Research ArticleClinical Studies

NX-PVKA Levels Before and After Hepatectomy of Hepatocellular Carcinoma as Predictors of Patient Survival: A Preliminary Evaluation of An Improved Assay for PIVKA-II

ATSUSHI NANASHIMA, TAKAFUMI ABO, NAOTA TAURA, HIDEKI SHIBATA, TATSUKI ICHIKAWA, KATSUNORI TAKAGI, JUNICHI ARAI, SHOUSABUROU OYAMA and TAKESHI NAGAYASU
Anticancer Research June 2013, 33 (6) 2689-2697;

Abstract

Although the protein-induced vitamin K absence or antagonist-II (PIVKA-II) is used as a prognostic marker in hepatocellular carcinoma (HCC), a newly-improved assay, NX-PVKA (PIVKA-II measured using P-11 and P-16 antibodies) and NX-PVKA-R (ratio of PIVKA-II and NX-PVKA), are more accurate markers of PIVKA-II. We conducted a prospectively preliminary analysis of the relationship between NX-PVKA-R and clinicopathological parameters and prognosis in 22 patients with HCC who underwent hepatectomy and measured changes of this marker's levels after treatment. Median value of PIVKA-II (80 mAU/ml), NX-PVKA (60 mAU/ml), NX-PVKA-R (1.5) and NX-PVKA-D (difference of markers, 15 mAU/ml) were determined. Tumor relapse was observed in six patients, and the one year relapse-free survival rate was 88%. Correlation between PIVKA-II or alpha-fetoprotein levels and NX-PVKA, NX-PVKA-R or -D levels was significant (p<0.001). NX-PVKA-R was significantly correlated with tumor size (p<0.05). In patients who underwent pre-treatment before hepatectomy, PIVKA-II, NX-PVKA and NX-PVKA-R tended to be higher than in patients without pre-treatment, but this difference was not significant (p>0.10). For macroscopic findings, NX-PVKA-R for the confluent-nodular type was significantly higher than that for the simple-nodular type (p<0.05). The tumor-free survival rate in the group with a high NX-PVKA-R was significantly lower than that in the group with a low NX-PVKA-R group (p<0.05). In patients with tumor recurrence, postoperative NX-PVKA-R increased again. We conclude that a high value of NX-PVKA-R after hepatectomy for HCC reflects malignant potential and predicts early recurrence in patients with HCC.

Hepatocellular carcinoma (HCC) is a common malignant disease worldwide and patient prognosis has recently improved due to advances in treatment modalities (1, 2). Sensitive diagnostic markers are necessary to diagnose HCC and to estimate the biological malignant behavior and prognosis. Alpha-fetoprotein (AFP) is a classical marker for HCC and, subsequently, protein-induced by vitamin K absence or antagonist-II (PIVKA-II; des-γ-carboxy prothrombin) is a more specific marker for the diagnosis of HCC, as it reflects tumor aggressiveness and prognosis (3, 4). Specificity for diagnosis of HCC by PIVKA-II was higher than that of AFP and was not influenced by the degree of chronic hepatitis or cirrhosis (5). However, PIVKA-II levels are influenced by other factors, such as a lack of vitamin K, use of anti-coagulants, or biliary occlusion (6). Recently, the number of elderly patients with HCC has increased and the prevalence of co-morbidity, such as coronary or cerebral diseases, has also increased, including our institute as well (7, 8). These patients are often administrated anti-coagulants, and therefore the results of PIVKA-II tests are not reliable. To increase specificity in all situations, improvement of this assay is necessary.

There are various types of PIVKA-II resulting from variations in the number of glutamine residues, and these variations are different in patients with increased PIVKA-II, due to HCC and those who have been administered vitamin K antagonists (9). Based on these differences, an improved PIVKA-II assay has been developed, which detects the variants of des-γ-carboxy prothrombin (DCP) with fewer glutamine-acid residues, using combined P-11 and P-16 antibodies to PIVKA-II (NX-PVKA) (Sekisui Medical Co., Ltd., Tokyo, Japan). According to the company's results and a previous report (10), NX-PVKA was not increased in patients treated with a vitamin K antagonist. Therefore, NX-PVKA is a promising tool for diagnostic purposes. Previous reports, including our study, indicated that normalization of initially high PIVKA-II correlated with better prognosis (11-13). Increased PIVKA-II levels, during follow-up after treatments, are associated with tumor recurrence and poor survival, and thus might be useful to predict tumor recurrence (4, 6, 14).

