Chinese Medical Journal 2003;116(5):772-776
Vascular endothelial growth factor expression in serum of patients with hepatocellular carcinoma

ZHAO Jianjun 赵建军,  HU Jingqun 胡敬群,  CAI Jianqiang 蔡建强,  YANG Xiaojie 杨晓洁,  YANG Zhihua 杨治华

ZHAO Jianjun 赵建军 (Department of Abdominal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China)

HU Jingqun 胡敬群 (Department of Abdominal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China)

CAI Jianqiang 蔡建强 (Department of Abdominal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China)

YANG Xiaojie 杨晓洁 (Department of Abdominal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China)

YANG Zhihua 杨治华 (Department of Abdominal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China)

Correspondence to:, (Tel: . Fax:. E-mail:)
Keywords
liver neoplasms;carcinoma;hepatocellular;neoplasm metastasis;vascular endothelial
Abstract
Objectives To determine the pre-therapeutic serum level of vascular endothelial growth factor (VEGF) in patients with hepatocellular carcinoma (HCC) and to elucidate the relation between the serum level and clinical characteristics and metastasis of HCC.
Methods One-hundred and fifteen HCC patients, 40 patients with benign liver lesions, and 30 healthy control subjects were included in this study. The serum VEGF level was measured with the quantitative sandwich enzyme linked immunosorbent assay (ELISA, R&D systems).
Results The serum VEGF levels in the HCC group (465.62±336.24 pg/ml) was significantly elevated as compared with those in patients with benign liver lesions (159.54±120.58 pg/ml) and those in normal controls (123.53±51.84 pg/ml). The VEGF levels were not significantly different between the patients with benign liver lesions and the normal controls. The serum VEGF concentration showed a positive rate of 77.4%, 25%, and 3.3% in the HCC patients, benign liver lesion patients and normal controls, respectively. In the 115 HCC patients, the serum VEGF levels in patients with portal vein (PV) emboli (n=26, 582.76±441.89 pg/ml), with metastasis (n=43, 548.29±438.57 pg/ml) or with large HCC lesions (≥5 cm in diameter) (n=69, 554.43±369.99 pg/ml) were significantly higher than those without PV-emboli (n=89, 431.39±292.84 pg/ml), without metastasis (n=72, 416.24±247.27 pg/ml) or with small HCC lesions (n=42, 328.67±227.47 pg/ml). The serum VEGF levels in stage Ⅰ, Ⅱ, Ⅲ, Ⅳa and Ⅳb HCC patients were 340.6 pg/ml, 451.55±307.84 pg/ml, 397.44±257.18 pg/ml, 486.10±397.73 pg/ml and 647.93±344.56 pg/ml, respectively.
Conclusion The pre-therapeutic serum VEGF levels in HCC patients appear to reflect the disease’s potential activity of vascular invasion and metastasis.

Vascular endothelial growth factor (VEGF), a glycoprotein which can combine with heparin and has a relative molecular weight of 34?000-46?000, is one of the most potent known angiogenesis factors.[1] Most tumor cells can secrete VEGF, which can induce angiogenesis in tumor tissues, increase vascular permeability, assist the entry of tumor cells into the vascular system and facilitate invasion and metastasis of tumor cells.[2] More and more studies show that there is a very close relationship between the high expression of VEGF and tumor metastasis, recurrence and poor prognosis.[3,4] We studied the expression of VEGF in patients with hepatocellular carcinoma (HCC) and benign liver diseases and in healthy people by the enzyme linked immunosorbent assay (ELISA) method in order to prospectively elucidate the relationship between VEGF level and clinical pathological characteristics of HCC as well as the recurrence and metastasis of tumors. Our aim was to select a more easily-tested, more sensitive and specific biological marker that can early reflect the metastasis and recurrence of HCC, thus providing a basis for the integrated treatment of HCC.

METHODS

Immunological reagents
Enzyme-labeling kit (96 wells) with anti-human VEGF monoclonal antibody prepared from mice (R&D System, USA).

Experimental samples
HCC serum samples and full clinical records were obtained from 115 patients with confirmed clinical diagnosis of primary HCC registered from August 1998 to January 2000. Peripheral venous blood was collected from in patients, and the separated serum samples were stored at -70℃ before examination. In the patients, the ratio of male to female was 101∶14, and the average age was 54 years (range: 28-84 years. The percentages of patients with infections of HBV and HCV and cirrhosis were 96.5% (111/115), 8.7% (10/115) and 80.0% (92/115), respectively.

Forty serum samples were taken from patients with benign liver diseases, including 11 patients with benign liver tumors at our hospital and 29 patients with cirrhosis at the Beijing Second Infectious Diseases Hospital (BSIDH). The ratio of male to female was 28∶12 and the average age was 49 years (range: 20-70 years). The percentages of patients with infections of HBV and HCV and cirrhosis were 85.0% (34/40), 7.5% (3/40) and 77.5% (31/40), respectively. Thirty normal serum samples from healthy volunteers were taken as the control. The ratio of male to female was 22∶8, and the average age was 26 years (range: 20-32 years.

