Clinicopathological And Prognostic Implications Of CD105 Expression In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC) is a common and aggressive human tumor in Chian,and is one of the major causes of morbidity and mortality.Even after careful surgical excision,this tumor shows a high percentage of recurrence.Angiogenesis, the formation of new capillaries from a preexisting vasculature,is implicated in tumor development,growth,progression,and metastasis.HCC is a highly vascularized tumor,so angiogenesis is very important for it's biological behaviour.VEGF is a member of a family of structurally related proteins that act as ligands for VEGF receptors.VEGF has effects on the development of angiogenesis and the survival of immature blood vessels.The role of VEGF in tumor angiogenesis has been extensively studied and has been shown to be a key mediator of angiogenesis in cancer.One often-quantified aspect of tumor vasculature is intratumoral microvessel density(MVD).It has been extensively investigated and was found to be a useful prognostic marker in many types of cancers.But some studies reported contrary findings,with nil or negative correlation between MVD and tumor recurrence or survival.A possible reason for these discrepancies may be the different methods used in the evaluation of MVD.Previous reports have demonstrated that MVD-CD34 is a better prognostic marker for HCC than IMVD assessed by many other endothelial markers,such as von Willebrand marker(vWF)and CD31.However,CD34 is a pan-endothelial cell marker,it react not only with proliferating vessels but also with established vessels in the tumor.therefore,it may not be able to reflect the exact angiogenesis activity in a tumor.Endoglin(CD105),a cell membrane glycoprotein,is up-regulated in endothelial cells of denovo blood vessels of various tumors compared to those of normal tissues.It appears to react only with the endothelial cells in the newly formed vessels,and in particular,the immature tumor blood vessels.CD105 has been demonstrated to be a superior angiogenesis marker in breast cancer, malignant melanoma,non-small cell lung cancer,etc.Moreover,studies have suggested that scores of MVD-CD105 might be superior to those of MVD-CD34 in terms of prognostic information in breast cancer,non-small cell lung cancer,colon cancer,etc.But,to date,not much is known about the implications of CD105 expression in HCC.Objective:This study was designed to identify the clinicopathological implications of CD105 expression in HCC and its adjacent non-tumorous liver tissues.And investigate whether CD105 or CD34 could provide better prognostic information in HCC.METHODS:1.Patients and tissue samples:Paraffin blocks of tumor and adjacent nontumorous liver tissues from 100 patients who underwent curative resection of HCC in the Zhu Jiang Hospital Affiliated Southern Medical University from 1999,1 to 2003,12 were used for this study.All tumors were pathologically confirmed to be HCC.Before and after operation,no chemotherapy or other treatment such as chemoembolization was given,no patient was found to have extrahepatic metastasis. All patients were followed-up by monitoring serumα-fetoprotein levels,X-ray and ultrasonography every 3-6 month after resection;for suspicious cases,computed tomography or magnetic resonance imaging were used to verify the recurrence. 2.All clinicopathological data and follow-up information were collected.Time limited for the research is 40 months after surgery.Disease-free survival time was calculated as the period from surgery until recurrence.3.Constructing tissue microarrays.Two array blocks were established with 10×15 cylindrical cores of 1.1 mm in diameter.Two cores of tumor and one core of adjacent nontumorous liver tissues were punched out from each donor block and placed in recipient blocks.4.Immunohistochemical staining and microvessel counting.Serial sections were stained for CD105,CD34 and VEGF respectively.The quantitation of microvessels (MVD-CD105,MVD-CD34) identified by anti-CD105 and anti-CD34 monoclonal antibodies,the semiquantitation of VEGF expression identified by anti-VEGF monoclonal antibody.All data were analyzed in conjunction with the clinicopathological characteristics and disease-free survival time of the patients from whom the specimens were obtained.5.Statistical analysis.Student's t-test was used for comparison of continuous variables between groups.Pearson's correlation coefficient was used to assess the relationship between continuous variables;Spearman's correlation coefficient was used to assess the relationship between categorical variables.Disease-free survival was computed using the Kaplan-Meier method and comparison between groups was done by the logrank test.Statistical analyses were performed using the SPSS(version 11.5) statistical software,P≤0.05 was considered statistically significant.RESULTS:1.Both CD105 and CD34 were expressed in the vascular endothelial cells of HCC tissue of all specimens(100%100/100).However,in the adjacent non-tumorous liver sections,CD105 showed positive microvessel staining in 27 of 100 cases(27%27/100),while CD34 showed positive staining only in 5 cases(5 %,5/100).69 cases of cirrhosis were found in the adjacent non-tumorous liver tissue (69%,69/100),and CD105 showed positive microvessel staining in 24 cases(34.8 %,24/69).The expression intensity of VEGF in tumor sections were asses??in 4 grades:negative in 12 cases(12%,12/100),weak in 15 cases(15%,15/100), moderate in 43 cases(43%,43/100),strong in and 30 cases(30%,30/100).2.There was a significant correlation between the MVD-CD105 and the MVD-CD34 in tumor sections(r=0.575,P＜0.001).The MVD-CD105 in tumor was positively associated with the expression of CD105 in the adjacent non-tumorous liver tissues(r=0.405,P＜0.001).And MVD-CD105 was positively associated with the expression intensity of VEGF(r=0.281,P=0.005),but the correlation was not significant for MVD-CD34(r=0.095,P=0.348).3.Correlation with clinicopathological data demonstrated that both MVD-CD34 (t=-2.362,P=0.020) and MVD-CD105(t=-2.220,P=0.029)in tumor sections were positively associated with the advanced TNM tumor stage.The correlation between microscopic venous invasion and MVD-CD105(t=2.468,P=0.015),but not MVD-CD34(t=0.768,P=0.444).4.When median scores of MVD were used as cut-off points,the patients with higher score of MVD-CD105 had a significantly poorer prognosis than those with lower score in disease-free survival analysis(x~2=16.32,P＜0.001),but no prognostic significance of MVD-CD34 was found(x~2=3.34,P=0.068).In the other hand,positive staining of CD105 in the adjacent non-tumorous liver tissues predicted a poorer disease-free survival(x~2=5.73,P=0.017).CONCLUSION:1.CD105 is an ideal angiogenesis marker in HCC,a higher MVD-CD105 is associated with more advanced stage and more aggressive tumors.2.MVD-CD105 in HCC is a prognostic indicator for recurrence in HCC patients.3.Positive staining of CD105 in the adjacent non-tumorous liver tissues is a prognostic indicator for recurrence in HCC patients.4.CD105 is a possible target for anti-angiogenetic therapy in HCC.