Combination of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates cumulative recurrence of hepatocellular carcinoma

Journal of Hepatology 51 (2009) 315–321 www.elsevier.com/locate/jhep

Combination of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates cumulative recurrence of hepatocellular carcinomaq Hitoshi Yoshiji*, Ryuichi Noguchi, Masahisa Toyohara, Yasuhide Ikenaka, Mitsuteru Kitade, Kosuke Kaji, Masaharu Yamazaki, Junichi Yamao, Akira Mitoro, Masayoshi Sawai, Motoyuki Yoshida, Masao Fujimoto, Tatsuhiro Tsujimoto, Hideto Kawaratani, Masahito Uemura, Hiroshi Fukui Third Department of Internal Medicine, Nara Medical University, Shijo-cho 840, Kashihara, Nara 634-8522, Japan

Background/Aims: No chemopreventive agent has been approved against hepatocellular carcinoma (HCC) yet. Since neovascularization plays a pivotal role in HCC, an angiostatic agent is considered as one of the promising approaches. The aim of this study was to elucidate the combined effect of the clinically used vitamin K2 (VK) and angiotensin-converting enzyme inhibitor (ACE-I) on cumulative recurrence after curative treatment on a total of 87 patients, especially in consideration of neovascularization. Methods: VK (menatetrenone; 45 mg / day) and/or ACE-I (perindopril; 4 mg/day) were administered for 36–48 months after curative therapy for HCC. The cumulative recurrence and several indices were analyzed. Results: A 48-month follow-up revealed that the combination treatment with VK and ACE-I markedly inhibited the cumulative recurrence of HCC in association with suppression of the serum level of the vascular endothelial growth factor (VEGF); a central angiogenic factor. The serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with VEGF. These beneficial effects were not observed with single treatment using VK or ACE-I. Conclusions: The combination treatment of VK and ACE-I may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the VEGF-mediated neovascularization. Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Angiogenesis; Angiotensin-converting enzyme inhibitor; Hepatocellular carcinoma; Vascular endothelial growth factor; Vitamin K2

Received 22 January 2009; received in revised form 31 March 2009; accepted 5 April 2009; available online 15 May 2009 Associate Editor: J.M. Llovet q The authors declared that they do not have anything to disclose regarding funding from industries or conflict of interest with respect to the manuscript. * Corresponding author. Tel.: +81 744 223051x2314; fax: +81 744 247122. E-mail address: [email protected] (H. Yoshiji). Abbreviations: EC, endothelial cells; ACE, angiotensin-converting enzyme; ACE-I, angiotensin-converting enzyme inhibitor; AT-II, angiotensin-II; AFP-L3, lectin-reactive a-fetoprotein; HCC, hepatocellular carcinoma; IFN, interferon; PIVKA-II, des-gamma-carboxyprothrombin; RFA, percutaneous radiofrequency ablation; TACE, transarterial chemoembolization; VK, vitamin K2; VEGF, vascular endothelial growth factor.

1. Introduction Hepatocellular carcinoma (HCC) is now a major health problem. The incidence of HCC is increasing not only in Asia but also in Europe and the United States, and is currently the leading cause of death among cirrhotic patients. Although the recent advances in treatment of HCC have significantly improved the prognosis of patients with HCC, the overall survival rate is still unsatisfactory; the 5-year survival rate is only 50–70% even after curative treatment such as hepatic resection and percutaneous radiofrequency ablation (RFA) [1]. One of the reasons for the poor prognosis of HCC is

0168-8278/$36.00 Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2009.04.011