In the present study, we prospectively performed a preliminary analysis measuring NX-PVKA before and after hepatectomy in 22 patients with HCC in order to evaluate the clinical significance in HCC treatment.

Materials and Methods

Patients. Data of 22 patients with HCC were collected. These patients were diagnosed with HCC at the Division of Surgical Oncology, Nagasaki University Hospital (NUH) between 2011 and 2012. All hepatic tumors were completely resected without macroscopic exposure of the resected section of the liver. The patients were followed-up every three months, and enhanced computed tomography of the liver was obtained every six months after hepatectomy to detect tumor recurrence. The minimum follow-up period after hepatic resection of HCC was three months (range=3-24 months). All patients survived and attended a follow-up visit. Six (27%) patients developed tumor recurrence after treatment. The study design was approved by the Ethics Review Board of NUH including collection of data from the medical records approval. The role of the Authors was as follows: Dr A.N. as a main author organized the entire research, Dr. T.A., Dr. K. T, and Dr. J.A. as the staff members of the liver surgery group collected data on NX-PVKA, Dr. N.T. as a liver physician managed patients before and after surgery and collected data on NX-PVKA, and Professor T.N. as a supervisor managed all clinical research in the Department of Surgery.

Measurement of serum samples. A 4-ml peripheral blood sample was collected from each patient before and every three months after hepatectomy. The sample was then centrifuged at 3,000 rpm (1,000 ×g) for 10 min. PIVKA-II was assayed by an enzyme-linked immunoassay using Eitest® PIVKA-II (Sanko Junyaku Co., Tokyo, Japan). The normal value of AFP for HCC in our hospital is less than 20 ng/ml. The reported normal value of PIVKA-II is <40 mAU/ml (14, 15). Elevating of PIVKA-II was defined as those exceeding this level.

P-16 monoclonal antibody-coated magnetic beads were prepared by the following procedure: 1 ml of 30 mg/ml magnetic bead suspension (Dynabeads M-450 Epoxy; Life Technologies Corporation, CA, USA) was placed into a test tube and the magnetic beads were trapped by a magnet in order to discard the supernatant. After the supernatant was discarded, 1 ml of the P-16 monoclonal antibody (0.5 mg/ml in 0.15 mol/l phosphate buffered saline, pH 7.8; Sekisui Medical Co., Ltd.) was added to the magnetic beads, and the mixture stirred at 25°C for 18 h. After washing the magnetic beads, 2 ml of 1% bobine serum albumin in 0.15 mol/l phosphate buffered saline (pH 7.8) was added and stirred at 25°C for 18 h for the blocking of the beads. These beads were diluted to 1 mg/ml using bead dilution reagent [0.05 mol/l Tris buffer (pH 7.5), 0.15 mol/l NaCl, 0.01% Tween20, 0.1% NaN 3, 10% normal rabbit serum, and 0.1% mouse serum] when in use.

For preparation of Ru-conjugated P-11 monoclonal antibody, 68 μl of Ru-complex compounds (10 mg of Ru [II] tris [bipyridyl]-NHS ester in 1 ml of dimethyl sulfoxide) was added to 1 ml of P-11 monoclonal antibody (1 mg/ml in 0.15 mol/l phosphate buffered saline, pH7.8; Sekisui Medical Co., Ltd.) for the conjugation, and stirred at 25°C for 30 min before adding 50 μl of 2 mol/l glycine solution to terminate the conjugate reaction. The Ru-conjugated P-11 monoclonal antibody was then isolated by collecting the Ru-bound protein fraction using Sephadex G-25 (previously equilibrated with 10 mmol/l phosphate buffer saline, pH 6.0). The obtained Ru-conjugated P-11 monoclonal antibody was diluted to 1 μg/ml using Ru dilution reagent [0.015 mol/l HEPES buffering solution [pH 7.8], 0.15 mol/l NaCl, 0.013 mol/l CaCl 2, 0.1% Tween20, 0.1% NaN 3, 5% normal rabbit serum, and 0.1% mouse serum] when in use.

