Abstract 2504: Determining tumour homologous recombination repair status in PDX and clinical samples using automated RAD51 scoring
Authors: Jack Robertson, Jack Ashton, Gemma N. Jones, Gordon Mills, Tugba Y. Ozmen, Matthew J. Rames, Soonyoung Park, Paul Waring, Natalia Lukashchuk, Elizabeth Harrington
Journal: Cancer Research (2024)

Abstract PARP inhibitors (PARPi) as monotherapy are approved in a wide variety of cancers that show genetic alterations that result in homologous recombination repair (HRR) deficiency. Current genomic scar tests for HRR deficiency (HRD) are predictive of response to PARPi on a population scale but they may fail to provide a real-time functional status of HRR in an individual. RAD51 is a critical DNA repair factor that functions in HRR to safeguard the integrity of the genome and could be a useful patient selection biomarker for PARPi response.During HRR, RAD51 forms filamentous structures to facilitate repair, which can be visualised by direct immunofluorescence (IF) as nuclear foci. Quantification of these RAD51 foci has been shown to be a functional readout of HRR status, however current manual counting of foci is challenging and can result in high inter-observer variability. In order to standardise RAD51 quantification, we aimed to develop an image analysis solution.Tumour samples from patient derived xenograft (PDX) studies and human tissues from commercial sources and clinical trials were co-stained with IF for RAD51 and geminin, as a marker of cells in S/G2 phase of the cell cycle. Manual scoring was performed as reported previously. Visiopharm was used to create and image analysis pipeline in allowing delineate tumour epithelial areas, detection of the geminin positive nuclei and RAD51 foci within these and the quantification of the foci.We have successfully developed an image analysis solution to select geminin positive proliferating tumour cells from PDX and clinical material, then quantify the RAD51 foci within these cells. There is a good correlation with manual scores with a similar accuracy to the manual inter-observer variability (PDX: R2 = 0.76, F1 = 0.95 [n=34]; breast cancer samples: R2 = 0.72, F1 = 0.92 [n=33]). Developing and image analysis solution has challenges due to several factors such as pan-nuclear staining of non-focus forming RAD51 being variable across samples, manual scoring using a wider Z-plane, susceptibility of RAD51 to pre-analytical factors and the fact that a duplex IF stain with geminin is required to mark actively replicating cells.The frequent inactivation of HRR is known to sensitise cancer cells to PARP inhibitors, we demonstrate a quantitative functional readout of HRR competency using RAD51 foci staining and an image analysis algorithm. This solution may enable the use of RAD51 foci as a patient selection biomarker for PARP inhibitors by providing an objective, robust and reproducible algorithm for scoring. Citation Format: Jack Robertson, Jack Ashton, Gemma N. Jones, Gordon Mills, Tugba Y. Ozmen, Matthew J. Rames, Soonyoung Park, Paul Waring, Natalia Lukashchuk, Elizabeth Harrington. Determining tumour homologous recombination repair status in PDX and clinical samples using automated RAD51 scoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2504.