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

TL;DR

This paper introduces a reliable pyrosequencing test to detect BRCA1 promoter methylation in ovarian cancer tissue, which could help predict treatment response.

Contribution

The study validates a pyrosequencing method for BRCA1 promoter methylation detection with high accuracy and reproducibility.

Findings

The pyrosequencing assay showed a high correlation (R2 = 0.9945) between predicted and observed methylation levels.

The assay demonstrated 100% concordance with TCGA methylation data and real-time PCR results.

The test was approved for clinical use by the New York State Department of Health.

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.

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