title: [A new quantitative DNA-methylation analysis of MSI colorectal cancers helps to separate sporadic colorectal cancers from HNPCC-candidates].
abstract: OBJECTIVE
  Promoter hypermethylation is a common mechanism for epigenetic control of gene expression and occurs frequently in tumors silencing tumor suppressor genes. Our aim was to establish a quantitative and precise method to analyze promoter methylation of tumor samples in order to identify HNPCC candidates.
  
  
  METHODS
  We established a new methylation specific relative quantitative real-time PCR technique for analysis of the methylation status of the hMLHI promoter in colorectal cancers (CRC). We determined methylation status of both the distal and proximal hMLH1-promoter region. The methylation quantification (MQ) was performed with cell line DNA and archival paraffinized tissue sections.
  
  
  RESULTS
  The accuracy of our analysis was validated with spiking experiments of methylated and unmethylated DNA. We assessed the hMLH1 methylation status 56 CRC patients with known microsatellite status and hMLH1 IHC. The methylation analysis divided the MSI-H CRC into two groups: Methylation positive sporadic CRC patients with a median age of 78.5 years and frequent BRAF mutations (82 %, p < 0.0001) and the unmethylated cancers from HNPCC candidates with a median age of 48 years. All hMLH1 positive sporadic MSS CRC were methylation negative. In all samples, the degree of methylation was mirrored by the shift of the melting points to higher temperatures.
  
  
  CONCLUSIONS
  In summary we introduced a quantitative and qualitative technique to analyze DNA methylation that can be performed with any dense CpG island. Our methylation analysis provides a potent diagnostic tool to differentiate between sporadic MSI-H cancers showing MLH1 methylation and MLH1 unmethylated HNPCC candidates.
authors.affiliation: Institut für Pathologie, Universität Regensburg.
authors.forename: M
authors.initials: M
authors.lastname: Bettstetter
authors.forename: P
authors.initials: P
authors.lastname: Rümmele
authors.forename: F
authors.initials: F
authors.lastname: Hofstädter
authors.forename: W
authors.initials: W
authors.lastname: Dietmaier
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D003937:Diagnosis, Differential; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D004273:DNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf