Triple positive tumor markers predict recurrence and survival in early stage hepatocellular carcinoma

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1 See discussions, stats, and author profiles for this publication at: Triple positive tumor markers predict recurrence and survival in early stage hepatocellular carcinoma Article in Hepatology Research November 21 Impact Factor: 2.74 DOI: /hepr Source: PubMed CITATIONS READS authors, including: Toru Beppu Kumamoto University 1 PUBLICATIONS 2,89 CITATIONS Kosuke Mima Kumamoto University 7 PUBLICATIONS 7 CITATIONS SEE PROFILE SEE PROFILE Takatoshi Ishiko Kumamoto University 124 PUBLICATIONS 1,175 CITATIONS Hideo Baba Kumamoto University 988 PUBLICATIONS 11,51 CITATIONS SEE PROFILE SEE PROFILE Available from: Toru Beppu Retrieved on: 11 May 216

2 bs_bs_banner Hepatology Research 214 doi: /hepr Original Article Triple positive tumor markers predict recurrence and survival in early stage hepatocellular carcinoma Shigeki Nakagawa, 1 Toru Beppu, 1,2 Hirohisa Okabe, 1 Keita Sakamoto, 1 Hideyuki Kuroki, 1 Kosuke Mima, 1 Hidetoshi Nitta, 1 Katsunori Imai, 1 Hiromitsu Hayashi, 1 Yasuo Sakamoto, 1,2 Daisuke Hashimoto, 1 Akira Chikamoto, 1 Takatoshi Ishiko, 1 Masayuki Watanabe 1 and Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, and 2 Department of Multidisciplinary Treatment for Gastroenterological Cancer, Kumamoto University Hospital, Kumamoto, Japan Aim: Hepatectomy is feasible for patients with hepatocellular carcinoma (HCC) with good hepatic function who meet the Milan criteria. Several studies have indicated that tumor markers of HCC, α-fetoprotein (AFP), Lens culinaris agglutininreactive fraction of AFP percentage and protein induced by vitamin K absence/antagonist-ii were good predictors of malignant potential. It is important to identify highly malignant cases of HCC, and the aim of this study was to clarify the impact of triple positive tumor markers as the prognostic factors for early stage HCC within the Milan criteria. Methods: This study investigated 199 patients who underwent hepatectomy for HCC within the Milan criteria between January 21 and May 29. Cumulative recurrence-free survival (RFS), overall survival (OS) and clinicopathological parameters were analyzed according to the number of positive tumor markers. Results: In patients with triple positive tumor markers, 5-year RFS and OS was poor (17.1 and 61.4%, respectively). Multivariate analyses revealed independent risk factors for recurrence to be hepatitis C virus antibody positivity, non-initial treatment for HCC and triple positive tumor markers, and the independent risk factors for OS were high indocyanine green retention rate at 15 min value, maximum tumor size and triple positive tumor markers. Pathologically invasive growth, microvascular invasion and moderate to poor differentiation were significantly related to the number of the three tumor markers. Conclusion: Triple positive tumor markers for early stage HCC within the Milan criteria showed poor prognosis and malignant characteristics. These markers could be a useful predictor for the degree of malignant potential in early stage HCC. Key words: alpha-fetoprotein, des-γ-carboxy prothrombin, hepatocellular carcinoma, protein induced by vitamin K absence/antagonist-ii, prognosis Correspondence: Professor Hideo Baba, Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Honjo, Kumamoto , Japan. hdobaba@kumamoto-u.ac.jp Received 25 July 21; revision 17 October 21; accepted 1 November 21. INTRODUCTION HEPATOCELLULAR CARCINOMA (HCC) is a common malignancy with a particularly high prevalence in Asia, and its incidence is also rising in Western countries. 1,2 The most potent diagnostic methods for HCC are imaging studies, such as ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI). Tumor staging is predominantly conducted based on tumor number, size, macrovascular invasion and extrahepatic metastasis, and the therapeutic strategy is determined by the tumor stage and liver function. The various treatments for HCC may be curative, including local ablation therapy, hepatectomy and liver transplantation.,4 Liver transplantation can treat both the liver cancer and the background liver pathology, yielding an excellent survival rate for selected patients with liver dysfunction. 5,6 The most well-established criteria for liver transplantation for HCC is the Milan criteria: namely, a solitary tumor of 5 cm or less in diameter or three lesions or fewer with a largest diameter of cm, no macroscopic vascular invasion and no extrahepatic involvement. 5 Following the widespread acceptance of living donor liver transplantation in the last 1 years, viral hepatitis-induced HCC 21 The Japan Society of Hepatology 1

3 2 S. Nakagawa et al. Hepatology Research 214 has become the main indication for liver transplantation in Japan. 7 However, hepatectomy is still suggested as a first-line treatment for HCC in patients eligible for transplantation if they have good liver function. 6,8 HCC that fulfill the Milan criteria has been identified as early stage HCC and is reported to have a good prognosis and low recurrence rate compared with HCC that does not fulfill the Milan criteria. 9 Nevertheless, there are some HCC patients with poor prognosis and high recurrence rates after hepatic resection (HR) also within the Milan criteria. Although a few reports suggest prognostic factors for HCC patients within the Milan criteria, 9,1 identification of the highly malignant group before HR remains an important issue. For diagnosis of HCC, in addition to imaging-related diagnoses, the three tumor markers are clinically examined in Japan; alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L) and des-γcarboxy prothrombin (DCP). These tumor markers are not only used tor diagnosis of HCC, but also to predict the degree of HCC malignancy. 11,12 Recently, a few studies indicated that the number of positive tumor markers can predict the degree of HCC malignancy and patient prognosis. 1,14 However, the importance of triple positive tumor markers in early stage HCC is still unclear. In this study, we focused on early stage HCC patients who met the Milan criteria, and analyzed the influence of positive expression of these three tumor markers on patient prognosis and clinicopathological parameters. METHODS Patients and diagnosis THE STUDY POPULATION consisted of 199 HCC patients meeting the Milan criteria who underwent HR. All patients were treated at the Department of Gastroenterological Surgery, Kumamoto University Hospital, between January 21 and May 29. The patients underwent imaging studies, such as ultrasonography, dynamic CT and enhanced MRI and CT angiography for diagnosis and staging of HCC before surgery. The final diagnosis was confirmed pathologically in the resected specimens. The severity of liver disease (liver fibrosis staging and hepatitis activity grading) was also evaluated according to the criteria proposed by the Inuyama classification. 15 This study was a retrospective, non-interventional, observational study, approved by the institutional ethics committee of Kumamoto University Hospital and performed in accordance with the Helsinki Declaration of Written informed consent was obtained from all patients. Measurement of HCC tumor markers α-fetoprotein, AFP-L, and DCP were measured immediately before HR. The serum AFP level was determined by chemiluminescent enzyme immunoassay (Siemens Immulite AFP IV; Mitsubishi Chemical Medience, Tokyo, Japan), and the serum AFP-L level was expressed as a percentage of the total AFP (AFP-L / total AFP 1) by lectin affinity electrophoresis coupled with antibody-affinity blotting (AFP-L Test Wako; Wako Pure Chemical Industries, Osaka, Japan). Serum DCP was determined by chemiluminescent enzyme immunoassay (Lumipulse PIVKA-II Eisai; Eisai, Tokyo, Japan). The upper limits of the normal range of AFP, AFP-L, and DCP in our institution were 2 ng/ml, 1% and 4 mau/ml, respectively. Tumor markers higher than the upper limit of the normal range were defined as positive tumor markers for HCC. Patients were individually classified according to the number of positive tumor markers as negative, single positive, double positive and triple positive. Treatment and surveillance The surgical procedure was selected based on the tumor location, extent of the tumor, parenchymal liver function and the patient s general condition. HR was considered as the first choice treatment for patients with good liver functional reserve. If the liver function allowed, anatomical resection was employed. The reasons for undergoing radiofrequency ablation rather than HR included insufficient liver functional reserve, high operative risk associated with general condition, and refusal of HR. Anatomical resections (segmentectomy, sectionectomy and hemi-hepatectomy) were performed in 16 patients. Non-anatomical resections were performed in 9 patients. The follow-up program included AFP, AFP-L and DCP assays every 1 2 months, and ultrasonography, dynamic CT or dynamic MRI every 4 months after surgery. When a recurrence of HCC was detected, patients received further treatment for HCC, such as repeated hepatectomy, ablation therapy or transcatheter arterial chemoembolization. After treatment for recurrent lesions, the same surveillance was continued. Estimated risk factors for recurrence and survival A total of 18 variables were analyzed as follows: age, sex, presence of hepatitis B surface antigen and hepatitis C 21 The Japan Society of Hepatology

4 Hepatology Research 214 Triple positive tumor markers in early HCC antibody (HCVAb), Child Pugh score, serum level of albumin, total bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), prothrombin activity, indocyanine green dye retention rate at 15 min (ICG-R15), platelet count (PLT), number of tumors, maximum tumor size, procedure of liver resection, initial treatment for HCC and triple positive tumor marker expression. The cut-off values of albumin, total bilirubin and prothrombin activity, PLT, ICG-R15, AST and ALT were based on the median values. Statistical analysis Dichotomous variables were compared using the χ 2 - test, and continuous variables were compared using Student s t-tests. Recurrence-free survival and diseasespecific survival were analyzed using the Kaplan Meier method and differences were analyzed using the log rank test. To estimate the risk factors for recurrence and survival by Cox proportional hazards regression analysis, continuous variables were converted into binary. All analyses were performed using the JMP program (SAS Institute, Cary, NC, USA). P <.5 were considered to be significant. RESULTS Patient characteristics and tumor markers AMONG THE 199 patients, there were 161 men and 8 women ranging in age from 4 to 81 years, with a mean 1 standard deviation of years. The median follow up was 47.7 months (range, ). During the follow-up period, 8 patients died of recurrence or progression of HCC, two patients died of hepatic failure, one patient died of renal failure and one patient died of acute aortic dissection. The median values of serum AFP, AFP-L and DCP levels were 12.7 ng/ml, less than.5% and 46. mau/ml, respectively. Serum AFP, AFP-L and DCP levels over the upper limits of normal range were observed in 8 (41.9%), 41 (2.6%) and 11 patients (55.%), respectively. Table 1 shows the patients backgrounds and clinical characteristics according to the number of positive tumor markers. Qualitative evaluation of these three tumor markers revealed that 74 patients were single positive (AFP, n = 22; DCP, n = 52; AFP-L, n = ), 8 patients were double positive (AFP and DCP, n = 25; AFP and AFP-L, n = 8; DCP and AFP-L, n = 5), 28 patients were triple positive and 59 patients were all negative. Among these four groups, maximum tumor size was different and no significant differences were observed in the other factors. Patient prognosis and the number of positive tumor markers The overall survival and recurrence-free survival of the patients were demonstrated (Fig. 1). Recurrence-free survival rates at 5 years were 17.1% in triple positive, 2.6% in double positive,.% in single positive and.1% in all negative patients. Overall survival rates at 5 years after hepatectomy were 61.4% in triple positive, 74.2% in double positive, 79.% in single positive and 86.% in all negative patients. Single and double positive and all negative patients were defined as the nontriple positive group. Survival analysis between triple positive and non-triple positive were also analyzed. Recurrence-free survival rates at 5 years were 17.1% in triple positive patients and 29.5% in non-triple positive patients with statistical significance (P =.81). Overall survival rates at 5 years were 61.4% in triple positive and 8.16% in non-triple positive patients, respectively (P =.128). Next, we evaluated recurrence-free and overall survival rates of each combination of double positive and triple positive compared with the group of all negative and single positive (Fig. S1 and Table S1). Recurrence-free survival rates at 5 years were 1.5% in the all negative and single positive group; 68.5% for AFP + AFP-L, 6.% for AFP-L + DCP and 18.6% for AFP + DCP in each double positive group (P =.598,.459 and.51 respectively); and 17.1% in the triple positive group (P =.177). Overall survival rates at 5 years were 82.1% in all negative and single positive groups, 64.2% for AFP + AFP-L, 8.% for AFP-L + DCP and 74.% for AFP + DCP in each double positive (P =.2974,.7621 and.5619 respectively) and 61.% in triple positive groups (P =.82). Postoperative survival rates based on the combination of three tumor markers The factors associated with recurrence-free survival were also evaluated by univariate and multivariate analyses (Table 2). Univariate analysis revealed that HCVAb positivity, PLT less than , ICG-R15 higher than 12.6%, non-initial treatment for HCC and triple positive tumor markers were significant variables for poor recurrence-free survival. Multivariate analysis demonstrated that HCVAb positivity (relative risk [RR], 1.65; P =.154), non-initial treatment for HCC (RR, 1.87; P =.47) and triple positive tumor markers for HCC (RR, 1.68; P =.76) were independent risk factors for recurrence. 21 The Japan Society of Hepatology

5 4 S. Nakagawa et al. Hepatology Research 214 Table 1 Patients characteristics Variable Negative (n = 59) Single positive (n = 74) Double positive (n = 8) Triple positive (n = 28) P-value Sex Male/female 47/12 6/14 /8 24/4.92 Age 66 (44 8) 68 (4 81) 64.5 (6 79) 62.5 (48 7).85 HBsAg Negative/positive 47/12 54/2 24/14 15/1.597 HCVAb Negative/positive 4/25 6/8 18/2 14/ Child Pugh classification Class A/B 42/17 62/12 /5 26/2.54 Albumin 4.9 (.2 4.6).95 ( ) 4. (.4 5.).85 ( 4.5).6867 Total bilirubin.7 (. 1.7).8 (. 1.6).8 (. 2.).75 (. 1.6).8795 Prothrombin activity 91 (61 127) 9.5 (6 14) 9 (7 16) 87 (74 127).55 PLT ( 1 4 /mm ) (7.5 7.) 15. (4 46.8) 14. ( ) 12.1 (5.5 2.).488 ICG-R15 (%) 12.5 (.1 4.5) 14.8 ( ) 11.5 ( ) 1.5 (.9 7.2).628 Liver damage grade Grade A/B 5/6 69/5 5/ 24/ No. of tumors Single/multiple 45/14 68/6 28/1 21/7.42* Tumor size 2 cm/> cm 46/1 45/29 22/16 1/15.226* Liver resection Anatomical/non-anatomical /29 9/5 2/18 17/ Treatment for HCC initial/non-initial 41/18 58/16 29/9 21/ *P <.5. HCC, hepatocellular carcinoma; HCVAb, hepatitis C antibody; HBsAg, hepatitis B surface antigen; ICG-R15, indocyanine green retention rate at 15 min; PLT, platelets. 21 The Japan Society of Hepatology

6 Hepatology Research 214 Triple positive tumor markers in early HCC 5 (a) (b) Recurrence free survival rate (%) Recurrence free survival rate (%) Number at risk Years Negative Single Double Triple Years after hepatectomy Number at risk Years Not triple Triple Years after hepatectomy (c) (d) 1 1 Overal survival rate (%) Overal survival rate (%) Years after hepatectomy Number at risk Years Negative Single Double Triple Number at risk Years Not triple Triple Years after hepatectomy Figure 1 Recurrence-free and overall survival curves according to the number of positive tumor markers. (a) The triple positive group had significantly lower recurrence-free interval compared with the all negative and single positive groups, but not compared with the double positive groups (P =.1,.89 and.275, respectively). (b) The triple positive group had a significantly worse recurrence-free survival compared to the non-triple positive groups (P =.77). (c) The triple positive group had significantly worse overall survival compared with the all negative, single positive and double positive groups (P =.2,.28 and.226, respectively). (d) The triple positive group had a significantly worse overall survival compared with the non-triple positive groups (P =.6). (a) ( ) triple negative, ( ) single positive, ( ) double positive, ( ) triple positive; (b) ( ) not triple positive, ( ) triple positive; (c) ( ) triple negative, ( ) single positive, ( ) double positive, ( ) triple positive; d: ( ) not triple positive, ( ) triple positive. 21 The Japan Society of Hepatology

7 6 S. Nakagawa et al. Hepatology Research 214 Table 2 Univariate and multivariate analysis for disease-free survival Variable Univariate analysis Multivariate analysis RR 95% CI P-value RR 95% CI P-value Sex Female 1 Male Age < HBsAg Negative 1 Positive HCVAb Negative 1 1 Positive * * Child Pugh classification Class A 1 Class B Albumin > Total bilirubin > Prothrombin activity >91% 1 291% PLT > /mm /mm * ICG-R % 1 1 >12.6% No. of tumors Single 1 Multiple Tumor size 2 cm 1 > cm Liver surgery Non-anatomical 1 Anatomical Treatment for HCC Initial 1 1 Non-initial * * Tumor markers Non-triple positive 1 1 Triple positive * * *P <.5. Cut-off value was defined as the median value. CI, confidence interval; HCC, hepatocellular carcinoma; HCVAb, hepatitis C antibody; HBsAg, hepatitis B surface antigen; ICG-R15, indocyanine green retention rate at 15 min; PLT, platelets; RR, relative risk. 21 The Japan Society of Hepatology

