA2 interact with RAD51 to control its activity on DNA. Defects in homologous recombination in tumors are clinically relevant, with evidence of synthetic lethality of such cancers to poly ADP ribose polymerase (PARP) inhibitors.

Mutations in RAD51 are uncommon in cancer, but aberrant over-expression of RAD51 has been reported as a mechanism to overcome a recombination defect in in-vitro models. However there are no large scale studies on RAD51 expression in clinically annotated data sets. Here we report a series of experiments to study RAD51 protein expression in ovarian cancer, a tumor type where recombination defects are prominent and being evaluated in several clinical trials of PARP inhibition. Analysis of DNA repair protein expression in formalin fixed clinical tissue is challenging, and our experiments highlight recent advances in quantitative microscopy that are generalizable to other clinical scenarios as well.

We first evaluated several commercially available monoclonal antibodies to RAD51 using siRNA depleted cell blocks, to identify an appropriate reagent and staining conditions for formalin fixed paraffin embedded (FFPE) material. We then analyzed RAD51 expression in a collection of 600 ovarian cancer samples obtained through the British Columbia Cancer Agency (BCCA) OvCare consortium, using a combination of multiplexed immunofluorescence staining and automated spectral microscopy to quantify staining on a per-cell basis. We find that RAD51 expression displays a Gaussian distribution in this cohort of high grade epithelial cancers, with no obvious correlation to BRCA mutation status. RAD51 sub-nuclear foci, which are a commonly used measure of homologous recombination in-vitro, did not prove to be accurately quantifiable in FFPE material.

We then stained for RAD51 in a collection of approximately 250 ovarian cancer samples obtained from the SCOTROC4 trial. This clinical trial enrolled women with ovarian cancer into two treatment arms with fixed dose and escalated doses of carboplatin. Platinum sensitivity has been shown to correlate with sensitivity to PARP inhibitors, due to the requirement of homologous recombination for repair of platinum adducts. Therefore this trial offers a unique opportunity to study the predictive significance of biomarkers of homologous recombination in-vivo. In our analysis of this data set, we find that samples in the lowest quartile of RAD51 expression displayed a significantly improved survival after platinum chemotherapy, consistent with decreased HR in these cases. These are the first data on RAD51 in a clinical trial dataset of platinum sensitivity.

Our study highlights methods and technical challenges for the quantitative analysis of DNA repair proteins in human clinical trial specimen, and describes a potential biomarker for synthetic lethal approaches targeting homologous recombination deficiency.

Citation Format: Michal M. Hoppe, David SP Tan, Diana GZ Lim, Anthony Karnezis, David Huntsman, Jennifer Steel, Xinxue Liu, James Paul, Liz-Anne Lewsley, Nadeem Siddiqui, Robert Brown, Anand D. Jeyasekharan. RAD51 expression as a biomarker of homologous recombination deficiency in ovarian cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B04.