For preparation of the standardized antigen, warfarin plasma (45,000 mAU/ml) quantified by Picolumi PIVKA-II(Eidia Co., Ltd., Tokyo, Japan) was diluted with normal plasma (20 mAU/ml) to a concentration of 38,000, 20,000, 8,000, 1,000, 200, and 20 mAU/ml as the standardized antigen.

The samples were analyzed using an automated PicolumiIIIanalyzer (Eidia Co., Ltd.). The twenty-five microliters of P-16 monoclonal antibody-coated magnetic beads (1 mg/ml) and 150μL of Ru conjugated P-11 monoclonal antibody (1μg/mL) were added to samples at 30°C for 9 min and the value of the variants of des-γ-carboxy prothrombin (DCP) with fewer glutamine acid residues using combined P-11 and P-16 antibodies to PIVKA-II (NX-PVKA) was then obtained. The NX-PVKA-R value was calculated by comparing the value of standardized antigen NX-PVKA with the value of Picolumi PIVKA-II measured independently using the following equation: Picolumi PIVKA-II/NX-PVKA.

We also calculated the value of NX-PVKA-D as the difference between Picolumi PIVKA-II and NX-PVKA.

Patients were then divided into two groups based on these levels at the cut-off level as i) patients with normal range (the normal group), and ii) patients with levels over the upper limit (the high-level group). The tumor-related factors were compared with the results from histopathological examination of the resected specimen. For assessment, we applied the criteria of the Liver Cancer Study Group of Japan by the Classification of Primary Liver Cancer (16).

Statistical analysis. Differences in categorical data between groups or prevalence were assessed by the chi-square, Fischer's exact or Dunnett's multiple comparison tests. Differences in continuous data between groups were evaluated by the Student's test or Mann Whitney test. Correlation between continuous data was evaluated by Pearson's test. The disease-free survival rates were calculated using the Kaplan Meier method, and differences between groups were tested for significance using the log-rank test. A two-tailed p-value of <0.05 was considered significant. Statistical Package for the Social Science (SPSS) version 18.0 software (SPSS, Chicago, IL, USA) was used for all statistical analyses.

Results

Patients' demographics. The mean age of the patients at the time of surgery was 68.1±18.4 years (median=74 years; range=45-85 years), and there were 17 males (77%) and five females. The background liver abnormalities included chronic hepatitis in 18 (81%) patients, cirrhosis in three (14%), and normal liver in one (5%), and these were associated with hepatitis virus B (n=5), hepatitis virus C (n=6), alcoholic disease (n=4) or other diseases (n=6). According to the Child-Pugh classification, all 22 patients were classified as A, and liver damage grade by Japanese classification was grade A in 19 and B in three. The number of tumors was solitary in 17 (76%) and multiple in five (24%) cases. Tumor size was less than 2 cm in three cases (14%), 2-5 cm in 15 (67%) and more than 5 cm in four (19%). Vascular infiltration was observed in seven (33%) cases. Macroscopic findings according to the Liver Cancer Study Group of Japan (16) was single-nodular (SN) disease in six (23%) cases, single-nodular with extranodular growth (SNEG) in 14 (67%) and confluent-multinodular (CMN) type in two (10%). Pre-treatment with chemoembolization or ablation therapy was performed in six patients (27%). Histological evaluation showed well-differentiated tumors in four (18%) cases, moderately-differentiated in 17 (77%) and poorly-differentiated in one (5%). The pathological tumor node metastasis (TNM) stage of HCC according to the Liver Cancer Study Group of Japan (16) was stage I in 2 (9%), stage II in 9 (41%), stage III in 6 (27%) and stage IVA in 5 (23%). The degree of fibrosis by Knodell's classification was 0 in two (10%), 1 in three (15%), 2 in five (20%), 3 in nine (45%) and 4 in two (10%). All patients underwent surgical resection. The surgical resections included limited hepatic resections (n=6), segmentectomy (n=6), sectionectomy (n=5) and hemi-hepatectomy (n=4). Tumor relapse during follow-up was observed in six (29%) cases, including recurrence in the liver (n=5) and lung (n=2). In patients with relapse, three patients underwent re-hepatectomy, two underwent transarterial chemoembolization and one underwent sorafenib administration. The one-year relapse-free survival rate was 88% and the median relapse-free survival period was 570 days.

