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10.1245/s10434-007-9366-z
Annals of Surgical Oncology 14:1835-1845 (2007)
© 2007 Society of Surgical Oncology
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Original Article

High Serum Vascular Endothelial Growth Factor Levels Predict Poor Prognosis after Radiofrequency Ablation of Hepatocellular Carcinoma: Importance of Tumor Biomarker in Ablative Therapies

Ronnie T. P. Poon, MS, PhD1, Cecilia Lau, MPhil1, Roberta Pang, PhD2, Kelvin K. Ng, PhD1, Jimmy Yuen, MBBS3 and Sheung Tat Fan, MS, MD, PhD, DSc1

1 Department of Surgery, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong, China
2 Department of Medicine, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong, China
3 Department of Radiology, Queen Mary Hospital, The University of Hong Kong, 102 Pokfulam Road, Hong Kong, China

Correspondence: Address correspondence and reprint requests to: Ronnie T. P. Poon, MS, PhD; E-mail: poontp{at}hkucc.hku.hk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Radiofrequency ablation (RFA) is a recently developed treatment for hepatocellular carcinoma (HCC). Thus far, the prognostic impact of tumor biomarkers has not been evaluated in this treatment. High serum level of vascular endothelial growth factor (VEGF) has been shown to predict microscopic vascular invasion and metastasis in HCC. This study investigated the prognostic significance of pre-treatment serum VEGF level in patients with HCC undergoing RFA treatment.

Methods: Serum VEGF levels were measured using enzyme-linked immunosorbent assay in 120 patients with HCC undergoing RFA, and in 15 healthy controls. Serum VEGF levels were correlated with clinicopathological features of the HCC patients. The prognostic significance of serum VEGF levels was assessed by univariate and multivariate analyses.

Results: The median serum VEGF level in the HCC patients was 240 pg/mL (range 17–1162), significantly higher than that of healthy controls (p = .024). The serum VEGF levels were significantly correlated with platelet counts (r = .487, p < .001) but not other clinicopathological features. Patients with serum VEGF level > 240 pg/mL had worse overall and recurrence-free survival compared with those with serum VEGF level > 240 pg/mL (p = .005 and .002, respectively). By multivariate analysis, serum VEGF level was a significant prognostic factor of both overall and recurrence-free survival.

Conclusions: High pre-treatment serum VEGF levels predict poor prognosis after RFA of HCC. This study highlights the importance of tumor biomarker as a prognostic predictor in ablative therapy for HCC, which has an intrinsic problem of unavailability of histopathological prognostic features.

Key Words: Vascular endothelial growth factor • Radiofrequency ablation • Hepatocellular carcinoma • Tumor biomarker


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third leading cause of cancer death in the world.1 The resectability rate of HCC is limited to 20% to 30% as a result of various factors such as multifocal disease and poor liver function reserve as a result of underlying cirrhosis.2,3 Liver transplantation is an alternative curative treatment for early HCC, but severe organ shortage limits its application.4 Hence, various local ablative therapies have been developed for the treatment of unresectable HCC confined to the liver.5

Radiofrequency ablation (RFA) is one of the most widely used ablative therapies for HCC, with a high complete ablation rate of 85% to 100% for tumors ≤5 cm in diameter.6–8 RFA is now considered a curative treatment for small HCC not amenable to resection or transplantation. Recently, favorable long-term survival results with 5-year survival rates of 40% to 55% have been reported.9–11 RFA has been shown to be superior to ethanol injection in complete tumor ablation rate and survival results in prospective randomized trials,12,13 and a recent randomized trial suggested that RFA may even achieve long-term survival outcomes comparable with those after hepatic resection for HCC ≤5 cm in diameter.14 With the growing evidence of its efficacy in prospective clinical trials, RFA is rapidly gaining popularity as the treatment of choice for unresectable HCC ≤5 cm.15

Although favorable long-term survival after RFA for HCC has been reported, similar to hepatic resection, RFA is associated with a high incidence of postoperative recurrence.15 In recent studies with long-term follow-up data, recurrence rates of 50% to 65% have been reported.9,11,16,17 One study reported a 4-year disease-free survival of only 14%.18 The most common type of recurrence is distant intrahepatic recurrence related to undetected microscopic intrahepatic metastasis or multicentric recurrence.9,10,17 Microscopic venous invasion is one of the most important prognostic factors for recurrence after resection or transplantation for HCC, suggesting the importance of microscopic metastasis in tumor recurrence.19–21 However, in the case of ablative therapies, histopathological data such as microscopic venous invasion and microsatellite nodules are not available for prognostic prediction.

