Recurrence of Barcelona Clinic Liver Cancer Stage A Hepatocellular Carcinoma After Hepatectomy

Author’s Accepted Manuscript Recurrence of Barcelona Clinic Liver Cancer Stage A Hepatocellular Carcinoma after Hepatectomy Hao Zou, Cheng-zhan Zhu, Chang Wang, Zu-sen Wang, Xiang Ma, Bing Han, Li-qun Wu www.elsevier.com

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S0002-9629(17)30283-5 http://dx.doi.org/10.1016/j.amjms.2017.05.014 AMJMS469

To appear in: The American Journal of the Medical Sciences Received date: 1 February 2017 Revised date: 23 May 2017 Accepted date: 23 May 2017 Cite this article as: Hao Zou, Cheng-zhan Zhu, Chang Wang, Zu-sen Wang, Xiang Ma, Bing Han and Li-qun Wu, Recurrence of Barcelona Clinic Liver Cancer Stage A Hepatocellular Carcinoma after Hepatectomy, The American Journal of the Medical Sciences, http://dx.doi.org/10.1016/j.amjms.2017.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original Article Recurrence of Barcelona Clinic Liver Cancer Stage A Hepatocellular Carcinoma after Hepatectomy Hao Zou1, MD, Cheng-zhan Zhu1，MD, PhD, Chang Wang2, MD, Zu-sen Wang1, MD, PhD, Xiang Ma1, MD, Bing Han1, MD, PhD, Li-qun Wu1 , MD, PhD 1. Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, No.16 Jiangsu Road, Qingdao City 266003, Shandong Province, China. 2. Department of Gynecology, The Affiliated Hospital of Qingdao University, No.1677 Wutaishan Road, Qingdao City 266555, Shandong Province, China. Conflict-of-interest statement: The authors have no conflict of interest to report. Correspondence to: Professor Li-qun Wu, MD, PhD, Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qingdao University, No.16 Jiangsu Road, Qingdao City 266003, Shandong Province, China. E-mail:[email protected]: +86-532-82911323Fax: +86-532-82911323 Funding Sources：No Funding of Sources to state. Keywords: hepatocellular carcinoma; hepatectomy; recurrence; risk factors Running title: Risk factors for BCLC A HCC recurrence

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Abstract Background：The Barcelona Clinic Liver Cancer (BCLC) staging system is widely used to classify hepatocellular carcinoma (HCC). This study is to investigate the prognostic factors for BCLC Stage A HCC patients after R0 hepatectomy. Methods: A total of 592 BCLC Stage A HCC patients following R0 hepatectomy from 1997 to 2012 were enrolled in this study. Kaplan-Meier analysis and Cox regression were used to analyze the risk factors associated with recurrence. Receiver operating characteristic (ROC) curves were used to establish a new scoring system to evaluate the independent risk factors for recurrence. Furthermore, subgroup analyses were performed to evaluate surgical margins on tumor recurrence between the anatomical and non-anatomical resection group. Results: Independent risk factors for BCLC Stage A HCC recurrence were preoperative alanine transaminase (ALT)>40U/L, liver cirrhosis, surgical margin <5mm, non-anatomic resection, and maximum tumor diameter>5cm. Based on these 5 risk factors, we established a new scoring system in this study, named “HCC recurrence scoring system”. Patients with a high score (≥3 points, 1 point for each factor) composed the high recurrence risk group. Moreover, the subgroup analyses demonstrated that different surgical margins had no significant effect on tumor recurrence in the anatomical resection group (P=0.408), while it had a significant effect in the non-anatomical resection group (P=0.000). Conclusions: For BCLC Stage A patients with scores ≥3 points, close postoperative

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follow-up and positive measures to prevent recurrence are particularly important. Anatomical resection is preferred for BCLC Stage A patients. Adequate surgical margins are necessary for patients with poor liver function.

Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, with the second-highest mortality rate1. Curative treatments currently include hepatectomy, radiofrequency ablation, and liver transplantation, with hepatectomy considered to be the optimal strategy because of the limitations of radiofrequency ablation and liver transplantation. However, about 50% of tumors recur during the first 3 years, and >70% recur during the first 5 years after hepatectomy2, with such recurrences being the major cause of patient death. It is therefore important to investigate the clinicopathological factors related to disease-free survival (DFS). The Barcelona Clinic Liver Cancer (BCLC) staging system is a highly validated staging system that is widely accepted in clinical settings. It can be used to classify tumors, and takes account of the patient’s general condition, liver function, and systemic condition. The BCLC staging system is also widely recommended as a treatment allocation guideline in European countries3. BCLC Stage A HCC refers to patients with early liver cancer, including patients with single tumors or up to 3 tumors <3 cm that are for radical therapies4. Progress in diagnostic techniques and enhanced physical examinations mean that increasing numbers of patients in China are being identified

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with early stage liver cancer. Most resectable early stage HCCs are BCLC Stage A, for which the BCLC staging system recommends hepatectomy as the best option 4. R0 resection, defined as no cancer cells in the surgical margin, is a necessary requirement for a good prognosis. Numerous studies have investigated the factors affecting patient prognosis after hepatectomy, including tumor size and alpha-fetoprotein (AFP) levels5, 6. However, the patient selection criteria in most previous studies have been wide, and we therefore focused on the independent risk factors in patients with BCLC Stage A HCC, and also aimed to quantify the risk level based on these independent risk factors following R0 hepatectomy. In the present study, we retrospectively analyzed independent risk factors that may affect recurrence after R0 hepatectomy in BCLC Stage A HCC patients, and established an HCC recurrence scoring system to guide clinical treatments and improve the prognosis of HCC.

Patients and Methods Patients and follow-up This is a retrospective study. The study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. All study participants or their legal guardians provided informed written consent prior to study enrollment. A total of 592 patients with BCLC Stage A HCC who underwent R0 resection were enrolled in this study from January 1997 to December 2012. All the patients were

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consecutive. The patients included 496 males and 96 females, aged 17–83 years (median age 55 years). All patients underwent R0 hepatectomy involving either anatomical or non-anatomical resection. All patients were followed up after surgery. Serum alanine transaminase(ALT), aspartate transaminase (AST), total bilirubin (TBIL), Albumin and AFP, liver ultrasound examination were performed monthly within the 3 months after surgery, and once every 3 months thereafter. Computed tomography examination of the lungs and enhanced computed tomography or magnetic resonance imaging of the liver was performed every 3 months during the first 2 years, and once every 6 months thereafter. The median follow-up period was 57.0 months (5.7–205.7 months). Follow-up ended on 31 August 2016 or at the time of death. HCC recurrence was confirmed by imaging examination. Statistical analysis Data were analyzed by the Kaplan–Meier method and Cox regression using SPSS 18 software (IBM Corporation, Armonk, NY, USA). Receiver operating characteristic (ROC) curves were used to establish the recurrence scoring system. P<0.05 was set as the level of statistical significance. Results Risk factors affecting HCC recurrence following R0 resection The clinicopathological characteristics of all 592 patients with BCLC Stage A HCC are provided in Table 1. During the follow-up period, 354 patients (59.8%) experienced