View this table:
Table I.

Relationship between the continuous parameters and protein-induced by vitamin K absence or antagonist-II (PIVKA-II), NX-PVKA (detecting variants of PIVKA-II with fewer glutamic acid residues using P-11 and P-16 antibodies), ratio between NX-PVKA and PIVKA-II (NX-PVKA-R), and difference between NX-PVKA and PIVKA-II (NX-PVKA-D).

Tumor marker levels and their correlations. Median and mean values were: PIVKA-II: 80 and 541±901 mAU/ml; NX-PVKA: 58 and 254±493 mAU/ml; NX-PVKA-R: 1.3 and 2.3±2.6; and NX-PVKA-D: 15 and 287±517 mAU/ml. We applied a median cut-off value in the subsequent analysis. Correlation between PIVKA-II and NX-PVKA, NX-PVKA-R or NX-PVKA-D was significant with r=0.887 (p<0.001), r=0.409 (p=0.048), and r=0.898 (p<0.001), respectively. Correlations between AFP and NX-PVKA, NX-PVKA-R or NX-PVKA-D were r=−0.038 (p=0.87), r=0.764 (p<0.001) and r=0.662 (p=0.0015), respectively.

Relationship between NX-PVKA, NX-PVKA-R, NX-PVKA-D and clinicopathological features in patients with HCC who underwent hepatectomy. Table I shows the correlation between patient age, the number of tumors and tumor size and PIVKA-II, NX-PVKA, NX-PVKA-R and NX-PVKA-D. Only NX-PVKA-R was significantly correlated with tumor size (p<0.05). Table II shows the relationship between the categorical clinicopathological features and PIVKA-II, NX-PVKA, NX-PVKA-R and NX-PVKA-D levels. These parameters were not significantly associated with patient background, such as age, gender, background liver and liver function. In patients who underwent prior local treatment before hepatectomy, PIVKA-II, NX-PVKA and NX-PVKA-R tended to be higher than those in patients without pre-treatment, but this result was not statistically significance (p>0.10). For p-values regarding pre-treatments, that for NX-PVKA-R was the lowest between these markers. No markers were significantly associated with the number of tumors, tumor size, vascular infiltration, TNM stage, histological differentiation or degree of liver fibrosis. By macroscopic findings, NX-PVKA-R in CMN type was significantly higher than that in the SN type (p<0.05). No markers were significantly associated with the prevalence of tumor recurrence after hepatectomy.

Relationship between disease-free rates and changes in tumor markers. Six out of 22 patients (27%) showed tumor recurrence during this period and tumor-free survival was 570 days, and 1- or 2-year survival rates were 88 and 35%, respectively. In all patients that survived, tumor-free survival according to tumor markers was analyzed as follows. The tumor-free survival rate according to a high PIVKA-II level tended to be significantly lower than that in the low PIVKA-II group, but this difference was not statistically significant [Figure 1a; mean survival period (median survival period; MSP) was 478 and 645 days, respectively]. The tumor-free survival rate according to NX-PVKA was the same as the one according to PIVKA-II levels (not shown). Tumor-free survival rate in the high NX-PVKA-R group was significantly lower than that in the low NX-PVKA-R group (Figure 1b; 457 and 677 days, respectively) (p<0.05). Tumor-free survival rate in the high NX-PVKA-D group tended to be significantly lower than that in the low NX-PVKA-D group, but this difference was not statistically significant (Figure 1c; 500 and 677 days, respectively).

View this table:
Table II.

