Detection and Clinical Significance of Methylation of Peripheral Plasma DNA in Patients with Hepatocellular Carcinoma_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WANG Ping ¹ , GENG Xiaoping ¹ , ZHU Lixin ¹ , LI Xiaoming ² , LI Dong. ¹

1. Center of Organ Transplant,The First Affiliated Hospital of Anhui Medical University, Heifei 230022, Anhui Province, China;;
2. Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong, China;

Corresponding author：

Keywords：

Hepatocellular carcinoma; Methylation; Plasma; SLIT2; Death-associated protein kinase

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Objective 　To evaluate the effect of methylation determination about the peripheral plasma DNA in diagnose of hepatocellular carcinoma （HCC） and select the highly sensitive and specific methylated cancer suppressor genes.
Methods 　Methylation-specific PCR （MSP） was used to detect the degree of methylation about SLIT2 and DAPK genes in peripheral plasma and associated cancer tissues of 34 patients with HCC confirmed by pathology, then analyzed their relationship to clinicopathologic feature.
Results 　The positive rate of the promoter methylation of SLIT2 and DAPK genes in cancer tissues in 34 cases were 70.6% （24/34） and 79.4% （27/34）, while the relevant promoter methylation rate in plasma were 44.1% （15/34） and 50.0% （17/34） correspondingly. The sensitivity of detection of DNA methylation about SLIT2 and DAPK genes in plasma was 62.5% and 63.0%, respectively；both of the specificity for them were 100%. The negative predicted value was 52.6% and 41.2%, respectively；while both of the positive predicted value were 100%. There were no significant correlation between the clinicopathologic features and the methylation rate in cancer tissues and plasma （P＞0.05）. In plasma of patients whose AFP＜400 μg/L, the positive rate of combined detection of DNA methylation of SLIT2 and DAPK was 61.1% （11/18）.
Conclusions 　The detection rate of DNA methylation of SLIT2 and DAPK genes in plasma is higher, and there is a significant correlation between the DNA methylation in HCC tissue and plasma, based on MSP method. DNA methylation in plasma, as an non-invasive method, could be used to diagnose HCC, especially for the patients whose AFP is negative. HBV infection may be only associate with DNA methylation of part gene.

Citation： WANG Ping,GENG Xiaoping,ZHU Lixin,LI Xiaoming,LI Dong.. Detection and Clinical Significance of Methylation of Peripheral Plasma DNA in Patients with Hepatocellular Carcinoma. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2012, 19(7): 727-732. doi: Copy

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6.	黄朝晖，胡瑜，华东，等. 定量PCR 检测肝细胞癌患者循环血浆DNA 及其临床意义 [J]. 中国癌症杂志， 2010， 20(9)：663-667.
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12.	张敬杰，欧阳涛，万文徽，等. 乳腺癌患者外周血浆中游离的肿瘤相关DNA 检测及其临床价值 [J]. 中华肿瘤杂志， 2007，29(8)： 609-613.
13.	Iyer P， Zekri AR， Hung CW， et al. Concordance of DNA methylation pattern in plasma and tumor DNA of Egyptian hepatocellular carcinoma patients [J]. Exp Mol Pathol， 2010， 88(1)：107-111.
14.	Wong IH， Lo YM， Zhang J ， et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients [J].Cancer Res， 1999， 59(1)： 71-73.
15.	An Q， Liu Y， Gao Y， et al. Detection of p16 hypermethylation in circulating plasma DNA of non-small cell lung cancer patients [J]. Cance Lett， 2002， 188(1)： 109-114.
16.	Fujiwara K， Fujimoto N， Tabata M， et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer [J]. Clin Cancer Res， 2005，11(3)： 1219-1225.
17.	Chang H， Yi B， Li L， et al. Methylation of tumor associated genes in tissue and plasma samples from liver disease patients [J]. Exp Mol Pathol， 2008， 85(2)： 96-100.
18.	Leerapun A， Suravarapu SV， Bida JP， et al. The utility of lens culinaris agglutinin reactive alpha-fetoprotein in the diagnosis of hepatocellular carcinoma：evaluation in a United States referral population [J]. Clin Gastroenterol Hepatol， 2007， 5(3)： 394-402．.
19.	Nakagawa T， Seki T， Shiro T， et al. Clinicopathologic significance of protein induced vitamin K absence or antagonist Ⅱ and alpha-fetoprotein in hepatocellular carcinoma [J]． Int J Oncol，1999， 14(2) ： 281-286.
20.	Nishida N， Nagasaka T， Nishimura T， et al. Aberrant methylation of multiple tumor suppressor genes in aging liver， chronic hepatitis， and hepatocellular carcinoma [J]. Hepatology，2008， 47(3)： 908-918.
21.	Shim YH， Yoon GS， Choi HJ， et al. p16 Hypermethylation in the early stage of hepatitis B virus associatedhepatocarcinogenesis [J]. Cancer Lett， 2003， 190(2)： 213-219.

