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Original article
Prognostic significance and potential therapeutic target of VEGFR2 in hepatocellular carcinoma
  1. Jianfei Huang1,2,
  2. Xialing Zhang1,
  3. Qi Tang1,
  4. Feng Zhang1,
  5. Yuhua Li1,
  6. Zhenqing Feng1,
  7. Jin Zhu1,3
  1. 1Key Laboratory of Antibody Technique of Ministry of Health, Department of Pathology, Nanjing Medical University, Nanjing, China
  2. 2Surgical Comprehensive Laboratory, Department of Pathology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China
  3. 3Huadong Medical Institute of Biotechniques, Nanjing, Jiangsu, China
  1. Correspondence to Professor Zhenqing Feng, Cancer Center and Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu, China; fengzhenqing{at}njmu.edu.cn; and Jin Zhu Ph.D, Department of Pathology, Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University. Huadong Medical Institute of Biotechniques, Nanjing, Jiangsu, 210002, China; zjsimmons{at}yahoo.com.cn

Abstract

Background Vascular endothelial growth factor receptor 2 (VEGFR2) has been suggested to play an important role in solid tumours. Although several reports have shown the relationship between VEGFR2 expression and hepatocellular carcinoma (HCC), the expression pattern of VEGFR2 in HCC parenchyma or stroma, as well as the relationship between VEGFR2 expression and clinicopathological characteristics in HCC, are yet to be satisfactorily defined.

Methods One-step real-time PCR, western blotting and immunohistochemistry were used to characterise the expression of VEGFR2 in HCC using a self-made anti-VEGFR2 monoclonal antibody (A8H1).

Results Expression of VEGFR2 in HCC cells was higher than in hepatic cells (p<0.001). Comparison of clinicopathological characteristics and immunohistochemistry by χ2 test analysis showed that the high expression of VEGFR2 in HCC was related to large tumour diameter (p=0.012), poor differentiation (p=0.007), high serum α-fetoprotein (p=0.029), multifocal gross classification (p=0.007), and less than 5 years' survival (p=0.029). Kaplan–Meier survival and Cox regression analyses showed that high VEGFR2 expression (p=0.009) and stage grouping with TNM classification (p=0.004) were independent prognotic factors.

Conclusions The efficacy of A8H1 in immunohistochemistry using HCC tissues was confirmed. There was a correlation of high VEGFR2 expression with prognostic significance in HCC. Additionally, the self-made anti-VEGFR2 monoclonal antibody could be used for future anti-HCC-targeted therapy research.

  • VEGFR2
  • HCC
  • monoclonal antibody
  • immunohistochemistry
  • prognostic significance
  • therapeutic target
  • antibodies
  • cancer research
  • immunohistochemistry
  • liver
  • tumour markers

https://doi.org/10.1136/jcp.2010.085142

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    Introduction

    Liver cancer, especially hepatocellular carcinoma (HCC), is the sixth most common neoplasm worldwide and the third most prevalent cause of cancer-related mortality globally.1 According to the statistics of the WHO in 2006, these relate to around 662 000 deaths per year. Most patients who develop liver cancer will die within 1 year.2 HCC with cirrhosis is most often attributable to chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV); in the Asia-Pacific region HCC is a major public health problem.2 3 China alone accounts for 55% of cases of HCC worldwide,1 and the overall prevalence of HCC for 2000–05 has been reported as 26–32 per 100 000 persons.2 The incidence of HCC in Qidong of Jiangsu Province of China is among the highest in the world4 5; almost all cases of HCC in this area are related to HBV infection.4 6 7

    The treatment of HCC is multidisciplinary and involves pathologists, surgeons, oncologists and interventional radiologists. However, HCC diagnosis most usually results in a poor patient prognosis.8 9 The early manifestation of HCC is silent; it is slow growing, and shows few characteristic symptoms. Most patients present with an advanced tumour stage, often with bilobar tumours, portal vein invasion or metastasis10 11; traditional treatment is less effective10 12 at the time of diagnosis. Although there have been advances in surgery, liver transplantation and systemic chemotherapy, the recovery rate has increased little over recent decades.2 8 13

    Vascular endothelial growth factor receptor 2 (VEGFR2) has been suggested to play an important role in the formation of new blood vessels in tumours.14 15 The VEGF/VEGFR pathway is considered to be one of the most important regulators of angiogenesis and a key target in anti-cancer treatment.16–18 Inhibition of angiogenesis has already led to some therapeutic efficacy in animal tumour models.10 19–22 Therefore, VEGFR2 is always considered as highly expressed in tumour cells and tumorous blood vessels when compared to normal tissues.

