The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks

Abstract

PARP inhibitor (PARPi) sensitivity is related to tumor-specific defects in homologous recombination (HR). Therefore, there is great clinical interest in tests that can rapidly and reliably identify HR deficiency (HRD). Functional HRD tests determine the actual HR status by using the (dis)ability to accumulate RAD51 protein at sites of DNA damage as read-out. In this study, we further improved and calibrated a previously described RAD51-based functional HRD test on 74 diagnostic formalin-fixed paraffin-embedded (FFPE) specimens (RAD51-FFPE test) from endometrial cancer (EC n = 25) and epithelial ovarian cancer (OC n = 49) patients. We established optimal parameters with regard to RAD51 foci cut-off (≥ 2) and HRD threshold (15%) using matched endometrial and ovarian carcinoma specimens for which HR status had been established using a RAD51-based test that required ex vivo irradiation of fresh tissue (RECAP test). The RAD51-FFPE test detected BRCA deficient tumors with 90% sensitivity and RECAP-HRD tumors with 87% sensitivity, indicating that it is an attractive alternative to DNA-based tests with the potential to be applied in routine diagnostic pathology.

The Evolution of Ovarian Carcinoma Subclassification

“…Even the cut-offs for commercial HRD tests have been shifting, highlighting the challenges in establishing a threshold for a continuous variable that informs a binary treatment decision [ 73 ]. It remains to be seen whether signatures can reproducibly predict the response to PARP inhibitors [ 74 , 75 ], particularly since Brenton and colleagues have depicted the genomic entropy of HGSC with several copy number signatures present in any individual patient [ 69 ]. It may be worth considering giving PARP inhibitors to all patients with HGSC and then identifying the molecular characteristics of the patients that do not respond (negative predictive testing).…”

Tumor BRCA Testing in Epithelial Ovarian Cancers: Past and Future—Five-Years’ Single-Institution Experience of 762 Consecutive Patients

“…To date, methods for HRD detection are limited to genetic profiling of homologous recombination repair genes or genomic assays evaluating HRD-induced genomic scar, even if emerging evidence supports the evaluation of RAD51 foci formation [ 56 , 57 , 58 ]. As recently reported, an NGS profiling including HR genes ( BARD1 , BRIP1 , PALB2 , RAD51C , and RAD51D ) could improve the rate of tumor with HRD by 5–6%, identifying patients who may mostly benefit from PARPi therapy [ 29 ].…”

Toward More Comprehensive Homologous Recombination Deficiency Assays in Ovarian Cancer, Part 1: Technical Considerations

“…Though originally focused on BC samples, the assay was subsequently directly performed on FFPE samples, showing a correlation between RAD51 foci and HRD status [ 182 , 183 ]. Recently, an adaptation to ovarian and endometrial cancer FFPE tissues, entitled RAD51-FFPE, was developed [ 184 ]. Interestingly, RAD51-FFPE paved the way for clinical applications by optimizing the test via calibration of a threshold corresponding to functional HRD.…”