Comparison Of Ki67,CD34and VEGF Expression Between HBV And HCV Related Hepatocellular Carcinoma And Its Clinical Significance

Background&ObjectiveHepatocellular carcinoma (HCC) is the fifth most common type of cancer affecting over one million people annually worldwide, with a mortality rate almost equal to its incidence. In our country, the incidence and death of HCC are nearly54%of the world each year. HCC is the fourth most prevalent tumor type and the second cause of cancer-related death in China which is just less than lung cancer.At present, the incidence of hepatocellular carcinoma is rising and becoming much younger. In recent year, with the intensive research and the progress of treatment technology on HCC, we have a deeper understanding of the occurrence, development and transfer of liver cancer. On the basic of it, some new strategies and methods of diagnosis, treatment and prevention about HCC emerged, which further improved the effect of the HCC treatment, and prolonged the survival of the patients. But the case fatality rate of HCC is still high, and the prevention of HCC still faces severe challenges. The improvement of HCC treatment may mainly rely on the basic studies of HCC, especially some intensive research on the carcinogenesis, recurrence and metastasis of HCC.Human hepatitis virus infection plays an important role in the pathogenesis of HCC. In our country, hepatitis B virus (HBV) is the main reason, but with the application of hepatitis b vaccine and the improvement of the health conditions, HBV infection rate is declined and HBV related hepatocellular carcinoma (B-HCC) also reduced. However, hepatitis C virus related hepatocellular carcinoma (C-HCC) were significantly increased. After the hepatitis infection cause liver damage associated with the hepatocellular proliferation. The cell proliferation will accumulate the random variation of genes and the process of DNA mutation. Finally lead to the occurrence of HCC. Nearly80%of HCC due to HBV or HCV related chronic liver disease (CLD) and the incidence trend in HCC is parallel with the change of HBV and HCV infection. But the clinical features, prognosis and pathogenic mechanism of HBV and HCV related HCC are different. The contrast research of is still less. We will focus on the difference between B-HCC and C-HCC, try to find out some meaningful result between them.The occurrence and development of HCC is the result of the combined action of polygene, multi-channel and multi-stage. The cell cycle regulation disorder and tumor angiogenesis is the two important aspects in the process of HCC. Ki-67is a kind of nuclear antigens, which appears in the cell cycle of S, G, M, and in late Gl nucleus. It is one of the most representative indicators in the process of tumor cell proliferation. The proliferation of tumor cells activity is related to the tumor biological behavior. Therefore, Ki-67can be used as a biomarker to judge the tumor malignant degree and prognosis. Cluster of differentiation34(CD34) is a heavy glycosylated type I transmembrane protein, belongs to the saliva sticky protein family, which express in all small vascular endothelial cells and vascular origin cellsin malignant tumor. In most solid tumors, CD34was negative, but a large number of studies have shown that in HCC tumor tissue, CD34staining was strong, and the high-density area is surrounding the tumor. CD34expression is consistent with the tumor microvascular number and density in HCC. CD34can be used to calculate tumor blood vessels.When the tumor blood vessels is rich, it will be easy to metastasis and invasion and the postoperative recurrence rate will also increase. Vascular endothelial growth factor (VEGF), a signal protein produced by cells that stimulates vasculogenesis and angiogenesis, is one of the strongest angiogenic factors in our body.it firstly binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, and then activate a series of signal transduction pathways, promote the migration, proliferation and survival of the neovascular endothelial cells. Vascular endothelial growth factor receptors(VEGFR) widely exist in the surface of the endothelial cells and cancer cells in the tumor, and its expression level is significantly higher than normal tissue. VEGF expression is higher in and around the HCC or hepatic metastatic carcinoma tissues. The malignant degree of HCC is correlated with the expression of VEGF. So VEGF can be used as a marker of recurrence after the resection of HCC.These molecular markers associated with the occurrence, development and prognosis of HCC, plays an important role in the different pathophysiological mechanisms in HCC formation process. As it is still not clear about Ki67, CD34and VEGF expression in HCC tissue caused by HBV and HCV, this research will adopt the immune histochemical method to detect the expression of Ki-67, CD34and VEGF in B-HCC and C-HCC, and then explore its relationship with the biological behavior of HCC and its difference expression between B-HCC and C-HCC in order to provide valuable judgment index for the two different HCC. Methods1.Eighteen cases of C-HCC samples were obtained from the Department of Hepatobiliary Surgery of Nanfang Hospital during the period of December2003to October2010,and surgically excised.As a comparison,thirty-four cases of B-HCC samples were obtained from the Department of Hepatobiliary Surgery of Nanfang Hospital during the period of March2011to December2012.All samples were fixed within2hours by Neutral formaldehyde, or refrigerated With a ice box,then quickly transferred to the low temperature refrigerator. All tissue samples were made paraffin section. And The diagnosis of HCC was confirmed, which checked by two pathology with random double blind method.2. The clinical pathological parameters of B-HCC and C-HCC are collected in order to observe the disease-free survival in the two groups, and analyze the