Hepatocellular carcinoma (HCC) is the fifth

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1 Original Article / Liver Risk factors for early recurrence of small hepatocellular carcinoma after curative resection Yan-Ming Zhou, Jia-Mei Yang, Bin Li, Zheng-Feng Yin, Feng Xu, Bin Wang, Wen Xu and Tong Kan Shanghai, China BACKGROUND: Poorer prognosis is seen in patients with hepatocellular carcinoma (HCC) after curative hepatic resection with early recurrence ( 1 year) than in those with late recurrence (>1 year). This study aimed to identify risk factors for postoperative early recurrence of small HCC ( 3 cm in diameter). METHODS: The study population consisted of 158 patients who underwent curative resection for small HCC between January 2002 and July Risk factors for early recurrence were analyzed. RESULTS: Thirty-three (20.8%) patients developed early recurrence after surgery. Univariate analysis showed the following significant risk factors for early recurrence in small HCC: serum alpha-fetoprotein (AFP) level >100 ng/ml, lack of tumor capsule formation, microscopic vascular invasion, high Edmonson-Steiner grades, and cytokeratin-19 (CK-19) expression (P<0.05). Multivariate stepwise logistic regression analysis showed that serum AFP level >100 ng/ml (odds ratio 2.561, 95% confidence interval to 6.206, P=0.037) and microscopic vascular invasion (odds ratio 4.549, 95% confidence interval to , P=0.001) were independent factors. CONCLUSIONS: Postoperative early recurrence is related to serum AFP level >100 ng/ml and microscopic vascular invasion in patients with small HCC. Adjuvant therapy and careful follow-up are required for patients with these risk factors. (Hepatobiliary Pancreat Dis Int 2010; 9: 33-37) KEY WORDS: carcinoma, hepatocellular; risk factors; recurrence Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, responsible for deaths globally every year, and its incidence is increasing because of hepatitis B and C virus infection. [1] Hepatic resection is regarded as a potentially curative treatment modality for patients with HCC. [2] Despite remarkable improvement in surgical techniques and perioperative management, the long-term outcome after resection of HCC is far from satisfactory because of the high postoperative recurrence of either intrahepatic metastatic or metachronous multicentric HCC. [3] According to the time point of recurrence from the date of hepatectomy, recurrences are classified into early ( 1 year) and late (>1 year) recurrences. The prognosis of patients with early recurrence is worse than that of patients with late recurrence. [4] Large tumor size is one of the risk factors for recurrence. [3] Nevertheless, even in a small HCC after curative resection, early recurrence is a frequent manifestation. [5] In this study we analyzed clinical and pathological factors associated with early recurrence of small HCC after curative resection. Author Affiliations: Department of Hepato-Biliary-Pancreato-Vascular Surgery, First Xiamen Hospital, Fujian Medical University, Xiamen , China (Zhou YM and Li B); Department of Special Treatment (Yang JM, Xu F and Kan T), Department of Molecular Oncology (Yin ZF and Xu W), Department of Pathology (Wang B), Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai , China Corresponding Author: Tong Kan, MD, Department of Special Treatment, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai , China (Tel: ; Fax: ; kanto @vip.163.com) 2010, Hepatobiliary Pancreat Dis Int. All rights reserved. Methods Patients From January 2002 and July 2004, 164 consecutive patients underwent a curative hepatic resection for small HCC at the Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China. Curative resection was defined as removal of all recognizable tumor with a clear microscopic margin. The enrollment criteria included tumor size 3 cm in diameter and single tumor, no extrahepatic metastasis, Hepatobiliary Pancreat Dis Int,Vol 9,No 1 February 15,

2 Hepatobiliary & Pancreatic Diseases International no radiologic evidence of macrovascular invasion, and no therapy for HCC. One hundred fifty-eight patients were analysed. Histologically HCC was graded according to the criteria of Edmondson and Steiner (G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated; G4, undifferentiated), [6] and the histological pattern of HCC was classified by the criteria of the World Health Organization. [7] Microscopic vascular invasion was histopathologically defined by the presence of clusters of cancer cells floating in the vascular space. [8] The expression of biliary/progenitor cell markers cytokeratin-19 (CK-19) and p53 in HCC was assessed by immunohistochemistry using a polymer-based method with the anti-ck-19 antibody (diluted at 1 50, NeoMarkers, Fremont, CA, USA) or anti-p53 antibody (1 50, NeoMarkers). Surgical procedures were classified into anatomical or non-anatomical resections. Anatomical resection involves resection of the tumor together with the tumorcontaining portal vein and corresponding hepatic territory and includes hemihepatectomy, sectorectomy, and segmentectomy. Other resections such as limited resection and tumor enucleation are