The representative tumor markers for HCC, AFP, and PIVKA-II are not satisfactory in terms of sensitivity and specificity in the early diagnosis of HCC. In search for novel markers for HCC, three-step proteome analyses were carried out in serum samples obtained from 12 patients with HCC and 10 with LC. As a first step, serum samples were subjected to antibody-based immunoaffinity column system that simultaneously removes twelve of abundant serum proteins. The concentrated flow-through was then fractionated using reversed-phase HPLC. Proteins obtained in each fraction were separated by SDS-PAGE. Serum samples obtained from patient with HCC and with LC were analyzed in parallel and their protein expression patterns were compared. A total of 83 protein bands were found to be upregulated in HCC serum. All the protein bands, the intensity of which was different between HCC and LC groups, were identified. Among them, clusterin was most significantly overexpressed ( ). The overexpression of serum clusterin was confirmed by ELISA using another validation set of HCC samples. Furthermore, serum clusterin was elevated in 40% of HCC cases in which both AFP and PIVKA-II were within their cut-off values. These results suggested that clusterin is a potential novel serum marker for HCC. 1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is a leading cause of death in many countries. Chronic infection by hepatitis B virus (HBV) or hepatitis C virus (HCV) and cirrhosis are major risk factors for HCC development [1, 2]. At present, HCC surveillance with tumor markers and imaging studies such as ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI) have been recommended for patients with cirrhosis [3, 4]. These imaging studies are expensive and the ultrasound is highly dependent on the ability of the operator. Therefore, more sensitive and specific serum biomarkers for early detection of HCC are desirable. Serum tumor markers for detecting HCC could be divided into 4 categories: oncofetal and glycoprotein antigens, enzymes and isoenzymes, genes, and cytokines. Alpha-fetoprotein (AFP) and protein induced by vitamin-K absence or antagonist-II (PIVKA-II) also called des-gamma-carboxyprothrombin (DCP) are representative tumor markers for the diagnosis of HCC. The elevated level of AFP is observed in only 50–70% of patients with HCC and also frequently in patients with cirrhosis or exacerbations of chronic hepatitis [5], and its sensitivity is low in patients with earlier/small tumors [6–8].
References
[1]
J. O. Ogunbiyi, “Hepatocellular carcinoma in the developing world,” Seminars in Oncology, vol. 28, no. 2, pp. 179–187, 2001.
[2]
L. J. Lopez and J. A. Marrero, “Hepatocellular carcinoma,” Current Opinion in Gastroenterology, vol. 20, no. 3, pp. 248–253, 2004.
[3]
S. Fujiyama, M. Tanaka, S. Maeda, H. Ashihara, R. Hirata, and K. Tomita, “Tumor markers in early diagnosis, follow-up and management of patients with hepatocellular carcinoma,” Oncology, vol. 62, no. 1, pp. 57–63, 2002.
[4]
J. Szklaruk, P. M. Silverman, and C. Charnsangavej, “Imaging in the diagnosis, staging, treatment, and surveillance of hepatocellular carcinoma,” American Journal of Roentgenology, vol. 180, no. 2, pp. 441–454, 2003.
[5]
P. J. Johnson, “The role of serum alpha-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma,” Clinics in Liver Disease, vol. 5, no. 1, pp. 145–159, 2001.
[6]
D. S. Chen, J. L. Sung, and J. C. Sheu, “Serum α-fetoprotein in the early stage of human hepatocellular carcinoma,” Gastroenterology, vol. 86, no. 6, pp. 1404–1409, 1984.
[7]
F. Trevisani, S. De Notariis, G. Rapaccini et al., “Semiannual and annual surveillance of cirrhotic patients for hepatocellular carcinoma: effects on cancer stage and patient survival (Italian experience),” American Journal of Gastroenterology, vol. 97, no. 3, pp. 734–744, 2002.
[8]
F. Nomura, K. Ohnishi, and Y. Tanabe, “Clinical features and prognosis of hepatocellular carcinoma with reference to serum alpha-fetoprotein levels: analysis of 606 patients,” Cancer, vol. 64, no. 8, pp. 1700–1707, 1989.
