Functional Ex Vivo Assay Reveals Homologous Recombination Deficiency in Breast Cancer Beyond BRCA Gene Defects

Abstract

Tumors of germline 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 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 irradiation of fresh breast cancer tissue: the recombination REpair CAPacity (RECAP) test. Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring deficiencies, mutational signatures, tumor-infiltrating lymphocytes (TIL), and microsatellite instability (MSI). RECAP was completed successfully in 148 of 170 samples (87%). Twenty-four tumors showed HRD (16%), whereas six tumors were HR intermediate (HRi; 4%). HRD was explained by 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 ( = 0.023) compared with HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in breast cancer (1%; = 0.017). RECAP is a robust functional HR assay detecting both -deficient and-proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.

Role of the DNA damage response in prostate cancer formation, progression and treatment

“…Besides blood-based germline mutation and biopsy based somatic mutation testing, new studies are looking into circulating tumor cells (CTC) or cell-free DNA based detection of a panel of clinically actionable genes to select eligible patients [90,94,95]. Moreover, the efforts made for identifying tumors with HR deficiency by using mutational signatures (HRDetect) or functional HRD tests will guide us to a more personalized cancer management approach [96][97][98][99][100].…”

Prevention of DNA Replication Stress by CHK1 Leads to Chemoresistance Despite a DNA Repair Defect in Homologous Recombination in Breast Cancer

“…Therefore, the established methods to successfully identify HR deficient tumors should be extended by the detection of CHK1 activation. This could be made possible by extending BRCA1/2 testing by genes of the intra S-phase damage response or the combined detection of RAD51 with RPA in S-phase cells [25,26,58].…”

“…The ex vivo tissue slice culture assay analyzes primary tumor samples for their ability to perform HR. This is assessed by the formation of RAD51 foci after DNA damage, where absence of foci formation indicates HR deficiency [25,26]. It remains unclear in both approaches: (i) whether the observed genetic alteration leads to a functional repair defect, (ii) whether the loss of the RAD51 foci formation provides sufficient information about the functionality of HR, and (iii) how overexpression of RAD51 in interaction with CHK1, in its multiple functions, impacts on these processes.…”

The RECAP Test Rapidly and Reliably Identifies Homologous Recombination-Deficient Ovarian Carcinomas

“…As an alternative to DNA-based HRD tests, functional assays that assess the ability of replicating tumor cells to accumulate RAD51 protein at DNA DSBs were developed for use in breast, ovarian and endometrial cancer [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. A major advantage of this approach is that RAD51-based tests detect the current HR status of the tumor, irrespective of underlying genetics.…”

“…The REcombination CAPacity test (RECAP) test is a RAD51-based functional test which was previously successfully used for the analysis of HR function in solid breast and endometrial carcinomas [ 36 , 37 ]. In contrast to other RAD51-based tests, the RECAP test enables the assessment of HR on primary tumor tissue without the need to dissociate tissue and culture tumor cells, greatly reducing the time required to perform the test.…”