Identification of Cell Clonal Origin of Multinodular Hepatocellular Carcinoma by Analyzing Mitochondrial DNA D-Loop Region Variations_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LI Shilai ¹ , LI Lequn ² , PENG Tao ² , SU Ming ²

1.Department of Emergency, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China;;
2.Department of Hepatobiliary Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China;

Corresponding author：

Keywords：

Mitochondrial DNA; D-Loop region; Multinodular hepatocellular carcinoma; Clonal origin

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Objective To explore the feasibility of identifying clonal origin of hepatocellular carcinoma (HCC) by analyzing the mitochondrial DNA D-Loop region variations. Methods Forty-two patients with a total of 112 HCC nodules consequentially hospitalized for radical resection of HCC in the department of hepatobiliary surgery of the First Affiliated Hospital of Guangxi Medical University from April 2004 to August 2007 were included for study group (multinodular HCCs). Control group included 20 cases of HCC (40 samples) hospitalized in the same period that consisted of two sub-groups: control groupⅠconsisted of 16 cases of single nodular HCC that each had two pieces of inconsecutive tumor tissues and control groupⅡconsisted of 4 cases of HCC with portal vein tumor embolus whose tumor tissues and portal vein tumor embolus were collected simultaneously. Normal control included 5 patients who were donors for liver transplantation or underwent liver trauma without any liver disease. Polymerase chain reaction (PCR) and direct sequencing were applied to study the mtDNA D-Loop region. The sequences of multinodular lesions were compared among different groups. Results For all the 42 cases of the study group, basic group variations appeared in 131 sites (131/1 122, 11.7%, the number 1 122 was the length of mtDNA D-Loop) with point mutation in 15 sites, insert in 9 sites, and deletion in 16 sites. And of all the variations in the study group, 98 were polymorphism. In study group, 20 cases were categorized as multicentric occurrence (MO) based on their variant mtDNA D-Loop sequences in each nodule from the same patient. And 22 cases were characterized as intrahepatic metastasis (IM) based on the identical mtDNA D-Loop sequences found in each nodule from the same patient. In all 20 cases in the control group, the inconsecutive tumor tissues or the portal vein tumor embolus and original tumors shared identical mtDNA D-Loop sequences. For the normal control group, basic group variations appeared in 14 sites, and they were all polymorphism including a new polymorphism (NT 479 A gt;G). Conclusions There is a high rate of changes in mtDNA D-Loop region. And our study speculates a novel discrimination of MO and IM origins among multinodular HCCs using PCR and direct sequencing of the mtDNA D-Loop sequences.

Citation： LI Shilai,LI Lequn,PENG Tao,SU Ming. Identification of Cell Clonal Origin of Multinodular Hepatocellular Carcinoma by Analyzing Mitochondrial DNA D-Loop Region Variations. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(6): 567-572. doi: Copy

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20.	Yamamoto H, Tanaka M, Katayama M, et al. Significant existence of deleted mitochondrial DNA in cirrhotic liver surrounding hepatic tumor [J]. Biochem Biophys Res Commun, 1992; 182(2): 913-920.
21.	Nishikawa M, Nishiguchi S, Shiomi S, et al. Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma [J]. Cancer Res, 2001; 61(5): 1843-1845.
22.	Lee HC, Li SH, Lin JC, et al. Somatic mutations in the D-Loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma [J]. Mutat Res, 2004; 547(1-2): 71-78.
23.	Ikeda K, Arase Y, Kobayashi M, et al. Significance of multicentric cancer recurrence after potentially curative ablation of hepatocellular carcinoma: a longterm cohort study of 892 patients with viral cirrhosis [J]. J Gastroenterol, 2003; 38(9): 865-876.
24.	Utsunomiya I. Pathological study on multicentric occurrence of hepatocellular carcinoma [J]. Kurume Med J, 2005; 52(4): 133-138.
25.	Fliss MS, Usade1 H, Caballero OL, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids [J]. Science, 2000; 287(5460): 2017-2019.
26.	Okochi O, Hibi K, Uemura T, et al. Detection of mitochondrial DNA alterations in the serum of hepatocellular carcinoma patients [J]. Clin Cancer Res, 2002; 8(9): 2875-2878.

