Assessment of Methylation Events during Colorectal Tumor Progression by Absolute Quantitative Analysis of Methylated Alleles

Abstract

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm 2 of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R z 0.966, P < 0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P < 0.0001 by Kolmogorov-Smirnov test

Identification of a Quantitative MINT Locus Methylation Profile Predicting Local Regional Recurrence of Rectal Cancer

“…Before and after sodium bisulfite modification, double-stranded and single-stranded DNA were quantified using PicoGreen and OliGreen assays (Molecular Probes; Invitrogen), respectively. Sufficient input DNA for AQAMA was determined as described (17). To assess background signal, a salmon sperm DNA sample without tumor DNA was included in all assays in triplicate.…”

“…Primary tumor methylation levels of MINT loci 1, 2, 3, 12, 17, 25, and 31 were assessed by AQAMA in triplicate (17). Controls for specificity of AQAMA for both methylated and unmethylated sequences, as well as controls for nonspecific amplification, were included (17,27).…”

“…Primary tumor methylation levels of MINT loci 1, 2, 3, 12, 17, 25, and 31 were assessed by AQAMA in triplicate (17). Controls for specificity of AQAMA for both methylated and unmethylated sequences, as well as controls for nonspecific amplification, were included (17,27). As a final outcome of the analysis, the methylation index (MI) of a sample was the mean value of three MI measurements that were calculated for each well as follows: [copy number methylated alleles /(copy number methylated alleles + copy number unmethylated alleles )].…”

“…In this study, we investigated epigenetic changes in rectal cancer and specifically methylation of methylatedin-tumor (MINT) loci. MINT loci are CpG dinucleotiderich regions located in nonprotein-encoding DNA regions and have been reported to become methylated in a tumorspecific manner in (colo)rectal cancer (17,18), gastric cancer (19), and recently malignant melanoma (20).…”

TS gene polymorphisms are not good markers of response to 5-FU therapy in stage III colon cancer patients

“…Nevertheless, there is increasing evidence that age affects normal colonic mucosa and tumors. For instance, DNA methylation has been shown to increase with age in normal colonic mucosa [30,35] and different protein expression patterns have been found in the colonic mucosa of the elderly compared to that of younger people [26]. Moreover, Morris et al have also shown that the molecular aberrations in tumors differ according to age [34].…”