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recurrence or metastasis. The 3- and 5-year overall survival (OS) rates were 77.8% and 63.6%, respectively, and the 3- and 5-year DFS rates were 53.3% and 40.1%, respectively. Univariate analysis revealed that male gender, preoperative serum gamma-glutamyl transpeptidase>64 U/L, alanine transaminase >40 U/L, liver cirrhosis, surgical margin <5 mm, non-anatomic resection, intraoperative blood loss >1000 mL, intraoperative blood transfusion, preoperative AFP >20 µg/L, and maximum tumor diameter >5 cm were risk factors for poor DFS (P<0.05). Multivariate analysis identified preoperative ALT >40 U/L, liver cirrhosis, surgical margin <5 mm, non-anatomic resection, and maximum tumor diameter >5 cm as independent prognostic factors (Table 2). The DFS curves for each independent risk factor are illustrated in Figure 1. Establishment of HCC recurrence scoring system The independent risk factors that influenced HCC recurrence following R0 hepatectomy were further analyzed using ROC curves, with the scores as the test variable and recurrence as the status variable. Patients were allocated 1 point for each factor, giving a maximum total of 5 points, with 3 points taken as the cut-off value. The area under the curve (AUC) was 64.5% (P=0.000) and the Youden index was 0.211 (specificity=66.1% and sensitivity=55.0%) (Figure 2). The scoring distribution is summarized in Table 3. The patients were divided into high-risk (≥3 points, n=341) and low-risk groups (≤2 points, n=251), and DFS rates were analyzed in both groups (Figure 3). The 1-, 2- and 5-year DFS rates for the low-risk group were 86.9%, 72.5%, and 51.8%, compared with 77.7%, 57.5%, and 32.3%, respectively, for the high-risk group (P=0.000).

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Effect of surgical margin on tumor recurrence We further analyzed the surgical factors affecting prognosis by determining if the effect of surgical margin differed between patients in the anatomical and non-anatomical resection groups. Surgical margin was not a risk factor for tumor recurrence following R0 resection in the anatomical resection group (median DFS 50.8 vs 79.0 months, P=0.408) (Figure 4), but surgical margin <5 mm significantly influenced tumor recurrence following R0 resection in the non-anatomical resection group (median DFS 24.0 vs 50.0 months, P=0.000) (Figure 4).

Discussion Postoperative recurrence is the major factor affecting survival in patients with HCC7. It is therefore necessary to understand the factors influencing tumor recurrence to improve patient prognosis. In this study, we identified 5 independent risk factors for recurrence following R0 hepatectomy in BCLC Stage A HCC patients, and used these to establish an HCC recurrence scoring system to evaluate risk level. Subgroup analysis was performed to detect the influence of surgical margins on tumor recurrence. The current results indicated that preoperative ALT >40 U/L, liver cirrhosis, surgical margin <5 mm, non-anatomic resection, and maximum tumor diameter >5 cm were independent risk factors for recurrence after R0 hepatectomy. Elevated preoperative ALT was also shown to be an independent risk factor for postoperative HCC recurrence.8 Increased ALT is an indicator of impaired hepatocytes, which could be

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related to hepatitis B virus infection. Cirrhosis is also a widely recognized independent risk factor for HCC recurrence after surgery9-11, and the 1-, 3-, and 5-year DFS rates in the present study were 80.7%, 52.3%, and 39%, respectively, for patients with cirrhosis, and 89.8%, 62.6%, and 49.5%, respectively, for patients without cirrhosis. In China, hepatitis B virus infection is the main cause of damaged liver cell and liver cirrhosis. Patients who underwent antiviral therapy were found to survive longer than those without antivirals12. Large tumors with rich blood supplies are generally thought to grow rapidly and invade the surrounding liver tissues more easily, though large tumors are also more prone to microvascular invasion13, 14. Moreover, about 50% of tumors with diameters >4 cm involve microvascular invasion,15 which is an important factor associated with the recurrence of liver cancer10, 16, 17. Given the number of independent risk factors for tumor recurrence in patients with BCLC Stage A tumors, we assigned points based on the ROC curve and used these to establish an HCC recurrence scoring system. We then classified patients as high-risk (≥3 points) or low-risk (≤2 points) patients, according to their scores. Although the AUC was <70%, we did not use the scores directly for diagnosis, but rather used the ROC curve to find the cut-off value. A cut-off value of 3 points revealed a significant difference in DFS between the 2 groups (P=0.000). The HCC recurrence scoring system was validated in 89 BCLC Stage A patients who underwent R0 hepatectomy in our institution during 2013 (P=0.011). Accordingly, scores ≥3 were associated with a high risk of recurrence among patients with BCLC Stage A, suggesting that these patients

