Abstract
Background: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%–80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy.
Methods: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing.
Results: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively.
Conclusions: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.
Acknowledgments
We would like to thank Drs. David H. Walker, Don W. Powell, Cornelis Elferink, Allan R. Brasier, and Animesh Chandra for helpful discussions and critical review of the manuscript. Supported in part by grants from the National Institute of Environmental Health Sciences (NIEHS) Center Grant P30 ES006676 and a Clinical and Translational Science Award (8UL1 TR000071) from the National Center for Advancing Translational Sciences, National Institutes of Health.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research support played no role in thestudy design; in the collection, analysis, and interpretationof data; in the writing of the report; or in the decision tosubmit the report for publication.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
References
1. Eheman C, Henley SJ, Ballard-Barbash R, Jacobs EJ, Schymura MJ, Noone AM, et al. Annual report to the nation on the status of cancer, 1975–2008, featuring cancers associated with excess weight and lack of sufficient physical activity. Cancer 2012;118:2338–66.10.1002/cncr.27514Search in Google Scholar
2. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012;379:1245–55.10.1016/S0140-6736(11)61347-0Search in Google Scholar
3. Hoshida Y, Toffanin S, Lachenmayer A, Villanueva A, Minguez B, Llovet JM. Molecular classification and novel targets in hepatocellular carcinoma: recent advancements. Semin Liver Dis 2010;30:35–51.10.1055/s-0030-1247131Search in Google Scholar
4. Ozturk M, Arslan-Ergul A, Bagislar S, Senturk S, Yuzugullu H. Senescence and immortality in hepatocellular carcinoma. Cancer Lett 2009;286:103–13.10.1016/j.canlet.2008.10.048Search in Google Scholar
5. Lowe SW, Sherr CJ. Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 2003;13:77–83.10.1016/S0959-437X(02)00013-8Search in Google Scholar
6. Sherr CJ. The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2001;2:731–7.10.1038/35096061Search in Google Scholar PubMed
7. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, et al. 5’ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med 1995;1:686–92.10.1038/nm0795-686Search in Google Scholar PubMed
8. Nishida N, Kudo M, Nagasaka T, Ikai I, Goel A. Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma. Hepatology 2012;56: 994–1003.10.1002/hep.25706Search in Google Scholar PubMed
9. Wang Y, Cheng J, Xu C, Liu S, Jiang S, Xu Q, et al. Quantitative methylation analysis reveals gender and age differences in p16INK4a hypermethylation in hepatitis B virus-related hepatocellular carcinoma. Liver Int 2011;32:420–8.10.1111/j.1478-3231.2011.02696.xSearch in Google Scholar PubMed
10. Harder J, Opitz OG, Brabender J, Olschewski M, Blum HE, Nomoto S, et al. Quantitative promoter methylation analysis of hepatocellular carcinoma, cirrhotic and normal liver. Int J Cancer 2008;122:2800–4.10.1002/ijc.23433Search in Google Scholar PubMed
11. Kaneto H, Sasaki S, Yamamoto H, Itoh F, Toyota M, Suzuki H, et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut 2001;48:372–7.10.1136/gut.48.3.372Search in Google Scholar PubMed PubMed Central
12. Liew CT, Li HM, Lo KW, Leow CK, Chan JY, Hin LY, et al. High frequency of p16INK4A gene alterations in hepatocellular carcinoma. Oncogene 1999;18:789–95.10.1038/sj.onc.1202359Search in Google Scholar PubMed
13. Nishida N, Nagasaka T, Nishimura T, Ikai I, Boland CR, Goel A. Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology 2008;47:908–18.10.1002/hep.22110Search in Google Scholar PubMed PubMed Central
14. Wong IH, Lo YM, Zhang J, Liew CT, Ng MH, Wong N, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res 1999;59:71–3.Search in Google Scholar