1. Gautschi O， Bigosch C， Huegli B， et al. Circulating deoxyribonucleic acid as prognostic mark in non-small-cell lung cancer patients undergoing chemotherapy [J]. J Clin Oncol， 2004，22(20)：4157-4164.
2. Schlechte HH， Stelzer C， Weickmann S， et al. TP53 gene in blood plasma DNA of tumor patients [J]. Ann NY Acad Sci，2004， 1022(6)：61-69.
3. Mandel P， Metais P. Les acides nucleiques du plasma sanguin chez l’homme [J]. CR Acad Sci Paris， 1948， 142：241-243.
4. Leon SA， Shapiro B， Sklarof DM， et a1. Free DNA in the serum of cancer patients and the efect of thempy [J]. Cancer Res，1977， 37(3)： 646-650．.
5. Vaart MVD， Pretorius PJ. Is the role of circulating DNA as a biomarker of cancer being prematurely overrated? [J]. Clinical Biochemistry， 2010， 43(1)： 26-36.
6. 黄朝晖，胡瑜，华东，等. 定量PCR 检测肝细胞癌患者循环血浆DNA 及其临床意义 [J]. 中国癌症杂志， 2010， 20(9)：663-667.
7. Jackson PE， Qian GS， Friesen MD， et al. Specific p53 mutations detection in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry [J]. Cancer Res， 2001， 61(1)：33-35.
8. Chang YC， Ho CL， Chen HHW， et al. Molecular diagnosis of primary liver cancer by microsatellite DNA analysis in the serum [J]. Brit J Cancer， 2002， 87(12)： 1449-1453.
9. Board RE， Williams VS， Knight L， et al. Isolation and extraction of circulating tumor DNA from patients with small cell lung cancer [J]. Ann NY Acad Sci， 2008， 1137(8)： 98-107.
10. Chan KC， Yeung SW， Lui WB， et al. Effects of preanalytical factors on the molecular size of cell-free DNA in blood [J]. Clin Chem， 2005， 51(4)：781-784.
11. Thierry AR， Mouliere F， Gongora C， et al. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts [J]. Nucleic Acids Res， 2010， 38(18)： 6159-6175.
12. 张敬杰，欧阳涛，万文徽，等. 乳腺癌患者外周血浆中游离的肿瘤相关DNA 检测及其临床价值 [J]. 中华肿瘤杂志， 2007，29(8)： 609-613.
13. Iyer P， Zekri AR， Hung CW， et al. Concordance of DNA methylation pattern in plasma and tumor DNA of Egyptian hepatocellular carcinoma patients [J]. Exp Mol Pathol， 2010， 88(1)：107-111.
14. Wong IH， Lo YM， Zhang J ， et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients [J].Cancer Res， 1999， 59(1)： 71-73.
15. An Q， Liu Y， Gao Y， et al. Detection of p16 hypermethylation in circulating plasma DNA of non-small cell lung cancer patients [J]. Cance Lett， 2002， 188(1)： 109-114.
16. Fujiwara K， Fujimoto N， Tabata M， et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer [J]. Clin Cancer Res， 2005，11(3)： 1219-1225.
17. Chang H， Yi B， Li L， et al. Methylation of tumor associated genes in tissue and plasma samples from liver disease patients [J]. Exp Mol Pathol， 2008， 85(2)： 96-100.
18. Leerapun A， Suravarapu SV， Bida JP， et al. The utility of lens culinaris agglutinin reactive alpha-fetoprotein in the diagnosis of hepatocellular carcinoma：evaluation in a United States referral population [J]. Clin Gastroenterol Hepatol， 2007， 5(3)： 394-402．.
19. Nakagawa T， Seki T， Shiro T， et al. Clinicopathologic significance of protein induced vitamin K absence or antagonist Ⅱ and alpha-fetoprotein in hepatocellular carcinoma [J]． Int J Oncol，1999， 14(2) ： 281-286.
20. Nishida N， Nagasaka T， Nishimura T， et al. Aberrant methylation of multiple tumor suppressor genes in aging liver， chronic hepatitis， and hepatocellular carcinoma [J]. Hepatology，2008， 47(3)： 908-918.
21. Shim YH， Yoon GS， Choi HJ， et al. p16 Hypermethylation in the early stage of hepatitis B virus associatedhepatocarcinogenesis [J]. Cancer Lett， 2003， 190(2)： 213-219.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Detection and Clinical Significance of Methylation of Peripheral Plasma DNA in Patients with Hepatocellular Carcinoma

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