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should be treated with anti-relapse therapy and more rigorous follow-up. The high incidence of HCC recurrence may be related to intrahepatic metastases and the multicentric occurrence of de novo HCC18. The surgical approach and surgical margin are, thus, important prognostic factors. We accordingly analyzed the prognostic effects of the surgical margin in patients who underwent anatomical and non-anatomical resection, and found that the surgical margin had no effect on DFS among patients who underwent anatomic resection, but did influence survival in the non-anatomical resection group. Anatomical resection was previously shown to improve OS and DFS rates significantly in patients with single small liver carcinomas. 19 However, other studies indicated that anatomical and non-anatomical resection had similar curative effects2,20, and found no relationship between the surgical margin and postoperative tumor recurrence21. Anatomical resection has the advantage of removing small sub-clinical metastases in the liver segment, and is also associated with a lower risk of complications22, 23. In contrast, the main purpose of non-anatomical resection is to achieve a functional residual liver volume2,

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. The importance of

anatomical resection and surgical margin thus remain controversial. However, the current results suggest that anatomical resection should be the first choice in patients with good liver function or if residual liver volume can be ensured. Adequate surgical margins should be ensured in patients with poor liver function.

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In conclusion, we established a scoring system based on preoperative ALT >40 U/L, liver cirrhosis, surgical margin <5 mm, non-anatomic resection, and maximum tumor diameter >5 cm, and demonstrated its usefulness for indicating BCLC Stage A HCC recurrence after R0 hepatectomy. Surgical margin is an important factor following non-anatomic resection for HCC. High-risk patients with scores ≥3 should undergo close postoperative follow-up and treatment.

References 1. Maluccio M, Covey A. Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma CA Cancer J Clin 2012;62:394-9. 2. Marubashi S, Gotoh K, Akita H, et al. Anatomical versus non-anatomical resection for hepatocellular carcinoma The British journal of surgery 2015;102:776-84. 3. Bruix J, Sherman M, American Association for the Study of Liver D. Management of hepatocellular carcinoma: an update Hepatology 2011;53:1020-2. 4. Llovet JM, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classification Semin Liver Dis 1999;19:329-38. 5. Vauthey JN, Lauwers GY, Esnaola NF, et al. Simplified staging for hepatocellular carcinoma Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2002;20:1527-36. 6. Ma WJ, Wang HY, Teng LS. Correlation analysis of preoperative serum alpha-fetoprotein (AFP) level and prognosis of hepatocellular carcinoma (HCC) after

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hepatectomy World journal of surgical oncology 2013;11:212. 7. Kaibori M, Ishizaki M, Saito T, et al. Risk factors and outcome of early recurrence after resection of small hepatocellular carcinomas American journal of surgery 2009;198:39-45. 8. Lin CL, Kao JH. Risk stratification for hepatitis B virus related hepatocellular carcinoma J Gastroenterol Hepatol 2013;28:10-7. 9. Wang Q, Fiel MI, Blank S, et al. Impact of liver fibrosis on prognosis following liver resection for hepatitis B-associated hepatocellular carcinoma Br J Cancer 2013;109:573-81. 10. Gui H, Yang H, Zhang S, et al. Mirroring Tool: The Simplest Computer-Aided Simulation Technology? J Craniofac Surg 2015;26:2115-9. 11. Fukuda S, Itamoto T, Amano H, et al. Clinicopathologic features of hepatocellular carcinoma patients with compensated cirrhosis surviving more than 10 years after curative hepatectomy World J Surg 2007;31:345-52. 12. Yang T, Lu JH, Zhai J, et al. High viral load is associated with poor overall and recurrence-free survival of hepatitis B virus-related hepatocellular carcinoma after curative resection: a prospective cohort study Eur J Surg Oncol 2012;38:683-91. 13. Sumie S, Kuromatsu R, Okuda K, et al. Microvascular invasion in patients with hepatocellular carcinoma and its predictable clinicopathological factors Annals of surgical oncology 2008;15:1375-82. 14. Pawlik TM, Delman KA, Vauthey JN, et al. Tumor size predicts vascular invasion