    Although the relationship between VEGFR2 expression in HCC and clinicopathological characteristics has been studied,14 23–29 the high expression of VEGFR2 protein in HCC tissue and peritumoral tissue is still controversial. It is hoped that the controversy will be resolved with our proposed method.

    We determined the expression levels of VEGFR2 mRNA in four HCC cell lines and a human liver cell line using one step quantitative real-time PCR. We also characterised the expression of VEGFR2 protein in those cell lines by western bolt with a self-made murine anti-VEGFR2 monoclonal antibody (named A8H1).30 We examined the relationship between VEGFR2 expression and clinicopathological significance in HCC by immunohistochemistry with A8H1. Therefore, the aim of this study was also to clarify the potential feasibility of the anti-VEGFR2 monoclonal antibody in anti-HCC treatment. Further studies on the chimeric murine–human fragment from murine monoclonal antibody, for targeted therapy of HCC will be summarised in our next study.

    Methods

    A panel of formalin-fixed paraffin-embedded HCC tissues and tumour-adjacent (with non-malignant tissues) undergoing surgical therapy was obtained from the First Affiliated Hospital of Nanjing Medical University, between 2003 and 2006, consisting of 121 HCC samples. The mean age of patients at the time of surgery was 52 years (range 22–80 years). Clinical data (including age, tumour size, serum level of α-fetoprotein (AFP) and 5 years' follow-up survival records) and other information were obtained from each patient's records. All tumours were classified according to WHO.4 Before surgical therapy, none of the patients had received neoadjuvant chemotherapy, radiation therapy or immunotherapy. Survival was calculated from the date of surgery until the date of death or last follow-up. Ethics approval to perform this study was obtained from the Human Research Ethics Committee of the Nanjing Medical University.

    HCC cell lines (BEL-7402, SMMC-7721, HepG2 and SK-HEP-1), human liver cell line LO-2, and human umbilical vein-derived endothelial cell line (HUVEC) were obtained from the cell bank of the Chinese Academy of Science in Shanghai, China. They were cultured in DMEM medium (Gibco, Invitrogen, Carlsbad, California, USA) containing 10% fetal calf serum (FCS) at 37°C, 5% CO2.

    Total RNA from the six cell lines mentioned above was extracted using the Trizol reagent (Invitrogen). To avoid possible genomic DNA contamination, all RNA samples were treated with RNase-free DNase (Promega, Madison, Wisconsin, USA). A one-step quantitative real-time reverse transcription–PCR (one-step qPCR) was performed according to the standard protocol using the Real Time PCR (Bio-Rad IQ5, Hercules, CA, USA) with SensiMixTM One-Step Kit (Quantace, UK). The curve of the VEGFR2 mRNA level was shown for the qPCR results. VEGFR2 specific oligonucleotide primers, forward 5′-TGTCCTGATGTGATATGTCTGAG-3′ and reverse 5′-GGGTGAGAGTGGGTTGGG-3′, were designed to give a 125 bp PCR product. Amplification conditions consisted of 30 min at 42°C for reverse transcription and 2 min at 94°C for Taq activation followed by 40 cycles of 94°C for 20 s, 55°C for 20 s, and elongation at 72°C for 30 s.

