Anatomic Pathology / MICROVESSEL DENSITY AND VEGF IN LIVER CANCER

Assessment of angiogenesis may yield important information for an effective antiangiogenic treatment for hepatocellular carcinoma (HCC) because HCC is characteristically hypervascular. We examined the relationship of microvessel density (MVD), vascular endothelial growth factor (VEGF), and VEGF receptors Flt-1 and Flk-1/KDR in 50 patients with HCC and in 3 hepatoma cell lines. VEGF messenger RNA (mRNA) was overexpressed in 26 tumors (52%), and the 3 VEGF isoforms (121, 165, and 189) were present in high frequencies. Flt-1 mRNA was overexpressed in 34 tumors (68%), with levels significantly increased in HCCs compared with the nontumorous livers. Tumor Flt-1 mRNA significantly correlated with tumor VEGF mRNA levels. Within the group of tumors 8.5 cm or less in diameter, tumors with intrahepatic metastasis in the form of tumor microsatellite formation had significantly higher VEGF mRNA levels. MVD assessed by immunohistochemical analysis with CD34 antibody was inversely related to tumor size. Angiogenesis as assessed by MVD and tumor VEGF expression seems to have a more important role in tumor growth and intrahepatic metastasis in smaller HCCs. The differential up-regulation of Flt-1 suggests that it may have an important role in angiogenesis in HCC. Extensive experimental and clinical data have clearly established that tumor growth is angiogenesis dependent.1 Angiogenesis is essential for solid tumors to grow beyond 1 or 2 mm in diameter. In addition, angiogenesis must occur for metastasis formation and growth. Specific factors must be expressed and the appropriate receptors must be present on the target endothelium to initiate basement membrane degradation, endothelial cell proliferation and migration, and capillary tubule formation. The dynamic process of angiogenesis is regulated by a balance of positive and negative regulators, and the induction and maintenance of an angiogenic response occur through the release of various angiogenic factors from tumor cells. Vascular endothelial growth factor (VEGF) seems to have a central role in the process of tumor-associated angiogenesis.2,3 It has been found to be overexpressed in many tumors, including carcinomas of the colon,4 breast,5,6 endometrium,7 and ovary.8 Four isoforms of the VEGF gene are encoded through alternate exon splicing, and the 121and 165-amino acid forms dominate in tumors. Two of the 4 isoforms are secreted and bind to specific, high-affinity receptor tyrosine kinases, Flt-1 and Flk-1/KDR, which are found almost exclusively on endothelial cells.9 Via interaction with the receptors, the VEGF protein signals the endothelial cells to proliferate, migrate, and form capillary tubules. In contrast, deletion of 1 of the 7 extracellular domains of Flt-1 completely abolishes the binding of VEGF.10 Such a VEGFreceptor system has been demonstrated in the liver.11 Transcripts of Flt-1 and Flk-1/KDR are strongly expressed in rat sinusoidal cells but are barely detectable in hepatocytes, whereas VEGF transcript is weakly produced in hepatocytes and is not expressed in sinusoidal cells. Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2001;116:838-845 839 © American Society of Clinical Pathologists Hepatocellular carcinoma (HCC) is one of the most common malignant neoplasms in the world and is the second most common fatal cancer in Hong Kong and Southeast Asia. It is characteristically a highly vascular tumor, and its diagnosis often relies on imaging techniques making use of its hypervascularity. It has a high risk of spontaneous rupture, leading to massive hemorrhage, and venous permeation and spreading of the tumor by venous route are found commonly. There have been a few reports on VEGF expression in HCC, but the results are conflicting.12-19 Moreover, the expression of the receptors of VEGF in human HCC remains to be elucidated.14 Intratumoral microvessel density (MVD), highlighted by endothelial markers, has been reported to be an independent predictor of prognosis in patients with cancers. However, there are few reports on the relationship between MVD and VEGF expression in HCC. We report our findings on MVD and expression of VEGF and its receptors, Flt-1 and Flk-1/KDR, in human HCC and HCC cell lines and their clinicopathologic significance. Materials and Methods Patients and Tissue Samples Fifty patients with HCC who underwent surgical resections between March 1990 and December 1994 at Queen Mary Hospital, Hong Kong, were studied. None of the patients had received other treatments for HCC, such as percutaneous ethanol injection or transcatheter arterial chemoembolization, before surgical resection. None had apparent distant metastatic disease. Of the patients, 43 were men and 7 were women; age ranged from 33 to 71 years (mean, 52.7 years). Serum hepatitis B surface antigen and anti–hepatitis B surface antigen were assayed by an enzyme immunoassay test (Abbott Laboratories, Chicago, IL). In each case, tumor and nontumorous liver tissues were obtained immediately after resection, snap-frozen in liquid nitrogen, and kept at –70°C. The tumor tissues were obtained randomly from the tumor masses, but necrotic areas were avoided. The nontumorous liver tissues were obtained at sites away from the tumors.