Risk factors for early recurrence of smallhepatocellular 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

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

(Hepatobiliary Pancreat Dis Int 2010; 9: 33-37)

carcinoma, hepatocellular;risk factors;recurrence

Introduction

Hepatocellular carcinoma (HCC) is the fi fth most common cancer in the world, responsible for 500 000 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 classi fi ed 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.

MethodsPatients

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 de fi ned 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,no radiologic evidence of macrovascular invasion,and no therapy for HCC. One hundred fi fty-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 classi fi ed by the criteria of the World Health Organization.[7]Microscopic vascular invasion was histopathologically de fi ned by the presence of clusters of cancer cells fl oating 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 classi fi ed 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 classi fi ed 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 fi ndings of ultrasound or computed tomography were inconclusive, hepatic angiography with infusion of iodized oil was performed. Biopsy was not required to con fi rm 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 de fi ned 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 signi fi cant.

Results

The 158 patients consisted of 138 males (87.3%) and 20 females (12.7%) with mean and median ages of49.5 years (range 23-82) and 49 years, respectively.One hundred fi fty- fi ve patients (98.1%) were positive for hepatitis B surface antigen. Of these, 99 had histologically con fi rmed 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.

Table 1. Univariate analysis of risk factors of early recurrence after resection of small HCC

Table 2. Results of a multivariate analysis by logistic regression

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 signi fi cant 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% con fi dence interval 1.057 to 6.206, P=0.037)and microscopic vascular invasion (odds ratio 4.549,95% con fi dence interval 1.865 to 11.097, P=0.001) were independent factors (Table 2).

Discussion

Currently, the de fi nition of small HCC varies among investigators. Tumor sizes of 5 cm, 4.5 cm, 4 cm, and 3 cm are commonly used to de fi ne 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 de fi ned 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 de fi ned 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 fi ndings of other investigators,[24]microscopic vascular invasion was also an independent risk factor in our analysis. This fi nding 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 signi fi cant effect of the width of the resection margin or anatomical resection on postoperative recurrence,[25,26]which is consistent with our fi nding.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 signi fi cant 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 fi laments 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 signi fi cant 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 signi fi cant within the fi rst postoperative year. In this study, however, we did not fi nd a signi fi cant 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 identi fi cation of signi fi cant risk factors for early recurrence may better select patients for postoperative surveillance. Our fi ndings suggest that in patients with small HCC and serum AFP level ＞100 ng/ml, microscopic vascular invasion should be closely monitored in the fi rst year for early tumor recurrence. If early recurrence arises from intrahepatic metastasis, adjuvant therapies are necessary as chemoembolization-lipiodolization,internal radiation, chemotherapy, or adoptive immunotherapy.[34]A previous meta-analysis found that postoperative transarterial chemotherapy may reduce the incidence of early recurrence after hepatectomy.[35]

In conclusion, 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.

Funding: None.

Ethical approval: Not needed.

Contributors: ZYM wrote the fi rst draft. All authors contributed to the intellectual context and approved the fi nal version. KT is the guarantor.

Competing interest: No bene fi ts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

1 Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma.Lancet 2003;362:1907-1917.

2 Lau WY, Lai EC. Hepatocellular carcinoma: current management and recent advances. Hepatobiliary Pancreat Dis Int 2008;7:237-257.

3 Tung-Ping Poon R, Fan ST, Wong J. Risk factors, prevention,and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 2000;232:10-24.

4 Poon RT, Fan ST, Ng IO, Lo CM, Liu CL, Wong J. Different risk factors and prognosis for early and late intrahepatic recurrence after resection of hepatocellular carcinoma. Cancer 2000;89:500-507.

5 Adachi E, Maeda T, Kajiyama K, Kinukawa N, Matsumata T, Sugimachi K, et al. Factors correlated with portal venous invasion by hepatocellular carcinoma: univariate and multivariate analyses of 232 resected cases without preoperative treatments. Cancer 1996;77:2022-2031.

6 Edmondson HA, Steiner PE. Primary carcinoma of the liver.A study of 100 cases among 48900 necropsies. Cancer 1954;7:462-503.

7 Ishak KG, Anthony PP, Sobin LH. Histological typing of tumours in the liver. WHO International Histological Classi fi cation of Tumours. 2nd ed. New York: Springer Verlag;1994.

8 Tsai TJ, Chau GY, Lui WY, Tsay SH, King KL, Loong CC, et al. Clinical signi fi cance of microscopic tumor venous invasion in patients with resectable hepatocellular carcinoma. Surgery 2000;127:603-608.

9 Park JH, Koh KC, Choi MS, Lee JH, Yoo BC, Paik SW, et al.Analysis of risk factors associated with early multinodular recurrences after hepatic resection for hepatocellular carcinoma. Am J Surg 2006;192:29-33.

10 Shimada M, Takenaka K, Gion T, Fujiwara Y, Kajiyama K,Maeda T, et al. Prognosis of recurrent hepatocellular carcinoma:a 10-year surgical experience in Japan. Gastroenterology 1996;111:720-726.

11 Cong WM, Wu MC. The biopathologic characteristics of DNA content of hepatocellular carcinomas. Cancer 1990;66:498-501.

12 He P, Tang ZY, Ye SL, Liu BB. Relationship between expression of alpha-fetoprotein messenger RNA and sone clinical parameters of human hepatocellular carcinom. World J Gastroenterol 1999;5:111-115.

13 Mizejewski GJ. Biological role of alpha-fetoprotein in cancer:prospects for anticancer therapy. Expert Rev Anticancer Ther 2002;2:709-735.