Relationship between the categorical clinicopathological features, and protein-induced by vitamin K absence or antagonist-II (PIVKA-II), NX-PVKA (detecting variants of PIVKA-II with fewer glutamic acid residues using P-11 and P-16 antibodies), ratio between NX-PVKA and PIVKA-II (NX-PVKA-R) and the difference between NX-PVKA and PIVKA-II (NX-PVKA-D).

The tumor marker levels after hepatectomy in 15 of 22 patients (68%) were examined and five patients with postoperative tumor recurrence could be followed (Table III). In comparison with the preoperative NX-PVKA-R, that at one month after hepatectomy had significantly decreased in 14 out of 15 patients (93%). One patient with low preoperative NX-PVKA-R did not have a decrease of NX-PVKA-R at one month. Mean NX-PVKA-R level at 1 month (0.42±0.24) was significantly lower than that preoperatively (1.95±1.97) (p<0.01). In three patients with tumor recurrence, postoperative NX-PVKA-R increased over nine months, but was less than 1.5 (median preoperative NX-PVKA-R) in two patients, even at the time of recurrence. Out of 10 patients without tumor recurrence, an increase of NX-PVKA-R for over six months occurred in eight, but with no tumor recurrence. In these patients, NX-PVKA-R at one month in patients with tumor recurrence (0.60±0.41) was not significantly higher than the one in patients without recurrence (0.37±0.12) (p=0.199).

Discussion

Specific HCC markers such as PIVKA-II and AFP L3 fraction are commonly used in Japan for the diagnosis or evaluation of tumor aggressiveness, and high values of these markers reflect patient prognosis after treatment (4-6, 11-15, 17-21). PIVKA-II is a precursor of prothrombin, and a vitamin K-dependent serum protein (22). This protein is activated by vitamin K-dependent carboxylase. The proposed mechanism of production of PIVKA-II by HCC is i) aberration of carboxylase, ii) overproduction of precursor proteins, or iii) decrease of vitamin K concentration (9, 23, 24). Our previous reports showed that the preoperative PIVKA-II level is an independent prognostic marker in patients with HCC who undergo hepatectomy (13, 15, 25). Furthermore, monitoring of PIVKA-II is useful for predicting tumor recurrence after hepatectomy at an earlier period compared to using AFP and our study also showed that changes in PIVKA-II significantly correlated with prognosis in patients with HCC undergoing hepatectomy (11-13, 26, 27). Thus, normalization of high levels of these sensitive tumor markers suggests curability with local treatments. With respect to PIVKA-II, however, this marker increases based on a lack of or decrease of vitamin K, such as the one during the use of anti-coagulants, chronic hepatic dysfunction or jaundice, as described above (9, 10, 28-31). Other physiological conditions may also influence the PIVKA-II level and the true level produced by HCC is necessary to evaluate the relationship with tumor aggressiveness before and after local treatments. An assay measuring NX-PVKA has recently been developed by the same company that developed the assay for PIVKA-II (9, 10). The difference between these two assays is in their recognition of the aberration in carboxylation of the carboxyglutamate residue in PIVKA-II. Serum PIVKA-II markers in patients with HCC can be identified by using a MU-3 antibody (10, 32), in which the antibody mainly reacts with the 9-10 glutamic acid residues of the conventional PIVKA-II, although the expression of DCP variants with fewer than six glutamine-acid residues was relatively higher among patients with non-malignant liver diseases, such as those with chronic hepatitis and liver cirrhosis, than among those with HCC (33). Other variants of DCP with fewer glutamine-acid residues can be detected using P-11 and P-16 antibodies (NX-PVKA). HCC-associated PIVKA-II contains two kringle domains similar to those of hepatocyte growth factor (HGF) which is thought to induce proliferation of HCC cells (34, 35). A serum level of NX-PVKA >100 mAU/ml was the best predictive marker after treatment in patients with HCC in comparison with the serum levels of conventional PIVKA-II, AFP or AFP-L3 fraction (32). Therefore, evaluation of NX-PVKA is promising to evaluate aggressiveness of HCC more sensitively. To compare conventional PIVKA-II and NX-PVKA levels, the company Eidia applied the ratio of both levels as NX-PVKA-R, and a cut-off level of >1.5 was used to diagnose HCC (company data, unpublished). In the present study, we also proposed the difference between those levels, as NX-PVKA-D, as described above. We then examined the relationship among these four types of PVKA-II values, clinicopathological factors, and early prognosis in a small number of patients with HCC, as a preliminary study.