    To collect total cellular protein from HCC cell lines and human liver cell line for western blot (WB), cells maintained in a culture flask were washed with phosphate-buffered saline (PBS) and lysed with RIPA Lysis Buffer (Santa Cruz Biotechnology, USA) on ice, separately. Then the cell lysates were centrifuged and the total cellular protein in centrifuge supernatant was collected. Total cellular protein was subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was probed with A8H1 and β-actin (Santa Cruz Biotechnology), followed by a secondary horseradish peroxidise (HRP) conjugated goat anti-mouse antibody (Dako, USA). The reaction was detected by ECL WB Substrate (Pierce, USA) in accordance with the manufacturer's instructions. The expression of VEGFR2 protein level was measured by Gel Pro Analyser Software (Media Cybernetics, USA).

    All liver cancer tissues were formalin fixed and paraffin embedded. All serial sections were cut at 4 μm on a manual rotary microtome (Leica RM2235, Germany). The sections for H&E staining were stained by Automated Slide Stainer (Leica ST5020, Germany) and coverslipped with Robotic Coverslipper (Leica CV5030, Germany). Other sections for immunohistochemistry (IHC), using Autostainer Universal Staining System (LabVision, USA), were deparaffinised, and peroxidase was quenched with methanol and H2O2 3% for 15 min. For antigen retrieval, those sections were boiled under pressure in citrate buffer, pH 6.0, for 3 min. Non-specific binding was blocked by 5% goat serum in PBS for 15 min, and the tissues were incubated with A8H1 containing 1% bovine serum albumin for 1 h. Following washing with PBS, sections were incubated with HRP-conjugated goat anti-human antibody (Dako-Cytomation, USA) for 15 min and washed. The colour was developed by 15 min incubation with DAB solution (Kem-En-Tec Diagnostics, Denmark) and sections were weakly counterstained with haematoxylin. Negative controls were included by replacement of the primary antibody with PBS.

    Quantisation of immunostaining was as follows. All sections used for quantisation were immunostained at the same time under the same conditions. The evaluation of immunostaining of VRGFR2 was made blind to us by an independent observation simultaneous design. Percentages of VRGFR2 positive cells were scored into four categories according to staining: 0 for 0%, 1 for 1–33%, 2 for 34–66%, and 3 for 67–100%. The VRGFR2 staining intensities were also scored into four grades: 0, 1, 2, and 3. The sum of the percentages and intensity scores was used as the final VRGFR2 staining score as described earlier,31 and has been defined as follows: 0 to 2, low expression; 3 to 6, high expression.

    The following clinical data were evaluated: gender, age, tumour size, preoperative serum AFP level and other clinicopathological information, as well as outcome. The relationship between VRGFR2 expression and relevant patient and tumour characteristics was assessed, using a χ2 test. The Kaplan–Meier method was used to estimate survival rate. Univariate and multivariate analysis was carried out using the Cox regression method. A p-value <0.05 was considered to be statistically significant. Data were analysed using STATA V.9.0.

    Results

    The expression of VEGFR2 mRNA in the six cell lines was measured by one-step qPCR. It was confirmed that the VEGFR2 gene expression was present in all cell lines at different levels. The VEGFR2 mRNA level in HCC cell lines, especially in HepG2 or BEL-7402, was higher than that in LO-2. The VEGFR2 mRNA level is shown in figure 1.

    Figure 1
    Figure 1

    Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA level for the one-step qPCR result. Normalised by the VEGFR2 mRNA level of HUVEC, the VEGFR2 mRNA level of hepatocellular carcinoma (HCC) cells are all higher than that in hepatic cells.

    To confirm the expression of VEGFR2 in HCC cell lines, WB was performed with four human HCC cell lines and a human liver cell line, LO-2. The results, measured by Gel Pro Analyser Software, showed the expression of VEGFR2 protein in order of highest to lowest: BEL-7402, HepG2, SK-HEP-1, SMMC-7721 and LO-2. Though the VEGFR2 protein has three forms, including 230 kDa of mature form, 200 kDa of glycosylated intermediate form, and 150 kDa of non-glycosylated form, the hepatic cells of LO-2 showed a very weak band at 230 kDa (figure 2).

    Figure 2
    Figure 2

    Evaluation of vascular endothelial growth factor receptor 2 (VEGFR2) protein expression by western blot. Three VEGFR2 bands at approximately 230 kDa, 200 kDa and 150 kDa, corresponding with those of mature form, glycosylated intermediate form and non-glycosylated form of VEGFR2 are shown at different levels in the cell line. Line 1: LO-2; line 2: HepG2; line 3: SMMC-7721; line 4; SK-HEP-1; line 5: BEL-7402.

