Disease Progression from Chronic Hepatitis C to Cirrhosis and Hepatocellular Carcinoma is Associated with Increasing DNA Promoter Methylation

  • Published : 2013.11.30


Background: Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. Materials and Methods: 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. Results: In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%), respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. Conclusions: The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA methylation signatures with potential clinical applications in diagnosis and prognosis. In addition, methylation frequency could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC. We can conclude that methylation processes are not just early events in hepatocarcinogenesis but accumulate with progression to cancer.


HCV;cirrhosis;hepatocellular carcinoma;tumor suppressor gene methylation;progression


  1. Altekruse SF, McGlynn KA, Reichman ME (2009). Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol, 27, 1485-91.
  2. Anzola M, Cuevas N, Lopez-Martinez M, et al (2004). P14ARF gene alterations in human hepatocellular carcinoma. Eur J Gastroenterol Hepatol, 16, 19-26.
  3. Chang H, Yi B, Li L, et al (2008). Methylation of tumor associated genes in tissue and plasma samples from liver disease patients. Exp Mol Pathol, 85, 96-100.
  4. Ferlay J, Shin HR, Bray F, et al (2010). GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide: IARC CancerBase No 10. Lyon, France: International Agency for Research on Cancer.
  5. Fukai K, Yokosuka O, Imazeki F, et al (2005). Methylation status of p14ARF, p15INK4b, and p16INK4a genes in human hepatocellular carcinoma. Liver Int, 25, 1209-16.
  6. Herbst A, Rahmig K, Stieber P, et al (2011). Methylation of NEUROG1 in serum is a sensitive marker for the detection of early colorectal cancer. Am J Gastroenterol, 106, 1110-8.
  7. Iakova P, Timchenko L, Timchenko NA (2011). Intracellular signaling and hepatocellular carcinoma. Semin Cancer Biol, 21, 28-34.
  8. Iwata N, Yamamoto H, Sasaki S, et al (2000). Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma. Oncogene, 19, 5298-302.
  9. Iyer P, Zekri AR, Hung CW, et al (2009). Concordance of DNA methylation pattern in plasma and tumor DNA of Egyptian hepatocellular carcinoma patients. Exp Mol Pathol, 88, 107-11.
  10. Jung N, Won JK, Kim BH, et al (2012). Pharmacological unmasking microarray approach-based discovery of novel DNA methylation markers for hepatocellular carcinoma. J Korean Med Sci, 27, 594-604.
  11. Lavanchy D (2011). Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect, 17, 107-15.
  12. Lee S, Lee HJ, Kim JH, et al (2003). Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol, 163, 1371-8.
  13. Liu WJ, Wang L, Wang JP, et al (2006). Correlations of CpG island methylator phenotype and OPCML gene methylation to carcinogenesis of hepatocellular carcinoma. Ai Zheng, 25, 696-700.
  14. Matsukura S, Soejima H, Nakagawachi T, et al (2003). CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection. Br J Cancer, 88, 521-9.
  15. McGivern DR, Lemon SM (2011). Virus-specific mechanisms of carcinogenesis in hepatitis C virus associated liver cancer. Oncogene, 30, 1969-83.
  16. Nomoto S, Kinoshita T, Kato K, et al (2007). Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma. Br J Cancer, 97, 1260-5.
  17. Parkin DM, Bray F, Ferlay J (2005). Global cancer statistics, 2002. CA Cancer J Clin, 25, 74-108.
  18. Peng CY, Chen TC, Hung SP, et al (2002). Genetic alterations of INK4alpha/ARF locus and p53 in human hepatocellular carcinoma. Anticancer Res, 22, 1265-71.
  19. Salhab M, Canelo R (2011). An overview of evidence-based management of hepatocellular carcinoma: a meta-analysis. J Can Res Ther, 7, 463-75.
  20. Schutte K, Bornschein J, Malfertheiner P (2009). Hepatocellular carcinoma--epidemiological trends and risk factors. Dig Dis, 27, 80-92.
  21. Shih YL, Shyu RY, Hsieh CB, et al (2006). Promoter methylation of the secreted frizzled-related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma. Cancer, 107, 579-90.
  22. Whittaker S, Marais R, Zhu AX (2010). The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene, 29, 4989-5005.
  23. Yamashita T, Honda M, Kaneko S (2011). Molecular mechanisms of hepatocarcinogenesis in chronic hepatitis C virus infection. J. Gastroenterol. Hepatol, 26, 960-4.
  24. Yang B, Guo M, Herman JG, Clark DP (2003). Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol, 163, 1101-7.
  25. Zhang YJ, Wu HC, Shen J, et al (2007). Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA. Clin Cancer Res, 13, 2378-84.
  26. Zhao ZH, Geng XP, Zhu LX, Li HM, Liew CT (2005). Hypermethylation of Ras association domain family protein 1A, hypermethylated in cancer 1 and p73 genes in hepatocellular carcinoma. Zhonghua Wai Ke Za Zhi, 43, 1528-32.

Cited by

  1. Clinical Significance of Joint Detection of Serum VEGF, SIL-2R and HGF in Patients with Primary Hepatocellular Carcinoma before and after Percutaneous Microwave Coagulation Therapy vol.15, pp.11, 2014,
  2. Clinical Study of Hepatectomy Combined with Jianpi Huayu Therapy for Hepatocellular Carcinoma vol.15, pp.14, 2014,
  3. Role of P14 and MGMT Gene Methylation in Hepatocellular Carcinomas: a Meta-analysis vol.15, pp.16, 2014,
  4. IL28B rs12979860 Gene Polymorphism in Egyptian Patients with Chronic Liver Disease Infected with HCV vol.15, pp.17, 2014,
  5. Hepatitis C Virus Prevalence and Genotyping among Hepatocellular Carcinoma Patients in Baghdad vol.15, pp.18, 2014,
  6. Expression in Colorectal Cancer is Linked to Ethnic Origin vol.15, pp.5, 2014,
  7. DNA Methylation Profile of Genes Involved in Inflammation and Autoimmunity in Inflammatory Bowel Disease vol.93, pp.28, 2014,
  8. Hepatoprotective Effects of Curcumin Against Diethyl Nitrosamine Induced Hepatotoxicity in Albino Rats vol.16, pp.1, 2015,
  9. Plasma Nuclear Factor Kappa B and Serum Peroxiredoxin 3 in Early Diagnosis of Hepatocellular Carcinoma vol.16, pp.4, 2015,
  10. Hepatitis C Virus (HCV) genotypes distribution among hepatocellular carcinoma patients in Southern Italy: a three year retrospective study vol.12, pp.1, 2017,
  11. Molecular Mechanisms of Hepatocarcinogenesis Following Sustained Virological Response in Patients with Chronic Hepatitis C Virus Infection vol.10, pp.10, 2018,