classified as nonanatomical resections. [9] Follow-up After discharge, the patients were followed up every one month by tumor marker analysis (alpha-fetoprotein, AFP) and ultrasound or computed tomography at least every 3 months for more than a year. Tumor recurrence was characterized by new lesions on imaging with appearances typical of HCC or a rising AFP level. When findings of ultrasound or computed tomography were inconclusive, hepatic angiography with infusion of iodized oil was performed. Biopsy was not required to confirm the recurrence. Intrahepatic recurrent HCCs were divided into nodular ( 3 nodules) or multinodular (>4 nodules) ones according to the number of intrahepatic nodules. [10] Patients who had tumor recurrence were treated with re-resection when possible or by transcatheter arterial chemoembolization, percutaneous ethanol injection, and radiofrequency ablation. The end point of this study was time to early tumor recurrence, which was defined as the period between surgery and the diagnosis of recurrence as being 1 year. Statistical analysis Quantitative data were expressed as mean±sd. Groups were compared by univariate analysis using the Chi-square test, and by multivariate analysis using logistic regression analysis with backward and forward step selection. Analyses were made using statistical software (SPSS, Inc, Chicago, IL). A P<0.05 was considered statistically significant. Results The 158 patients consisted of 138 males (87.3%) and 20 females (12.7%) with mean and median ages of Table 1. Univariate analysis of risk factors of early recurrence after resection of small HCC Variables Recurrence (n=33) No recurrence (n=125) P value Gender Male/Female 29/4 109/ Age (yr) >60/ 60 8/25 17/ AFP (ng/ml) >100/ /13 40/ Total bilirubin (μmol/l) >20/ 20 7/26 20/ ALT (U/L) >50/ 50 11/22 38/ AST (U/L) >50/ 50 10/23 23/ Tumor location Right lobe/left lobe 25/8 109/ Resection margin (cm) >1/ 1 24/9 98/ Tumor capsule Absent/Present 14/19 30/ Microscopic vascular invasion Absent/Present 14/19 101/ Edmonson-Steiner grade Ⅰ-Ⅱ/Ⅲ-Ⅳ 2/31 31/ Histological pattern Trabecular Pseudoglandular 3 4 Compact 0 7 Scirrhous 2 2 Clear cell 2 2 Liver histology Normal Chronic hepatitis Cirrhosis Type of surgical resection Anatomical/Non-anatomical 18/15 73/ CK-19 expression Positive/Negative 8/25 10/ p53 expression Positive/Negative 6/27 17/ ALT: alanine aminotransferase; AST: aspartate aminotransferase. 34 Hepatobiliary Pancreat Dis Int,Vol 9,No 1 February 15,2010

3 Risk factors for early recurrence of small HCC after curative resection Table 2. Results of a multivariate analysis by logistic regression Variables 49.5 years (range 23-82) and 49 years, respectively. One hundred fifty-five patients (98.1%) were positive for hepatitis B surface antigen. Of these, 99 had histologically confirmed cirrhosis, and the other 56 had evidence of chronic hepatitis. On immunohistochemical examination, 18 tumors (11.4%) were stained positive for CK-19 and 23 (14.5%) were positive for p53 protein. Thirty-three patients (20.8%) developed early tumor recurrence after surgery. All patients were found to have tumor recurrence by ultrasound, computed tomography, and hepatic angiography. Eight patients had nodular recurrence and 24 had multinodular recurrence. Univariate analysis showed that the following significant risk factors for early recurrence in small HCC: serum AFP level >100 ng/ml, lack of tumor capsule formation, microscopic vascular invasion, high Edmonson-Steiner grades, and CK-19 positive expression (P<0.05) (Table 1). Multivariate stepwise logistic regression analysis revealed that serum AFP level >100 ng/ml (odds ratio 2.561, 95% confidence interval to 6.206, P=0.037) and microscopic vascular invasion (odds ratio 4.549, 95% confidence interval to , P=0.001) were independent factors (Table 2). Discussion Regression Odds ratio (95% Wald coefficient confidence interval) Standard error P value AFP ( ) Tumor capsule ( ) Microscopic ( ) vascular invasion Edmonson ( ) Steiner grade CK ( ) Overall model Chi-square=32.034; P< Currently, the definition of small HCC varies among investigators. Tumor sizes of 5 cm, 4.5 cm, 4 cm, and 3 cm are commonly used to define a small HCC. [11] Cong et al [11] found that the period when HCC is about 3 cm in size may be important as changes occur in DNA stemline and biological characteristics, and could be a key point between relatively benign and malignant HCC. Pathologically, small HCC is thus defined a tumor less than or equal to 3 cm in diameter. The time interval from resection to recurrence of HCC is thought to be an independent prognostic factor of survival, and a year is the most discriminant time point of HCC recurrence when compared with other points such as 0.5, 2, and 3 years. [10] The prognosis of patients with early recurrent ( 1 year) tumors was worse than that of patients with late recurrence despite a similar treatment regimen. Even after re-resection, survival results of early recurrence are inferior to those of late recurrence. [4] Therefore, we defined early recurrence as being 1 year in this study. Our results showed that a serum AFP level >100 ng/ml was an