Figure 1.
Figure 1.

Disease-free survival rates two years after hepatectomy for hepatocellular carcinoma (HCC) according to levels of tumor markers: According to levels of protein-induced by vitamin K absence or antagonist-II (PIVKA-II) with cut-off level of 80 mAU/ml (a); The PIVKA-II variants with fewer glutamate residues can be detected using P-11 and P-16 antibodies (NX-PVKA) and survival is shown using PIVKA-II as a ratio of this (NX-PVKA-R), with a cut-off level of 1.5 (b) and using the difference between NX-PVKA-II and PIVKA-II (NX-PVKA-D) with a cut-off level of 15 mAU/ml.

View this table:
Table III.

Relationship between post-operative changes of the ratio of PIVKA-II to NX-PVKA (NX-PVKA-R) after hepatectomy and tumor recurrence.

First of all, correlations between the conventional markers such as PIVKA-II or AFP and the new markers were examined. As a result, a significant correlation between parameters was observed; however, the degree of correlation varied. In the present series, anti-coagulant was not administered to all patients, but these patients did have various background liver dysfunctions. The observed variation in correlation might be influenced by background liver status, as suggested in other reports (29, 30). To define the cut-off level for the markers, the median and mean values were calculated in the present series. The mean values ranged widely and the number of patients with each marker in excess of the mean level was low; therefore, we applied the median value as a cut-off level in the next step of the study. With respect to NX-PVKA-R, the median value was close to 1.5 according to the company's criteria, and therefore, this level was applied.

By examining the relationships with clinicopathological parameters, only NX-PVKA-R was significantly correlated with tumor size and the irregular macroscopic findings of HCC, but not PIVKA-II, NX-PVKA and NX-PVKA-D, although NX-PVKA was strongly-associated with tumor size, tumor number, and portal vein infiltration by Takeji et al.'s report (31). Tumor size and macroscopic findings were powerful prognostic factors in patients with HCC in previous reports (36, 37) and, therefore, NX-PVKA-R would be an additional marker for these malignant characteristics. These markers were not associated with patients' demographics, background liver and liver functional grade, and can, therefore, be applied in patients influenced by these factors. Serum levels of PIVKA-II and NX-PVKA, and NX-PVKA-R tended to be associated with a history of pre-treatment, such as ablation or transarterial chemoembolization; however, the period from these treatments was more than one month. In these cases, HCC was not cured by the pre-treatments, and therefore, increased marker levels might reflect tumor aggressiveness accelerated by the pre-treatments or surrounding liver damage. The existence of pre-treatments alone did not reflect poor prognosis in the present series. NX-PVKA and NX-PVKA-R were not associated with other tumor-related factors, staging or histological parameters, although NX-PVKA-R was correlated with increased size, vascular infiltration, stage III-IV, poor differentiation and increased fibrosis, but these results were not statistically significant. This may be due to the small sample size. Takeji et al. reported that the NX-PVKA level was associated with platelet counts, prothrombin time, gender, size or number of tumors and venous infiltration in 197 patients with HCC, including those undergoing various treatments (31). These markers were not significantly associated with tumor relapse rate, although NX-PVKA-R or PIVKA-II levels might be increased in patients with tumor relapse, but this difference was not significant either. The proposed NX-PVKA-D was not associated with any parameters and, therefore, NX-PVKA-R was the best tool for evaluating NX-PVKA to reflect the malignancy of HCC.