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its high rate of recurrence. Increasing evidence indicates that the high rate of recurrence, even after the curative therapy, is due to intrahepatic metastasis or multi-centric development of each respective neoplasm clone. Since the high-risk group of either primary or secondary HCC development seems to be clearer than in the other types of tumors, it is likely that chemopreventive agents will be beneficial in improving the prognosis of HCC. Some of the clinically available agents, such as interferon (IFN), reportedly exert a chemopreventive effect against primary and/or secondary HCC development [2,3]. Long-term administration, however, of these agents to cirrhotic patients sometimes leads to severe side effects, such as bone marrow suppression. Furthermore, the suppressive effect of IFN on the first recurrence of HCC was not significant. Moreover, the beneficial effect of IFN could be observed only in the virological responders [4,5]. Tumor growth, including HCC, is angiogenesisdependent. Any solid tumor that has not acquired its own new blood supply cannot grow to more than only a few millimeters in size [6,7]. Therapies targeting the tumor vessels have proven very successful for cancer treatment in experimental models [8]. We previously reported that angiogenesis plays a pivotal role in murine HCC development, and that suppression of the angiogenic-signaling pathway markedly attenuates HCC growth [9–11]. In addition to assessment of tumor growth, recent studies have demonstrated that angiogenesis has already begun at a very early stage when the tumor consists of just 100–300 cells [12]. Moreover, several reports have shown that angiogenesis is involved in the early steps of carcinogenesis [13–15]. A recent study on the endothelial cell markers in the dysplastic lesions of the liver has suggested that in HCC, alterations in the hepatic microcirculation already occur at a very early stage of liver carcinogenesis [16]. We also previously proved that angiogenesis plays a pivotal role in murine hepatocarcinogenesis [17]. Collectively, antiangiogenic agents are promising candidates for chemoprevention of HCC. Several anti-angiogenic agents have already been employed in clinical practice, including sorafenib [18]. Sorafenib is an oral multi-kinase inhibitor that acts against several tyrosine kinases (VEGFR1, PDGFR, ckit receptors), and serine/threonine kinases (b-Raf, p38) [19]. A recent report has revealed that sorafenib has survival benefits in patients at advanced stages of HCC, and that this drug will become the standard of cure in advanced stages of HCC [20]. However, there seem to be several serious concerns in employing this agent in the chemoprevention of HCC. As long-term administration is required and the drug metabolism is usually hypoactive in patients with cirrhosis, an agent that has been proven safe would be preferable for chemoprevention of HCC. Almost all patients show adverse

effects with sorafenib, and several symptoms are very severe [21,22]. Also, considering long-term administration, cost-performance is a problem since this drug is very expensive [23]. An alternative approach may be to find a clinically available compound that also exhibits anti-angiogenic activity for which the safety of longterm administration has been proven. We previously reported that the clinically used angiotensin-converting enzyme inhibitor (ACE-I) and vitamin K2 (VK) exert strong anti-angiogenic activities, and that the combination treatment of VK and ACE-I at clinically comparable low doses showed a marked suppressive effect against the development of HCC in rats via angiogenesis suppression [24]. We also previously reported that the combined treatment of ACE-I and VK ameliorated hepatic dysplastic nodules in one patient with liver cirrhosis [25]. In this study, we examined whether this combination regimen suppressed HCC development in clinical practice. We investigated the effects of ACE-I and VK on disease recurrence in patients with HCC after they had received curative RFA therapy.

2. Patients and methods 2.1. Patients and study design This study was conducted on a total of 100 patients with HCC who were admitted to our hospital between July 2002 and July 2005. A previous report showing the role of VK in the development of HCC in women with viral cirrhosis described 25 patients in each of the VK-treated and control groups [26]. Another study on VK and the recurrence of HCC was performed on a similar number of patients [27]. We referred to these studies to determine the suitable number of patients in each group for our current study. We first started the study to examine the effect of the combination treatment of ACE-I and VK, which is designated as ‘‘study-I”. As shown in the demographics of this study (Fig. 1), we first enrolled 60 patients. Among them, we were able to follow up with 50 patients in study-I. The patients were randomly divided into either the control group (Group 1, G1: n = 25) or combination-treated group (Group 2, G2: n = 25). We assigned the patients to either the treatment or control group using sealed envelopes. No placebo was used in G1. The patients in G2 were given oral ACE-I (perindopril: 4 mg/day) and VK (menatetrenone: 45 mg/day) continuously for 48 months. The respective dose of both compounds is a standard dose in clinical practice. We next started ‘‘study-II” one year after study-I to examine the effect of single treatment with VK or ACE-I. In study-II, we enrolled 40 patients. Among them, we were able to follow up with 37 patients until the end of the study (Fig. 1). The patients in Group 3 (G3: n = 19) and Group 4 (G4: n = 18) were given oral ACE-I or VK continuously for 36 months at the same doses as G2, respectively. The clinical profiles, laboratory data, and characteristics of the patients are shown in Table 1. Patients who received medication that could potentially influence the VK metabolism such as warfarin, and those who received other types of anti-hypertensive agents were excluded from this study. We recommended all patients to stop alcohol intake completely. Until the end of the observation of recurrence, no patients received any additional therapy for HCC, such as IFN. A definite diagnosis of HCC was made through a combination of several imaging modalities, such as hepatic angiography, enhanced computed tomography (CT), and magnetic resonance imaging (MRI). Since we did not encounter any unusual lesions in this study