independent risk factor for early recurrence of small HCC. AFP is a mammalian tumorassociated fetal glycoprotein with approximately 72 kd molecular weight. Under physiological conditions, AFP is synthesized and secreted by fetal hepatocytes, gastrointestinal cells, and yolk sac cells, and its levels decline gradually after birth to <10 ng/ml in 300 days. AFP has served as a representative tumor marker of HCC for more than 40 years. Elevated AFP levels were found in 60% to 70% of the HCC patients. [12] The biological role of AFP has been widely investigated for many years. A myriad of studies have found that AFP plays an important role in the regulation of both oncogenic and ontogenetic growth. [13] Although early studies indicated that AFP and its derived peptide fragments were able to inhibit oncogenic growth, [14-16] more recent reports showed AFP can promote growth of HCC cells. [17-19] AFP-positive HCC has a higher cell proliferative activity, as measured by the Ki-67 index. [20] Downregulation of AFP is able to suppress the growth of HCC cells. [21, 22] In addition, a high AFP level correlates with more aggressive behavior and poor prognosis of HCC patients. [23] Therefore, the high level of AFP in the serum of HCC subjects attributes to tumor development rather than serves merely as a tumor marker. Postoperative HCC recurrence is thought to take place in two ways, intrahepatic metastasis in the residual liver and metachronous, multicentric hepatocarcinogenesis based on chronic hepatitis. [4] Tumor venous invasion is the direct evidence of intrahepatic metastasis. Similar to the findings of other investigators, [24] microscopic vascular invasion was also an independent risk factor in our analysis. This finding strongly supports the hypothesis that early recurrence is mainly attributable to intrahepatic metastasis. [4] Because HCC often spreads within the intrahepatic vasculature, some authors advocate a wide surgical margin (>1 cm) or anatomical resection to achieve a successful resection and increase the chance of a cure. [8] However, other studies have found no significant effect of the width of the resection margin or anatomical resection on postoperative [25, 26] recurrence, which is consistent with our finding. Hepatobiliary Pancreat Dis Int,Vol 9,No 1 February 15,

4 Hepatobiliary & Pancreatic Diseases International Shirabe et al [24] found that only a small proportion of intrahepatic recurrences occur near the resection margin. Furthermore, microsatellites and histologic vascular invasion beyond 1 cm are very common with both small and large HCC. [27] High Edmonson-Steiner grades and lack of tumor capsule formation were significant risk factors shown by univariate analysis, but not by multivariate analysis. Given that high grade tumor and tumor without encapsulation are more likely to be associated with microscopic vascular invasion, [9, 28] it is cleary explained why the two factors were eliminated from multivariate analysis that also included microscopic vascular invasion. Most HCCs are derived from mature hepatocytes, however, growing evidence has indicated that HCC may originate from hepatic progenitor cells that undergo malignant changes. [29] CKs are cytoskeletal intermediate filaments present in both normal and malignant epithelial cells. CK-19 is considered to be a marker of biliary cells and hepatic progenitor cells. Clinically, patients with CK19-positive HCC are associated with early recurrence and poor outcome. [30] Although CK-19 expression is a significant risk factor of early recurrence shown by univariate analysis, this is not the case under multivariate analysis in our study. This is due to the small number of patients with a positive CK-19 (n=18, 11.3%) in our study. Further research is needed to clarify the mechanism by which CK-19 enhances tumor recurrence. p53 is one of the most commonly inactivated tumor suppressor genes associated with the development of human cancers including HCC. It has multiple functions in several central cellular processes, including gene transcription, DNA repair, cell cycling, genomic stability, chromosomal segregation, senescence, and apoptosis. Inactivation of the p53 gene abrogates its normal function, leading to genomic instability and loss of growth control. [31] Hayashi et al [32] demonstrated that p53 mutation is an unfavorable prognostic factor for recurrence, which is especially significant within the first postoperative year. In this study, however, we did not find a significant correlation between expression of p53 and early recurrence of small HCC. Because p53 protein is detected more frequently in large tumors, these abnormalities probably might occur after the initial stages of hepatocellular carcinogenesis as a relatively late event in tumor progression. [33] The identification of significant risk factors for early recurrence may better select patients for postoperative surveillance. Our findings suggest that in patients with small HCC and serum AFP level >100 ng/ml, microscopic vascular invasion should be closely monitored in the first year for early tumor recurrence. 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