Tumor expression of vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, has been shown to be related to microscopic venous invasion and metastasis of HCC.22–24 VEGF is a secreted protein that can be detected in the circulation. A previous study from our group demonstrated that high serum VEGF levels predicted the presence of microscopic venous invasion and intrahepatic metastasis in HCC.25 Furthermore, a high preoperative serum VEGF level has been reported to be an independent unfavorable prognostic factor in patients undergoing resection for HCC,26,27 and high pre-treatment circulating VEGF levels predict poor response and survival in patients with advanced HCC undergoing transarterial chemoembolization.28,29 However, a substantial proportion of patients undergoing resection or chemoembolization in those studies had large advanced HCCs, some even with macroscopic venous invasion. The most widely accepted indications for RFA are small HCC ≤5 cm and ≤4 tumor nodules without macroscopic vascular invasion.15 It remains unclear whether the serum VEGF level has a prognostic value in patients with small HCCs undergoing RFA for HCC. Hence, we conducted a prospective study to test the hypothesis that high pretreatment serum VEGF levels predict poor prognosis in patients undergoing RFA for HCC.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Between September 1, 2001 and December 30, 2004, a total of 143 patients with newly diagnosed HCC underwent RFA treatment at Queen Mary Hospital, The University of Hong Kong, Hong Kong, China. Of these, 120 patients who underwent RFA for HCC ≤5 cm in diameter with a curative intent were recruited into this prospective study. Two patients with RFA for a palliative intent, seven patients with incomplete ablation of the tumor and 14 patients with tumor diameter larger than 5 cm were excluded from this study. General criteria for exclusion from RFA in our center include the presence of extrahepatic metastasis, imaging finding of portal vein or hepatic vein tumor thrombus, more than four tumor nodules, Child-Pugh class C liver cirrhosis, and gross ascites. The diagnosis of HCC was suggested by imaging findings and serum alfa-fetoprotein (AFP) levels. For the purpose of this prospective study, diagnosis of HCC was confirmed by fine-needle aspiration cytology (n = 78), or in some cases of open RFA, by Trucut core biopsy (n = 42). None of the patients received any adjuvant therapy. The study was approved by the institutional review board of the hospital, and informed consent was obtained from the patients.

RFA Procedure
All RFA procedures were performed by a dedicated team of hepatobiliary surgeons and interventional radiologists, with a standard protocol by a cool-tip RFA system (Radionics Inc., Burlington, MA). A single electrode with a 2- or 3-cm exposed tip was used for small tumors, and a cluster electrode consisting of three parallel electrodes was used for medium-size tumors. The ablation was performed by an automatic impedance control mode. Each ablation cycle lasted for 6 to 12 minutes. We aimed at ablation of all tumors with a curative intent, with a margin of 1 cm if feasible, in a single session of RFA. Details of the RFA procedure in our unit has been described previously.30

Each patient was discussed in a team meeting before the appropriate approach was decided. Patients with tumors located in a position amenable to percutaneous RFA were treated by this approach by interventional radiologists. Surgical approach was o3ered in the following circumstances: tumor morphology requiring multiple ablations even with the use of the cluster electrode; tumors located near the dome of the liver; or tumors located near visceral organs such as the gallbladder, colon, or stomach. In selected patients who had not undergone a previous upper abdominal surgery, a laparoscopic approach was used if the tumor position was favorable.

Patient Follow-up
Clinical data, including long-term follow-up results, were collected prospectively. All patients underwent contrast computed tomography scan 1 month after RFA to assess the completeness of ablation. Patients who had incomplete ablation were excluded from this study to avoid the confounding e3ect of incomplete ablation on long-term prognosis. All patients were followed in a HCC clinic and underwent serial monitoring of serum AFP, chest radiograph, and helical contrast computed tomographic scan every 3 months for detection of any intrahepatic recurrence or distant metastasis. Local recurrence is defined as tumor recurrence within or at the periphery of the ablated lesion on subsequent computed tomographic scans after complete ablation was documented. Distant intrahepatic recurrence is defined as a new tumor that appeared in the liver separate from the ablated area. Extrahepatic recurrence refers to any tumor recurrence outside the liver. By the time of censoring of data for analysis (June 30, 2006), follow-up was complete for all patients, and the last patient in the cohort had been followed for 18 months.

