The RAD51 test is emerging as a promising biomarker for the assessment of functional homologous recombination deficiency (HRD). Yet, the robustness and reproducibility of the immunofluorescence... Show moreThe RAD51 test is emerging as a promising biomarker for the assessment of functional homologous recombination deficiency (HRD). Yet, the robustness and reproducibility of the immunofluorescence-based RAD51 test, in different academic laboratories, have not been systematically investigated. Therefore, we tested the performance of the RAD51 assay in formalin-fixed paraffin-embedded (FFPE) high-grade serous ovarian carcinoma (HGSOC) samples in four European laboratories. Here, we confirm that subtle differences in staining procedures result in low variability of RAD51 and γH2AX scores. However, substantial variability in RAD51 scoring was observed in some samples, likely due to complicating technical and biological features, such as high RAD51 signal-to-noise ratio and RAD51 heterogeneity. These results support the need to identify and perform additional quality control steps and/or automating image analysis. Altogether, resolving technical issues should be a priority, as identifying tumours with functional HRD is urgently needed to guide the individual treatment of HGSOC patients. Follow-up studies are needed to define the key tissue quality requirements to assess HRD by RAD51 in FFPE tumour samples, as this test could help in guiding the individual treatment of HGSOC patients. Show less
