High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis.
High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O(6)-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh or formalin-fixed paraffin-embedded human cancer cell lines. Then we used these assays for the analysis of MGMT and APC promoter methylation in a subset of archival formalin-fixed paraffin-embedded colorectal tumor specimens. All samples with promoter methylation of MGMT or APC and randomly selected samples without promoter methylation were analyzed twice. All results generated by HRM were validated with MGMT and APC MethyLight assays. APC and MGMT promoter methylation data were consistent and reproducible throughout the dilutions and all three replicates in the methylated DNA dilution matrix and between two experiments in clinical samples. There was high concordance between HRM and MethyLight results. HRM for APC promoter methylation revealed consistent results between fresh and formalin-fixed paraffin-embedded human cancer cell line DNA. The methylation status in archival tumor specimens from patients with colorectal cancer can therefore be determined with high quality by HRM. The ability to analyze archival tissues greatly facilitates further research and its clinical implementation.
Balic
Marija
M
Division of Oncology, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, 8036, Graz, Austria. marija.balic@meduni-graz.at
Pichler
Martin
M
Strutz
Jasmin
J
Heitzer
Ellen
E
Ausch
Christoph
C
Samonigg
Hellmut
H
Cote
Richard J
RJ
Dandachi
Nadia
N
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D013048:Specimen Handling; D016707:Tissue Fixation; D044366:Transition Temperature