Table 5.

Main strengths and weaknesses of the HRD assays.

Platform	Strengths	Weaknesses
Genomic HRD assay
BRCA mutation + GIS score (or LOH score)	FDA-approved companion test Rapid analysis	Unable to detect non-BRCA HRR gene mutations Indeterminate cutoff threshold defining HRD status Intratumor heterogeneity between specimen biopsy site and other tumor-invasive or metastatic sites Unable to represent the functional HR
Targeted gene panel	Customized gene panel More rapid analysis Fewer variant of uncertain significance (VUS) results	Report limited to the customized genes of the panel Unable to detect noncoding and structural variants Heterogeneous coverage based on library preparation and enrichment method Unable to represent the functional HR
Whole exome sequencing	Analysis of all coding exons (2% of genome) Detection of novel somatic mutations	Unable to detect noncoding and structural variants Heterogeneous coverage based on library preparation and enrichment method Unable to represent the functional HR
Whole genome sequencing	Analysis of all coding and noncoding regions in the genome Detect CNV, variants, and structural rearrangements with high sensitivity Mutational signatures: “Signature 3” is associated with BRCA mutations in ovarian cancer	Expensive and time consuming Difficult interpretation of results (much VUS) Unable to represent the functional HR
Promoter methylation	Hypermethylation of BRCA1 and RAD51C in association with HRD has been reported	Unable to detect HRR gene mutations Conflicting results for HRR gene methylation in predicting PARPi response Unresolved technical problems, including sample purity, quantitative protocols, and definition of gene copy number changes Unable to represent the functional HR
Functional HRD assay
RAD51 foci formation assay	Represents the functional HR	Unable to detect HRR gene mutations Unresolved technical problems, including timing, tissue sampling, DNA damage induction, methods of measurement, and definition of HRD Unsuitable for slowly proliferating tumors Unable to identify defects in RAD51 downstream or RAD51-independent mechanisms
DNA fiber assay	Represents the functional HR	Unable to detect HRR gene mutations Requires fresh, viable tissues Indeterminate definition of replication fork degradation Indeterminate correlation with clinical response