Methylation Sensitive High Resolution Melting (MS-HRM) Assay for the Detection of BRCA1 and BRCA2 Promoter Hypermethylation


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Diana Pelka1,Jack Grant1,Sasha Hansel1,David Moore1,Phil Bennett1,Geraldine Thomas2,Gareth Gerrard1
1Sarah Cannon Molecular Diagnostics, London, UK,2Imperial College London, London, UK

Abstract

Background

BRCA1 & BRCA2 genes encode key components of the DNA double-strand break repair pathway. Cancers driven by loss of BRCA1/2 are associated with sensitivity to PARP inhibitors (PARPi), such as olaparib, through the synthetic-lethality of concomitantly blockading the single-strand repair pathways mediated by PARP. Monoallelic BRCA1/2 mutations require a ‘second-hit’ to the unaffected allele, since only tumours with complete abrogation of BRCA1/2 are targetable with PARPi. One recognised second-hit mechanism is gene promoter hypermethylation, which can also cause gene silencing in the absence of mutations. We sought to use extant MS-HRM protocols (based on a validated MLH1 assay) using primer and control kits from MethylDetect ApS, Denmark, to implement a BRCA1/2 promoter hypermethylation assay.

Method

14 triple-negative, grade 3 invasive ductal breast carcinoma (BC) and 13 prostate adenocarcinoma (PA) FFPE samples were sourced from the Imperial College Healthcare Tissue Bank. They were macro-dissected to obtain DNA from paired tumour and normal tissue. 100ng DNA from each was bisulphite-treated (EpiTect Fast, Qiagen) in a 20μL reaction and 3μL used in the MS-HRM reaction, along with CpG-flanking primers for either BRCA1 or BRCA2. Kit provided methylation controls were used for IQC. The MS-HRM reactions were run on a Qiagen RotorGeneQ, using EpiTect HRM mastermix (Qiagen) and analysed with the RotorGene v2.3.1.49 software.

Results

7/13 (53.8%) and 0/12 BC samples showed BRCA1 and BRCA2 promoter hypermethylation, respectively; none of the 13 PA samples were hypermethylated for either gene. 1 BRCA1 and 2 BRCA2 samples failed to yield usable results (both BC).

Conclusion

The detection of BRCA1 hypermethylation in over half of the BC samples in this limited-scale implementation of a low-cost, rapid and sensitive assay, demonstrates the potential utility of this approach for stratifying patients for PARPi therapy.