15. Fleischhacker M, Schmidt B. Cell-free DNA resuscitated for tumor testing. Nat Med 2008;14:914–5.10.1038/nm0908-914Search in Google Scholar PubMed
16. Warren JD, Xiong W, Bunker AM, Vaughn CP, Furtado LV, Roberts WL, et al. Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer. BMC Med 2011; 9:133.10.1186/1741-7015-9-133Search in Google Scholar PubMed PubMed Central
17. Iizuka N, Sakaida I, Moribe T, Fujita N, Miura T, Stark M, et al. Elevated levels of circulating cell-free DNA in the blood of patients with hepatitis C virus-associated hepatocellular carcinoma. Anticancer Res 2006;26:4713–9.Search in Google Scholar
18. Wong IH, Lo YM, Lai PB, Johnson PJ. Relationship of p16 methylation status and serum alpha-fetoprotein concentration in hepatocellular carcinoma patients. Clin Chem 2000;46:1420–2.10.1093/clinchem/46.9.1420Search in Google Scholar
19. Zhang C, Li Z, Cheng Y, Jia F, Li R, Wu M, et al. CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma. Clin Cancer Res 2007;13:944–52.10.1158/1078-0432.CCR-06-2268Search in Google Scholar PubMed
20. Zhang YJ, Wu HC, Shen J, Ahsan H, Tsai WY, Yang HI, et al. Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA. Clin Cancer Res 2007;13:2378–84.10.1158/1078-0432.CCR-06-1900Search in Google Scholar PubMed
21. Minguez B, Lachenmayer A. Diagnostic and prognostic molecular markers in hepatocellular carcinoma. Dis Markers 2010;31:181–90.10.1155/2011/310675Search in Google Scholar
22. Zhou J, Shi YH, Fan J. Circulating cell-free nucleic acids: promising biomarkers of hepatocellular carcinoma. Semin Oncol 2012;39:440–8.10.1053/j.seminoncol.2012.05.013Search in Google Scholar PubMed
23. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996;93:9821–6.10.1073/pnas.93.18.9821Search in Google Scholar PubMed PubMed Central
24. Mikeska T, Candiloro IL, Dobrovic A. The implications of heterogeneous DNA methylation for the accurate quantification of methylation. Epigenomics 2010;2:561–73.10.2217/epi.10.32Search in Google Scholar PubMed
25. Lof-Ohlin ZM, Nilsson TK. Pyrosequencing assays to study promoter CpG site methylation of the O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A, and APC1A genes. Oncol Rep 2009;21:721–9.Search in Google Scholar
26. Holdenrieder S, Stieber P, Chan LY, Geiger S, Kremer A, Nagel D, et al. Cell-free DNA in serum and plasma: comparison of ELISA and quantitative PCR. Clin Chem 2005;51:1544–6.10.1373/clinchem.2005.049320Search in Google Scholar PubMed
27. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010;17:1471–4.10.1245/s10434-010-0985-4Search in Google Scholar PubMed
28. Song do S, Bae SH. Changes of guidelines diagnosing hepatocellular carcinoma during the last ten-year period. Clin Mol Hepatol 2012;18:258–67.10.3350/cmh.2012.18.3.258Search in Google Scholar PubMed PubMed Central
29. Tellez CS, Shen L, Estecio MR, Jelinek J, Gershenwald JE, Issa JP. CpG island methylation profiling in human melanoma cell lines. Melanoma Res 2009;19:146–55.10.1097/CMR.0b013e32832b274eSearch in Google Scholar
30. Rotolo S, Diotti R, Gordon RE, Qiao RF, Yao Z, Phelps RG, et al. Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild-type INK4A in melanoma cells. Int J Cancer 2005;115:164–9.10.1002/ijc.20865Search in Google Scholar PubMed
31. Zainuddin N, Kanduri M, Berglund M, Lindell M, Amini RM, Roos G, et al. Quantitative evaluation of p16(INK4a) promoter methylation using pyrosequencing in de novo diffuse large B-cell lymphoma. Leuk Res 2011;35:438–43.10.1016/j.leukres.2010.10.001Search in Google Scholar PubMed
32. Wong IH, Zhang J, Lai PB, Lau WY, Lo YM. Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients. Clin Cancer Res 2003;9:1047–52.Search in Google Scholar
33. Chu HJ, Heo J, Seo SB, Kim GH, Kang DH, Song GA, et al. Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma. J Korean Med Sci 2004;19:83–6.10.3346/jkms.2004.19.1.83Search in Google Scholar PubMed PubMed Central
