The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co‐segregation, family cancer history profile, co‐occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case‐control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene‐specific calibration of evidence types used for variant classification.

Aims: Next Generation Sequencing (NGS)-based BRCA tumour tissue testing poses several challenges. As a first step of its implementation within a regional health service network, an in-house validation study was compared with published recommendations. Methods: Epithelial ovarian cancer (EOC) formalin-fixed paraffin-embedded specimens stored in the archives of the eight regional pathology units were selected from a consecutive series of patients with known BRCA germline status. Two expert pathologists evaluated tumour cell content for manual macrodissection. DNA extraction, library preparation and NGS analyses were performed blinded to the germinal status. Parameters used in the study were confronted with guidelines for the validation of NGS-based oncology panels and for BRCA tumour tissue testing. Results: NGS analyses were successful in 66 of 67 EOC specimens, with good quality metrics and high reproducibility among different runs. In all, 19 BRCA pathogenic variants were identified: 12 were germline and 7 were somatic. A 100% concordance with blood tests was detected for germline variants. A BRCA1 variant showed a controversial classification. In different areas of two early stage EOCs showing somatic variants, intratumour heterogeneity not relevant for test results (variant allele frequency >5%) was observed. Compared with expert recommendations, main limitations of the study were absence of controls with known somatic BRCA status and exclusion from the validation of BRCA copy number variations (CNV). Conclusions: A close collaboration between pathology and genetics units provides advantages in the implementation of BRCA tumour tissue testing. The development of tools for designing and interpreting complex testing in-house validation could improve process quality.

Epithelial ovarian cancers (EOCs) harboring germline or somatic pathogenic variants in BRCA1 and BRCA2 genes show sensitivity to poly(ADP-ribose) polymerase inhibition. It has been suggested that BRCA1 promoter methylation is perhaps a better determinant of therapy response, because of its intrinsic dynamic feature, with respect to genomic scars or gene mutation. Conflicting evidence was reported so far, and the lack of a validated assay to measure promoter methylation was considered a main confounding factor in data interpretation. To contribute to the validation process of a pyrosequencing assay for BRCA1 promoter methylation, 109 EOCs from two Italian centers were reciprocally blindly investigated. By comparing two different pyrosequencing assays, addressing a partially overlapping region of BRCA1 promoter, an almost complete concordance of results was obtained. Moreover, the clinical relevance of this approach was also supported by the finding of BRCA1 transcript down-regulation in BRCA1-methylated EOCs. These findings could lead to the development of a simple and cheap pyrosequencing assay for diagnostics, easily applicable to formalin-fixed, paraffin-embedded tissues. This technique may be implemented in routine clinical practice in the near future to identify EOCs sensitive to poly(ADP-ribose) polymerase inhibitor therapy, thus increasing the subset of women affected by EOCs who could benefit from such treatment.