Homologous recombination deficiency testing implementation in the routine clinical setting: real-life experience and lessons learned.

BACKGROUND: Homologous recombination deficiency (HRD) is a critical biomarker for predicting response to poly (ADP-ribose) polymerase (PARP) inhibitors in high-grade serous ovarian cancer (HGSOC). Hence, genomic scar-based HRD testing must be implemented in routine clinical labs. This study aims to obtain a head-to-head comparison of some of the applications available for HRD status determination in a real-world clinical cohort to guide HRD testing standardization. MATERIALS AND METHODS: HRD scores were obtained from HGSOC formalin-fixed, paraffin-embedded samples from the Molecular Prescreening Program at Vall d'Hebron Institute of Oncology (VHIO) using two approaches: VHIO-HRD (n = 229) and shallow sequencing-based HRD score (LSTsh-HRD; n = 123). Large-scale transitions (LST), telomeric allelic imbalance (TAI), and genomic loss of heterozygosity (LOH) were calculated using data from a custom hybrid-capture panel (VHIO-HRD score), and LST was determined from shallow whole-genome sequencing of a genomic library (LSTsh-HRD score). Validation was obtained by benchmarking against established and commercially available platforms. Tumor fraction (TF) was estimated using several computational tools. RESULTS: Optimized HRD status cutoff for VHIO-HRD (LSTVHIO-HRD + TAIVHIO-HRD + LOHVHIO-HRD) was set to ≥47 based on comparison with commercially available tools, whereas HRD status cutoff for LSTsh-HRD was predefined at ≥20. Despite differences in chemistry and scoring metrics, strong concordance in HRD classification was observed across assays. TF strongly influenced HRD performance, with optimal sensitivity at TF ≥0.4 for VHIO-HRD and ≥0.2 for LSTsh-HRD. CONCLUSIONS: We validated that HRD scar quantification can be implemented across testing laboratories using assays with different library preparation chemistries and biomarker calculation algorithms. TF is a critical determinant of accurate HRD scoring; LSTsh-HRD quantification offers improved sensitivity in low-cellularity contexts, and VHIO-HRD enables status classification across the continuum of score values in samples with TF ≥0.4. A multiplatform, standardized HRD testing strategy may enhance biomarker-driven patient selection for PARP inhibitor therapies in HGSOC and other HRD-associated tumors.