Precision Medicine and Imaging Functional Ex Vivo Assay Reveals Homologous Recombination Deficiency in Breast Cancer Beyond BRCA Gene Defects Titia G. Meijer 1 , Nicole S.Verkaik 1 , Anieta M. Sieuwerts 2 , Job van Riet 3,4 , Kishan A.T. Naipal 1 , Carolien H.M. van Deurzen 5 , Michael A. den Bakker 6 , Hein F.B.M. Sleddens 5 , Hendrikus-Jan Dubbink 5 ,T. Dorine den Toom 5 , Winand N.M. Dinjens 5 , Esther Lips 7 , Petra M. Nederlof 7 , Marcel Smid 2 , Harmen J.G. van de Werken 3,4 , Roland Kanaar 1 , John W.M. Martens 2 , Agnes Jager 2 , and Dik C. van Gent 1 Abstract Purpose: Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double-strand break (DSB) repair and high sensitivity to PARP inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In this study, we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancer tissue: the recom- bination REpair CAPacity (RECAP) test. Experimental Design: Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by explor- ing BRCA deficiencies, mutational signatures, tumor-infiltrat- ing lymphocytes (TIL), and microsatellite instability (MSI). Results: RECAP was completed successfully in 125 of 170 samples (74%). Twenty-four tumors showed HRD (19%), whereas six tumors were HR intermediate (HRi; 5%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas seven HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (P ¼ 0.023) compared with HR proficient (HRP) tumors and MSI was more frequent- ly observed in the HRD group (2/20, 10%) than expected in breast cancer (1%; P ¼ 0.017). Conclusions: RECAP is a robust functional HR assay detect- ing both BRCA1/2-deficient and BRCA1/2-proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results. Clin Cancer Res; 24(24); 6277–87. Ó2018 AACR. Introduction Breast cancer is the most common malignancy in women with the second highest cancer-related mortality rate (1). Approximate- ly 3% of all breast cancer cases are due to germline mutations in BRCA1/2 (2), and in triple-negative breast cancers (TNBC) this percentage is even 10% to 20% (3). The BRCA proteins play an important role in the homologous recombination (HR) pathway, the error-free DNA double-strand break (DSB) repair pathway that operates during the S- and G 2 -phase of the cell cycle. HR deficiency (HRD) leading to impaired DNA DSB repair is fre- quently caused by, but not limited to, defects in BRCA1/2 (4). Therapies specifically targeting tumor cells with impaired HR capacity are PARP inhibitors (PARPi), as well as classical che- motherapies such as platinum-derivates and alkylating agents (5). PARPi causes persistence of single-strand DNA breaks (SSB) by trapping PARP1 on DNA, whereas platinum-derivates cause DNA interstrand crosslinks. Both types of lesions result in replication fork stalling and/or collapse, frequently leading to DSBs that need HR for their repair (5). The targeted approach of PARPi kills tumor cells lacking HR, whereas normal cells remain unharmed, due to their normal DSB repair capacity, a phenomenon often referred to as synthetic lethality. Recently, FDA approval was granted for the use of Olaparib in germline BRCA mutated breast cancer based on the results of the Olympiad trial (6). Although evidence is emerging that the use of PARPi could be extended beyond germline BRCA1/2 mutated cancers to sporadic cancers with BRCA-like features, a gold standard test for predicting response to treatments targeting HR is not yet available (7). Several different HRD tests exist, mostly based on genomic patterns or transcriptional predictors of BRCAness (8–12). These genomic tests measure the accumulation of mutations and chromosomal aberrations over time, but not necessarily reflect the real-time HR status. Beyond mutational status, several 1 Department of Molecular Genetics and Oncode Institute, Erasmus MC University Medical Center Rotterdam, the Netherlands. 2 Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus MC University Medical Center Rotterdam, the Netherlands. 3 Erasmus MC University Medical Center Rotterdam, Cancer Computational Biology Center, Rotterdam, the Netherlands. 4 Department of Urology, Erasmus MC University Medical Center Rotterdam, the Netherlands. 5 Department of Pathology, Erasmus MC University Medical Center Rotterdam, the Netherlands. 6 Maasstad ziekenhuis, Rotterdam, the Netherlands. 7 Depart- ment of Pathology, Netherlands Cancer Institute, Amsterdam, the Netherlands. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corrected online March 27, 2019. Corresponding Author: Dik C. van Gent, Erasmus MC University Medical Center Rotterdam and Oncode Institute, P.O. Box 2040, Rotterdam 3000 CA, the Netherlands. Phone: 31 10 704 39 32; Fax: 31 10 704 10 03; E-mail: d.vangent@erasmusmc.nl doi: 10.1158/1078-0432.CCR-18-0063 Ó2018 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 6277 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/24/24/6277/2049550/6277.pdf by guest on 21 June 2022