Pyrosequencing Assay for BRCA1 Methylation Analysis: Results from a Cross-Validation Study

Abstract

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.