Abstract 2796: Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity
Authors: Kochupurakkal Bose S. , Parmar Kalindi , Lazaro Jean-Bernard , Unitt Christine , Zeng Qing , Reavis Hunter , Ganesa Chirag , Zhou Shan , Liu Joyce , Palakurthi Sangeetha , Strickland Kyle , Howitt Brooke , Konstantinopoulos Panagiotis , Kirschmeier Paul , Geradts Joseph , Drapkin Ronny , Matulonis Ursula , D'Andrea Alan , Shapiro Geoffrey 
Published: July 2017

Abstract
Homologous recombination (HR) repair deficiency confers sensitivity to inhibitors of poly(ADP-ribose) polymerase (PARP). To date, the identification of tumors with impaired HR has relied on genomic features, including mutational signature, LOH-based HRD assays or gene expression analyses defining ‘BRCAness’. These tests analyze history of the tumor rather than providing a functional assessment of HR status at the time of diagnosis. Therefore, development of a functional assay for HR status in tumors is essential to make accurate treatment decisions. Here, we describe a RAD51-based immunohistochemical (IHC) assay that identifies HR status. We first screened commercial anti-RAD51 antibodies and identified a monoclonal antibody that detects RAD51 foci in HR-competent normal fibroblasts and shows no evidence of foci in HR-deficient (BRCA2-/-) VU423 fibroblasts after γ-irradiation. Conditions for detecting RAD51 foci in FFPE samples were identified using HR-deficient and HR-proficient triple-negative breast cancer cell lines. HR-deficient, PARP inhibitor-sensitive cell lines exhibited high levels of nuclear RAD51 and no evidence of foci, whereas HR-proficient, PARP inhibitor-resistant cells had low levels of nuclear RAD51 and foci. This result was confirmed in a BRCA1-mutated, PARP inhibitor-sensitive PDX model, where there was no evidence of foci although RAD51 levels were high. We further evaluated the pattern of RAD51 staining in 13 high-grade serous ovarian cancer (HGSOC) PDX models, for which sensitivity to olaparib had been characterized. The only olaparib-sensitive model demonstrated complete absence of RAD51 staining. Among the other 12 olaparib-resistant models, RAD51 foci were detectable, both before and after irradiation. The presence or absence of RAD51 foci correlated with olaparib sensitivity and not with BRCA mutation status. Therefore, tumors that are HR-deficient and PARP inhibitor-sensitive are characterized by either high RAD51 nuclear staining without foci, or absence of RAD51 staining. To validate these findings, we analyzed RAD51 staining patterns in a cohort of 50 primary HGSOCs from patients subsequently treated with platinum-based chemotherapy. Among these 50 samples, 45 demonstrated either RAD51 nuclear staining without foci or an absence of RAD51 staining. Five samples demonstrated RAD51 staining with foci. The median survivals of these groups were 6.1 and 1.5 years, respectively. In conclusion, we have developed a robust IHC assay for determining the functional HR-status in tumor samples. Further work will be required to determine if the staining patterns observed predict PARP inhibitor sensitivity among primary patient samples. Funded by a Biomarker Supplement to UM1 CA186709, NIH Grant P50 CA168504, SU2C Ovarian Cancer Dream Team and BCRF grant.
Citation Format: Bose S. Kochupurakkal, Kalindi Parmar, Jean-Bernard Lazaro, Christine Unitt, Qing Zeng, Hunter Reavis, Chirag Ganesa, Shan Zhou, Joyce Liu, Sangeetha Palakurthi, Kyle Strickland, Brooke Howitt, Panagiotis Konstantinopoulos, Paul Kirschmeier, Joseph Geradts, Ronny Drapkin, Ursula Matulonis, Alan D'Andrea, Geoffrey Shapiro. Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2796. doi:10.1158/1538-7445.AM2017-2796