Abstract
The aim of this study was to investigate whether menatetrenone (MNT) suppresses hepatocellular carcinoma (HCC) recurrence in patients undergoing hepatectomy. Between January 2005 and September 2009, 101 patients who underwent curative hepatectomy for primary HCC were enrolled in the study. Patients were divided into two groups: a non-MNT group (n=51), and an MNT group (n=50) that was administered 45 mg of MNT daily. During the observation period, recurrence was observed in 33 patients in the non-MNT group and in 28 patients of the MNT group (p=0.545). In patients with a preoperative Des-γ-carboxy-prothrombin (DCP) level lower than 40 AU/l (n=38), the cumulative disease-free survival rates at 12, 36, and 60 months in the non-MNT group, were 81.3%, 0.0%, and 0.0%, respectively, while those in the MNT group were 78.3%, 58.1%, and 31.0%, respectively (p=0.060). MNT has a moderately suppressive effect on HCC recurrence after hepatectomy, especially in patients with a normal preoperative DCP level.
- Des-γ-carboxy-prothrombin
- disease-free survival
- hepatocellular carcinoma recurrence
- menatetrenone
- vitamin K2 analog
Hepatocellular carcinoma (HCC) is a major cause of death worldwide. Surgical resection is the most reliable therapeutic modality, however, it is still associated with a high incidence of postoperative recurrence (1). Although some chemotherapeutic agents, and a molecular targeting agent, are being used to inhibit the recurrence of HCC, results, so far. have not been satisfactory (2).
Vitamin K is a fat-soluble vitamin that acts as a co-enzyme of carboxylase, which produces clotting factors. Carboxylase catalyzes the formation of γ-carboxyglutamic acid from glutamic acid residues (3). Because γ-carboxyglutamic acid is a major component of the bone matrix, vitamin K has been used for the treatment of osteoporosis.
Abnormal uncarboxylated prothrombin (Des-γ-carboxy-prothrombin; DCP), also known as protein induced by vitamin K absence or antagonist II (PIVKA-II), is increased in the serum of patients with HCC (3). Administration of vitamin K reduces the serum concentration of DCP. Recently, it has been suggested that a vitamin K2 analog, menatetrenone (MNT), would be therapeutically useful for suppressing the development and recurrence of HCC (4-6). However, no previous studies have assessed whether MNT suppresses the recurrence of HCC in patients undergoing surgical resection as the only therapeutic modality. To clarify this issue, we conducted a prospective randomized controlled trial.
Patients and Methods
Patients. Patients who had undergone curative hepatectomy for the first time under a preoperative diagnosis of HCC, and whose surgical specimens had been histologically confirmed to be HCC, were recruited for this study. Patients who had been taking warfarin or other vitamin K analogs were excluded.
Study design. Patients were recruited between September 2005 and October 2009, and gave their written informed consent prior to study participation. The study, approved by the Ethics Committee of Dokkyo Medical University Hospital, was registered to the University Hospital Medical Information Network (UMIN, number 000001815), and conducted in accordance with the Declaration of Helsinki. Follow-up periods ranged from 17 to 68 months (median=34 months).
Sample size calculation. Recurrence rates after hepatectomy at our Department were 37.6% at 1 year, 68.8% at 3 years, and 80.7% at 5 years. We assumed that MNT would reduce the recurrence rate by 50% in each of the above periods. Use of the nQuery Advisor 6.01 software package (Saugus, MA, USA) revealed that both the control group and the study group required at least 48 patients each.
Trial profile.
Treatment allocation. After histological diagnosis of HCC, the patients were stratified according to four parameters: age (65 years or less), sex (male or female), surgical method (anatomical or non-anatomical hepatectomy), and the Japan Integrated Staging Score (JIS; 0 and 1, or 2 and 3). Anatomical hepatectomy included lobectomy, segmentectomy, and sub-segmentectomy, and non-anatomical hepatectomy included partial resection of the liver. The JIS was calculated from the Child-Pugh score and the TMN classification (7).
Following recruitment, the patients were randomly assigned to two groups: a non-MNT (control) group who were not given MNT, and an MNT group who were administered 15 mg of MNT (Eisai, Tokyo, Japan) three times a day, orally, every day. In the non-MNT group, no placebo was used.
Follow-up principle. The primary end-point of the study was recurrence of HCC after surgical resection. Screening for recurrence was conducted by ultrasonography and computed tomography, which were performed two months after hepatectomy and every three months thereafter.
Statistical analysis. Differences in proportions between the two groups were evaluated using the χ2 test. Continuous data were analyzed using the Student's t-test for means, or the Mann–Whitney U-test for medians. Mean values are expressed along with standard deviation, and median values are expressed with the range. The disease-free survival curves obtained according to the Kaplan–Meier method were compared by log-rank test. Differences at p<0.05 were regarded as being statistically significant.
Results
Among 109 potentially eligible patients, 105 were randomized for group allocation (Figure 1). Among the 54 patients allocated to the MNT group, three developed skin erythema and one newly-developed cerebral infarction; these four patients were excluded from the study. Among patients who completed the study protocol, 51 were allocated to the non-MNT group and 50 to the MNT group.