8 Hepatology Research 214 Triple positive tumor markers in early HCC 7 The factors associated with overall survival were evaluated by univariate and multivariate analyses (Table ). Univariate analysis revealed that ICG-R15 higher than 12.6%, maximum tumor diameter larger than cm and triple positive tumor markers were significant variables for poor survival. Multivariate analysis revealed that ICG-R15 higher than 12.6% (RR, 2.46; P =.146), maximum tumor diameter larger than cm (RR, 2.71; P =.5) and triple positive tumor markers for HCC (RR, 2.57; P =.198) were independent risk factors for overall survival. Comparison of pathological findings among number of positive tumor markers Pathological findings obtained from the resected specimens were compared according to numbers of positive tumor markers (Fig. 2). Invasive growth (negative, %; single positive, 8.22%; double positive, 1.5%; triple positive, 17.86%), microvascular invasion (negative, 18.64%; single positive, 27.%; double positive, 9.47%; triple positive, 5.57%), and moderate to poor tumor differentiation (negative, 71.19%; single positive, 87.84%; double positive, 89.47%; triple positive, 1%) were significantly different among these four groups. DISCUSSION IN THIS STUDY, we clearly demonstrated that patients who were triple positive for three tumor markers of HCC (AFP, AFP-L and DCP) had significantly shorter survival, pathologically more invasive tumors and more poorly differentiated characteristics in early stage HCC within the Milan criteria. α-fetoprotein is the most widely used tumor marker for HCC and its high serum levels correlate with poor prognosis Furthermore, AFP-L positive status has been reported to correlate with a large number of HCC tumors, those with high malignant potential 12 and poor prognosis and high recurrence rate. 24,25 We have previously reported that the doubling time of preoperative AFP is a significant predictor for both recurrence-free and overall survival. 26 A high level of serum DCP was reported to correlate with portal vein invasion and indicate a poor prognosis in HCC patients. 2,27 An elevated preoperative DCP has been demonstrated to be associated with a high recurrence rate after liver transplantation. 28 Recently, the utility of the combined measurement of these three tumor markers (AFP, AFP-L and DCP) was demonstrated in predicting the outcome and recurrence for HCC patients treated with HR. 1,14 These studies investigated early to advanced stage HCC and reported that triple positive tumor markers are associated with invasive tumor growth. The combination of AFP-L and DCP was also reported to correlate with poor differentiation and poor prognosis in early to advanced stage HCC. 1 In early stage HCC, a few reports 9,1 indicated effective prognostic markers for patients for HCC who underwent curative HR. Sakaguchi et al. investigated the utility of DCP to predict outcome after hepatectomy in HCC patients within the Milan criteria. 1 They set the cut-off value of DCP as 1 mau/ml, and described that a high serum DCP level can predict poor prognosis and high recurrence rate. We evaluated various cut-off values including 1 mau/ml, such as 8 mau/ml, 16 mau/ml, tertile values and quartile values as shown in Table S2. The cut-off value of 1 mau/ml did not have a significant difference in recurrence-free and overall survival rate in our study, and 16 mau/ml and 2 mau/ml (the first quartile) could predict a significant worth recurrence-free survival rate in high DCP patients, but could not predict the difference of overall survival rate. No cut-off value had a significant difference in both recurrence-free and overall survival rate. We also evaluated the meaning of the combination of each two tumor markers as a prognostic factor. The patients double positive for AFP and DCP had an early recurrence rate similar to the patients with triple positive tumor markers, but combination of all three of double positives did not have significant difference compared with the negative and single positives in overall survival rate. Previous reports have set the various cut-off values as median value, value calculated from receiver operator curve analysis, upper or lower normal limit value, and values based on other reports. In our study, we defined the cut-off values of the three tumor markers as the upper normal limit based on the other reports. 1,14 The merit of using this cut-off value was that the value was objective and common between different institutions. This study also revealed pathological differences among the number of positive tumor markers. Several studies revealed that high serum levels of tumor markers were predictive of portal vein invasion. 28 In addition to the previous study, our pathological findings revealed that there is increasing invasiveness and poorer differentiation characteristics in HCC according to the number of positive tumor markers. It is necessary to modify the treatment strategy and follow up of HCC patients based on tumor stage, liver function and the type of viral hepatitis. 2 Postoperative 21 The Japan Society of Hepatology