Data analysis
According to documentation of the clinical and pathological conditions and prognosis, 17 non-repetitive criteria were selected for classification, including gender, age, HBV infection, HCV infection, portal vein (PV) emboli before operation, tumor size, tumor position, liver capsule invasion, alpha-fetoprotein (AFP) level before operation, cirrhosis, hypersplenia, Child’s class, lymph node metastasis, histological grading, ascites, metastasis or not, and TNM stage. The data were processed with SPSS statistics software, and the χ2 test was used for enumerative data to calculate sensitivity and specificity. Student’s t test and analysis of variance were used for the measurement of data.

RESULTS

Relationship between VEGF and pathological characteristics of HCC
Expression of VEGF in liver diseases
Significant differences (P=0.0001) in VEGF expression were found between the HCC group and the benign liver disease group, and between the HCC group and the normal control group. No significant difference (P=0.995) was found between 11 patients with benign liver tumor and 29 patients with cirrhosis in the benign liver disease group. The difference between the benign liver disease group and the normal control group was not significant (P=0.594, Table 1 ).


Determination of positive VEGF rates
According to the mean level of VEGF in normal human serum (123.53 pg/ml), the normal level of VEGF was calculated as 227.20 pg/ml. Taking this as the standard, the VEGF expression positive rates for HCC patients, benign liver disease patients and normal controls were 77.4% (89/115), 25% (10/40), and 3.3% (1/30), respectively, ( Table 2 ). Therefore, the sensitivity of HCC was 77.4%, and the specificity was 96.7%. Combined with imaging analysis, VEGF was used as an accessory marker in the differential diagnosis of benign and malignant liver tumors.

Relationship between VEGF and pathological characteristics of HCC
Statistically, no correlation was observed between the expression of VEGF with clinical pathological criteria such as gender, age, HBV infection, HCV infection, cirrhosis, hypersplenia, Child’s class, tumor position, histological grading, AFP, ascites, lymph node metastasis, liver function (ALT and AST value, normal level <40 pg/μl), and the liver capsule was invaded.

Relationship between VEGF and metastasis and recurrence of HCC
Relationship between VEGF and HCC with PV tumor emboli, tumor size and TNM stage
PV emboli and tumor size were determined by preoperative computerized (CT), MRI, hepatic arteriography or ultrasonography. The HCC stage was classified according to the international TNM standard (1987).

Relationship between VEGF and PV tumor emboli
Of the 115 HCC patients, 26 were found with PV tumor emboli and 89 without PV tumor emboli. There was a significant difference in serum VEGF level (P=0.016) between the two groups. Of the 89 patients without PV tumor emboli, intrahepatic and remote metastasis occurred in 30 patients. Of the 26 patients with PV emboli, metastasis within liver occurred in 13 patients whose average VEGF level (656.71 pg/ml) was significantly higher than that of the other 13 patients (441.24 pg/ml) with PV tumor emboli but no metastasis (P=0.002, Table 3 ). HCC metastasis did occur unless tumor cells invaded the vessels and created emboli. In this study, the levels of VEGF in the patients without PV tumor emboli were significantly lower than that those with PV tumor emboli. The level of VEGF in patients with PV emboli but no metastasis was significantly lower than that in the patients with PV tumor emboli and metastasis. This finding suggested that high-level expression of VEGF predicts a higher level of vessel invasiveness and higher metastasis tendency of the tumor.

Relationship between VEGF and tumor size
According to clinical standards, 42 patients were classified as having small HCC (<5 cm) with a VEGF level of 328.67±227.47 pg/ml. Sixty-nine patients were classified as having large HCC (≥5 cm), with a VEGF level of 554.43±369.99 pg/ml. In the two groups, the serum level of VEGF expression was significantly different (P=0.001), and there was a positive correlation between the serum level of VEGF and tumor size ( Table 3 ). In the 42 patients with small HCC, the positive rate of VEGF was 64.3% (27/42), and the metastasis rate was 30.9% (13/42). In the 69 patients with large HCC, the positive rate of VEGF was 85.5% (59/69), and the metastasis rate was 40.6% (28/69).

Relationship between VEGF and TNM stage of HCC
The serum VEGF level in HCC patients increased with the progress of the disease: at stage Ⅰ (340.60 pg/ml), stage Ⅱ (451.55±307.84 pg/ml), stage Ⅲ (397.44±257.18 pg/ml), stage Ⅳa (486.10±397.73 pg/ml), and Ⅳb (647.93±344.56 pg/ml). The VEGF level in the stage Ⅳb group with remote metastasis was significantly higher than that in groups of other stages. But there was no significant difference in VEGF level among patients in stages Ⅰ, Ⅱ and Ⅲ.