Assay of Serum VEGF Level
Peripheral venous blood samples were taken from the 120 patients with HCC before RFA treatment, and from 15 sex- and age-matched healthy controls. Blood samples were drawn into a serum separator tube and centrifuged at 3000 rpm for 10 minutes, then stored at –80°C until analysis. The levels of serum VEGF were quantified by the Quantikine human VEGF Immunosorbent assay kit (R&D Systems, Minneapolis, MN). The assay had been shown to be reproducible in previous studies.25,31,32 The assay uses the quantitative sandwich immunoassay technique that uses immobilized murine monoclonal antibody and horseradish peroxidase–linked polyclonal antibody, both of which are specific against human VEGF and exhibit no marked cross-reactivity with other angiogenic factors.

The assay was performed according to manufacturer’s instructions. First, 100-µL aliquots of the serum samples were serially diluted, and a pipette was used to transfer them to a 96-well microtiter plate coated with the murine monoclonal antibody, which was incubated for 2 hours at room temperature. Any VEGF present in the sample was bound by the immobilized monoclonal antibody. After washing away any unbound substances, the horseradish peroxidase–linked polyclonal antibody was then added to each well to sandwich the VEGF. After further washings, the enzyme reaction was carried out by adding the substrate tetramethylbenzidine. The intensity of the color developed was measured by reading optical absorbance at 450 nm with a microtiter plate reader. The VEGF level was determined by interpolation to a standard curve generated from standards of VEGF recombinant protein that came with the kit. All samples were assayed in duplicate by an investigator blinded to the clinical data. The sensitivity of the assay for VEGF was 9 pg/mL, and the coefficients of variation of intraassay and interassay measurement were in the range given by the manufacturer (4.5%–6.7% and 6.2%–8.8%, respectively).

Statistical Analysis
Continuous data were expressed as median and range. Groups were compared by the {chi}2 test (or Fisher exact test where appropriate) for nominal variables and the Mann-Whitney U-test for continuous data. Correlations of continuous data were performed by the Spearman rank correlation coefficient (r). Survival rates were estimated by the Kaplan-Meier method and compared by the log rank test. Multivariate analysis was performed by the Cox proportional hazard model. All statistical analyses were performed by SPSS 11.5 for Windows statistical software (SPSS, Chicago, IL). A P value <.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Baseline clinical characteristics of the 120 patients are shown in Table 1Go. One hundred patients (83%) had hepatitis B virus (HBV)-related HCC, and the other 20 patients had hepatitis C virus (HCV)-related HCC. None of the patients had coinfection of the two hepatitis viruses. All patients had evidence of underlying cirrhosis. The study population included 68 patients with unresectable HCC and 52 patients who had potentially resectable HCC but who were randomized to RFA in an ongoing randomized trial comparing RFA and resection for small HCC ≤5 cm in diameter. RFA was performed by percutaneous approach in 57 patients, laparoscopic approach in 11 patients, and open approach in 52 patients. There were no RFA-related deaths.


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  		TABLE 1. Baseline clinical data
 
Correlation of Serum VEGF Levels and Clinicopathological Features
The median serum VEGF level of the 120 patients with HCC was 240 pg/mL (range, 17–1162 pg/mL), significantly higher than that of the 15 healthy controls (median, 152 pg/mL, range, 41–396) (P = .024). No significant difference of serum VEGF levels was observed between male and female patients with HCC (median, 238 vs. 246 pg/mL, P = .543). A significant positive correlation between serum VEGF levels and platelet count was observed (r = .487, P < .001, Figure 1Go). There were no significant correlations between serum VEGF levels and age (r = .088, P = .341), serum albumin (r = .122, P = .120), bilirubin (r = –.129, P = .069), aspartate aminotransferase (r = –.178, P = .085), alanine aminotransferase (r = –.118, P = .206), or creatinine (r = .123, P = .180) levels. No significant difference in serum VEGF levels was observed between patients with HBV-related HCC and those with HCV-related HCC (median, 248 vs. 232 pg/mL, P = .496), or between patients with Child-Pugh class A cirrhosis and those with Child-Pugh class B cirrhosis (median, 236 vs. 247 pg/mL, P = .254).