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and histologic grade: Implications for selection of surgical treatment for hepatocellular carcinoma Liver Transpl 2005;11:1086-92. 15. Esnaola NF, Lauwers GY, Mirza NQ, et al. Predictors of microvascular invasion in patients with hepatocellular carcinoma who are candidates for orthotopic liver transplantation Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract 2002;6:224-32; discussion 32. 16. Roayaie S, Blume IN, Thung SN, et al. A system of classifying microvascular invasion to predict outcome after resection in patients with hepatocellular carcinoma Gastroenterology 2009;137:850-5. 17. Sumie S, Nakashima O, Okuda K, et al. The significance of classifying microvascular invasion in patients with hepatocellular carcinoma Annals of surgical oncology 2014;21:1002-9. 18. Sakon M, Nagano H, Nakamori S, et al. Intrahepatic recurrences of hepatocellular carcinoma after hepatectomy: analysis based on tumor hemodynamics Archives of surgery 2002;137:94-9. 19. Yamashita Y, Taketomi A, Itoh S, et al. Longterm favorable results of limited hepatic resections for patients with hepatocellular carcinoma: 20 years of experience Journal Of the American College Of Surgeons 2007;205:19-26. 20. Kang CM, Choi GH, Kim DH, et al. Revisiting the role of nonanatomic resection of small (<= 4 cm) and single hepatocellular carcinoma in patients with well-preserved liver function J Surg Res 2010;160:81-9.

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21. Poon RT, Fan ST, Ng IO, et al. Significance of resection margin in hepatectomy for hepatocellular carcinoma: A critical reappraisal Annals of surgery 2000;231:544-51. 22. Hasegawa K, Kokudo N, Imamura H, et al. Prognostic impact of anatomic resection for hepatocellular carcinoma Annals of surgery 2005;242:252-9. 23. Ochiai T, Sonoyama T, Kikuchi S, et al. Anatomic wide hepatectomy for treatment of hepatocellular carcinoma Journal of cancer research and clinical oncology 2007;133:563-9. 24. Cho YB, Lee KU, Lee HW, et al. Anatomic versus non-anatomic resection for small single hepatocellular carcinomas Hepato-gastroenterology 2007;54:1766-9.

Figure Legends Figure 1. DFS curves for each independent risk factor. A: Preoperative ALT levels. B: Preoperative cirrhosis status. C: Surgical margins. D: Anatomical hepatectomy status. E: Maximum tumor diameters Figure 2. ROC curve for the different scores. Figure 3. DFS curves for the high-risk and low-risk groups. Figure 4. DFS curves for different surgical margins in anatomical resection group or non-anatomical resection group. A: DFS curves for the different surgical margins in the anatomical resection group. B: DFS curves for the different margins in the non-anatomical resection group.

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Table 1. Clinical and pathological characteristics of the patients Factors Gender (Male, Female)

n (%) 496 (83.8), 96 (16.2)

Age ( ≤ 60years, > 60years)

419 (70.8), 173 (29.20)

Alcohol abuse (No, Yes) *

381 (64.4), 149 (25.2)

Preoperative TACE (No, Yes)

514 (86.8), 78 (13.2)

HBsAg (Negative, Positive)

86 (14.5), 506 (85.5)

Anti-HCV (Negative, Positive)

577 (97.5), 15 (2.5)

ALT ( ≤ 40U/L, > 40U/L)

322 (54.4), 270 (45.6)

GGT ( ≤ 64U/L, > 64U/L)

408 (68.9), 184 (31.1)

Child-Pugh classification (A, B)

573 (96.8), 19 (3.2)

Cirrhosis (No, Yes)

59 (10.0), 533 (90.0)

Portal hypertension (No, Yes)

493 (83.3), 99 (16.7)

Liver resection range ( ≤ 2 segment, > 2 segment)

371 (62.7), 221 (37.3)

Surgical margin ( < 5mm, ≥ 5mm)

201 (34.0), 391 (66.0)

Anatomic resection (Yes, No)

162 (27.4), 430 (72.6)

Hepatic inflow occlusion (No, Yes)

280 (47.3), 312 (52.7)

Intraoperative blood loss ( ≤ 1000ml, > 1000ml)

