Clinical Value Of RASSF1A And APC DNA Methylation In Plasma For Primary Liver Cancer

Objective: RASSF1A and APC genes belong to the tumor suppressorgene of Ras and Wnt signaling pathway. It was confirmed that aberrantmethylation can be detected in tissues of primary liver cancer, but theconclusions were unconformity. Fresh tissues were applicated widly forRASSF1A and APC DNA methylation in oncology research for, but plasmaspeciments are most convenient to methylation detection, some studies hadshown that there had a high consistency for DNA methylation changesbetween tumor tissue and plasma samples which are in paired, indicating thatdetection of DNA methylation in plasma has potential utility as a tumormarker. In this study, we assess the methylation status of RASSF1A and APCgenes in plasma of liver cancer, cirrhosis and healthy controls withmethylation specific PCR, their relationship with clinical data are alsoanalyzed.Methods:1Patients:102cases of plasma from liver cancer patients including8cases of stage Ⅰ,41cases of stage Ⅱ, and53cases of stage Ⅲ, who werefrom the Fourth hospital of Hebei Medical University between May2007andSeptember2011,80cases plasma of cirrhosis and100cases plasma of healthypeople were collected. There were82males and20females in group of livercancer, the age from29to84, the average age was56.62±10.66. There were57males and23females in group of cirrhosis, the age from23to78, theaverage age was58.68±10.28. According to the domestic standard ofclinical diagnosis and stages (Guangzhou,2001) to make clinical diagnosisand stages for liver cancer. Then the clinical information of all the patientswere recorded, and the survival status were followed up through telephonecontact. 2Methods of detection for suppressor gene promoter methylation:Asplasma samples, the supernatant was obtained after centrifugaling of completeblood which were anticoagulated by EDTA for2minites, DNA from plasmawas extracted with QIAamp DNA Blood Mini Kit, and purified with WizardDNA kit after it was modified with sulfite methods, all steps according to theinstructions of the kits, methylation specific PCR(MSP) of suppressor geneswas run. The sequence analysis of RASSF1A gene methylation: the forward5'-GGGTTTTGCGAGAGCGCG-3', and reverse primers5'-GATAACAAACGCGAACCG-3', the sequence analysis of RASSF1A geneunmethylation: the forward5'-GGGGTTTTGTGAGAGTGTG-3', and reverse primers5'-ACTAACAAACACAAACCAAAC-3'; the sequence analysis of APC genemethylation: the forward5'-TATTGCGGAGTGCGGGTC-3', and reverse primers5'-TCGACGAACTCCCGACGA-3', the sequence analysis of APC geneunmethylation: the forward5'-GTGTTTTATTGTGGAGTGTGGGTT-3', and reverseprimers5'-CCAATCAACAAACTCCCAACAA-3'. the methylation status wasassessed subsequently.3Statistics analysis: All statistical analyses were performed by SPSS13.0software. P<0.05was considered to indicate a statistically significantdifference.Results:1Comparing the methylation status of RASSF1A, APC genes in plasmaof liver cancer, cirrhosis and healthy peoplePositive ratio of RASSF1A gene methylation in plasma of liver cancerand cirrhosis were53of102(51.96%) versus12of80(15%), no methylationwas found in healthy people, the difference was statisticallysignificant(χ~2=80.944,P=0.000<0.05). Comparing the methylation status ofliver cancer and cirrhosis, there were significantly diffrences among thegroup(sχ~2=26.677,P=0.000). Comparing the healthy control with liver cancerand cirrhosis, there were significantly diffrences among the groups(P=0.000,P=0.000).Positive ratio of APC gene methylation in plasma of liver cancer and cirrhosis were48of102(47.05%) versus18of80(22.5%), no methylation wasfound in healthy people, the difference was statistically significant(χ~2=62.429,P=0.000<0.05). Comparing the methylation status of APC gene in liver cancerand cirrhosis, there were significantly diffrences among the groups(χ~2=11.700,P=0.001). Comparing the healthy