Figure 1
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  		FIG. 1. Scatter plot showing the correlation between the serum vascular endothelial growth factor (VEGF) levels and platelet count (r = .487, P < .001).

 
The serum VEGF levels in patients with HCC >3 cm in diameter (median, 246 pg/mL; range, 17–1162 pg/mL) were higher than those in patients with HCC ≤3 cm in diameter (median, 202 pg/mL; range, 42–656 pg/mL), but the difference was not statistically significant (P = .213). There was no significant correlation between serum VEGF and tumor size when the two were correlated as continuous variables (r = .146, P = .217). Patients with solitary tumor had comparable serum VEGF levels as patients with multiple tumors (median, 238 vs. 246 pg/mL, P = .223). There was no significant difference in patients with potentially resectable HCC compared with those with unresectable HCC (median, 237 vs. 248 pg/mL, P = .205). There was no significant correlation between serum VEGF and AFP levels (r = .072, P = .434). Of the 42 patients who had undergone core biopsy, histological evaluation of tumor differentiation by Edmonson grade33 was possible in 24 patients with adequate tissue samples. There was no significant difference in serum VEGF levels between 14 patients with low-grade (grade 1 or 2) tumors and 10 patients with high-grade (grade 3 or 4) tumors (median, 254 vs. 230 pg/mL, P = .124).

Long-Term Tumor Recurrence and Survival Results After RFA
Figure 2Go shows the overall and recurrence-free survival of the 120 patients from the time of RFA, with a median follow-up of 40 months (range, 18–57 months). The 1-, 2-, 3-, and 4-year overall survival rates were 89%, 82%, 68%, and 50%, respectively. The 1-, 2-, 3-, and 4-year recurrence-free survival rates were 67%, 46%, 43%, and 40%, respectively.


Figure 2
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  		FIG. 2. O verall survival (A) and recurrence-free survival (B) curves after radiofrequency ablation (RFA) in the 120 patients.

 
By the time of censoring of data for analysis, 66 patients (55%) had developed recurrent tumors, including 56 patients with intrahepatic recurrence alone, 6 patients with concurrent intrahepatic and extrahepatic recurrences, and 4 patients with extrahepatic recurrence alone. Of the 62 patients with intrahepatic recurrence, 15 developed local recurrence at the ablated site, whereas the other 47 patients developed distant intrahepatic recurrence. The organs of distant metastasis in the 10 patients with extrahepatic recurrence included the lung (n = 4), bone (n = 3), lymph node (n = 2), and adrenal gland (n = 1). None of the patients developed needle-track tumor seeding during follow-up. Of the 66 patients with tumor recurrence, the recurrence occurred within 1 year after RFA in 40 patients (61%).

Forty-two (35%) of 120 patients had died by the time of data analysis. Of these 42 patients, 33 died of tumor recurrence, 6 died of liver failure without tumor recurrence, 1 died of variceal bleeding, 1 died of renal failure, and 1 died of cardiac failure.

Prognostic Significance of Serum VEGF Levels
Figure 3Go shows the prognostic influence of serum VEGF levels on overall and recurrence-free survival by using the median serum VEGF level of 240 pg/mL in the whole study population as the cutoff value for high and low serum VEGF levels, in accordance with previous studies.26,32,34 Patients with high serum VEGF levels had significantly worse overall and recurrence-free survival compared with those with low serum VEGF levels (P = .005 and .002, respectively).


Figure 3
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  		FIG. 3. Prognostic influence of serum vascular endothelial growth factor (sVEGF) level on overall (A) and recurrence-free (B) survival after radiofrequency ablation (RFA), using the median sVEGF level of 240 pg/mL of the whole study population as the cutoff value for low and high sVEGF levels.