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In this study, we employed the therapeutic modalities according to the algorithm of hepatocellular carcinoma treatment by the Liver Cancer Study Group of Japan (LCSGJ). The LCSGJ scoring system is a reliable system that is comparable to those of the Cancer of the Liver Italian Program (CLIP) and the Barcelona Clinic Liver Center (BCLC), and the evidence-based clinical practice guidelines for HCC in Japan supported by the Japanese Ministry of Health, Labor, and Welfare that have been recently published [28–30]. In this algorithm, the therapeutic strategy is chosen based on the degree of liver damage as determined by LCSGJ and the characteristics of the tumor itself. The indications of RFA of LCSGJ are three or fewer tumors measuring 3 cm or less, or a solitary tumor with a major axis of 5 cm or less. In our university, an inter-department conference is held every week. We discuss the selection of therapeutic modalities with surgeons and radiologists in each case of HCC. When RFA is selected in this conference, the patient is admitted to our department. After RFA, follow-ups were conducted with patients using enhanced CT and US once a month for the first three months. If any viable HCC was detected during this period, the patient was excluded from this study. All patients gave informed written consent before participating in this study. This protocol was approved by the Institutional Review Board (IRB) of Nara Medical University, and the study was conducted in compliance with ethical and humane principles. We carefully followed the CONSORT Statement for randomized studies to perform the current analysis.

2.2. Detection of HCC recurrence

Fig. 1. The study design profile for evaluation of the effects of ACE-I and VK on cumulative recurrence after curative therapy. The effect of combination treatment of ACE-I and VK was investigated in ‘‘study-I”. Among the enrolled 60 patients, 50 were randomly divided into either the control group (Group 1, G1: n = 25) or combination-treated group (Group 2, G2: n = 25). The patients in G2 were given oral ACE-I (perindopril: 4 mg/day) and VK (menatetrenone: 45 mg/day) continuously for 48 months. Study-II was conducted to examine the effect of single treatment using VK or ACE-I. Among the enrolled 40 patients, 37 were divided into either the ACE-I-treated group 3 (G3: n = 19) or VKtreated group (G4: n = 18). The patients in G3 and G4 were given oral ACE-I or VK continuously for 36 months at the same doses as G2, respectively. We were able to conduct follow-ups with all enrolled patients in each group for analysis.

that require needle biopsy for histological confirmation, we did not have any histological proof of the diagnosis of patients in both study-I and study-II.

Follow-ups were conducted with all patients every 4 weeks at our hospital. To find the recurrent HCC nodules, imaging studies such as US and CT were performed every 3 months. The serum tumor markers; namely, a-fetoprotein (AFP), lectin-reactive AFP (AFP-L3), and des-gamma-carboxyprothrombin (PIVKA-II), were measured every 2 months with routine laboratory methods. We also examined the alteration of the vascular endothelial growth factor (VEGF, a central angiogenic factor) [31] in the serum prior to and at 12 months after starting drug administration using an enzyme-linked immunosorbent assay (ELISA) kit (R & D systems), according to the manufacturer’s instructions as described previously [17]. If any recurrence was detected, the patient was excluded from the protocol. Immediately, such patients were treated as having secondary HCC according to the algorithm of LCSGJ. We conducted follow-ups with patients every month during the observation period, and all recurrent cases met the aforementioned RFA criteria (namely, three or fewer tumors: <3 cm or a solitary tumor: <5 cm). Accordingly, all patients with the first recurrence were treated with RFA. Nevertheless, several patients with a secondary recurrence received transarterial chemoembolization (TACE) and/or arterial infusion chemotherapy afterward. Liver transplantation is uncommon in Japan for patients with recurrent HCC.

