Evaluative Methodology for HRD Testing: Development of Standard Tools for Consistency Assessment.

Abstract

Homologous recombination deficiency (HRD) has emerged as a critical prognostic and predictive biomarker in oncology. However, current testing methods, especially those reliant on targeted panels, are plagued by inconsistent results from the same samples. This highlights the urgent need for standardized benchmarks to evaluate HRD assay performance. In phases IIa and IIb of the Chinese HRD Harmonization Project, we developed ten pairs of well-characterized DNA reference materials derived from lung, breast, and melanoma cancer cell lines and their matched normal cell lines, each paired with seven cancer-to-normal mass ratios. Reference datasets for allele-specific copy number variations (ASCNVs) and HRD scores were established and validated based on three sequencing methods and nine analytical pipelines. The Genomic Instability Scores (GIS) of the reference materials ranged from 11 to 96, enabling validation across various thresholds. The ASCNV reference datasets covered a genomic span of 2340 to 2749 Mb, equivalent to 81.2% to 95.4% of the autosomes in the 37d5 reference genome. These benchmarks were subsequently utilized to assess the accuracy and reproducibility of four HRD panel assays, revealing significant variability in both ASCNV detection and HRD scores. The concordance between panel-detected GIS and reference GIS ranged from 0.81 to 0.94, and only two assays exhibited high overall agreement with Myriad MyChoice CDx for HRD classification. This study also identified specific challenges in ASCNV detection in HRD-related regions and the profound impact of high ploidy on consistency. The established HRD reference materials and datasets provide a robust toolkit for objective evaluation of HRD testing.