 
Forty-two patients (70%) in the high serum VEGF group and 24 patients (40%) in the low serum VEGF group developed recurrence after similar median follow-up of 40 months (P = .003). Twenty-seven patients (45%) in the high serum VEGF group and 13 patients (22%) in the low serum VEGF group developed recurrence within 1 year after RFA (P = .007). Table 2Go shows the pattern of recurrences in the high and low serum VEGF groups, respectively, according to the sites of recurrence. The incidences of local recurrence was similar, but the incidences of distant intrahepatic recurrence and extrahepatic recurrence were significantly higher in the high serum VEGF group than in the low serum VEGF group (P = .015 and .048, respectively).


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  		TABLE 2. Pattern of recurrences in high and low serum VEGF groups
 
Tables 3 and 4GoGo show univariate analyses of clinical variables and serum VEGF levels for their prognostic influence on overall and recurrence-free survival, respectively. All continuous variables, including serum VEGF levels, were dichotomized by using the respective median values as the cutoff. Significant unfavorable prognostic factors for overall survival included Child-Pugh class B cirrhosis (P = .029), serum albumin ≤ 35 g/L (P = .013), multiple tumors (P = .025), and high serum VEGF level (P = .005). Significant unfavorable prognostic factors for recurrence-free survival included HCV etiology (P = .012), serum albumin ≤ 35 g/L (P = .034), multiple tumors (P = .034), and high serum VEGF level (P = .002).


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  		TABLE 3. Univariate analysis of prognostic factors for overall survival
 

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  		TABLE 4. Univariate analysis of prognostic factors for recurrence-free survival
 
Significant prognostic factors in the univariate analyses were entered into multivariate analyses. Table 5Go shows the results of multivariate analyses of prognostic factors for overall and recurrence-free survival, respectively. High serum VEGF level (P = .002) and low serum albumin level (P = .016) were independent unfavorable prognostic factors of overall survival, whereas high serum VEGF level (P < .001) and HCV etiology (P = .001) were independent unfavorable prognostic factors of recurrence-free survival. Serum VEGF level was the most significant prognostic factor for both overall and recurrence-free survival.


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  		TABLE 5. Prognostic factors for overall and recurrence-free survival by multivariate analysis
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Angiogenesis is critical to the growth and metastasis of cancers.35 VEGF is one of the most potent angiogenic factors expressed in various human cancers.36,37 Studies have shown that high serum VEGF levels predicted poor survival results independent of clinicopathological features in patients with different types of cancer undergoing resection or receiving chemotherapy.31,32,34,38 HCC is a highly vascular cancer, and it has been well established that angiogenesis plays an important role in its biological aggressiveness.39 VEGF is a critical mediator in angiogenesis of HCC.24,40 Recently, a study from our group has demonstrated that VEGF has an autocrine effect in stimulating cancer cell proliferation in HCC in addition to its angiogenic effect, further underscoring the importance of VEGF in HCC.41 Overexpression of VEGF in HCC has been shown to correlate with tumor progression and poor prognosis.22,24,40

The evaluation of tumor VEGF expression requires tumor tissue, which may not be available in patients with HCC who are not candidates for resection or transplantation. Hence, there has been a great interest in the study of circulating VEGF as a surrogate marker of tumor VEGF expression. Previously, there has been debate regarding whether the circulating VEGF levels reflect tumor expression of VEGF.38 A recent study by our group has clearly demonstrated a positive quantitative correlation between serum VEGF levels and tumor expression of VEGF at either mRNA or protein levels in patients with HCC, indicating that serum VEGF level is a valid circulating biomarker of tumor VEGF expression.42 Our group subsequently showed that high preoperative serum VEGF level had a negative prognostic impact independent of conventional pathological parameters in patients undergoing resection of HCC.26 Another group also reported that high preoperative serum VEGF level was the most important adverse prognostic factor in patients undergoing resection.27 Furthermore, other studies have demonstrated that high pretreatment circulating VEGF levels predicted poor response and survival in patients undergoing transarterial chemoembolization for advanced HCC.28,29