Table 1 Demographic characteristics of the enrolled patients. Characteristics

Control (G1)

Combination (G2)

ACE-I (G3)

VK (G4)

No of patients Age (range) (yrs) Gender (M/F) Etiology (HCV/HBV/other) Alcohol (<40 g/>40 g/day) Tumor stage (I/II/III) Tumor mean size (mm) No of tumors AFP (ng/ml) PIVKA-II (mAU/ml) ALT (IU/l) Child–Pugh (A/B)

25 60.5 ± 8.5a 17/8 1/3/1 12/13 13/10/2 18.7 ± 9.5 1.59 ± 0.92 88.5 ± 253.3 41.3 ± 66.8 66.8 ± 31.5 20/5

25 63.4 ± 7.6 (0.217)b 15/7 (0.989) 19/2/1 (0.754) 10/12 (0.861) 10/11/1 (0.495) 16.9 ± 8.6 (0.381) 1.45 ± 0.87 (0.513) 101.3 ± 452.5 (0.906) 66.5 ± 82.6 (0.254) 62.3 ± 42.6 (0.684) 19/3 (0.562)

19 59.4 ± 8.3 (0.466) 10/9 (0.869) 15/1/1 (0.736) 10/9 (0.783) 8/9/2 (0.519) 20.1 ± 8.0 (0.230) 1.50 ± 0.88 (0.433) 92.4 ± 303.1 (0.804) 58.8 ± 71.2 (0.399) 74.8 ± 36.6 (0.527) 16/3 (0.722)

18 62.8 ± 7.4 (0.282) 10/8 (0.808) 15/0/3 (0.818) 8/10 (0.688) 10/7/1 (0.781) 17.9 ± 9.2 (0.454) 1.62 ± 0.9 (0.471) 79.8 ± 221.4 (0.792) 60.2 ± 64.6 (0.451) 56.6 ± 28.8 (0.610) 16/2 (0.441)

a b

The data represent means ± SD. p Value as compared with the control group.

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2.3. Statistical analysis The variables of the characteristics of enrolled patients were analyzed with the Mann–Whitney U test and the Fisher exact probability test. The cumulative recurrence of HCC was plotted using the Kaplan– Meier method, and the differences in recurrence curves were tested using the log-rank test.

3. Results 3.1. Patients’ characteristics The characteristics of the enrolled patients are shown in Table 1. There were no significant differences among the patients of all groups in age, gender, etiology of the disease, or background liver function (Child–Pugh score). Also, the tumor baseline, such as stage, number of tumors, AFP, AFP-L3, PIVKA-II levels, was no different among the groups. Both ACE-I and VK are widely used without serious adverse effects, and the safety of their long-term administration has been proven in clinical practice. In this study, there were no severe toxic effects in either group, and no abnormal laboratory data were found that could likely be related to treatment with either ACE-I or VK. Accordingly, after randomization, we could follow up with all enrolled patients in each group until the detection of recurrence. 3.2. HCC recurrence The results of study-I are shown in Fig. 2. The combination treatment of ACE-I and VK (G2) significantly suppressed the cumulative recurrence of HCC as compared with the control group (G1). The cumulative recurrence rates of HCC in G1 at 12, 24, 36, and 48 months were 24% (6/25), 48% (12/25), 68% (17/25), and 72% (18/25), respectively. On the other hand, the corresponding cumulative recurrence rates in G2 were 12% (3/25), 28% (7/25), 32% (8/25), and 36% (9/25), respectively. We compared several markers among the enrolled patients following the 12-month treatment (Table 2). Administration of VK is known to decrease the PIVKA-II level, and we noticed the suppression of the PIVKA-II level at a similar magnitude in the combination- and VK-treated groups. The mean blood pressure decreased in the combination- and ACE-I-treated groups, but there was no significant difference between the groups. Also, there were no significant differences in the ALT level between the combination-treated and other groups, indicating that the suppressive effect of this combination treatment on cumulative recurrence was not due to anti-hypertensive and cytoprotective activities. We next examined whether or not the alteration of AFP and AFP-L3 levels correlated with the inhibitory effect on cumulative recurrence of HCC in

