Clinicopathological Significance Of DNA Methylation And Its Relationship With Genetic Mechanism In Sporadic Colorectal Cancer

Objectives: To explore the correlation between the promotor hypermethylation of suppressor genes and clinicopathological features as well as genetic changes including microsatellite instability(MSI), chromosomal instability(CIN) and the expression of several oncogenes and suppressor genes in sporadic colorectal cancers(SCRC).Methods: Detecting the promotor hypermethylation of five genes including p14^ARF, hMLH1, p16^INK4a, MGMT and MINT1 with methylation specific PCR, evaluating the microsatellite instability status with two microsatellite loci of BAT25 and BAT26, analyzing the ploidy with flow cytometry and detecting the protein expression of nine genes (p14^ARF, hMLH1, p16^INK4a, MGMT, k-ras, APC, p53, BAX and TGF β R II) among 71 SCRC patients and exploring the correlation between genes promotor hypermethylation and clinicopathological characteristics as well as the genetic mechanism of SCRCs.Results: Part 1: The positive rates of genes promotor hypermethylation of p14^ARF, hMLH1, p16^INK4a, MGMT , MINT1 and CIMP were 28. 2%(20/71), 9. 9%(7/71), 25. 4%(18/71), 36. 6%(26/71), 49. 3%(35/71)and 21.1%(15/71) respectively. SCRCs with hMLH1 gene promotor hypermethylation were more likely to happen to the right hernicolon(P — 0.018) and to be poorly-differentiated adenocarcinomas(P=0.032). p16^1NK4a gene promotor hypermethylation was significantly correlated with the proneness of colonic location (P=0.043), poor differentiation (P=0.000), lymph node metastasis(P=0.034)and later stages (P=0.046). The proportion of right-sided colonic cancers (40 %vs12.5%, p=0.24), poorly-differentiated cancers(46.7%vs14.3%, P=0.012), cancers with lymph node metastasis (86.7%vs48.2%, P=0.008) and cancers with TNM III/IV stage (86.7%vs50.0%, P=0.011) in SCRCs showing CIMP was significantly higher than that of those without CIMP.Part 2: 18 and 50 cases showed diploidy and aneuploidy respectively among 71 patients. 3 cases with CV value higher than 8 percent were excluded from the ploidy analysis. The positive rate of MSI among SCRC patients was 9. 9 percent (7/71). p14^ARF(P=0.062), hMLH1 (P=0.074), p16^INK4a(P=0.053) and MINT1 (P=0.002) gene promotor hypermethylation and positive CIMP(P=0.003) displayed the inclination of diploidy. SCRCs with hMLH1 (P=0.001) or MINT1 (P=0.055) gene promotor hypermethylation were significantly more likely to be MSI. The positive rate of MSI in positive CIMP patients was higher than that of negative CIMP ones in the present study, but the difference was not statistically significant(P=0.158). CIMP was significantly more frequent in microsatellite and chromosomal stable (MACS) SCRCs (diploid and microsatellite stable)than that in CIN phenotype (aneuploid and microsatellite stable) (46.2%vs8.3%,P=0.004). The hMLH1 promotor hypermethylation was less prevalent in MACS SCRCs than that in MSI phenotype (MSI regardless of the ploidy) (7.7%vs57.1 %, P=0.031). Both the MINT1 (69.2%vs35.4%,P=0.029) and the p16^INK4a(46.2%vs16.7%, P=0.057) gene promotor hypermethylation were more common in MACS SCRCs than that in CIN phenotype. Part 3: The positive rates of protein expression of p14^ARF, hMLH1, p16^INK4a, MGMT, k-ras, APC, p53, BAX and TGF β R II were 68. 6% (48/70), 76. 8% (53/69), 61. 8% (42/68), 87. 3% (62/71), 43. 7% (31/71), 42. 3% (30/71), 47.9% (34/71), 71.4% (50/70) and 59. 2% (42/71) respectively. Promotor hypermethylation of hMLH1 (P=0.046) or MGMT (P=0.010) gene was remarkably associated with the loss of protein. Promotor hypermethylation of MINT1 gene was significantly correlated with the accumulation of mutant-type p53 protein(P=0.024). There was a tendency of activated k-ras protein expression in SCRCs with MGMT gene promotor hypermethylation(P=0.070) or positive CIMP(P=0.043) compared with the counterpart.Conclusions: Part 1: SCRCs with hMLH1 gene promotor hypermethlation have an obvious inclination to locate on the right semicolon and to be poorly-differentiated. SCRCs with p16^INK4a gene promotor hypermethlation are more likely to be colonic cancers, to bepoorly-differentiated, to metastasize to lymph nodes and to be in the later stages. SCRCs with positive CIMP are significantly inclined to occur to the right hemicolon, to have poor differentiation, to have lymph node metastasis and to be in the later stages. Part 2: There was an intimate relationship between epigenetic changes and genetic instability. Cancers with MINT1 gene promotor hypermethylation or positive CIMP are significantly more likely to be diploid while cancers with hMLH1 gene promotor hypermethylation are significantly inclined to demonstrate MSI. MACS SCRCs may compose a unique phenotype with distinct clinicopathological characteristics and gene expression profile from CIN and MSI phenotype. MACS SCRCs have a significantly lower rate of hMLH1 gene promotor hypermethylation than MSI phenotype and a remarkable higher rate of MINT1 gene promotor hypermethylation or CIMP than CIN phenotype, indicating that MACS SCRCs may develop along a pathway characterized by CIMP shared by MSI phenotype but hMLH1 gene promotor hypermethylation may not play an important role in the carcinogenesis of MACS SCRCs. The correlation among CIN, MSI and CIMP may be complicated, which reflects the competent and dependent relationship between epigenetic and genetic mechanisms. CIN mechanism may be independent of MSI and epigenetic mechanisms. A considerable part of SCRCs with MSI, but not all, may develop from a background of epigenetic instability. Part3: Promotor hypermethylation of hMLH1 or MGMT gene may cause the loss of protein. SCRCs with MINT1 gene promotor hypermethylation has a significant lower rate of accumulated mutant-type p53 protein, while those with MGMT gene promotor hypermethylation(P=0.070) or positive CIMP(P=0.043) have a relatively higher rate of activated k-ras protein expression compared with the counterpart. That the loss of MGMT protein can cause k-ras gene mutation may explain the close relation among CIMP, MGMT gene promotor hypermethylation and k-ras protein expression.