control with liver cancer andcirrhosis, there were significantly diffrences among the groups(P=0.000,P=0.000).2Comparing RASSF1A, APC gene's methylation status with the clinicalcharacter in plasma of liver cancer.The methylation status of RASSF1A and APC were not related to age,sex, Child pugh score, the value of AFP, and treatment. The methylation statusof RASSF1A had correlation with tumor number, with or without cirrhosis,portal vein tumor embolus, and clinical stage.The methylation frequency ofRASSF1A gene in multiple tumors and single tumor is60%vs36.54%,P=0.018,in cirrhosis and without cirrhosis is56.18%vs23.08%, P=0.026,with and without portal vein tumor emlolus is67.65%vs38.24%,P=0.005, inclinical stage Ⅲ and in clinical stage(Ⅰ+Ⅱ) is62.26%vs40.82%, P=0.030.The methylation frequency of APC gene with HBsAg positive and withHBsAg negative is55.81%vs31.25%, P=0.003, with and without portal veintumor emlolus is64.71%vs38.24%, P=0.012.The diagnosis value of RASSF1A and APC methylation in AFP negativeliver cancer patients is evaluated subsequently, in58patients, RASSF1A genemethylation occurred in31cases(53.4%), APC gene methylation occurred in27cases(46.5%).3The internal relationships among the two suppressor gene'smethylatiom status in plasma of liver cancerIn the102cases of patients with liver cancer, the frequency fornon-methylation of all the two suppressor genes, two genes methylation andone gene methylation was29.41%(30/102),70.59%(72/102) and28.43%(29/102), respectively. No internal relationships of the two genemethylation status could be found among these two genes with Pearson's test. 4The diagnostic value by detection of RASSF1A, APC gene methylationin plasmaAmong the282plasma samples of liver cancer, cirrhosis, and healthycontrol, the sensitivity for RASSF1A gene methylation to predict liver canceris52%,the specificity is93.3%, repectively. The sensitivity for APC genemethylation to predict liver cancer is47.1%,the specificity is90%, repectively.The combination of the two genes methylation to predict liver cancer is70.6%,the specificity is86.7%, repectively.5The relationship between RASSF1A,APC gene methylation andsurvival in plasma of liver cancerThe survival rate of RASSF1A gene's methylation status wassignificantly different between the patients with positive(mean survivaltime:15.28±2.85months) and negative (mean survival time:25.86±3.77months)(P=0.011<0.05).There was no significant difference in survival rate of APC gene'smethylation status between patients with positive(mean survival time:17.16±2.81months) and negative(mean survival time:25.86±3.77months)(P=0.175>0.05).The survival rate of RASSF1A or APC gene's methylation status wassignificantly different between the patients with positive(mean survival time:10.80±2.59months) and negative (mean survival time:23.63±4.91months)(P=0.011<0.05).Multivariate analysis showed that AFP, clinical stage, treatment, with orwithout portal vein tumor embolus were independent prognosis factors, noneof the two genes methylation status were independent prognosis factors, Butthe combination of RASSF1A and APC gene methylation status was anindependent prognosis factors(P=0.019<0.05).Conclusions:1RASSF1A, APC gene methylation rate in plasma of liver cancer wasremarkable higher than that in plasma of cirrhosis and healthy people, itindicates that RASSF1A, APC gene methylation have some effection in formation process of liver cancer.2The combination of RASSF1A and APC gene methylation detectioncan be used for screening the HCC cancer risk in AFP negative patients.RASSF1A, APC gene methylation status have correlation with portal veintumor thrombus, RASSF1A gene methylation status has correlation withclinical stage. It suggests that RASSF1A and APC gene methylation statusmay reflect the condition and prognosis of liver cancer.3The combination of RASSF1A and APC gene methylation status wasan independent prognosis factors.