    Immunohistochemically positive staining was localised, mainly in the cytoplasma membrane of the HCC cells and hepatic cells (figure 3). Of the HCC tumours, high VEGFR2 expression was in 74 (61.16%) of 121 tumour tissues and 28 (35.86%) of 121 peritumoral tissues. These show statistical significance (p<0.001) when subjected to χ2 test analysis.

    Figure 3
    Figure 3

    H&E staining and expression pattern of vascular endothelial growth factor receptor 2 (VEGFR2) in hepatocellular carcinoma (HCC) and tumour-adjacent tissues with serial section. (A) HCC tissue pattern with H&E staining (magnification ×200). (B) Expression pattern of VEGFR2 in HCC with IHC positive staining. (C) Tumour-adjacent tissue pattern with H&E staining. (D) Expression pattern of VEGFR2 in HCC with immunohistochemistry negative staining.

    HCC specimens from 121 patients were investigated and the clinicopathological features were analysed in relation to VEGFR2 expression status (table 1). Comparison of clinicopathological characteristics and immunohistochemistry by χ2 test analysis showed that the high expression of VEGFR2 in HCC was related to large tumour diameter (p=0.012), poor differentiation (p=0.007), high serum AFP (p=0.029), multifocal gross classification (p=0.007) and less than 5 years' survival (p=0.029). In contrast, no statistically significant correlation was found for gender, age, liver cirrhosis or portal vein invasion.

    View this table:
    Table 1

    Correlation of high vascular endothelial growth factor receptor 2 expression with clinicopathological characteristics in hepatocellular carcinoma

    As determined by univariate Cox regressions for all factors considered, high VEGFR2 expression (p=0.049), portal vein invasion (p=0.009) and stage grouping with TNM classification (p=0.001) were significant factors for survival. However, the multivariate Cox regression model showed that high VEGFR2 expression (p=0.009) and stage grouping with TNM classification (p=0.004) were the strongest predictors of survival (table 2). Kaplan–Meier survival curves showed that the patients with a high VEGFR2 expression and stage grouping with TNM classification had a significantly shorter survival time (figure 4).

    View this table:
    Table 2

    Univariate and multivariate analysis of prognostic factors for survival in hepatocellular carcinoma

    Figure 4
    Figure 4

    Survival curves after surgical therapy in hepatocellular carcinoma. (A) Curves calculated by vascular endothelial growth factor receptor 2 (VEGFR2) expression with the Kaplan–Meier method. The high expression of the VEGFR2 group (red line) has significantly less survival than the low expression of the VEGFR2 group (blue line). (B) The curves calculated by portal vein invasion with the Kaplan–Meier method. The stage grouping III (blue line) has a significantly better survival rate than the stage grouping IV (red line).

    Discussion

    VEGFR2 is considered to be one of the most important regulators of angiogenesis, and is a key target in anti-cancer treatment.14 15 17 VEGFR2 expression has been reported in various types of solid tumour, such as human thyroid tumour,32 33 tumours of the digestive system,23–29 34 35 tumours of the female genital system36 37 and so on. When taken together, these results indicate that VEGFR2 expression showed encouraging signs as a potential therapeutic target and prognostic tool for the survival rate for solid tumours described in this report. However, the expression pattern of VEGFR2 in HCC parenchyma or stroma, and the relation between high or low VEGFR2 expression and clinicopathological characteristics in HCC are not yet well enough defined.