1. Yamamoto T, Kajino K, Kudo M, et al. Determination of the clonal origin of multiple human hepatocellular carcinomas by cloning and polymerase chain reaction of the integrated of hepatitis B virus DNA [J]. Hepatology, 1999; 29(5): 1446-1452.
2. Liver Cancer Study Group of Japan. Classification of primary liver cancer [S]. Frist English Edition, Kanehara Shuppan, Tokyo， 1997： 1-22.
3. Matsumoto Y, Fuji H, Matsuda M, et al. Multicentric occurrence of hepatocellular carcinoma: diagnosis and clinical significance [J]. J Hepatobiliary Pancreat Surg, 2001; 8(5): 435-440.
4. Esumi M, Aritaka T, Arri M, et al. Clonal origin of human hepatoma detemined by integration of hepatitis B virus DNA [J]. Cancer Res, 1986; 46(11): 5767-5771.
5. Chen PJ, Chen DS, Lai MY, et al. Clonal origin of recurrent hepatocellular carcinomas [J]. Gastroenterology, 1989; 96(2 Pt 1): 527-529.
6. Hsu HC, Chiou TJ, Chen JY, et al. Clonality and Clonal evolution of hepatocellular carcinoma with multiple nodules [J]. Hepatology, 1991; 13(5): 923-928.
7. Oda T, Tsuda H, Scarpa A, et al. Mutation pattern of the p53 gene as a diagnostic marker for multiple hepatocellular carcinoma [J]. Cancer Res, 1992; 52(13): 3674-3678.
8. Peng SY, Lai PL, Chu JS, et al. Expression and hypomethylation of alpha-fetoprotein gene in unicentric and multicentric human hepatocellular carcinomas [J]. Hepatology, 1993; 17(1): 35-41.
9. Chen YJ, Yeh SH, Chen JT, et al. Chromosomal changes and clonality relationship between primary and recurrent hepatocellular carcinoma [J]. Gastroenterology, 2000; 119(2): 431-440.
10. Okamoto M, Utsunomiya T, Wakiyama S, et al. Specific gene-expression profiles of noncancerous liver tissue predict the risk for multicentric occurrence of hepatocellular carcinoma in hepatitis C virus-positive patients [J]. Ann Surg Oncol, 2006; 13(7): 947-954.
11. Yamada S, Nomoto S, Fujii T, et al. Correlation between copy number of mitochondrial DNA and clinico-pathologic parameters of hepatocellular carcinoma [J]. Eur J Surg Oncol, 2006; 32(3): 303-307.
12. 胡义德, 钱桂生, 李淑平, 等. 人癌细胞线粒体DNA控制区序列特征分析 [J]. 遗传, 1999; 21(4): 1-6.
13. Nowell PC. The clonal evolution of tumor cell populations [J]. Science, 1976; 194(4260): 23-28.
14. Calabrese P, Tavaré S, Shibata D. Pretumor progression: clonal evolution of human stem cell populations [J]. Am J Pathol, 2004; 164(4): 1337-1346.
15. Tang ZY, Ye SL, Liu YK, et al. A decade’s studies on metastasis of hepatocellular carcinoma [J]. J Cancer Res Clin Oncol, 2004; 130(4): 187-196.
16. Copeland WC, Wachsman JT, Johnson FM, et al. Mitochondrial DNA alterations in cancer [J]. Cancer Invest, 2002; 20(4): 557-569.
17. Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis [J]. Annu Rev Pharmacol Toxicol, 2004; 44: 239-267.
18. Lièvre A, Blons H, Houllier AM, et al. Clinicopathological significance of mitochondrial D-Loop mutations in head and neck carcinoma [J]. Br J Cancer, 2006; 94(5): 692-697.
19. Han CB, Li F, Zhao YJ, et al. Variations of mitochondrial D-Loop region plus downstream gene 12S rRNA-Trna(phe) and gastric carcinomas [J]. World J Gastroenterol, 2003; 9(9): 1925-1929.
20. Yamamoto H, Tanaka M, Katayama M, et al. Significant existence of deleted mitochondrial DNA in cirrhotic liver surrounding hepatic tumor [J]. Biochem Biophys Res Commun, 1992; 182(2): 913-920.
21. Nishikawa M, Nishiguchi S, Shiomi S, et al. Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma [J]. Cancer Res, 2001; 61(5): 1843-1845.
22. Lee HC, Li SH, Lin JC, et al. Somatic mutations in the D-Loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma [J]. Mutat Res, 2004; 547(1-2): 71-78.
23. Ikeda K, Arase Y, Kobayashi M, et al. Significance of multicentric cancer recurrence after potentially curative ablation of hepatocellular carcinoma: a longterm cohort study of 892 patients with viral cirrhosis [J]. J Gastroenterol, 2003; 38(9): 865-876.
24. Utsunomiya I. Pathological study on multicentric occurrence of hepatocellular carcinoma [J]. Kurume Med J, 2005; 52(4): 133-138.
25. Fliss MS, Usade1 H, Caballero OL, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids [J]. Science, 2000; 287(5460): 2017-2019.
26. Okochi O, Hibi K, Uemura T, et al. Detection of mitochondrial DNA alterations in the serum of hepatocellular carcinoma patients [J]. Clin Cancer Res, 2002; 8(9): 2875-2878.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Identification of Cell Clonal Origin of Multinodular Hepatocellular Carcinoma by Analyzing Mitochondrial DNA D-Loop Region Variations

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