| BackgroundPrimary hepatocellular carcinoma (PHC) is the high worldwide incidence of malignant. Hepatocellular carcinoma (HCC) is a major health problem, being the sixth most common cancer worldwide with 626,000 new cases in 2002. HCC is the third most malignant tumor of digestive system and the second cause of death among the malignant tumors in china. Intrahepatic Cholangiocarcinoma (ICCA) is an epithelial cancer originating from the bile ducts with features of cholangiocyte differentiation, accounted for 10% of bile duct cancer cells. Advanced ICCA has a devastating prognosis, with a median survival of<24 months. The only curative therapy is surgical extirpation or liver transplantation, but unfortunately the majority of patients present with advanced stage disease, who are not amenable to surgical therapies.Alpha-fetoprotein (AFP) is the only serum marker currently recommended for HCC surveillance among patients with cirrhosis. AFP is not a very good screening test since it has a sensitivity of 39-64%, a specificity of 76-91% and a positive predictive value of 9-32%.Carbohydrate antigen 199 (CA 199) is the most common used tumor marker for ICCA. CA199 are not specific for intrahepatic cholangiocarcinoma. CA199 is also raised in pancreatic cancer, colorectal cancer, gastric cancer, and gynaecological malignancies. Additionally, CA199 may be elevated in patients with acute cholangitis. In a series of patients without primary sclerosing cholangitis, the sensitivity of a serum CA199 level of more than 100 U/mL in diagnosing cholangiocarcinoma was only 53%. Therefore, the diagnosis of ICCA requires to find better serological markers.Golgi protein-73 (GP73) is a newly identified serum marker for HCC. GP73 is widely expressed in normal epithelial cells from numerous tissues. Its function is unknown. Resolution of hepatocytes is paralleled by a reduction and normalization of GP73 expression, but upregulated in hepatocytes from patients with viral and non-viral liver disease and in HCC. These data indicate that GP73 may be a potential marker for the early detection of HCC.Based on its apparent role in liver disease and GP73 has barely been researched for hepatocellular carcinoma in Asians,our exploratory study were to determine if GP73 protein could be detected in the serum of patients with liver disease, to determine whether serum GP73 levels are the highest in patients with HCC and ICCA or not compared to those with cirrhosis, Chronic hepatitis B viral infection, stomach cancer, breast cancer, colon cancer and health control, and to compare the performance characteristics of GP73 and AFP for differentiating HCC. Then our study should compare the performance characteristics of GP73 and CA199 for ICAA.At last, we researched whether GP73 may detected by nested PCR or not.Methods:1 Detection of GP73 ptotein in serum by Western blotThere were 31 patients of every group with hepatitis B and HBV-related HCC.31 persons were healthy controls.20 patients was diagnosised HBV-related cirrhosis.13 patients was diagnosised cholangiocarcinoma. There were each 10 patients with stomach cancer, breast cancer and colon cancer.6 patients was diagnosised pancreatic cancer. A 5ml blood sample was drawn from each subject, spun, aliquoted, polyacrylamide gel electrophoresis, added first antibody, added second antibodies, dyeing, transmembrane and detected the result. For normalization, each gel also included a lane containing 0.5 ml of serum from a common pool of HCV and HBV negative sources. The result was relative unit(specimen sample/normal sera).2 Detection of AFP and CA199 in serum by Protein Chip.Experiment is divided into nine groups, which the groups is the first part of the same.3ml blood sample had been drawn from each group. Then the samples were spun, abstracted the serum, according to the manufacturer's protocols.3 Detection of GP73 mRNA in serum by nested PCR. There were each 31 patients with hepatitis B and HBV-related HCC. 31 persons were healthy controled.20 patients was diagnosised HBV-related cirrhosis.13 patients were diagnosised cholangiocarcinoma. The basis of Genbank sequence of human GP73 cDNA, primer was designed by Prime 5.0.0.5ml blood sample was drawn from each subject. Divided into 5 groups according to the concentration of GP73 and extracted and quantitated RNA which as a model of reverse transcription to synthesis cDNA chains, carried out PCR ampliation by primer. Products were detected by agarose gel electrophoresis, the amount of expression of GP73 mRNA was judged by intracontrast of photodensity. Densitometric analysis was performed to qualitative of GP73 mRNA by gel imaging system and Quantity One.Results:1 Serum GP73 protein could been detected in the serum of all groups in Asian by western blot analysis, but the levels were the highest in HBV-related HCC and Cholangiocarcinoma patients compared to HBV-related cirrhosis patients, hepatitis B patients, stomach cancer, breast cancer, colon cancer, pancreatic cancer and controls with healthy persons. Statistical analysis didn't show a significant difference between the controls with healthy persons and all other groups (stomach cancer, breast cancer,colon cancer,pancreatic cancer) 2 serum GP73 levels were 20.50±12.13 relative units in those with HBV-related HCC. The HBV-related HCC AUROC curve for GP73 was 0.82 (95%CI:0.71-0.92), with a sensitivity of 74.2%, specificity of 83.9%, and an optimal cutoff point of 8.4 relative units; while the AUROC for AFP was 0.65 (95%CI:0.50-0.79), with a sensitivity of 61.3%, specificity of 71.0%, and a cutoff of 20.97ng/ml. GP73 had a better sensitivity, specificity and AUROC compared to AFP.3 ICCA AUROC curve for GP73 was0.69(95%CI:0.49-0.88), with a sensitivity of 61.5%, specificity of 87.1%, and an optimal cutoff point of 9.1relative units; while the AUROC for CA199 was 0.40(CI:0.19-0.61), with a sensitivity of 23.1%, specificity of 90.3%, and a cutoff of 364.82U/ml.GP73 had a better AUROC compared to CA199. Serum GP73 and CA199 diagnosed ICAA together with sensitivity of 70.4%, specificity of 80.8%.4 Hepatocellular carcinoma and cholangiocellular carcinoma together constitute the primary liver cancer. Serum GP73 levels were 99.55±139.51 ng/ml in those with PHC. AFP levels were 20.50±12.13 relative units in those with PHC. In this study, Statistical analysis didn't show a significant difference between the controls with healthy persons and all other groups in the AFP of ICCA. Statistical analysis showed a significant difference between the controls with healthy persons and all other groups in the serum GP73 of ICCA, while in other tumors were also no significant increase in serum GP73.5 Nested PCR analysis showed that GP73 was detected in the serum of all patients but the levels were the highest in HCC compared to the other groups. Serum GP73 expression were 93.5%(29/31) in those with HBV-related HCC. Serum GP73 expression were 45.2% in those with healthy persons.Conclusion1 serum GP73 could been detected in the serum of all groups in Asian by western immunoblot analysis, but the levels were the highest in HBV-related HCC and ICAA patients compared to cirrhosis patients, hepatitis B patients and controls with healthy persons.2 The sensitivity, specificity and AUROC of sGP73 were better than AFP for the diagnosing HCC. SGP73 could replace AFP as a good tumor marker for HCC.3 The sensitivity and AUROC of sGP73 were better than CA199 for diagnosing ICAA. SGP73 can become a tumor marker for ICAA. Serum GP73 and CA199 diagnosed ICAA together can improved the sensitivity.4 Because the serum GP73 of ICC A was significantly higher than those with healthy persons, sGP73 could replace AFP as a good tumor marker for PHC.5 Nested PCR analysis showed that GP73 was detected in the serum of all patients but the levels were the highest in HCC compared to the other groups. Serum GP73 expression were 45.2% in those with healthy persons, nevertheless GP73 in nested PCR could not be taken a good way in the clinical testing of PHC. |
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