A Rapid and Reliable Test for BRCA1 Promoter Hypermethylation in Paraffin Tissue Using Pyrosequencing

<b>Background:</b> Ovarian cancers harboring inactivating mutations in <i>BRCA1</i> or <i>BRCA2</i> demonstrate increased sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPis). <i>BRCA1</i> promoter methylation could serve as a more precise biomarker for therapy response, as it reflects a dynamic mechanism, compared with genomic scarring, which remains persistent and lacks real-time prediction of sensitivity after prior lines of treatment. Additionally, the <i>BRCA1</i> promoter methylation may provide a more precise biomarker for identifying homologous recombination deficiency compared to genomic scars. In this study, we describe the validation of a pyrosequencing method to assess <i>BRCA1</i> promoter methylation status. <b>Methods:</b> Tumor DNA from high-grade serous ovarian carcinoma was tested targeting 11 CpG sites adjacent to the <i>BRCA1</i> transcription start site. All cases had concordant results compared with TCGA methylation data or real-time PCR results. To determine the sensitivity of this assay, we performed a dilution series experiment using seven mixtures of methylated DNA and unmethylated genomic DNA (100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.56%). <b>Results:</b> We observed a high degree of correlation (R² = 0.9945) between predicted and observed results. Intra- and inter-run reproducibility was established by performing six cases in triplicate in the same run and in three different runs. <b>Conclusions:</b> By applying 10% as the cutoff for detection of methylation, the PyroMark Q24 pyrosequencing assay demonstrated 100% concordance across all the ovarian cancer cases included in this validation. This assay has been approved by the New York State Department of Health as a laboratory-specific assay for clinical use.