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Quantitative Correlation of Serum Levels and Tumor Expression of Vascular Endothelial Growth Factor in Patients with Hepatocellular Carcinoma

Centre for the Study of Liver Disease and Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong, China

	ABSTRACT

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Recent studies have suggested that serum levels of vascular endothelial growth factor (VEGF) may provide useful prognostic information in patients with various types of cancers. However, there has been a debate on whether serum VEGF level is a true reflection of tumor angiogenic activity in cancer patients. This debate originates from the finding that most VEGF in the serum is released from platelets during clotting. It has been postulated that platelet may serve the role of storage for circulating VEGF derived from the tumors. We conducted a study to clarify whether the platelet load of VEGF in the circulation correlates with tumor expression of VEGF. We measured quantitatively the serum VEGF₁₆₅ levels and tumor cytosolic VEGF₁₆₅ concentration by an ELISA and tumor VEGF₁₆₅ mRNA by real-time quantitative reverse transcription-PCR in 60 patients with hepatocellular carcinoma. Serum VEGF₁₆₅ levels correlated significantly with platelet counts (r = 0.662, P < 0.001). When corrected for platelet count, serum VEGF₁₆₅/platelet correlated significantly with tumor cytosolic VEGF₁₆₅ concentration (r = 0.447, P = 0.006), which in turn correlated with VEGF₁₆₅ mRNA expression in the tumors (r = 0.315, P = 0.020). Advancing tumor stage was associated with a significant increase in tumor cytosolic VEGF₁₆₅ concentration (P = 0.006), tumor VEGF₁₆₅ mRNA expression (P = 0.012), serum VEGF₁₆₅/platelet (P = 0.001), and serum VEGF₁₆₅ levels (P = 0.003). In conclusion, our data showed that the platelet load of VEGF in the circulation correlated positively with tumor VEGF expression. This study provides strong evidence that supports the use of serum VEGF level as an indirect estimate of tumor VEGF expression.

	INTRODUCTION

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Tumor angiogenesis is fundamental to tumor growth and metastasis (1) . VEGF² is a potent angiogenic factor with five molecular isoforms generated by alternative splicing of VEGF mRNA, which are composed of 206, 189, 165, 145, and 121 amino acid residues, respectively (2) . VEGF₁₂₁ and VEGF₁₆₅ are both secreted into the circulation, but VEGF₁₆₅ is the predominant isoform secreted by most tumors (2) . The other isoforms do not enter the circulation in a significant amount because they are either bound to the extracellular matrix (VEGF₁₄₅) or not secreted (VEGF₁₈₉ and VEGF₂₀₆).

Numerous studies have shown that high-serum VEGF levels predict advanced disease in cancer patients (3, 4, 5, 6) . Serum VEGF may be a surrogate marker of tumor angiogenesis (7) . However, there has been a debate of whether serum VEGF level actually reflects tumor expression of VEGF (7, 8, 9, 10, 11, 12) . Megakaryocytes are known to express VEGF, which is stored in the granules of the platelets and released during platelet activation (13 , 14) . Comparison of serum and plasma VEGF levels in the same cancer patient has shown much higher serum levels (8 , 15) , suggesting that most VEGF in the serum is derived from the platelets during clotting (16) . Furthermore, a significant correlation between serum VEGF and platelet count has been observed in cancer patients (9 , 17) . Some authors suggested that serum VEGF might reflect platelet count rather than tumor VEGF secretion (16) . Others advocated that platelet may store VEGF secreted from the tumor based on the finding of several-folded higher cellular content of VEGF in platelets of cancer patients compared with healthy individuals (9 , 17 , 18) . A recent study further demonstrated that serum VEGF levels corrected for variation in platelet counts correlated with advancing tumor stage, and the authors recommended serum VEGF normalized by platelet count as a standardized measurement of circulating VEGF (11) . However, there is no evidence in the literature of a direct quantitative correlation between the levels of circulating VEGF and tumor expression of VEGF.