The current study shows that high serum VEGF level was an independent unfavorable prognostic factor for both overall and recurrence-free survival in a group of patients with HCC undergoing RFA. To our knowledge, this is the first report on the prognostic significance of a circulating tumor biomarker in patients with HCC undergoing an ablative therapy. This study is also unique in that the prognostic significance of serum VEGF level was assessed in a group of patients with early HCCs ≤5 cm without macroscopic vascular invasion. Previous studies that evaluated the prognostic significance of circulating VEGF in patients with HCC undergoing resection or chemoembolization included a substantial proportion of patients with advanced HCC.26–29 In several types of cancers, tumor angiogenic activity has been shown to predict metastasis or postoperative recurrence in patients with apparently early disease by conventional staging, such as node-negative colon cancer,43 stage A testicular tumor,44 and thin malignant melanoma.45 Hence, assessment of tumor angiogenic activity may offer an additional prognostic marker for more refined prognostic classification in patients with apparently early disease staged by conventional criteria. Previous studies by our group and others also showed that high tumor microvessel density was an independent predictor of tumor recurrence in patients with resection of HCC ≤5 cm in diameter.46,47 The prognosis after resection or ablation of HCCs can vary considerably for tumors of the same size, and the biologic characteristics of the tumors are critical in determining the invasiveness and prognosis even for small HCCs.48 Our study suggests that serum VEGF may be a useful biomarker for more refined prognostic prediction in patients with small HCC treated by ablative therapy.

Thus far, there have been few studies on prognostic factors of long-term survival after RFA of HCCs.10,11,18,49,50 Poor liver function was commonly identified as an adverse prognostic factor in previous studies.10,18,49,50 Similar to the study of Xu et al.,18 our study identified low serum albumin ≤ 35 g/L as an unfavorable prognostic factor for overall survival in the multivariate analysis. As shown in this study, tumor recurrence rather than liver failure is the main cause of long-term deaths after RFA. Other studies have reported similar or even higher recurrence rate after RFA.9,10,17,18 Hence, the elucidation of prognostic effect of tumor factors on tumor recurrence and survival is important. However, there are discrepancies in the tumor features identified as prognostic factors in previous studies. Kim et al.17 reported that tumor size greater than or smaller than 3 cm was an important factor of tumor recurrence, whereas other studies found no prognostic influence of tumor size on overall or recurrence-free survival.18,49,50 Tumor multiplicity was found to be an important prognostic factor of overall survival in one study,10 but not in the other studies.17,18,49 Likewise, conflicting data regarding whether serum AFP level was a prognostic factor of overall or recurrence-free survival have been reported.11,17,18,49

In the current study, we did not observe any important prognostic influence of tumor size or serum AFP level on overall or recurrence-free survival. Furthermore, as reported by previous studies,25,27,51 there was no correlation between serum VEGF and AFP levels, indicating that VEGF is an independent tumor biomarker for HCC. In fact, a recent study showed that the combined use of serum AFP and VEGF increased the sensitivity of detection of early HCC from 68.2% to 95.5% compared with AFP alone.52 Tumor multiplicity was an unfavorable prognostic factor for both overall and recurrence-free survival in the univariate analyses of this study, but not in the multivariate analyses. Interestingly, HCV etiology of HCC was found to be a risk factor for worse recurrence-free survival in the multivariate analysis. A previous study on patients with small HCCs treated by hepatic resection revealed a higher risk of intrahepatic recurrence associated with HCV as compared with HBV etiology, presumably related to a higher risk of multicentric recurrence in HCV cirrhosis.53

A major problem in prognostic prediction after ablative therapy for HCC is the lack of histopathological features such as microscopic vascular invasion or intrahepatic metastasis, which are the most important prognostic factors after resection or transplantation for small HCC.20,21,53 The only possible histological feature that may be available is tumor differentiation if a core biopsy is performed. However, in clinical practice, most HCCs are ablated on the basis of diagnosis made by imaging findings combined with raised serum AFP level, or by fine needle aspiration cytology. Except for the purpose of a prospective study like this one, core biopsy is seldom performed because of the concern of increased risk of bleeding and tumor cell seeding. Even if it is performed, adequate tissue for accurate grading may not be available. Hence, tumor differentiation is not a pathological feature that can be relied on for prognostic prediction in most patients, and whether it has a prognostic significance in ablative therapy remains unclear. For the same reason, it is not possible to directly evaluate the tumor expression of VEGF or other biomarkers in most cases. Recently, the need of incorporating molecular biomarkers for better prognostic prediction after resection or transplantation of HCC has been emphasized.4,54 With the lack of histopathological information and the inconsistent prognostic significance of conventional tumor parameters reported in the literature, a circulating biomarker such as VEGF that may serve as a prognostic predictor is even more important in the case of ablative therapy.

