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Poster Display session 3

2493 - Methylation analysis of MLH1 using droplet digital PCR and methylation sensitive restriction enzyme.

Date

30 Sep 2019

Session

Poster Display session 3

Topics

Translational Research

Tumour Site

Presenters

Celine De Rop

Citation

Annals of Oncology (2019) 30 (suppl_5): v574-v584. 10.1093/annonc/mdz257

Authors

C. De Rop1, G. Beniuga2, J. Radermacher2, K. Dahan3, P. Vannuffel1

Author affiliations

  • 1 Molecular Biology Department, Institut de Pathologie et de Génétique, 6041 - Gosselies/BE
  • 2 Anatomical Pathology Department, Institut de Pathologie et de Génétique, 6041 - Gosselies/BE
  • 3 Clinical Genetics Department, Institut de Pathologie et de Génétique, 6041 - Gosselies/BE

Resources

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Abstract 2493

Background

Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes being MLH1 and MSH2 the most commonly mutated. By contrast MLH1 inactivation as the result of promoter methylation is strongly indicative of a sporadic cancer, providing LS exclusion criteria for 85% of high microsatellite instability (MSI-H) tumours. Here we present a cost effective strategy enabling to test methylation of MLH1 promoter without bisulfite conversion and with minimal DNA quantity requirement.

Methods

After macrodissection from HE stained slides, DNA was extracted from FFPE tissue sections of colorectal carcinomas. A droplet digital PCR (ddPCR) was performed using two reactions mix. A 270-bp region of the MLH1 promoter (chr3:36993151-36993420) is amplified in the presence/absence of HinP1I, a methylation sensitive restriction enzyme, targeting 3 CpG islands. Analysis was made by the QuantaSoft™ (Bio-Rad) software which calculates amplicon concentrations between the 2 reactions to obtain a percentage of MLH1 methylation. Sensitivity of the technique was assigned to 5% of methylation with a minimal DNA concentration of 5 ng.

Results

Methylation analysis by ddPCR was compared to pyrosequencing combined with bisulfite conversion for 65 samples and a 100% concordance was obtained. Moreover 10 samples were analysed by ddPCR and MethylLight RealTime-PCR with the same concordance. After validation, the technique was implemented in the clinical diagnosis and, in one year, out the 79 MMR-deficient colorectal carcinomas analysed, 60 (76 %) were MLH1-methylated tumours.

Conclusions

This ddPCR sequencing combining methylation sensitive restriction enzyme is a cost-effective strategy, requiring less technical turn around time and minimal DNA quantity as compared to standard analysis. Moreover, this technique could be further used for other promoter methylation analysis (such as MGMT) and on circulating tumoral DNA.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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