HCC is a hypervascular tumor. Angiogenesis plays a significant role in the progression of HCC (19 , 20) . VEGF is an important mediator of angiogenesis in HCC (21 , 22) . Serum VEGF level has been found to predict venous invasion and metastasis in HCC (23 , 24) . To test the hypothesis that serum VEGF levels reflect tumor VEGF expression, we conducted a study to quantitatively evaluate the relationship between serum VEGF levels and tumor VEGF expression in patients with HCC.

	MATERIALS AND METHODS

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Patients and Specimens.
Between 1998 and 2001, 60 patients (44 men and 16 women, age 16–79 years) undergoing resection of HCC were recruited in this study with informed consents. The study protocol was approved by the Ethics Committee of our institution. Serum hepatitis B surface antigen was positive in 47 patients (78%). No patient had received any anticancer therapy before surgery. Tumor was staged according to the Tumor-Node-Metastasis staging (25) .

Before surgery, peripheral venous blood samples were taken from the patients into a serum separator tube and centrifuged at 3000 rpm for 10 min, then stored at -70°C. Fresh tumor tissue and nontumorous liver were obtained immediately after tumor resections.

Quantitation of Serum and Tumor Cytosolic VEGF₁₆₅ Protein Concentration.
VEGF₁₆₅ of both serum and tissue cytosolic samples were quantified by Quantikine human VEGF Immunosorbent assay kit (R&D Systems, Minneapolis, MN). The assay had been shown to be reproducible (4 , 6 , 14) . It exhibited no significant cross-reactivity with other angiogenic factors and had a sensitivity of 9 pg/ml. All samples were assayed in duplicate. To correct for variation in platelet counts, VEGF per platelet (pg/10⁶ platelets) was calculated by dividing serum VEGF concentration (picogram/milliliter) by the platelet count (x10⁶/ml; Refs. 10 , 11 , and 17 ).

Protein cytosolic fraction was obtained by homogenization of tissues as described (26) . Homogenates were centrifuged at 20,000 x g at 4°C for 10 min. The supernatants were collected for assay of the cytosolic VEGF₁₆₅ concentration, and the total protein concentration was determined using Bio-Rad total protein assay system (Bradford, Hercules, CA).

Immunohistochemical Staining for Tumor VEGF₁₆₅ Expression.
Paraffin-embedded, 4-µm sections were deparaffinized, rehydrated, treated with 3% H₂O₂ in methanol, and then microwaved for 10 min. Endogenous biotin was blocked. Sections were then incubated with normal rabbit serum for 30 min at 37°C. VEGF₁₆₅ mouse antihuman monoclonal antibody (R&D Systems) at 1:200 dilution was applied, and sections were then incubated with 1:100 rabbit antimouse biotinated IgG secondary antibody (DAKO Corp., Carpinteria, CA) for 1 h at 37°C. Finally, sections were incubated with 1:100 avidin-biotin complex method-horseradish peroxidase (DAKO) and developed with 3,3'-diamino-benzidine tetrachloride (DAKO). VEGF₁₆₅ expression was classified as strong if >30% of cells were stained positive and weak if <30% cells were stained positive.

Real-time Quantitative Reverse transcription-PCR for Tumor VEGF₁₆₅ mRNA Expression.
Total RNA from tumor and nontumor frozen tissues were extracted using RNeasy Mini Kit (Qiagen, Valencia, CA). RNA was pretreated with DNase I (Invitrogen, Carlsbad, CA), and cDNA was synthesized by SuperScript (Invitrogen). The VEGF₁₆₅ probe (5'-6Fam-TGA ATG CAG ACC AAA GAA AGA TAG AGC AAG-TAMRA-3'), forward primer (5'-AGC TTC CTA CAG CAC AAC AAA TG-3'), and reverse primer (5'-CAA GGC CCA CAG GGA TTT T-3') were designed using the ABI PRISM Primer Express Software (Applied Biosystems, Foster City, CA). Real-time quantitative PCR analysis was performed as described (27) using ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Ribosomal 18 s was used as an internal control.