Relationship between VEGF expression and HCC metastasis
In the 115 patients HCC, the difference was significant between the serum VEGF level (548.291±438.57 pg/ml) of the 43 metastasis patients and the serum VEGF level (416.24±247.27 pg/ml) of the 72 patients without metastasis (P=0.001). The difference was significant between the 36 patients of whom metastasis was found with a serum VEGF level of 560.86±467.35 pg/ml and the 79 patients, of whom metastasis was not found with a serum VEGF level of 422.22±247.31 pg/ml, (P=0.001). The VEGF positive expression rates in the metastasis group and the group without metastasis were 69.4% and 81.0%, respectively. There was no significant difference between the two groups.

DISCUSSION

Metastasis and recurrence are important characteristics of malignant tumors. Metastasis of tumor cells begins at the early stage of primary neoplasia generation. Remote metastasis of tumor cells depends on their invasiveness, the ability to escape immune surveillance and its adaptability in the new microenvironment. The gene variation during neoplasia development allows tumor cells to elaborate their potential abilities of invasion and metastasis.[5,6]

After HCC radical excision, incidences of intrahepatic metastasis and recurrence are increased to 45.2%-60.0% within five years. Most recurrence happens in the next two years. The high rates are noted even in small HCC. There are many causes of recurrence and metastasis. Many biological factors such as telomerase activity, cyclin A,[7] ALB-mRNA, AFP-mRNM, MMP-9[8] are related to the invasion and metastasis of HCC. AFP is a sensitive and specific biological marker, but it cannot be used to predict the metastasis and recurrence. VEGF is one of the most potent cytokines that can facilitate vascular endothelial growth. It can accelerate vascular endothelial growth and increase vessel permebility. Sufficient oxygen and nutrients are needed during tumor growth, and therefore the potential ability of angiogenesis is related to tumor growth. Angiogenesis is regulated by many factors. Although accelerated angiogenesis is a normal physiological reaction to wounds, inflammation, menstruation and embryogenesis, it is a pathological process for diabetic retinopathy, rheumatoid arthritis and solid tumors.[9] Angiogenesis is an important link in the process of the growth, invasion, and metastasis of tumors. The vessels in tumors provide not only nutrients and the route to excrete metabolism products, but a way for tumor cells to disseminate. It has been reported that tumors need the capillary network to provide nutrients when they grow to a size of 2-3 mm.[9] It has also been reported that VEGF expression can be found in many animal and human tumor cells, such as lung adenocarcinoma, bladder carcinoma, fibrocarcinoma, neuroglioma, gastric carcinoma, colon cancer, and pancreatic cancer.[10] VEGF expression is also a predictive index for liver metastasis of colon cancer, gastric cancer, and other tumors.

Jinno et al[11] found that serum VEGF levels increased gradually along with the disease progress in patients with hepatitis, cirrhosis and HCC, as well as in normal people. A significant difference was found between the HCC and other patient groups, but not among hepatitis patients, cirrhosis patients and normal controls. The plasma VEGF level is an effective marker for the determination of hepatic metastasis of tumors because of its specificity, sensitivity and precision satisfy clinical requirements. In our study, the serum VEGF concentration in HCC patients was significantly different from that in patients with benign hepatic diseases, and health controls (P=0.0001). The examination sensitivity was 77.4%, and its specificity was 96.7%. No difference was observed between the benign hepatic diseases group and the controls (P=0.594) and between benign hepatic disease patients and cirrhosis patients (P=0.995). Our results are similar to those reported. We also found that the serum VEGF level is closely related to the recurrence and metastasis of HCC, and that there is a significant difference between the recurrence group and the group without recurrence (P=0.001). It is suggested that the serum VEGF level of HCC patients before treatment can serve as an effective predictor for the recurrence and metastasis of HCC.

In addition, serum VEGF expression level is related to TNM stage (P=0.025). The serum VEGF level in HCC patients increases along with the progress of the disease from stage Ⅰ to Ⅳ. Although there is no significant difference in VEGF level in stage Ⅰ, stage Ⅱ and stage Ⅲ patients, the VEGF level increases significantly in stage Ⅳa with remote metastasis compared with that in other stages. The result shows that the later the stage, the higher the possibility of metastasis, and the higher the level of VEGF expression. Serum VEGF level is also closely related to the vessel’s degree of invasion (composite PV emboli) and tumor size. VEGF expression is higher in HCC patients with PV emboli or those with large HCC, which is in accordance with the results reported in the literature.[10]

In conclusion, the serum VEGF level in HCC patients is directly correlated with the metastasis and recurrence of tumors and increases gradually with the progression of HCC. The earlier the HCC metastasize, the higher the VEGF expression. Therefore, the pre-therapeutic serum VEGF level in HCC patients appears to reflect the disease’s potential activity of vascular invasion and metastasis.

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