DNA methylation in patients with colorectal cancer--correlation with some clinical and morphological features and with local tumour invasion.
OBJECTIVE
To study quantitatively the promoter methylation of hMLH1, p16INK, TIMP3 and TPEF genes in patients with colorectal cancer and synchronous polyps, and correlate it with some clinicomorphological features.


METHODS
DNA was extracted from all studied tumours and the corresponding normal mucosa. Microsatellite instability was analysed using two mononucleotide (BAT 25 and BAT 26) and three dinucleotide markers (D2S123, D5S356, D17S250) and automated DNA sequencing. Quantitative analysis of methylation was performed using DNA bisulfite modification, PCR with biotinylated primers, visualisation by 2% agarose gel electrophoresis and pyrosequencing.


RESULTS
High methylation levels of hMLH1 and p16INK were found in elderly patients (mean age 73.8 +/- 9.5 years and 65.7 +/- 16.6 years, p < 0.03, t-test). Proximal tumours were more often associated with microsatellite instability (p < 0.05, Fisher's test) and higher level of methylation of hMLH1, p16INK and TIMP3 (p < 0.02, Kruskal-Wallis test), while tumours with poor differentiation tended to have higher methylation of the p16INK gene (p < 0.02, Kruskal-Wallis test). Local tumour invasion was correlated with the level of methylation of hMLH1, TIMP3 and the CpG island methylator phenotype (CIMP) status. Tumours with liver metastases showed a lower level of TIMP3 methylation than tumours with no systemic invasion (p < 0.05, Kruskal-Wallis test). We found concordance of methylation in 56% of the cases with colonic cancer and synchronous adenomas; the remaining 44% were discordant.


CONCLUSIONS
Tumours with microsatellite instability, high level methylation and CIMP have distinctive clinical and morphological features. The level of hMLH1 and TIMP3 methylation and CIMP status can be correlated with the local tumour invasion. Different mechanisms, even for one and the same patient, can be responsible for the development of more than one third of the synchronous polyps and carcinomas.
Mirchev
Milko B
MB
Clinic of Gastroenterology, Medical University, Varna, Bulgaria. mbmirchev@yahoo.com
Kahl
Philip
P
Friedrichs
Nicolaus
N
Kotzev
Iskren A
IA
Buettner
Reinhard
R
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D019942:Genes, p16; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D053842:Microsatellite Instability; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D004273:DNA, Neoplasm; C501486:MLH1 protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C088390:TIMP3 protein, human; C423567:TMEFF2 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D000070957:MutL Protein Homolog 1