9 8 S. Nakagawa et al. Hepatology Research 214 Table Univariate and multivariate analysis for overall survival Variable Univariate analysis Multivariate analysis RR 95% CI P-value RR 95% CI P-value Sex Female 1 Male Age < HBsAg Negative 1 Positive HCVAb Negative 1 Positive Child Pugh classification Class A 1 Class B Albumin > Total bilirubin > Prothrombin activity >91% 1 291% PLT > /mm /mm ICG-R % 1 1 >12.6% * * No. of tumors Single 1 Multiple Tumor size 2 cm 1 1 > cm * * Liver surgery Non-anatomical 1 Anatomical Treatment for HCC Initial 1 Non-initial Tumor markers Non-triple positive 1 1 Triple positive * * *P <.5. Cut-off value was defined as the median value. CI, confidence interval; HCC, hepatocellular carcinoma; HCVAb, hepatitis C antibody; HBsAg, hepatitis B surface antigen; ICG-R15, indocyanine green retention rate at 15 min; PLT, platelets; RR, relative risk. 21 The Japan Society of Hepatology

10 Hepatology Research 214 Triple positive tumor markers in early HCC 9 1 * * 8 * 6 4 * 2 Invasive growth Fibrotic capsule formation Capsule infiltration Micro-vascular invasion * Tumor differentiation mod-poor Fibrous staging F-F4 Hepatiti activity A2- A Figure 2 Relationship between pathological parameters and number of positive tumor markers. *P <.5 by χ 2 -test. ( ) negative, ( ) single positive, ( ) double positive, ( ) triple positive. adjuvant chemotherapy for the remnant liver after HR has been reported to reduce recurrence with intrahepatic metastases in the early postoperative period, however, adjuvant chemotherapy is sometimes difficult for patients after a HR because of their insufficient hepatic functional reserve. Patients with a higher risk of early recurrence should be selected preoperatively to plan an adequate perioperative treatment strategy. If a precise prediction of early recurrence or poor prognosis can be achieved preoperatively, the surgeon could choose the alternative treatments (e.g. transplant or local ablation therapy) or give neoadjuvant therapies as a clinical trial. There has, however, been little evidence of the benefit of neoadjuvant regional or systemic therapies. In conclusion, a triple positive status for the three tumor markers of HCC is an independent risk factor for recurrence and survival, and indicates an increasing presence of microvascular invasion and poor differentiation, even for patients with early stage HCC that fulfills the Milan criteria. REFERENCES 1 Shaw JJ, Shah SA. Rising incidence and demographics of hepatocellular carcinoma in the USA: what does it mean. Expert Rev Gastroenterol Hepatol 211; 5: Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 24; 127: S5 S16. Burroughs A, Hochhauser D, Meyer T. Systemic treatment and liver transplantation for hepatocellular carcinoma: two ends of the therapeutic spectrum. Lancet Oncol 24; 5: Arii S, Yamaoka Y, Futagawa S et al. Results of surgical and nonsurgical treatment for small-sized hepatocellular carcinomas: a retrospective and nationwide survey in Japan. 21 The Japan Society of Hepatology

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12 Hepatology Research 214 Triple positive tumor markers in early HCC 11 protein induced by vitamin K absence II are potential indicators of a poor prognosis: a histopathological study of surgically resected hepatocellular carcinoma. J Gastroenterol 27; 42: Sasaki Y, Yamada T, Tanaka H et al. Risk of recurrence in a long-term follow-up after surgery in 417 patients with hepatitis B- or hepatitis C-related hepatocellular carcinoma. Ann Surg 26; 244: Shirabe K, Takenaka K, Gion T, Shimada M, Fujiwara Y, Sugimachi K. Significance of alpha-fetoprotein levels for detection of early recurrence of hepatocellular carcinoma after hepatic resection. J Surg Oncol 1997; 64: SUPPORTING INFORMATION ADDITIONAL SUPPORTING INFORMATION may be found in the online version of this article at the publisher s website: Figure S1 Comparison of recurrence-free and overall survival curves of the combination of each two tumor markers. (a) Compared with the group of all negative and single positive, only the combination of α- fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP) double positive had significantly lower recurrence-free survival rate within the double positive group (P-values are shown in Table S1). (b) Compared with the group of all negative and single positives, none of the three combinations of double positives had a significant difference in overall survival rate (P-values are shown in Table S1). Table S1 Univariate analysis of double-positive tumor markers (AFP + L, L + DCP, AFP + DCP) for RFS and OS. Table S2 Univariate analysis of various cut-off values of DCP (8 mau/ml, 1 mau/ml, 1 mau/ml, tertile values and quartile values) in RFS and OS. 21 The Japan Society of Hepatology