Study On The Correlation Between Angiogenesis And Intrahepatic Metastasis And PVTT In Hepatocellular Carcinoma

Part I : Distribution of Micrometastases surrounding SolitaryHepatocellular CarcinomaObjective To study the intrahepatic micrometastases distribution surrounding solitary hepatocellular carcinoma (HCC). Methods Fifty-four solitary HCC patients without tumor thrombus in the main portal vein or its first branch were collected. The specimens from them with sufficient surgical margins were made into multiple sections which were stained with HE and antibodies against alpha-fetoprotein(AFP) or melanoma-associated antigen(MAGE) to detect intrahepatic metastases. Results Three hundred and two micrometastases were found in 59.3% (32/54) HCC patients, and 59.4% (205/354) were form of microscopic tumor emboli in portal vein. Fourteen patients without hepatic vein microthrombi had portal vein microscopic tumor emboli. The spread distances were up to 4.7cm with the P₅₀ being 0.6cm, P₉₅ 2.5cm. With univariate analysis, preoperative serum AFP, the size, capsule status and Edmondson grade of primary tumor were found to be significant factors associating with intrahepatic micrometastases (P<0.05 or 0.01) . With multivariate analysis, the capsule status, serum AFP and Edmondson grade of primary tumor independently affected intrahepatic micrometastases (P< 0.01) . Among HE stain negative sections the micrometastases positive rate was 11.1% against AFP, 7.5% against MAGE and 18.5% against at least one of them. With univariate analysis, the 1-year recurrence free and overall survival rates in intrahepatic micrometastasis positive patients being 52.5%,62.5% respectively were higher than those in negative patients being 77.3% , 81.8% (P< 0.01);With multivariate analysis, the intrahepatic micrometastasis status was not the independent factor affecting the prognosis(P>0.05). Conclusions (1) Intrahepatic micrometastases are common in HCC with the main form of microscopic tumor emboli in portal vein. (2) The surgical margin of 2.5cm is ample for hepactomy with the adjustment according to the tumor's clinicopathological features. Most of patients need postoperative adjuvant therapy. (3) Immunohistochemistrical detection of intrahepatic micrometastases against AFP and MAGE is feasible in HCC patients. (4) Intrahepatic micrometastasis status is related to the prognosis but is not an independent factor.Part II: Expression and significant of four vascular specific growthfactors in hepatocellular carcinoma.Objective To find out the correlation between the expression of vascular endothelial growth factor(VEGF), angiopoietin 2 (Ang2), ephrinB2 and endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and the biological behaviors in human hepatocellular carcinoma (HCC). Methods Expression of VEGF, Ang2, ephrinB2 and EG-VEGF was detected by RT-PCR in 54 cases with HCC. The samples were also stained with CD34 by immunohistochemistry. Quantitation of microvessel density (MVD) and semi-quantitation of VEGF, Ang2, ephrinB2 and EG-VEGF expression were analyzed combining with clinicopathological features. In SMMC7721 cell line the expression level of each factor was dedected under hypoxic condition achieved by using 100umol/L cobalt chloride. Results (1) The expression levels of VEGF165, VEGF189, Ang2 and EG-VEGF mRNA were significantly higher in HCC than those in the normal liver tissue. (2) With univariant analysis MVD was significantly correlated with VEGF165, VEGF189, Ang2 and EG-VEGF mRNA with the rs being 0.789, 0.528, 0.645 and 0.309 respectively (PO.05 or 0.01). With multivariant analysis VEGF165, Ang2 and EG-VEGF mRNA independently affected MVD (PO.05 or0.01). (3) Positive serum AFP, poor differentiation, large diameter, not intact capsule and mtrahepatic metastasis were significantly related to the expression level of VEGF165mRNA, as well as poor differentiation and intrahepatic metastasis being to that of VEGF189mRNA;positive serum AFP, poor differentiation and intrahepatic metastasis being to that of ephrinB2 mRNA;positive serum AFP, poor differentiation, large diameter, not intact capsule and intrahepatic metastasis being to that of Ang2 mRNA;poor differentiation being to that of EG-VEGF mRNA (P<0.05 or 0.01) . (4) The expression of Ang2 mRNA positively associated with those of VEGF165, VEGF189 and ephrinB2 mRNA with rs being 0.504, 0.278, 0.402 respectively (P<0.05 or 0.01);and so did the expression ofVEGF165 with that of VEGF189mRNA (r,=0.691, P<0.01) . (5) With univariant analysis, the distance of intrahepatic spread of cancer was significantly correlated with VEGF165, VEGF189, and Ang2 mRNA (PO.05 or 0.01). With multivariant analysis, only tumor size and MVD were the independent factors(P<0.05 or 0.01). (6) In HCC VEGF165, VEGF189, and Ang2 mRNA associated with intra portal vein metastases(P<0.05 or 0.01), and so did ephrinB2mRNA with intra hepatic vein metastases (PO.05) , but EG-VEGF mRNA associated with neither of them(P>0.05). (7) With univariant analysis, the overall survival rate in VEGF 165 mRNA high level group was significantly higher than that in VEGF 165 mRNA lower level group ( *2=3.945, P=0.042). With multivariant analysis, VEGF 165 mRNA was not the independent factor to prognosis(P>0.05). (8) After Oh, 8h and 16h of hypoxic condition, VEGF165mRNA was upregulated (F=218.O, PO.01), and ephrinB2 mRNA was downregulated (F=47.28, PO.01) . Hypoxia did not effect the expression of Ang2mRNA significantly (F=0.912, P>0.05) .Conclusions VEGF, Ang2 and EG-VEGF mRNA may play a role in HCC angiogenesis and carcinogenesis. The expression of VEGF, ephrinB2, Ang2 and EG-VEGF mRNA is correlated with thebiological behaviors in HCC. VEGF 165 affect the prognosis but is not independent factor. Hypoxia stimulates the expression of VEGF mRNA and inhibits the expression of ephrinB2mRNA.Part m rEffect of four vascular specific growth factors on carcionogenesis and portal vein tumor thrombus formation in human hepatocellular carcinomaObjective To detect the correlation between the expression of vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang2), ephrinB2 and endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and carcionogenesis and portal vein tumor thrombus (PVTT) formation in human hepatocellular carcinoma (HCC). Methods Expression of VEGF, Ang2, ephrinB2 and EG-VEGF was detected by RT-PCR in 54 cases HCC without PVTT (group A), 9 cases HCC with PVTT (group B), 10 normal liver tissues (group D) and 10 cirrhosis tissues (group C). The samples were also stained with CD34 by immunohistochemistry. Quantitation of microvessel density (MVD) and semi-quantitation of VEGF, Ang2, ephrinB2 and EG-VEGF expression were analyzed to find the relations. Results The MVD was 146.69±77.79, 214.07±54.41, 32.85±8.49 and 34.83±8.29 in group A-D respectively with significant difference (PO.01). The MVD in group A was higher than that in group C (PO.01) , but lower than that in group B (PO.01). The expression levels of VEGF165, VEGF189, Ang2 and EG-VEGF mRNA were significant difference among the groups. The expression levels of VEGF 165, Ang2 and EG-VEGF mRNA in group A were all higher than those in group C, but lowerthan those in group B (P <0.05 or 0.01) . The MVD was significantly correlated with VEGF165, VEGF189, Ang2 and EG-VEGF mRNA with r, being 0.764, 0.510, 0.640 and 0.366 respectively in HCC (P< 0.01). Conclusions VEGF, Ang2 and EG-VEGF mRNA may play a role in HCC angiogenesis and carcinogenesis. They can promote PVTT formation in HCC by modulating angiogenesis.