A rapid and reliable test for BRCA1 promoter hypermethylation in paraffin tissue using pyrosequencing Journal Article
| Authors: | Bacares, R.; Soslow, R.; Olvera, N.; Levine, D. A.; Zhang, L. |
| Article Title: | A rapid and reliable test for BRCA1 promoter hypermethylation in paraffin tissue using pyrosequencing |
| Abstract: | Background: Ovarian cancers harboring inactivating mutations in BRCA1 or BRCA2 demonstrate increased sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPis). BRCA1 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 BRCA1 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 BRCA1 promoter methylation status. Methods: Tumor DNA from high-grade serous ovarian carcinoma was tested targeting 11 CpG sites adjacent to the BRCA1 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%). Results: 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. Conclusions: 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. © 2025 by the authors. |
| Keywords: | brca; pyrosequencing; hypermethylation |
| Journal Title: | Diagnostics |
| Volume: | 15 |
| Issue: | 5 |
| ISSN: | 2075-4418 |
| Publisher: | MDPI |
| Date Published: | 2025-03-01 |
| Start Page: | 601 |
| Language: | English |
| DOI: | 10.3390/diagnostics15050601 |
| PROVIDER: | scopus |
| PMCID: | PMC11898801 |
| PUBMED: | 40075848 |
| DOI/URL: | |
| Notes: | Article -- Source: Scopus |
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