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A genetic variant at KIF1B predicts clinical outcome of HBV-related hepatocellular carcinoma in Chinese
Cancer Epidemiology 38 (2014) 608–612
Contents lists available at ScienceDirect
Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net
A genetic variant at KIF1B predicts clinical outcome of HBV-related hepatocellular carcinoma in Chinese Mingde Huang a,1, Yun Pan b,1, Jibin Liu c, Fuzhen Qi d, Juan Wen b, Kaipeng Xie b, Hongxia Ma b,e, Hongbing Shen b,e, Yao Liu f,**, Juncheng Dai b,e,* a
Department of Oncology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, Jiangsu 223300, China Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166, China c Department of Hepatobiliary Surgery, Nantong Tumor Hospital, Nantong, Jiangsu 226361, China d Department of Hepatopancreatobiliary Surgery, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, Jiangsu 223300, China e State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, China f Pathology Center and Department of Pathology, Soochow University, Suzhou, Jiangsu 215123, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 April 2014 Received in revised form 22 July 2014 Accepted 28 July 2014 Available online 18 August 2014
Background: Recently, a genome-wide association study conducted in Chinese reported a single nucleotide polymorphism at KIF1B, rs17401966, associated with the susceptibility of hepatitis B virusrelated hepatocellular carcinoma. In this study, we aim to investigate the effect of rs17401966 on the prognosis of hepatitis B virus-related hepatocellular carcinoma patients at intermediate or advanced stages. Methods: The SNP rs17401966 was genotyped using the TaqMan allelic discrimination assay in 414 intermediate or advanced hepatocellular carcinoma patients. Log-rank test and Cox proportional hazard models were used for survival analyses. Results: Previous studies have identified that the G allele of rs17401966 demonstrated protective effect for the susceptibility of hepatitis B virus-related hepatocellular carcinoma. Here we found that subjects carrying the G allele of rs17401966 was significantly associated with a better survival compared with those carrying the A allele (adjusted hazard ratio = 0.82, 95% confidence intervals = 0.68–0.99, P = 0.044 in an additive genetic model). Conclusion: The variant G allele of rs17401966 may be a favorable biomarker for the prognosis of intermediate or advanced hepatitis B virus-related hepatocellular carcinoma patients in this Chinese population. ß 2014 Elsevier Ltd. All rights reserved.
Keywords: KIF1B Single nucleotide polymorphism Hepatocellular carcinoma Outcome
1. Introduction Primary liver cancer is the sixth most common cancer worldwide. An estimated 748,300 new liver cancer cases and 695,900 cancer deaths occurred worldwide in 2008 [1]. Half of these cases and deaths were estimated to occur in China [2]. Hepatocellular carcinoma (HCC) accounts for 70–85% of primary malignancies in liver [3]. Given its non-obvious symptoms at early
* Corresponding author at: Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, 211166, Jiangsu, China. Tel.: +86 25 8686 8437; fax: +86 25 8686 8439. ** Corresponding author at: Pathology Center and Department of Pathology, Soochow University, Suzhou 215123, Jiangsu, China. Tel.: +86 13605184096. E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Dai). 1 These authors contributed equally in this work. http://dx.doi.org/10.1016/j.canep.2014.07.012 1877-7821/ß 2014 Elsevier Ltd. All rights reserved.