526 (88.9), 66 (11.1)

Intraoperative blood transfusion (No, Yes)

458 (77.4), 134 (22.6)

AFP ( ≤ 20 ng/ml, > 20 ng/ml) *

308 (52.0), 276 (46.6)

Maximum tumor diameter ( ≤ 5cm, > 5cm)

411 (69.4), 181 (30.6)

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Number of lesions (One, Two, Three)

565 (95.4), 24 (4.1), 3 (0.5)

Histological differentiation (High, Middle and low, Necrosis)

88 (14.9), 482 (81.4), 22 (3.7)

Invasion of the liver capsule (No, Yes)*

208 (35.1), 378 (63.9)

Treatment after recurrence (Operation, TACE, Other)

84 (14.2), 173 (29.2), 97 (16.4)

Note: * indicates patients with missing data. Abbreviation: TACE, transcatheter arterial chemoembolization; ALT, alanine transaminase; GGT, gamma-glutamyl transpeptidase; AFP, alpha-fetoprotein.

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Table 2. Risk factors for recurrence in BCLC Stage A HCC patients following R0 resection Factors

Univariate

Multivariate analysis

analysis Median (months) Gender (Male , Female)

39.7, 205.7

Age ( ≤ 60years, > 60years)

48.1, 36.0

Alcohol (No, Yes)

49.2, 34.0

TACE (No, Yes)

43.9, 26.0

HBsAg (Negative, Positive)

49.0, 42.9

Anti-HCV (Negative, Positive)

42.9, 43.3

ALT ( ≤ 40U/L, > 40U/L)

56.0, 28.8

GGT ( ≤ 64U/L, > 64U/L)

53.8, 25.0

Child-Pugh classification (A, B)

43.3,

P-V

RR (95%Cl)

alue 0.0 07

P-V alue

0.722(0.520-1. 004)

0.0 53

0.2 38 0.1 72 0.1 11 0.1 93 0.9 32 0.0 00 0.0 00

1.278(1.016-1. 609) 1.227(0.956-1. 575)

0.0 36 0.1 08

0.0 16

26.0 Cirrhosis (No, Yes)

71.6, 40.0

Portal hypertension (No, Yes)

47.0, 33.9

Ascites (No, Yes)

43.3, 32.1

Liver resection range ( ≤ 2 segments, >2 segments) Surgical margin ( ≤5mm, >5mm)

43.0, 42.7 25.4, 52.0

Anatomic resection (Yes, No)

58.9, 36.8

Hepatic inflow occlusion (No, Yes)

45.0, 40.0

Intraoperative blood loss ( ≤ 1000ml, > 1000ml) Intraoperative blood transfusion (No, Yes) The preoperative AFP ( ≤ 20ng/ml, > 20ng/ml) Maximum tumor diameter ( ≤

48.1, 23.5 51.0, 27.0 51.7, 36.0 51.0,

73 0.0 15

1.670(1.127-2. 475)

0.0 11

0.1 38 0.2 13 0.9 27 0.0 00 0.0 07

0.720(0.577-0. 900) 1.494(1.154-1. 935)

0.0 04 0.0 02

0.3 89 0.0 06 0.0 04 0.0 11 0.0

1.231(0.844-1. 797) 1.098(0.814-1. 480) 1.195(0.962-1. 486) 1.330(1.046-1.

0.2 81 0.5 41 0.1 08 0.0 17

5cm, >5cm) Number of lesions ( < 2, ≥ 2)

25.0 42.9, 31.4

Histological differentiation

40.0,

(High, Middle and low , Necrosis)

43.1, 36.0

Invasion of liver capsule (No, Yes)

45.0, 41.1

01

691)

20

0.4 47 0.9 38 0.9 15

Abbreviation: TACE, transcatheter arterial chemoembolization; ALT, alanine transaminase; GGT, gamma-glutamyl transpeptidase; AFP, alpha-fetoprotein.

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Table 3. Score distribution of the BCLC Stage A patients Score

n

0

2

1

64

2

185

3

214

4

96

5

31