Fig. 2. The cumulative recurrence of secondary HCC after curative therapy by the combination treatment of ACE-I and VK for 48 months. The combination treatment of ACE-I and VK (G2: n = 25) significantly suppressed the cumulative recurrence of HCC compared with the control group (G1: n = 25). The cumulative recurrence rates of HCC in G1 at 12, 24, 36, and 48 months were 24% (6/25), 48% (12/25), 68% (17/25), and 72% (18/25), respectively. On the other hand, the corresponding cumulative recurrence rates in G2 were 12% (3/25), 28% (7/25), 32% (8/25), and 36% (9/25), respectively. *Statistically significant differences between the indicated groups (p < 0.01).

the combination-treated group. Before drug administration, the serum levels of AFP and AFP-L3 were no different between the combination-treated and control groups. No marked alteration was observed in the total AFP level during the 12-month period of administration in the combination-treated or control groups. On the other hand, the combination treatment of ACE-I and VK markedly attenuated the AFP-L3 level compared with that before drug administration, whereas the control group showed a significant increase after 12 months (Table 2). Since neovascularization reportedly plays a pivotal role in hepatocarcinogenesis, we next measured the VEGF expression level by ELISA in all patients of G1 and G2. The serum level of VEGF in G1 increased after 12 months, whereas the combination treatment of ACEI and VK (G2) markedly attenuated the VEGF level compared with that before administration (Fig. 3). Noteworthy was the finding that the alteration pattern of VEGF matched the results of AFP-L3. In study-II, contrary to the marked inhibitory effect seen in G2, single treatment with ACE-I (G3) or VK (G4) did not exert such an inhibitory effect during the 36-month follow-up compared with the control group (G1) (Fig. 4). The cumulative recurrence rates of HCC in G1 at 12, 24, and 36 months were 24% (6/25), 48% (12/25), and 68% (17/25), respectively. The corresponding cumulative recurrence rates in G3 were 16% (3/19), 37% (7/19), and 52% (10/19), respectively. In G4, the

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Table 2 Changes of several markers in the enrolled patients following 12-month treatment. Control (G1)

Mean BP (mm Hg) AFP (ng/ml) AFP-L3 (%) PIVKA-II (mAU/ml) ALT (IU/l) a

Combination (G2)

ACE-I (G3)

VK (G4)

Before

After

Before

After

Before

After

Before

After

130 ± 16a 88.5 ± 253.5 18.4 ± 3.6 41.3 ± 66.8 66.8 ± 31.5

128 ± 14 97.4 ± 1201.3 25.2 ± 6.1c 50.8 ± 60.1 67.4 ± 22.8

136 ± 18 101.3 ± 452.5 20.1 ± 5.2 66.5 ± 82.6 62.3 ± 42.6

124 ± 12b 88.7 ± 431.6 12.6 ± 3.1c 8.4 ± 4.2c 54.9 ± 33.1

136 ± 20 92.4 ± 303.1 17.8 ± 4.0 58.8 ± 71.2 74.8 ± 36.6

122 ± 13b 86.3 ± 251.2 14.7 ± 2.6b 49.7 ± 51.2 60.2 ± 34.4

141 ± 17 79.8 ± 221.4 16.8 ± 3.8 60.2 ± 64.6 56.6 ± 28.8

134 ± 15 70.4 ± 201.1 15.0 ± 2.9 7.6 ± 3.9 48.8 ± 30.3

The data represent means ± SD. Statistically significant as compared with before treatment (p < 0.05 and <0.01, respectively).

b, c

Fig. 3. The effect of combination treatment of ACE-I and VK on the serum VEGF level. The serum VEGF level in the control group (G1) increased after 12 months, whereas the combination treatment of ACE-I and VK (G2) markedly attenuated the VEGF level compared with the level before drug administration. (A) The VEGF level before and after administration of the combination treatment for 12 months. (B) Percentage of change in the VEGF level in each group. The data represent means ± SD (n = 25). *Statistically significant differences between the indicated groups (p < 0.01).

corresponding cumulative recurrence rates were 22% (4/ 18), 44% (7/18), and 61% (11/18), respectively. Although the primary end-point of this study was the cumulative recurrence rates, we also examined whether the survival curves of the patients were altered or not during the follow-up period. As shown in Fig. 5, no statistical differences could be observed among the groups.