In the present series, all patients survived and six patients had tumor recurrence, as the follow-up period was limited at this stage. The relationship between disease-free survival and preoperative markers PIVKA-II and NX-PVKA was examined. Only high values of NX-PVKA-R had a statistically significant correlation with poor disease-free survival in comparison with low values of NX-PVKA-R in the short-term period after hepatectomy, which was similar to findings of a previous study (31). The conventional PIVKA-II levels also tended to be associated with disease-free survival, but this result was not significant. As described above, some reports showed significance of PIVKA-II level as a predictor of patient prognosis after treatments (4-6, 11-15, 18-21, 26, 27), and therefore, the NX-PVKA-R value would be more sensitive in reflecting tumor malignancy and tumor relapse-free survival. We also focused on postoperative levels of sensitive markers. In 15 patients, including 5 with recurrent HCC, we measured NK-PVKA-R levels at one month and every 3-6 months after hepatectomy. As in our previous report, with respect to changes in PIVKA-II levels, normalization at one month was significantly associated with overall survival in patients with HCC who underwent hepatectomy in comparison with changes of AFP levels (13, 15). Kanazumi et al. reported that serum PIVKA-II levels decreased within 2 weeks after effective surgery (37). The half-life of PIVKA-II is less than one month (38), and therefore, the increase in PIVKA-II levels at one month is proposed to indicate viable HCC after treatment (i.e. not completely cured). For the same reason, the half-life by NX-PVKA may be similar to PIVKA-II, and calculation of NX-PVKA-R is important to evaluate treatment efficacy. All 15 patients showed NX-PVKA-R values that were less than the cut-off level of 1.5, and there was no significant difference in NX-PVKA-R at one month between patients with and without early tumor recurrence. During follow-up, NX-PVKA-R gradually increased in patients with tumor recurrence, but not in all patients. In patients with increased NX-PVKA-R, the conventional PIVKA-II levels also increased accordingly, but stayed below the cut-off level of 40 mAU/ml (39). At this stage, we could not determine which parameter was significant for follow-up, yet NX-PVKA-R may be better, considering the properties described above. We also found that patients with HCC who died at an earlier period after hepatectomy had high AFP and PIVKA-II levels after treatment (13, 15), and therefore, we plan to examine the influence of NX-PVKA-R on overall patient survival.

Nobuoka et al. reported that the postoperative AFP levels are useful to predict recurrence after hepatectomy by comparison with the postoperative PIVKA-II levels (11). Recent studies showed that AFP L3 fraction is a better specific marker for HCC than AFP (11, 35). The examination of both AFP and L3 fraction is necessary to improve the accuracy of predicting tumor relapse and survival in HCC (18, 40). A combination of AFP and PIVKA-II is currently recommended to diagnose tumor malignancy by the Japanese guidelines for HCC (41). Based on the results of the present preliminary study, NX-PVKA-R can also be considered a predictive marker after hepatectomy in patients with HCC. A subsequent analysis in a larger number of patients with HCC undergoing hepatectomy should be performed, and differences between PIVKA-II and NX-PVKA-R should be examined in those patients who undergo anti-coagulant therapy and who have had various chronic liver diseases or jaundice.

In summary, we conducted a prospective preliminary analysis to examine the relationship of the newly-developed PIVKA-II marker, NX-PVKA-R, and clinicopathological factors and early tumor recurrence in 22 patients with HCC who underwent curative hepatectomy. A value of NX-PVKA-R of more than1.5 reflected tumor aggressiveness, as also indicated by tumor size, macroscopic findings, and shorter disease-free survival. Increases of NX-PVKA-R during the follow-up period reflected early HCC recurrence. Careful follow-up is necessary for patients who fail to show a decrease of NX-PVKA-R during the early period after treatment. As NX-PVKA-R is promising for the prediction of malignant potential in HCC, further study in a larger number of patients is necessary.

  • Received March 27, 2013.
  • Revision received April 14, 2013.
  • Accepted April 15, 2013.

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NX-PVKA Levels Before and After Hepatectomy of Hepatocellular Carcinoma as Predictors of Patient Survival: A Preliminary Evaluation of An Improved Assay for PIVKA-II
ATSUSHI NANASHIMA, TAKAFUMI ABO, NAOTA TAURA, HIDEKI SHIBATA, TATSUKI ICHIKAWA, KATSUNORI TAKAGI, JUNICHI ARAI, SHOUSABUROU OYAMA, TAKESHI NAGAYASU
Anticancer Research Jun 2013, 33 (6) 2689-2697;
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