34. Zhang YJ, Rossner P Jr., Chen Y, Agrawal M, Wang Q, Wang L, et al. Aflatoxin B1 and polycyclic aromatic hydrocarbon adducts, p53 mutations and p16 methylation in liver tissue and plasma of hepatocellular carcinoma patients. Int J Cancer 2006;119: 985–91.10.1002/ijc.21699Search in Google Scholar PubMed
35. Chang H, Yi B, Li L, Zhang HY, Sun F, Dong SQ, et al. Methylation of tumor associated genes in tissue and plasma samples from liver disease patients. Exp Mol Pathol 2008;85:96–100.10.1016/j.yexmp.2008.07.001Search in Google Scholar PubMed
36. Iyer P, Zekri AR, Hung CW, Schiefelbein E, Ismail K, Hablas A, et al. Concordance of DNA methylation pattern in plasma and tumor DNA of Egyptian hepatocellular carcinoma patients. Exp Mol Pathol 2010;88:107–11.10.1016/j.yexmp.2009.09.012Search in Google Scholar PubMed PubMed Central
37. Whiley DM, Sloots TP. Sequence variation in primer targets affects the accuracy of viral quantitative PCR. J Clin Virol 2005;34:104–7.10.1016/j.jcv.2005.02.010Search in Google Scholar PubMed
38. Lang AH, Drexel H, Geller-Rhomberg S, Stark N, Winder T, Geiger K, et al. Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF. J Mol Diagn 2011;13:23–8.10.1016/j.jmoldx.2010.11.007Search in Google Scholar PubMed PubMed Central
39. Tougeron D, Lecomte T, Pages JC, Villalva C, Collin C, Ferru A, et al. Effect of low-frequency KRAS mutations on the response to anti-EGFR therapy in metastatic colorectal cancer. Ann Oncol 2013;24:1267–73.10.1093/annonc/mds620Search in Google Scholar PubMed
40. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011;11: 426–37.10.1038/nrc3066Search in Google Scholar PubMed
41. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74.10.1016/j.cell.2011.02.013Search in Google Scholar PubMed
42. Li B, Liu W, Wang L, Li M, Wang J, Huang L, et al. CpG island methylator phenotype associated with tumor recurrence in tumor-node-metastasis stage I hepatocellular carcinoma. Ann Surg Oncol 2010;17:1917–26.10.1245/s10434-010-0921-7Search in Google Scholar PubMed
43. Park BK, Park YN, Ahn SH, Lee KS, Chon CY, Moon YM, et al. Long-term outcome of chronic hepatitis B based on histological grade and stage. J Gastroenterol Hepatol 2007;22:383–8.10.1111/j.1440-1746.2007.04857.xSearch in Google Scholar PubMed
44. Baylin SB, Jones PA. A decade of exploring the cancer epigenome – biological and translational implications. Nat Rev Cancer 2011;11:726–34.10.1038/nrc3130Search in Google Scholar PubMed PubMed Central
45. Li J, Xu M, Yang Z, Li A, Dong J. Simultaneous inhibition of MEK and CDK4 leads to potent apoptosis in human melanoma cells. Cancer Invest 2010;28:350–6.10.1080/07357900903286966Search in Google Scholar
46. Zhao Y, Zhang Y, Yang Z, Li A, Dong J. Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis. Biochem Biophys Res Commun 2008;370:509–13.10.1016/j.bbrc.2008.03.148Search in Google Scholar PubMed
47. Feng Q, Stern JE, Hawes SE, Lu H, Jiang M, Kiviat NB. DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection. Exp Mol Pathol 2010;88:287–92.10.1016/j.yexmp.2010.01.002Search in Google Scholar PubMed PubMed Central
48. Katiyar SK, Singh T, Prasad R, Sun Q, Vaid M. Epigenetic alterations in ultraviolet radiation-induced skin carcinogenesis: interaction of bioactive dietary components on epigenetic targets(dagger). Photochem Photobiol 2012;88:1066–74.10.1111/j.1751-1097.2011.01020.xSearch in Google Scholar PubMed PubMed Central
49. Ma YT, Collins SI, Young LS, Murray PG, Woodman CB. Smoking initiation is followed by the early acquisition of epigenetic change in cervical epithelium: a longitudinal study. Br J Cancer 2011;104:1500–4.10.1038/bjc.2011.113Search in Google Scholar PubMed PubMed Central
50. Yang P, Ma J, Zhang B, Duan H, He Z, Zeng J, et al. CpG site-specific hypermethylation of p16INK4alpha in peripheral blood lymphocytes of PAH-exposed workers. Cancer Epidemiol Biomarkers Prev 2012;21:182–90.10.1158/1055-9965.EPI-11-0784Search in Google Scholar PubMed
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