CpG Island Methylator Phenotype Associated With Clinicopathologic Features In Hepatocellular Carcinoma

The development and progression of hepatocellular carcinoma (HCC) is a multistep processwhereby the normal hepatocytes undergo inflammation, fibrosis by the hepatitis virus or otherstimuli, followed by liver cirrhosis, which then progresses to HCC. But the understanding of themolecular pathways of hepatocarcinogenesis is limited, although recent molecular biologicalstudies have led to rapid progress in the understanding of the molecular events involved.Promoter hypermethylation has been found to frequently occur in tumor suppressor and cancergenes involved in many different signaling pathways and to be present in a different pattern indifferent tumor types. Cancer is believed to be due to an accumulation of DNA aberrations, andanalysis of DNA isolated from tumors has identified epigenetic and genetic alterations occurringat the target CpG of the (cytosine-5)-DNA methyltransferase (MTase) as one of the mostfrequent and consistent changes observed in tumor cells.Epigenetic silencing of tumor-related genes due to methylation of gene promoter regionsplays an important role in carcinogenesis. Most of the early studies in the field focused on theexamination of isolated genes. However, the pathophysiology of hypermethylation (why, when,where) remains obscure. The methylation pattern of multiple genes can provide different types ofuseful information about cancer cell. CpG island methylator phenotype (CIMP), in whichmultiple genes are concurrently methylated, is a novel marker for tumor progression.CIMP-associated cancers seem to have a distinct epidemiology, a distinct histology, distinctprecursor lesions and distinct molecular features. CIMP has also been observed in various typesof tumors and may contribute to cancer formation and progression by affecting the expression ofmultiple genes. Although it is important to analyze multiple genes to have an overall picture ofepigenetic alterations, there are only limited reports on such analysis in HCC.In this study, promoter methylation of P14 (p14ARF), P15 (p15INK4b), P16 (p16INK4a),P53, RB1 (retinoblastoma 1), ER (estrogen receptor), WTI (Wilms tumor 1), RASSF1A (Rasassociation domain family 1), and c-Myc were studied in HCC because they have been found tobe high frequency methylation in many malignancies. To gain insight into the role of epigeneticaberration of tumor-related genes in hepatocarcinogenesis, we examined the promotermethylation status of nine genes in 50 HCC, 50 paired non-tumor tissues, and 6 normal liver tissues by methylation-specific PCR and determined a hypermethylation profile in HCC. In thisstudy, CIMP status was classified as CIMP+ samples (with 5 or more methylated genes) and CIMP-(sampleswith 4 or fewer methylated genes).We found that the frequency of promoter methylation of nine genes in 50 HCC varied from10% in P53 to 94% in c-Myc. Methylation status of P14, P15, P16, ER, RASSF1A, WT1, andc-Myc was significantly correlated with HCC and non-tumor tissues (p＜0.05).Hypermethylation of one or more genes was found in 96% of HCC. CIMP was more frequent inHCC than in non-tumor tissues (70% and 12%, p＜0.001), no CIMP+ was present in normalliver tissues. There is a significant association between CIMP and methylation of P14, P15, P16,ER, RASSF1A and WT1 (p＜0.05) and serum alpha fetoprotein (AFP) level (p=0.001). CIMP+was more frequent in HCC with AFP≥30 ug/l than those with AFP＜30 ug/l (p=0.005). Also,the promoter hypermethylation of P15 and P16 was associated with elevated serum AFP levels in35 HCC samples with CIMP+ (p＜0.05). There was no statistically significant associationbetween CIMP status and age, gender, tumor size, smoking history, drinking alcohol, HBV, HCV,cirrhosis, node number, and metastasis.The results suggest that the simultaneous hypermethylation of multiple tumor-related genesmay be important for tumorigenesis. CIMP is also present in liver cancers and could be a criticalevent for malignant transformation from liver cirrhosis to HCC. Functional inactivation of P15and P16 mediated by promoter methylation may be required for aberrant expression of AFPduring hepatocarcinogenesis. Positive correlation of CIMP and AFP levels suggests that CIMPcan serve as a molecular marker of poor prognosis of in HCC.