Baseline demographics of the patients are shown in Table I. There were no significant inter-group differences in age, sex, presence of viral infection, presence of cirrhosis, Child-Pugh classification, JIS, ICG R15, total bilirubin, AFP, or DCP.
Table II shows the operative data. There were no significant inter-group differences in operative time, intraoperative blood loss, Pringle time, surgical method, number of tumors, or maximum tumor diameter.
Pathological findings are shown in Table III. There were no significant differences between the two groups in terms of tumor differentiation, Edmonson classification, presence of a tumor capsule, vessel invasion, intrahepatic metastasis, or tumor stage.
During the observation period, recurrence was observed in 33 patients (64.7%) in the non-MNT group and in 28 patients (56.0%) of the MNT group (p=0.545).
Figure 2A shows the cumulative disease-free survival rates in the two groups. Those at 12, 24, 36, 48, and 60 months in the non-MNT group were 70.6%, 49.6%, 26.3%, 26.3%, and 26.3%, respectively, whereas those in the MNT group were 71.9%, 51.9%, 44.5%, 33.0%, and 26.4%, respectively (p=0.483).
Among patients with a preoperative DCP level lower than 40 AU/l, recurrence was observed in 12 out of 16 (75.0%) in the non-MNT group and in 9 out of 22 (40.9%) in the MNT group (p=0.052). The cumulative disease-free survival rates at 12, 24, 36, 48, and 60 months in the non-MNT group were 81.3%, 44.7%, 0.0%, 0.0%, and 0.0%, respectively, whereas those in the MNT group were 78.3%, 66.4%, 58.1%, 40.0%, and 31.0%, respectively (p=0.060). Among patients with a preoperative DCP level equal to or higher than 40 AU/l, recurrence was observed in 22 out of 35 (62.9%) in the non-MNT group and in 18 out of 28 (64.3%) of the MNT group (p=1.000). The cumulative disease-free survival rates at 12, 24, 36, 48, and 60 months in the non-MNT group were 65.7%, 51.1%, 35.7%, 35.7%, and 35.7%, respectively, whereas those in the MNT group were 64.3%, 38.4%, 32.0%, 32.0%, and 21.3%, respectively (p=0.511).
Next, we evaluated the disease-free recurrence in patients who were hepatitis C virus (HCV)-positive without hepatitis B virus (HBV) positivity. Such patients accounted for 15 of the 51 in the non-MNT group and 14 out of the 50 in the MNT group. The disease-free survival rates of these patients at 12, 24, 36, 48, and 60 months were 93.3%, 60.0%, 17.1%, 17.1%, and 17.1% in the non-MNT group, and 71.4%, 40.8%, 40.8%, 27.2%, and 27.2% in the MNT group, respectively (p=0.637).
Figure 3 shows the overall survival rates in the two groups. The rates at 12, 24, 36, 48, and 60 months in the non-MNT group were 90.2%, 86.2%, 77.1%, 73.3%, and 73.3%, respectively, whereas those in the MNT group were 98.0%, 89.4%, 89.4%, 89.4%, and 89.4%, respectively (p=0.137).
Discussion
The present study represents the first randomized controlled trial aimed at evaluating the preventive effect of MNT on the recurrence of HCC in patients undergoing surgical resection-alone. The results revealed that MNT had a moderate, but non-significant, suppressive effect on HCC recurrence after surgical resection.
Baseline characteristics of patients.
Mizuta et al. (4) were the first to report the suppressive effect of MNT on HCC recurrence. In their study, they found that MNT significantly reduced the rate of HCC recurrence at 36 months after curative treatment. However, the treatment of the enrolled patients included not only surgical resection but also locoregional therapies, such as percutaneous ethanol injection and percutaneous radiofrequency ablation, in contrast to our present study, where all patients underwent curative surgical resection-alone. In the study by Mizuta et al., the 1, 2, and 3-year recurrence rates in the patients given MNT, were 12.5%, 20.6%, and 39.0%, whereas the corresponding rates in the present study were 20.6%, 36.9% and 43.6%, respectively. On the other hand, the corresponding rates for patients in the control groups were 55.2%, 83.2%, and 91.6% in the former study, and 30.0%, 52%, and 76.1% in the present one, respectively. Thus, the recurrence rate in the control group of the former study was higher than that in our present control group.
Operative data.
In our study, the disease-free survival rates in the non-MNT and MNT groups were similar until 15 months after surgery, but thereafter began to show an evident difference (arrow in Figure 1). We speculated that patients who suffered recurrence within 15 months after surgery might have included some who had intrahepatic metastasis or more invasive HCC. Therefore, we analyzed the cumulative disease-free survival rates in terms of the preoperative DCP levels (Figure 2B), as previous studies had indicated that DPC levels are a factor predictive of vessel invasion by HCC (4, 8). When the disease-free survival rate was re-evaluated by setting the cut-off preoperative DCP level at 40 AU/l (9), the difference between the non-MNT and MNT groups became more significant (Figure 2C). In patients with a preoperative DCP level lower than 40 AU/l, the disease-free survival rate tended to be better in the MNT group than in the non-MNT group.