    In the present study, our current data of qPCR, WB and IHC confirmed that VEGFR2 expression in HCC cells was higher than VEGFR2 expression in hepatic cells. The expression pattern of VEGFR2, mainly in the cytoplasm membrane of HCC cells, was in accordance with previous reports.14 25 26 To compare the expression levels of VEGFR2 mRNA, normalised by HUVEC, four HCC cell lines and a liver cell line were used. The results of qPCR showed that the mRNA expression of VEGFR2 in HepG2 and BEL-7402 was much higher than in hepatic cells. Furthermore, expression of VEGFR2 protein in the four HCC cell lines and a liver cell line were measured by WB and Gel Pro Analyser Software. As LO-2 only weakly showed a band of mature VEGFR2 form, high expression of VEGFR2 in BEL-7402 and HepG2 was conformed, with three thick bands corresponding to the different forms of VEGFR2. These data were similar to previous reports, which showed that the expression of VEGFR2 was higher in HCC than in liver tissues by RT-PCR23 24 and western blot.23

    On the contrary, Yamaguchi et al25 found VEGFR2 located in tumour mesenchyma, rather than in peritumour tissue, including some macrophages and endothelial cells, and HCC cells did not express VEGFR2. Moreover, Zeng et al26 reported that VEGFR2 was mainly associated with Kupffer cells and weakly expressed in some tumour endothelial cells, whereas all other structures were negative. Von Marschall et al27 observed that VEGFR2 was detected mainly in vascular endothelial cells of HCC, while VEGFR2 was detected much less in endothelial cells of cirrhotic livers. Moreover, Amaoka et al28 showed that high VEGFR2 expression in the cytoplasm membrane of HCC cells was closely related to the high differentiation of HCC, and VEGFR2 was frequently found more highly expressed in HCC tissues than in tumour-adjacent tissues. Jia et al29 reported that the expression of VEGFR2 in HCC tissues was lower than in peritumoral tissues and normal liver tissues; they also indicated that the expression of VEGFR2 was not related to the prognosis of HCC patients. However, we obtained the opposite results, as detailed below.

    We found that the high expression of VEGFR2 in HCC was related to progressive pathological factors, including large tumour diameter, poor differentiation, high serum AFP, multifocal gross classification and 5 years' survival. Kaplan–Meier survival curves showed that patients with high VEGFR2 expression had a shorter survival time than patients with low VEGFR2 expression; they also showed that patients with stage grouping IV had poorer prognosis than those patients without stage grouping III. The univariate and multivariate analysis showed high VEGFR2 expression and stage grouping with TNM classification to be independent poor prognostic factors for HCC patients.

    As mentioned previously, there are several complex relationships affecting high VEGFR2 expression, including diverse pattern and the clinicopathological characteristics in HCC. We considered the phenomenon to result from dissimilar antibodies and HCC samples from various areas. It is noteworthy that disparate anti-VEGFR2 antibodies, from different sources of monoclonal or polyclonal antibody, were used in previous investigations. This observation confirmed the efficacy of the self-made anti-VEGFR2 monoclonal antibody in IHC and also showed that VEGFR2 was a prognostic marker in HCC patients.

    Furthermore, VEGFR2 could be considered a potential therapeutic target from this investigation. A8H1 could be transformed to a chimeric murine–human fragment with gene engineering techniques as a vector for targeted therapy of HCC. These studies will be summarised in the near future.

    Take-home messages

    • Vascular endothelial growth factor receptor 2 (VEGFR2) is considered to be one of the most important regulators of angiogenesis, and is a key target in anti-cancer treatment.

    • The expression pattern of VEGFR2 in hepatocellular carcinoma (HCC) parenchyma or stroma, the relation between high or low VEGFR2 expression, and clinicopathological characteristics in HCC are not yet well defined.

    • VEGFR2 expression in HCC cells was higher than in hepatic cells. The expression pattern of VEGFR2, mainly in the cytoplasm membrane of HCC cells was in accordance with previous reports.

    • High expression of VEGFR2 in HCC was related to progressive pathological factors, including large tumour diameter, poor differentiation, high serum α-fetoprotein, multifocal gross classification and 5 years' survival.

    • High VEGFR2 expression and stage grouping with TNM were independent poor prognostic factors for HCC patients.

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    Footnotes

    • Funding This study was supported by the Natural Science Foundation (BS2007019) of Jiangsu Province and a grant (H200938) from the Health Department of Jiangsu Province, China.

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the Human Research Ethics Committee of Nanjing Medical University.

    • Provenance and peer review Not commissioned; externally peer reviewed.