Background: In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island\r\nmethylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential.\r\nMost experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines\r\ninto uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched\r\ncryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing.\r\nMethods: Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed.\r\nRates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured.\r\nResults: For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was\r\n1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and\r\n57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On\r\nfrozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA\r\nshowing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%)\r\nfor MGMT displayed significant differences in methylation levels.\r\nConclusions: Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE\r\nsamples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation\r\nlevels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended.\r\nThis can partly explain the conflicting results on the prognosis of CIMP colon cancers.
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