stage, most HCC patients are diagnosed at advanced stage, when they were not suitable for operation and most patients were in poor prognosis [4]. However, patients at the same stage may present different prognosis [5], because many factors can affect the long-term outcome, including tumor characteristics [6,7] and emerging biomarkers [8–11]. Here we explore the genetic factors affecting the long-term survival outcome in intermediate or advanced HCC patients. Recently, a single nucleotide polymorphism (SNP), rs17401966, locates at intron 24 of KIF1B, was identified associated with the susceptibility of hepatitis B virus (HBV)-related HCC from a genome-wide association study (GWAS) [12]. Several studies further confirmed that KIF1B was involved in the progression from hepatitis B virus (HBV) infection to HCC [12,13]. Moreover, frequent chromosomal loss at this locus was linked to several types of cancer, including basal cell carcinoma [14], gastric cancer [15], gastrointestinal stromal tumor [16], and neural and
M. Huang et al. / Cancer Epidemiology 38 (2014) 608–612
nonneural tumors [17]. However, no comprehensive analysis has been performed to explore this genetic variant on the prognosis of HCC patients. Here we hypothesize that rs17401966 at KIF1B may be associated with the prognosis of HCC patients, and we performed a case-only study to investigate the association between rs17401966 at KIF1B and the clinical outcome of HCC patients at intermediate or advanced stages. 2. Materials and methods 2.1. Participants The study was approved by the institutional review board of Nanjing Medical University. Written informed consent was obtained. The participants’ enrollment was described previously [18,19]. All the patients were treatment-naı¨ve newly diagnosis in consideration of prognostic modeling in HCC patients has a high complexity and should consider several tightly related aspects, including tumor stage, degree of liver function impairment, patient’s general condition, and treatment efficacy. In this study, we use the Barcelona Clinic Liver Cancer (BCLC) Stage System to evaluate the prognosis of HCC [20,21]. To construct a relatively homogenous population with similar treatment, our current study subjects were restricted to HCC patients at either intermediate (B) or advanced stage (C) without surgery and treatment-naive newly diagnosed. We recruited 414 HCC patients at intermediate or advanced stages with HBV positive plus hepatitis C virus (HCV) negative from Nantong Tumor Hospital and the first affiliated hospital of Nanjing Medical University, Jiangsu, China. All patients were followed up by personal or family contacts every 3 months from the time of enrollment until death or the last time of follow-up (last follow-up: January 2013). As a result, a total of 331 HCC patients at intermediate (B) or advanced stage (C) who had complete follow-ups and clinical information were enrolled in our study with a response rate of 80.0%. The maximum follow-up time (MFT) for the 331 patients was 60.7 months and the median survival time (MST) was 14.5 months. Demographic data, exposure information and clinical data of each patient were collected by a standard questionnaire. Individuals who smoked one cigarette per day for over one year were defined as smokers (ever-smokers were included) and those who consumed one or more alcohol drinks a week for over six months were categorized as alcohol drinkers (ever-drinkers were included). All the subjects included in the current study were not blood related.
609
2.4. Statistical methods Survival time was calculated from the date of HCC diagnosis to the date of patients dead or the last time of follow-up. Hardy– Weinberg equilibrium was assessed within patients by using a goodness-of-fit x2 test. Median survival time (MST) was calculated, and mean survival time was presented when the MST could not be calculated. Log-rank test was used to compare the survival time in different subgroups categorized by patient characteristics, clinical features and genotypes. The significance level was set at P < 0.05 and P values are given for two-sided testing. Univariate and multivariate Cox proportional hazard regression analysis were performed to estimate the crude or adjusted hazard ratio (HR) and their 95% confidence intervals (CIs), with adjustment of age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE (transcatheter hepatic arterial chemoembolization) status. The Chi-square-based Q test was applied to test the heterogeneity of associations between subgroups [22]. Analyses were carried out using Statistical Analysis System software (version 9.1.3; SAS Institute, Cary, NC). 3. Results 3.1. Patient characteristics and clinical features The demographic characteristics and clinical information for the 331 HCC patients in stage B or C recruited in the study were described in Table 1 and Supplementary Table 1. The median age was 53 years old. Of the 331 patients, 284 (85.8%) were males. 211(63.7%) and 204 (61.6%) were smokers or drinkers, respectively. 304 (91.8%) patients were in intermediate stage (B), 27 (8.2%) patients were in advanced stage (C). Among all patients, 240 (72.5%) received chemotherapy or TACE and 91 (27.5%) not. For the 331 HCC patients in stage B or C retained in our survival analysis, 258 died from HCC, and 2 died from other causes during a period of up to 60.7 months of follow-up. Drinking status and chemotherapy or TACE were significantly associated with the survival time (logrank P for drinking status = 0.006, for chemotherapy or TACE status <0.001). The drinkers had a higher risk of death than those who never drink (HR = 1.43, 95% CI = 1.11–1.84) and compared with those received neither chemotherapy nor TACE (MST = 3.4 months), patients received chemotherapy or TACE (MST = 16.8 months) had a 61% significantly decreased risk of death (HR = 0.39, 95% CI = 0.29–0.51).
2.2. SNPs genotyping 3.2. Genotypes of KIF1B polymorphisms and HCC patients’ survival Genomic DNA was extracted from a leukocyte pellet by traditional proteinase K digestion, phenol–chloroform extraction and ethanol precipitation. The SNP, rs17401966 A>G was genotyped using the TaqMan allelic discrimination assay on a 7900 system (Applied Biosystems). The primers and probes for rs17401966 were as follows. Primers: sense, 50 -TTTCCAGCACTTAATGAAAACACATAG, antisense, 50 -CAAAGTTAAATTTCCCTGCTTTGAA; Probes: allele A, FAM-TCTATGAGTCCGTATTGAGT-MGB, allele G, HEX-CTCTATGAGTCCATATTGAGT-MGB. The genotyping was performed blindly without knowing the subjects’ case or control status. Two blank (water) controls in each 384-well plate were used for quality control and more than 5% samples were randomly selected and repeated, yielding a 100% concordant.