[9]
S. L. Tsai, G. T. Huang, P. M. Yang, J. C. Sheu, J. L. Sung, and D. S. Chen, “Plasma des-γ-carboxyprothrombin in the early stage of hepatocellular carcinoma,” Hepatology, vol. 11, no. 3, pp. 481–488, 1990.
[10]
K. Soga, T. Watanabe, K. Aikawa, M. Toshima, K. Shibasaki, and Y. Aoyagi, “Serum des-gamma-carboxyprothrombin level by a modified enzyme immunoassay method in hepatocellular carcinoma: clinical significance in small hepatocellular carcinoma,” Hepato-Gastroenterology, vol. 45, no. 23, pp. 1737–1741, 1998.
[11]
F. Nomura, M. Ishijima, K. Kuwa, N. Tanaka, T. Nakai, and K. Ohnishi, “Serum des-gamma-carboxy prothrombin levels determined by a new generation of sensitive immunoassays in patients with small-sized hepatocellular carcinoma,” American Journal of Gastroenterology, vol. 94, no. 3, pp. 650–654, 1999.
[12]
M. Capurro, I. R. Wanless, M. Sherman et al., “Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma,” Gastroenterology, vol. 125, no. 1, pp. 89–97, 2003.
[13]
Y. Hippo, K. Watanabe, A. Watanabe et al., “Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma,” Cancer Research, vol. 64, no. 7, pp. 2418–2423, 2004.
[14]
T. Nakatsura, Y. Yoshitake, S. Senju et al., “Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker,” Biochemical and Biophysical Research Communications, vol. 306, no. 1, pp. 16–25, 2003.
[15]
Y. Midorikawa, S. Ishikawa, H. Iwanari et al., “Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling,” International Journal of Cancer, vol. 103, no. 4, pp. 455–465, 2003.
[16]
Y. K. Sung, S. Y. Hwang, M. K. Park et al., “Glypican-3 is overexpressed in human hepatocellular carcinoma,” Cancer Science, vol. 94, no. 3, pp. 259–262, 2003.
[17]
Z. L. Lü, D. Z. Luo, and J. M. Wen, “Expression and significance of tumor-related genes in HCC,” World Journal of Gastroenterology, vol. 11, no. 25, pp. 3850–3854, 2005.
[18]
R. Cui, J. He, F. Zhang et al., “Diagnostic value of protein induced by vitamin K absence (PIVKAII) and hepatoma-specific band of serum gamma-glutamyl transferase (GGTII) as hepatocellular carcinoma markers complementary to α-fetoprotein,” British Journal of Cancer, vol. 88, no. 12, pp. 1878–1882, 2003.
[19]
P. Tangkijvanich, P. Tosukhowong, P. Bunyongyod et al., “alpha-L-fucosidase as a serum marker of hepatocellular carcinoma in Thailand,” Southeast Asian Journal of Tropical Medicine and Public Health, vol. 30, no. 1, pp. 110–114, 1999.
[20]
H. Ishizuka, T. Nakayama, S. Matsuoka et al., “Prediction of the development of hepato-cellular-carcinoma in patients with liver cirrhosis by the serial determinations of serum alpha-L-fucosidase activity,” Internal Medicine, vol. 38, no. 12, pp. 927–931, 1999.
[21]
Z. Liu, L. Yan, T. Xiang, L. Jiang, and B. Yang, “Expression of vascular endothelial growth factor and matrix metalloproteinase-2 correlates with the invasion and metastasis of hepatocellular carcinoma,” Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, vol. 20, no. 2, pp. 249–254, 2003.
[22]
G. W. Huang, L. Y. Yang, and W. Q. Lu, “Expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in hepatocellular carcinoma: impact on neovascularization and survival,” World Journal of Gastroenterology, vol. 11, no. 11, pp. 1705–1708, 2005.