In this study, pretreatment serum VEGF level was found to be the most important prognostic factors of both overall and disease-free survival. To further elucidate the prognostic significance of serum VEGF levels, we performed a detailed analysis of its influence on the patterns of recurrence in terms of site of recurrence and early recurrence within 1 year. The patients with high serum VEGF levels had far higher incidences of distant intrahepatic recurrence and extrahepatic recurrence than those with low serum VEGF levels, suggesting that serum VEGF levels may predict the presence of microscopic metastasis in the liver and distant organs. Furthermore, high serum VEGF levels predicted early recurrence within one year after RFA. Intrahepatic recurrences within one year of RFA are more likely metastatic in origin rather than multicentric recurrence according to the experience in hepatic resection.55 Several previous studies have consistently reported that high serum VEGF levels predict venous invasion and metastasis in patients with HCC.25,51,56,57 The incidence of venous invasion is known to be higher in large HCCs >5 cm in diameter, but even for small HCC ≤5 cm in diameter, microscopic venous invasion is present in approximately 30% of cases.58 Because microscopic venous invasion is the most important prognostic factor after resection or transplantation of small HCC,20,21,53 it is intuitive to speculate that it may also be the most important prognostic factor after ablation of small HCCs. As histological assessment for microscopic venous invasion is not available in ablative therapies for HCC, serum VEGF may be useful as a surrogate biomarker of microscopic venous invasion in such cases.

Although the current study suggests that pretreatment serum VEGF level may be a useful prognostic biomarker for ablative therapy of HCC, further studies with larger patient populations are needed to validate its prognostic value and to determine the optimum cuto3 value before clinical application is possible. Furthermore, the biological significance of circulating VEGF in cancer patients remains to be clarified. The positive correlation of serum VEGF levels with platelet count in this study is consistent with the findings of previous studies.25,42,51 This is attributable to the fact that most VEGF in the peripheral circulation is carried in the platelets, and the VEGF is released when the platelets are activated during blood clotting. It has been postulated that platelets may serve as a scavenger or storage of VEGF from the tumor, and the release of VEGF from the platelets at distant metastasis may play a central role in the process of hematogenous dissemination of cancers.38,59 More recently, there is evidence that circulating VEGF may have a role in recruiting VEGF receptor positive endothelial progenitor cells from bone marrow, which seem to contribute to tumor angiogenesis.60,61 In fact, a study from our group has demonstrated increased levels of bone marrow derived endothelial progenitor cells in the peripheral circulation of patients with advanced HCC, and a high correlation between serum VEGF levels and levels of circulating endothelial progenitor cells was observed.62

The findings of this study may have some therapeutic implications. The pretreatment serum VEGF levels may be useful in selecting patients at high risk of recurrence for adjuvant therapies or combination treatment with chemoembolization.15 Currently, there is no proven effective adjuvant therapy after ablation of HCC. The prognostic significance of serum VEGF suggests that antiangiogenic therapy targeting VEGF may be a potentially effective adjuvant therapy to curtail the high incidence of recurrence after ablation of HCC. An anti-VEGF monoclonal antibody has been approved for treatment of cancer patients.63 Promising results that use the anti-VEGF antibody in combination with systemic chemotherapy in treating advanced HCC have recently been reported.64 Furthermore, several VEGF receptor antagonists currently in clinical trials have been shown to inhibit growth and metastasis of HCC in preclinical studies.39 It is worthwhile to investigate the efficacy of such anti-VEGF therapies in the adjuvant setting to prevent recurrence after ablative therapy for HCC, and to study whether preoperative serum VEGF levels may help select patients who are more likely to benefit from such therapies.

In conclusion, this study shows that a high pretreatment serum VEGF level predicts poor overall and recurrence-free survival after RFA of small HCC ≤5 cm in diameter. To our knowledge, this is the first study to demonstrate the prognostic significance of a tumor biomarker in ablative therapy for HCC. This may be of particular value because of the unavailability of conventional histopathological prognostic features inherent to the ablative nature of the treatment.


   ACKNOWLEDGMENTS
 
Supported by a Competitive Earmarked Research Grant from Research Grant Council of Hong Kong (HKU7337/02M).

Received for publication November 27, 2006. Accepted for publication January 9, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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