Calculation of Real-time Quantitative Reverse Transcription-PCR Data.
The relative amount of VEGF₁₆₅ mRNA, normalized to an internal control ribosomal 18 s and relative to a calibrator, was given by Livak and Schmittgen (28) :

where

Fig. 1, A and B are the amplification plots of VEGF₁₆₅ and ribosomal 18 s, respectively, in which changes in normalized reporter signal (Rn, Y axis) are plotted against cycle number (X axis). C_T was defined as the fractional cycle number at which the amount of amplified target reached a fixed threshold. The C_T value correlated with the input target mRNA levels, and a lower C_T value indicated a higher starting copy number. One of the samples was designated as the calibrator to compare the relative amount of target in different samples and adjust for the plate-to-plate variation in amplification efficiency.

View larger version (122K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, amplification plots of VEGF165 (multiplex) in different tissue samples. Emission intensity of the reporter (FAM) dye (Rn, Y axis) is plotted versus cycle number (X axis). The CT represents the fractional cycle number at which there is a significant increase in Rn above the chosen threshold (horizontal black line). B, amplification plots of ribosomal 18 s (multiplex) in different tissue samples. Emission intensity of the reporter (VIC) dye (Rn, Y axis) versus cycle number (X axis).

	RESULTS

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Serum VEGF₁₆₅ Level.
The median serum VEGF₁₆₅ level among the 60 patients was 248 pg/ml (140–425). The median platelet count was 162 x 10⁶/ml (133–235). The serum VEGF₁₆₅ levels correlated significantly with platelet counts (r = 0.662, P < 0.001; Fig. 2 ). The median serum VEGF₁₆₅/platelet was 1.4 pg/10⁶ platelets (0.75–2.44).

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Correlation of serum VEGF and platelet count in 60 HCC patients (r = 0.662, P < 0.001).

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Boxplot showing significant increase in platelet count with advancing tumor stage (P = 0.008). The top and bottom horizontal lines of the box indicate the 25th and 75th percentiles, respectively. The lines within the box indicate the median values. The top and bottom horizontal bars indicate data within 1.5 times the interquartile range.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Boxplots showing significant increase of serum VEGF levels (A, P = 0.003) and serum VEGF165/platelet (B, P = 0.001) with advancing tumor stage. The top and bottom horizontal lines of the box indicate the 25th and 75th percentiles, respectively. The lines within the box indicate the median values. The top and bottom horizontal bars indicate data within 1.5 times the interquartile range.

View larger version (160K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Immunohistochemical staining for VEGF165 in a tumor section. Positive staining (brownish staining) was noted mainly in the cytoplasm of tumor cells.

There was no significant correlation between tumor cytosolic VEGF₁₆₅ and tumor size (r = 0.151, P = 0.215), or between tumor VEGF₁₆₅ mRNA and tumor size (r = 0.118, P = 0.283). However, both tumor cytosolic VEGF₁₆₅ (P = 0.006) and VEGF₁₆₅ mRNA (P = 0.012) increased with advancing tumor stage. High tumor cytosolic VEGF₁₆₅ (P = 0.042) and VEGF₁₆₅ mRNA (P = 0.047) were also associated with tumor invasion of venous branch.