[23]
S. J. Kim, I. K. Choi, K. H. Park et al., “Serum vascular endothelial growth factor per platelet count in hepatocellular carcinoma: correlations with clinical parameters and survival,” Japanese Journal of Clinical Oncology, vol. 34, no. 4, pp. 184–190, 2004.
[24]
B. C. Song, Y. H. Chung, J. A. Kim et al., “Transforming growth factor-β1 as a useful serologic marker of small hepatocellular carcinoma,” Cancer, vol. 94, no. 1, pp. 175–180, 2002.
[25]
R. Sacco, D. Leuci, C. Tortorella, G. Fiore, F. Marinosci, and S. Antonaci, “Transforming growth factor β1 and soluble Fas serum levels in hepatocellular carcinoma,” Cytokine, vol. 12, no. 6, pp. 811–814, 2000.
[26]
X. P. Chen, H. Zhao, and X. P. Zhao, “Alternation of AFP-mRNA level detected in blood circulation during liver resection for HCC and its significance,” World Journal of Gastroenterology, vol. 8, no. 5, pp. 818–821, 2002.
[27]
X. Ding, L. Y. Yang, G. W. Huang et al., “Role of AFP mRNA expression in peripheral blood as a predictor for postsurgical recurrence of hepatocellular carcinoma: a systematic review and meta-analysis,” World Journal of Gastroenterology, vol. 11, no. 17, pp. 2656–2661, 2005.
[28]
N. Miura, Y. Maeda, T. Kanbe et al., “Serum human telomerase reverse transcriptase messenger RNA as a novel tumor marker for hepatocellular carcinoma,” Clinical Cancer Research, vol. 11, no. 9, pp. 3205–3209, 2005.
[29]
N. Miura, G. Shiota, T. Nakagawa et al., “Sensitive detection of human telomerase reverse transcriptase mRNA in the serum of patients with hepatocellular carcinoma,” Oncology, vol. 64, no. 4, pp. 430–434, 2003.
[30]
T. Hutchens and T. T. Yip, “New desorption strategies for the mass spectrometric analysis of macromolecules,” Rapid Communications in Mass Spectrometryvol, vol. 7, pp. 576–580, 1993.
[31]
F. Nomura, T. Tomonaga, K. Sogawa et al., “Identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry,” Proteomics, vol. 4, no. 4, pp. 1187–1194, 2004.
[32]
K. Sogawa, S. Itoga, T. Tomonaga, and F. Nomura, “Diagnostic values of surface-enhanced laser desorption/ionization technology for screening of habitual drinkers,” Alcoholism, vol. 31, supplement s1, pp. S22–S26, 2007.
[33]
V. Paradis, F. Degos, D. Dargère et al., “Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases,” Hepatology, vol. 41, no. 1, pp. 40–47, 2005.
[34]
S. Kanmura, H. Uto, K. Kusumoto et al., “Early diagnostic potential for hepatocellular carcinoma using the SELDI ProteinChip system,” Hepatology, vol. 45, no. 4, pp. 948–956, 2007.
[35]
N. L. Anderson and N. G. Anderson, “The human plasma proteome: history, character, and diagnostic prospects,” Molecular & Cellular Proteomics, vol. 1, no. 11, pp. 845–867, 2002.
[36]
N. Hattori, S. Oda, T. Sadahiro et al., “YKL-40 identified by proteomic analysis as a biomarker of sepsis,” Shock, vol. 32, no. 4, pp. 393–400, 2009.
[37]
K. Sogawa, Y. Kodera, M. Satoh et al., “Increased serum levels of pigment epithelium-derived factor by excessive alcohol consumption-detection and identification by a three-step serum proteome analysis,” Alcoholism, vol. 35, no. 2, pp. 211–217, 2011.
[38]
M. Kadowaki, T. Sangai, T. Nagashima et al., “Identification of vitronectin as a novel serum marker for early breast cancer detection using a new proteomic approach,” Journal of Cancer Research and Clinical Oncology, vol. 137, no. 7, pp. 1105–1115, 2011.