4. Discussion

Fig. 4. The cumulative recurrence of secondary HCC after curative therapy by single treatment with either ACE-I or VK for 36 months. Single treatment with either ACE-I (G3: n = 19) or VK (G4: n = 18) did not exert an inhibitory effect on the cumulative recurrence of HCC compared with the control group (G1: n = 25). The cumulative recurrence rates of HCC in G1 at 12, 24, and 36 months were 24% (6/25), 48% (12/ 25), and 68% (17/25), respectively. The corresponding cumulative recurrence rates in G3 were 16% (3/19), 37% (7/19), and 52% (10/19), respectively. In G4, the corresponding cumulative recurrence rates were 22% (4/18), 44% (7/18), and 61% (11/18), respectively.

In this study, we showed that the combination treatment with ACE-I and VK significantly suppressed the cumulative HCC recurrence after curative therapy along with suppression of VEGF and AFP-L3. Although a previous report has shown that VK decreased the recurrence rate of HCC [27], a recent multi-center Japanese clinical trial demonstrated that single treatment of VK failed to exert any inhibitory effect on the cumulative recurrence of HCC. Similarly, we did not observe any significant inhibitory effect of VK on the cumulative recurrence in this study. We also found that ACE-I mono-therapy could not exert such an inhibitory effect,

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In this study, we could not find a marked difference in the survival curves of the groups. Since the follow-up period of the current study might not have been long enough, no statistical differences could be observed among the groups. Further long-term and large-scale studies are also required to verify whether or not the suppressive effects of ACE-I and VK on cumulative recurrence will improve the prognosis in the future. In conclusion, we have shown that the combination treatment of ACE-I and VK significantly attenuated the cumulative recurrence of HCC after curative therapy along with the suppression of VEGF and AFP-L3. Although a large-scale long-term follow-up clinical control study is required, this combined treatment may represent a potential new strategy for secondary chemoprevention of HCC since both agents are widely used in clinical practice without serious side effects. Fig. 5. The Cumulative survival rates in all groups. No significant differences could be observed among the groups during the follow-up period. G1: control group (n = 25). G2: ACE-I and VK combined-treated group (n = 25). G3 and G4 received single treatment with either ACE-I (n = 19) or VK (n = 18), respectively. The follow-up period was 48 months for G1 and G2 and 36 months for G3 and G4.

either. Several recent studies have suggested that treatment using a single anti-angiogenic agent may not be sufficient to completely inhibit the tumor angiogenesis [32,33]. Recently, it has been reported that although the anti-angiogenic activity of single agents was negligible, the combination treatment with different angiostatic agents showed remarkable synergistic inhibitory effects on pathological neovascularization [34]. Since the actions of ACE-I and VK are mediated via different anti-angiogenic pathways [35], it is possible that the coordination of these different biological activities produced the combined suppressive effect of ACE-I and VK; an effect that is not present when either single agent is used. We also observed that this combination regimen attenuated the AFP-L3 level increment but not the total AFP level. Among the isoforms of AFP, AFP-L3 is known to indicate the presence of latent HCC cells in the cirrhotic liver, and the serum AFP-3 level is reportedly a useful biomarker in the chemoprevention of HCC [36,37]. Since the alteration pattern of VEGF matched the results of AFP-L3, the attenuation of AFP-L3 by the combination treatment with ACE-I and VK may reflect the suppression of hepatic neovascularization at an early stage of secondary hepatocarcinogenesis. However, we did not find any direct histological evidence of the alteration of neovascularization in the liver. Although the serum level of VEGF reportedly reflects the intrahepatic VEGF expression level in patients with chronic liver diseases [38], a chronological immunohistochemical observation would be required to confirm whether the alteration of VEGF really reflects intrahepatic neovascularization in the future.

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