Correlation between Serum VEGF Level and Tumor Expression of VEGF.
A trend toward a positive correlation between serum VEGF₁₆₅ and tumor cytosolic VEGF₁₆₅ was observed (r = 0.277, P = 0.103). Serum VEGF₁₆₅/platelet was significantly correlated with tumor cytosolic VEGF₁₆₅ (r = 0.447, P = 0.006; Fig. 6 ). There was no significant correlation of nontumorous cytosolic VEGF₁₆₅ with either serum VEGF levels (r = 0.111, P = 0.407) or serum VEGF₁₆₅/platelet (r = 0.021, P = 0.878). Strong tumor immunostaining of VEGF₁₆₅ was also associated with higher serum VEGF₁₆₅/platelet compared with weak VEGF₁₆₅ staining (median 1.75 versus 0.95/10⁶ platelets, P = 0.019). Serum VEGF₁₆₅/platelet also correlated with tumor VEGF₁₆₅ mRNA (r = 0.342, P = 0.023).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Correlation of serum VEGF/platelet and tumor cytosolic VEGF protein content (r = 0.447, P = 0.006).

	DISCUSSION

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Some indirect evidence has suggested that the tumor is a major source of serum VEGF in cancer patients. A remarkable decrease in serum VEGF levels after tumor resection has been reported (6 , 30) . One study demonstrated that serum VEGF levels in mesenteric venous blood draining from the tumors were several-fold higher compared with peripheral blood in colorectal cancer patients, indicating secretion of VEGF from the tumors into the circulation (31) . However, a more recent study found no significant increase in serum or plasma VEGF levels in the vein draining the tumors in patients with various carcinomas (32) . To validate the use of serum VEGF as an estimate of tumor angiogenic activity, it is imperative to show a direct quantitative correlation between serum VEGF level and tumor expression of VEGF.

In agreement with previous studies (9 , 11 , 17 , 23) , our data showed that serum VEGF₁₆₅ levels correlated with platelet counts. Although platelet is known to express VEGF (13 , 14) , several studies suggested that platelet probably has a more important role as a scavenger of the VEGF in the circulation. Although there was a significant correlation between serum VEGF and platelet count in cancer patients, such a correlation was not observed in healthy controls (10) . Furthermore, George et al. (11) showed that serum VEGF levels correlated with platelet counts in patients with colorectal cancer but not in patients with benign colorectal disease. Platelet is known to endocytose and store various circulating proteins (33) . The scavenger role of platelets may have a physiological function of restricting the angiogenic activity of circulating VEGF to sites where coagulation takes place, such as a healing wound. The leaky tumor vasculature may allow platelets to come into contact with the tumor and deposit VEGF to enhance tumor angiogenesis (34) . VEGF is known to stimulate the recruitment of endothelial progenitors from the bone marrow for physiological vasculogenesis (35) , which can be damped by the storage of VEGF in platelets. By storing VEGF, platelets may have a net proangiogenic effect on tumor endothelium (36) . Our study shows that serum VEGF₁₆₅/platelet correlated with tumor VEGF₁₆₅ protein level. This provides the most direct evidence thus far available for the role of platelet in the storage of VEGF released from the tumors.

We also evaluated the tumor expression of VEGF₁₆₅ at the transcriptional level using real-time quantitative reverse transcription-PCR. We demonstrated a significantly higher VEGF₁₆₅ mRNA expression in the tumors than in the nontumorous liver. VEGF₁₆₅ mRNA expression correlated significantly with VEGF₁₆₅ protein expression in the tumors. Both tumor cytosolic VEGF₁₆₅ protein and VEGF₁₆₅ mRNA increased significantly with advancing tumor stage. Serum VEGF₁₆₅/platelet also increased with advancing tumor stage. Neither serum VEGF₁₆₅/platelet nor tumor VEGF₁₆₅ expression was significantly related to tumor size. However, both serum VEGF₁₆₅/platelet and tumor VEGF₁₆₅ expression correlated significantly with venous invasion by the tumor, which is another determinant of tumor stage apart from tumor size.