[39]
M. Abulaizi, T. Tomonaga, M. Satoh, et al., “The application of a three-step proteome analysis for identification of new biomarkers of pancreatic cancer,” International Journal of Proteomics, vol. 2011, Article ID 628787, 13 pages, 2011.
[40]
H. Umemura, M. Nezu, Y. Kodera et al., “Effects of the time intervals between venipuncture and serum preparation for serum peptidome analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,” Clinica Chimica Acta, vol. 406, no. 1-2, pp. 179–180, 2009.
[41]
U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970.
[42]
L. Anderson and J. Seilhamer, “A comparison of selected mRNA and protein abundances in human liver,” Electrophoresis, vol. 18, no. 3-4, pp. 533–537, 1997.
[43]
C. R. M. Y. Liang, C. K. Leow, J. C. H. Neo et al., “Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry,” Proteomics, vol. 5, no. 8, pp. 2258–2271, 2005.
[44]
J. Kim, S. H. Kim, S. U. Lee et al., “Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins,” Electrophoresis, vol. 23, no. 24, pp. 4142–4156, 2002.
[45]
I. N. Lee, C. H. Chen, J. C. Sheu et al., “Identification of human hepatocellular carcinoma-related biomarkers by two-dimensional difference gel electrophoresis and mass spectrometry,” Journal of Proteome Research, vol. 4, no. 6, pp. 2062–2069, 2005.
[46]
O. L. Seung, S. J. Park, W. Kim et al., “Proteome analysis of hepatocellular carcinoma,” Biochemical and Biophysical Research Communications, vol. 291, no. 4, pp. 1031–1037, 2002.
[47]
J. T. Feng, Y. K. Liu, H. Y. Song et al., “Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis,” Proteomics, vol. 5, no. 17, pp. 4581–4588, 2005.
[48]
O. Blaschuk, K. Burdzy, and I. B. Fritz, “Purification and characterization of a cell-aggregating factor (clusterin), the major glycoprotein in ram rete testis fluid,” The Journal of Biological Chemistry, vol. 258, no. 12, pp. 7714–7720, 1983.
[49]
H. V. De Silva, W. D. Stuart, C. R. Duvic et al., “A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins,” The Journal of Biological Chemistry, vol. 265, no. 22, pp. 13240–13247, 1990.
[50]
I. P. Trougakos and E. S. Gonos, “Clusterin/Apolipoprotein J in human aging and cancer,” International Journal of Biochemistry and Cell Biology, vol. 34, no. 11, pp. 1430–1448, 2002.
[51]
T. L. Brown, B. C. Moulton, V. V. Baker, J. Mira, and J. A. K. Harmony, “Expression of apolipoprotein J in the uterus is associated with tissue remodeling,” Biology of Reproduction, vol. 52, no. 5, pp. 1038–1049, 1995.
[52]
C. Petropoulou, I. P. Trougakos, E. Kolettas, O. Toussaint, and E. S. Gonos, “Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts,” FEBS Letters, vol. 509, no. 2, pp. 287–297, 2001.
[53]
M. Redondo, E. Villar, J. Torres-Munoz, T. Tellez, M. Morell, and C. K. Petito, “Overexpression of clusterin in human breast carcinoma,” American Journal of Pathology, vol. 157, no. 2, pp. 393–399, 2000.
[54]
M. J. Xie, Y. Motoo, S. B. Su et al., “Expression of clusterin in human pancreatic cancer,” Pancreas, vol. 25, no. 3, pp. 234–238, 2002.
[55]
X. Chen, R. B. Halberg, W. M. Ehrhardt, J. Torrealba, and W. F. Dove, “Clusterin as a biomarker in murine and human intestinal neoplasia,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 16, pp. 9530–9535, 2003.
[56]
Y. K. Kang, S. W. Hong, H. Lee, and W. H. Kim, “Overexpression of clusterin in human hepatocellular carcinoma,” Human Pathology, vol. 35, no. 11, pp. 1340–1346, 2004.