Kakizaki et al. (5) investigated the effect of MNT on HCC recurrence exclusively in HCV-infected patients for up to 36 months, and found that it suppressed cancer recurrence significantly. As had been the case in the study conducted by Mizuta et al. (4), the enrolled patients underwent not only surgical resection but also locoregional therapy, and the recurrence rates in the control group were higher than those in our present control group. In our study, there were no significant differences in the disease-free survival rates between the non-MNT and MNT groups in patients who were HCV-positive but not HBV-positive (Figure 2D). Although it has been suggested that persistent HCV infection without associated HBV infection induces a chronic inflammatory state in the liver, and that MNT might prevent recurrence of HCC in such patients, we found no significant beneficial effect of MNT.
In a randomized controlled trial to assess the suppressive effect of MNT on HCC recurrence, after treatments that included surgical resection, transarterial chemoembolization, and percutaneous radiofrequency ablation, Hotta et al. (6) found no significant benefit of MNT.
Histological findings.
In another randomized controlled trial, Yoshiji et al. (10) found that combined administration of MNT along with angiotensin-converting enzyme inhibitor significantly decreased the 4-year recurrence rate of HCC after curative therapies including surgical resection, transcatheter arterial chemoembolization, and percutaneous radiofrequency ablation. They suggested that the anti-angiogenetic effect of the two drugs synergistically inhibited HCC recurrence. Another study has advocated the combined use of vitamin K and the multikinase inhibitor, sorafenib, for inhibiting the growth of HCC cells (11).
Cumulative disease-free survival rates. A: The cumulative disease-free survival rates at 12, 24, 36, 48, and 60 months in the non-menatetrenone (MNT) group were 70.6%, 49.6%, 26.3%, 26.3%, and 26.3%, respectively, whereas those in the MNT group were 71.9%, 51.9%, 44.5%, 33.0%, and 26.4%, respectively (p=0.483). B: In patients with a preoperative Des-γ-carboxy-prothrombin (DCP) level lower than 40 AU/l, the cumulative disease-free survival rates at 12, 24, 36, 48, and 60 months in the non-MNT group (squares) were 81.3%, 44.7%, 0.0%, 0.0%, and 0.0%, respectively, whereas those in the MNT group (triangles) were 78.3%, 66.4%, 58.1%, 40.0%, and 31.0%, respectively (p=0.060). C: In patients with a preoperative DCP level higher than 40 AU/l, the cumulative disease-free survival rates at 12, 24, 36, 48, and 60 months in the non-MNT group were 65.7%, 51.1%, 35.7%, 35.7%, and 35.7%, respectively, whereas those in the MNT group were 64.3%, 38.4%, 32.0%, 32.0%, and 21.3%, respectively (p=0.511). D. cumulative disease-free survival rates in patients positive for only HCV, and not for HBV. The disease-free survival rates at 12, 24, 36, 48, and 60 months for these patients were 93.3%, 60.0%, 17.1%, 17.1%, and 17.1% in the non-MNT group, and 71.4%, 40.8%, 40.8%, 27.2%, and 27.2% in the MNT group, respectively (p=0.637).
In vitro, MNT suppresses the growth of HCC cells by arresting the cell cycle in the G1-phase (12), through inhibition of Ikappa B (IκB) kinase activity and suppression of IκB phosphorylation and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, followed by inhibition of NF-κB-dependent cyclin-D1 expression. Another study has shown that in HCC cells, vitamin K2 suppressed the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases, resulting in inhibition of the expression of matrix metalloproteinase-1, -3 and -7 (13).
In the present study, MNT did not significantly reduce the recurrence rate of HCC after surgical resection; the disease-free survival rates in the MNT group tended to be higher than those in the non-MNT group until four years after surgical resection, especially in patients with a normal preoperative DCP level. Between 15 months and 4 years after surgical resection, multicentric recurrence may occur, and this may have been suppressed by administration of MNT.
Overall survival rates. The overall survival rates at 12, 24, 36, 48, and 60 months in the non-MNT group (squares) were 90.2%, 86.2%, 77.1%, 73.3%, and 73.3%, respectively, whereas those in the MNT group (triangles) were 98.0%, 89.4%, 89.4%, 89.4%, and 89.4%, respectively (p=0.137).
MNT is not a chemotherapeutic agent and does not have an aggressive cytotoxic effect on cancer cells. Although it is suggested that MNT may not be effective for preventing recurrence of HCC in the long term when used as a single adjuvant agent after surgery, it appears to have a moderately suppressive effect on HCC recurrence between 15 months and 4 years after surgical resection, especially in patients with a normal preoperative DCP level. Since MNT is already a safe and well-established drug for the treatment of osteoporosis, we recommend that MNT should be considered as an adjuvant agent for prevention of HCC recurrences after surgery.
Footnotes
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Conflicts of Interest
There are no conflicts of interest to declare.
- Received September 4, 2012.
- Revision received October 21, 2012.
- Accepted October 23, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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