14 Jacobson HI, Bennett JA, Mizejewski GJ. Inhibition of estrogen-dependent breast cancer growth by a reaction product of alpha-fetoprotein and estradiol. Cancer Res 1990;50:415-420.

15 Dudich E, Semenkova L, Dudich I, Gorbatova E, Tochtamisheva N, Tatulov E, et al. alpha-fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase-3-like proteases. Eur J Biochem 1999;266:750-761.

16 Gershwin ME, Castles JJ, Ahmed A, Makishima R. The in fl uence of alpha-fetoprotein on Moloney sarcoma virus oncogenesis: evidence for generation of antigen nonspeci fi c suppressor T cells. J Immunol 1978;121:2292-2298.

17 Wang XW, Xie H. Alpha-fetoprotein enhances the proliferation of human hepatoma cells in vitro. Life Sci 1999;64:17-23.

18 Li MS, Li PF, He SP, Du GG, Li G. The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line. World J Gastroenterol 2002;8:469-475.

19 Li MS, Li PF, Chen Q, Du GG, Li G. Alpha-fetoprotein stimulated the expression of some oncogenes in human hepatocellular carcinoma Bel 7402 cells. World J Gastroenterol 2004;10:819-824.

20 Fujioka M, Nakashima Y, Nakashima O, Kojiro M. Immunohistologic study on the expressions of alpha-fetoprotein and protein induced by vitamin K absence or antagonist II in surgically resected small hepatocellular carcinoma. Hepatology 2001;34:1128-1134.

21 Yang X, Zhang Y, Zhang L, Zhang L, Mao J. Silencing alphafetoprotein expression induces growth arrest and apoptosis in human hepatocellular cancer cell. Cancer Lett 2008;271:281-293.

22 Tang H, Tang XY, Liu M, Li X. Targeting alpha-fetoprotein represses the proliferation of hepatoma cells via regulation of the cell cycle. Clin Chim Acta 2008;394:81-88.

23 Peng SY, Chen WJ, Lai PL, Jeng YM, Sheu JC, Hsu HC. High alpha-fetoprotein level correlates with high stage, early recurrence and poor prognosis of hepatocellular carcinoma:signi fi cance of hepatitis virus infection, age, p53 and betacatenin mutations. Int J Cancer 2004;112:44-50.

24 Shirabe K, Kanematsu T, Matsumata T, Adachi E, Akazawa K, Sugimachi K. Factors linked to early recurrence of small hepatocellular carcinoma after hepatectomy: univariate and multivariate analyses. Hepatology 1991;14:802-805.

25 Yoshida Y, Kanematsu T, Matsumata T, Takenaka K,Sugimachi K. Surgical margin and recurrence after resection of hepatocellular carcinoma in patients with cirrhosis. Further evaluation of limited hepatic resection. Ann Surg 1989;209:297-301.

26 Kaibori M, Matsui Y, Hijikawa T, Uchida Y, Kwon AH,Kamiyama Y. Comparison of limited and anatomic hepatic resection for hepatocellular carcinoma with hepatitis C.Surgery 2006;139:385-394.

27 Lai EC, You KT, Ng IO, Shek TW. The pathological basis of resection margin for hepatocellular carcinoma. World J Surg 1993;17:786-791.

28 Kim BK, Han KH, Park YN, Park MS, Kim KS, Choi JS, et al.Prediction of microvascular invasion before curative resection of hepatocellular carcinoma. J Surg Oncol 2008;97:246-252.

29 Alison MR. Liver stem cells: implications for hepatocarcinogenesis. Stem Cell Rev 2005;1:253-260.

30 Uenishi T, Kubo S, Yamamoto T, Shuto T, Ogawa M, Tanaka H,et al. Cytokeratin 19 expression in hepatocellular carcinoma predicts early postoperative recurrence. Cancer Sci 2003;94:851-857.

31 Harris CC. Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J Natl Cancer Inst 1996;88:1442-1455.

32 Hayashi H, Sugio K, Matsumata T, Adachi E, Takenaka K,Sugimachi K. The clinical signi fi cance of p53 gene mutation in hepatocellular carcinomas from Japan. Hepatology 1995;22:1702-1707.

33 Stroescu C, Dragnea A, Ivanov B, Pechianu C, Herlea V,Sgarbura O, et al. Expression of p53, Bcl-2, VEGF, Ki67 and PCNA and prognostic signi fi cance in hepatocellular carcinoma. J Gastrointestin Liver Dis 2008;17:411-417.

34 Llovet JM, Schwartz M, Mazzaferro V. Resection and liver transplantation for hepatocellular carcinoma. Semin Liver Dis 2005;25:181-200.

35 Mathurin P, Raynard B, Dharancy S, Kirzin S, Fallik D, Pruvot FR, et al. Meta-analysis: evaluation of adjuvant therapy after curative liver resection for hepatocellular carcinoma. Aliment Pharmacol Ther 2003;17:1247-1261.

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 2004. Risk factors for early recurrence were analyzed.

RESULTS: Thirty-three (20.8%) patients developed early recurrence after surgery. Univariate analysis showed the following signi fi cant 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% con fi dence interval 1.057 to 6.206,P=0.037) and microscopic vascular invasion (odds ratio 4.549, 95% con fi dence interval 1.865 to 11.097, 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.

Author Af fi liations: Department of Hepato-Biliary-Pancreato-Vascular Surgery, First Xiamen Hospital, Fujian Medical University, Xiamen 361003,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 200438, China

Tong Kan, MD, Department of Special Treatment,Eastern Hepatobiliary Surgery Hospital, Second Military Medical University,Shanghai 200438, China (Tel: 86-21-25070808; Fax: 86-21-65562400; Email:kanto168.888@vip.163.com)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.

August 6, 2009

Accepted after revision November 1, 2009