The success rate of genotyping for rs17401966 was above 99%. We adopted Kaplan–Meier method and log-rank test were to examine the associations of KIF1B rs17401966 with HCC survival in different genetic models. Log-rank tests showed that patients carrying GG genotype of rs17401966 had an improved MST compared to those with AA genotype, although not reaching statistical significance (16.0 vs. 14.3 months, Table 2). In the Cox proportional hazard regression analyses, the G allele of rs17401966 was significantly associated with a better survival of intermediate or advanced HCC after adjusting for age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE status (adjusted HR = 0.82, 95% CI = 0.68–0.99, P = 0.044 in the additive model.
2.3. Serological testing 3.3. Stratified analysis HBsAg, anti-HBs, anti-HBc and anti-HCV were detected by the enzyme-linked immunosorbent assay (Kehua Bio-engineering Co., Ltd., Shanghai, China) following the manufacturer’s instructions as described previously [18].
The associations between KIF1B rs17401966 polymorphism and HCC survival were further investigated by stratified analysis of age, gender, smoking status, drinking status, BCLC stage, and
M. Huang et al. / Cancer Epidemiology 38 (2014) 608–612
610 Table 1 Patient characteristics and clinical features. Variables Age 53 >53 Gender Male Female Smoking status No Yes Drinking status No Yes BCLC stage stage B stage C Chemotherapy or TACE None Yes Ascitesa No Yes Hepatic encephalopathyb No Yes Vascular invasionc No Yes ALPd Normal (35–125 U/L) Abnormal (>125 U/L) PTe Normal (11–14 s) Abnormal (>14 s) GGTf Normal (0–50 U/L) Abnormal (>50 U/L) TBILIg Normal (4–20 mmol/L) Abnormal (>20 mmol/L) ALBh Normal (40–55 g/L) Abnormal (>55 g/L)
Patients (N = 331) (%)
Deaths (N = 258)
MST (months)
173 (52.3) 158 (47.7)
138 120
14.3 15.9
284 (85.8) 47 (14.2)
221 37
14.7 12.7
120 (36.3) 211 (63.7)
90 168
16 13.5
127 (38.4) 204 (61.6)
94 164
18 12.6
304 (91.8) 27 (8.2)
236 22
14.5 13
91 (27.5) 240 (72.5)
77 181
3.4 16.8
176 (53.2) 127 (38.4)
135 99
14.7 12.1
184 (55.6) 119 (40.0)
137 97
12.1 14.7
298 (90.0) 21 (6.3)
230 16
14.0 17.4
97 (29.3) 206 (62.2)
72 162
15.0 13.0
136 (41.1) 159 (48.4)
102 125
14.9 12.8
50 (15.1) 253 (76.4)
34 200
14.1 13.4
11 (3.3) 292 (88.2)
7 227
19.3 13.5
176 (53.2) 119 (36.0)
131 96
14.5 12.4
Log-rank P
HR (95% CI)
0.257 1 0.87 (0.68–1.11) 0.755 1 1.06 (0.75–1.50) 0.192 1 1.19 (0.92–1.53) 0.006 1 1.43 (1.12–1.84) 0.389 1 1.21 (0.78–1.88) <0.001 1 0.39 (0.29–0.51) 0.517 1 1.09 (0.84–1.41) 0.543 1 1.09 (0.83–1.42) 0.708 1 1.10 (0.66–1.83) 0.135 1 1.24 (0.94–1.63) 0.150 1 1.21 (0.93–1.58) 0.295 1 1.21 (0.84–1.75) 0.289 1 1.50 (0.71–3.19) 0.292 1 1.15 (0.86–1.50)
MST: median survival time; HR: hazard ratio; CI: confidence interval; HCC: hepatocellular carcinoma; BCLC stage: Barcelona Clinic Liver Cancer stage; TACE: transcatheter hepatic arterial chemoembolization; ALP: alkaline phosphatase; PT: prothrombintime; GGT: glutamyl transpeptidase; TBILI: total bilirubin; ALB: albumin. a Ascites information are unavailable in 28 patients. b Hepatic encephalopathy information are unavailable in 28 patients. c Vascular invasion information are unavailable in 12 patients. d ALP information are unavailable in 28 patients. e PT information are unavailable in 36 patients. f GGT information are unavailable in 28 patients. g TBILI information are unavailable in 28 patients. h ALB information are unavailable in 36 patients.
chemotherapy or TACE status. As shown in Table 3, the G allele of rs17401966 was still significantly associated with HCC survival in patients older than 53 years (adjusted HR = 0.72, 95% CI = 0.55– 0.95) and in males (adjusted HR = 0.79, 95% CI = 0.64–0.97). However, no significant heterogeneity was observed between different subgroups.