Because the platelet load of VEGF correlates with tumor VEGF expression, it is important to include the VEGF stored in the platelets in the measurement of circulating VEGF as an indirect estimate of tumor angiogenic activity. Our data support the use of serum VEGF level instead of plasma VEGF level in the evaluation of circulating VEGF. In an unpublished study by the authors, high pretreatment serum VEGF levels were found to predict poor prognosis in patients undergoing transarterial chemoembolization for HCC. Another group has demonstrated an increase in serum VEGF levels after transarterial embolization of HCC (37) . These data strengthen the clinical significance of serum VEGF in HCC patients. Because VEGF is a key mediator of angiogenesis secreted by most cancers (2) , it is reasonable to speculate that the results from the current study can be generalized to other common human cancers, but this needs to be confirmed with additional studies. A recent study that measured matched preoperative plasma and serum VEGF levels in patients with colorectal cancer showed that high-serum VEGF level but not plasma VEGF level was an independent predictor of survival (38) . It is unclear whether total serum VEGF level or serum VEGF/platelet provides better prognostic prediction. In our study, advanced tumor stage was associated with an increased platelet count. A study in colorectal cancer has demonstrated that thrombocytosis is associated with more advanced disease and worse prognosis (39) . Serum VEGF level reflects both the platelet load of VEGF and the platelet count; thus, it may be a better prognostic marker than serum VEGF/platelet. Some recent studies suggested that serum VEGF is not all tumor derived, and other cytokines secreted by the tumors, such as interleukin-6, may up-regulate the expression of VEGF in the platelet (17 , 32) . The relative contribution of VEGF directly secreted from the tumor and VEGF expressed by the platelet itself to the platelet load of VEGF in cancer patients need clarification with additional studies.

In conclusion, this study demonstrated for the first time that the platelet load of VEGF in the circulation of cancer patients correlated quantitatively with the expression of VEGF in the tumors. Our study provides strong evidence that supports the use of serum VEGF level as an indirect estimate of tumor VEGF expression.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Department of Surgery, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, China. Phone: (852) 28553641; Fax: (852) 28175475; E-mail: poontp{at}hkucc.hku.hk

² The abbreviations used are: VEGF, vascular endothelial growth factor; HCC, hepatocellular carcinoma; C_T, threshold cycle.

Received 11/ 7/02. Accepted 4/17/03.

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				X.-Y. Zhu, M. Rodriguez-Porcel, M. D. Bentley, A. R. Chade, V. Sica, C. Napoli, N. Caplice, E. L. Ritman, A. Lerman, and L. O. Lerman Antioxidant Intervention Attenuates Myocardial Neovascularization in Hypercholesterolemia Circulation, May 4, 2004; 109(17): 2109 - 2115. [Abstract] [Full Text] [PDF]

				P. Bono, A. Krause, M. von Mehren, M. C. Heinrich, C. D. Blanke, S. Dimitrijevic, G. D. Demetri, and H. Joensuu Serum KIT and KIT ligand levels in patients with gastrointestinal stromal tumors treated with imatinib Blood, April 15, 2004; 103(8): 2929 - 2935. [Abstract] [Full Text] [PDF]

				Y. Ren, R. T.-P. Poon, H.-T. Tsui, W.-H. Chen, Z. Li, C. Lau, W.-C. Yu, and S.-T. Fan Interleukin-8 Serum Levels in Patients with Hepatocellular Carcinoma: Correlations with Clinicopathological Features and Prognosis Clin. Cancer Res., December 1, 2003; 9(16): 5996 - 6001. [Abstract] [Full Text] [PDF]

				S. Ferrero and N. Ragni Prognostic Value of Plasma and Serum VEGF Levels in Patients With Resectable Hepatocellular Carcinoma Ann. Surg. Oncol., November 1, 2003; 10(9): 1123 - 1123. [Full Text] [PDF]

				R. T.-P. Poon, C. P.-Y. Lau, J. W.-Y. Ho, W.-C. Yu, S.-T. Fan, and J. Wong Tissue Factor Expression Correlates with Tumor Angiogenesis and Invasiveness in Human Hepatocellular Carcinoma Clin. Cancer Res., November 1, 2003; 9(14): 5339 - 5345. [Abstract] [Full Text] [PDF]

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