difference.3.1mmunohistochemical staining was employed in sections from all tumor tissues of HCC patients to determine the expression pattern of Ki-67. Correlation between the expression profile of Ki-67and the clinicopathological features of patients was analyzed. And analyzed the correlation of difference between B-HCC and C-HCC in the expression of Ki-67.4.1mmunohistochemical staining was employed in sections from all tumor tissues of HCC patients to determine the expression pattern of CD34. Correlation between the expression profile of CD34and the clinicopathological features of patients was analyzed. And analyzed the correlation of difference between B-HCC and C-HCC in the expression of CD34.5.1mmunohistochemical staining Was employed in sections from all tumor tissues of HCC patients to determine the expression pattern of VEGF. Correlation between the expression profile of VEGF and the clinicopathological features of patients was analyzed. And analyzed the correlation of difference between B-HCC and C-HCC in the expression of VEGF.6.Statistical analysis:The data were analyzed with SPSS software(SPSS version13.0; SPSS, Chicago, IL, USA). Student’s t test was used to analyze measurement data except the preoperative titer of AFP. Rank sum test (wilcoxon test) was used to analyze the preoperative titer of AFP.The comparison of the ratio in two samples was analyzed by Pearson Chi-Square test. Ranked data in two independent samples were analyzed by Mann-Whitney U test. The correlation of the variables were analyzed by Pearson correlation analysis method. Survival rates were calculated according to the Kaplan-Meier method, and differences were evaluated using the log-rank test. The Cox proportional hazards regression model was used to assess the hazard ratio (HR) and identify factors that independently predict survival.Differences were considered significant if the p value from a two-tailed test was<0.05.Results1. We first analyzed the clinicopathological features of patients with B-HCC and with C-HCC,showed that discrepancy of the clinicopathological features in B-HCC and C-HCC was not significantly associated with gender, the time From attacked hepatitis to found liver cancer,time of operation,the preoperative titer of AFP,the number of tumor,tumor location,liver cirrhosis,histodiagnosis, differentiation status, distant metastasis.However elevated mean age(P<0.001),mean length of stay(LOS)(P<0.001),mean postoperative length of stay(PLOS)(p=0.013), Preoperative child-pugh grades(p=0.001),tumor size(0.006)was strongly correlated with C-HCC compare with B-HCC. Median disease-free survival time for B-HCC and C-HCC were thirteen months and sixteen point five months.1-,2-disease-free survival rates were56.3%,32.0%for B-HCC respectively and75.0%,75.0%respectively for C-HCC,respectively. The result suggested C-HCC patients had a longer disease-free survival time and better outcome than B-HCC patients. The Cox proportional hazards regression model analysis showed the type of hepatic virus was a dependent of influential factors of postoperative recurrence. The postoperative death risk of HCC patients with hepatitis B virus was2.35times higher than that of the HCC patients with hepatitis C virus infection(P=0.108).2.Fifty-three samples of HCC were tested by immunohistochemical staining with Ki-67,CD34and VEGF. showed that the positive rate of Ki-67in B-HCC and C-HCC samples were79.4%(27/34) and44.4%(8/18), respectively(x2=6.540, P=0.011). VEGF was strongly elevated expression in C-HCC(U=179.5, P=0.006). However,expression of CD-34in B-HCC and C-HCC was not significant(x2=0.225,P=0.065).3.Expression of Ki-67in HCC tumor tissue was insignificantly associated with gender, age,the number of tumor, tumor size, tumor location, or distant metastasis However, elevated Ki-67expression was strongly correlated with the preoperative child-pugh grades (p=0.037), differentiation status (p<0.001), and liver cirrhosis (p=0.014)4.Expression of CD34in HCC tumor tissue was insignificantly associated with all the clinicopathological features of patients we collected.5.Expression of VEGF in HCC tumor tissue was not significantly associated with age, tumor size, tumor location, distant metastasis, the preoperative child-pugh grades,differentiation status, or liver cirrhosis. However, elevated VEGF expression was strongly correlated with gender (p=0.036) and the number of tumor (p=0.028).ConclusionsIn conclusion, our results highlight the fact that the clinic pathological profiles of B-HCC and C-HCC may be different. C-HCC correlates with more serious hepatic injury, longer LOS and PLOS. On the contrary, B-HCC correlates with young people, better liver function, shorter LOS and PLOS. It means that patients with B-HCC recovered earlier than C-HCC after operation. The causes of the result may be attributed to the patient of B-HCC is younger and better liver function compared with C-HCC. But the HCC patients with HBV, metastasis, portal vein tumor thrombus (PVTT) before the surgery have a higher postoperative recurrence rate (p<0.01). Then, the immunohistochemical staining test reveals that the positive rate of Ki-67in B-HCC samples were higher than C-HCC, the positive rate of VEGF in C-HCC samples were higher than B-HCC, but the positive rate of CD34has no significant difference between them. In HCC occurrence and development process, angiogenesis may be especially linked to HCV infection, and may play a more important role in tumor progression in C-HCC than in B-HCC. Therefore, it is very important for C-HCC to research some targeted therapy of angiogenesis. In B-HCC occurrence and development process, the disorder of the cell cycle may be especially linked to HBV infection, and may play a more important role in tumor progression in B-HCC than C-HCC. Further clinical drug research in this field will be conducive to control the B-HCC occurrence and development better.