4. Discussion Previous studies have identified that the G allele of rs17401966 demonstrated protective effect for the susceptibility of HBV-related HCC [12,13]. Here we found that the G allele of rs17401966 at KIF1B was significantly associated with a
Table 2 Genotypes of rs17401966 and hepatocellular carcinoma patients’ survival. Genotype
Patients
Deaths
MST (months)
1-year OS
3-year OS
Crude HR (95% CI)
Adjusted HR (95% CI)a
Pa
rs17401966 AA AG GG AG/GG Additive model
176 121 32 153
143 93 20 113
14.3 14.5 16.0 14.9
0.567 0.552 0.594 0.561
0.120 0.177 0.390 0.209
1 0.90 0.65 0.85 0.85
1 0.85 0.65 0.80 0.82
0.220 0.068 0.081 0.044
(0.70–1.17) (0.41–1.04) (0.66–1.08) (0.70–1.02)
(0.65–1.10) (0.40–1.03) (0.62–1.03) (0.68–0.99)
MST: median survival time; HR: hazard ratio; CI: confidence interval; TACE: transcatheter hepatic arterial chemoembolization; 1-year OS: 1-year cumulative overall survival rate; 3-year OS: 3-year cumulative overall survival rate. a Adjusted for age, gender, smoking and drinking status, BCLC stage, and chemotherapy or TACE status.
M. Huang et al. / Cancer Epidemiology 38 (2014) 608–612
611
Table 3 Stratified analysis of rs17401966 genotypes and hepatocellular carcinoma survival. Variables
Age 53 >53 Gender Male Female Smoking status No Yes Drinking status No Yes Chemotherapy or TACE None Chemotherapy TACE Both
rs17401966 (patients/deaths)
Adjusted HR (95% CI)a
P for heterogeneity
AA
AG
GG
90/77 86/66
63/49 58/44
18/10 14/10
0.72 (0.55–0.95) 0.92 (0.69–1.23)
0.227
147/121 29/22
110/85 11/8
25/13 7/7
0.79 (0.64–0.97) 1.23 (0.79–1.92)
0.077
66/50 110/93
38/29 83/64
16/11 16/9
0.95 (0.71–1.28) 0.79 (0.62–1.01)
0.345
68/53 108/90
47/34 74/59
12/7 20/13
0.80 (0.57–1.12) 0.89 (0.70–1.13)
0.614
47/41 50/32 57/52 22/18
38/31 30/18 35/30 18/14
6/5 10/4 12/8 4/3
0.85 0.69 0.79 1.14
0.554
(0.58–1.25) (0.45–1.07) (0.58–1.09) (0.66–1.97)
TACE: transcatheter hepatic arterial chemoembolization; HR: hazard ratio; CI: confidence interval. a Adjusted for age, gender, smoking and drinking status, BCLC stage, and chemotherapy or TACE status (excluded the stratified factor in each stratum).
favorable prognosis in HCC patients at intermediate or advanced stage. KIF1B locates on chromosome 1p36, which encodes two spliced isoforms of kinesin protein, KIF1Ba and KIF1Bb, both of which involved in the transport of organelles and vesicles [23]. For many years, KIF1B gene has been elucidated to act as a tumor suppressor in multiple cancers by acting a variety of inhibitors of cell proliferation and/or activators of apoptotic cell death, such as aggressive neuroblastoma, pheochromocytoma, colon, liver, brain, breast, and other cancers [24]. Recent study also demonstrated that KIF1B was associated with gastric cancer (GC) invasion [25], which indicated that KIF1B may play a role in the progression of cancers. Rs17401966 locates at intron 24 of KIF1B. By using the database of RegulomeDB (http://regulomedb.org), we found that rs17401966 might has an effect on the binding of motif YY1 and the expression of KIF1B [26,27], which indicating that rs17401966 might function by interacting with YY1 and regulating KIF1B expression. Recent GWAS and the following replication studies have shown that the G allele of rs17401996 was associated with decreased risk for HCC [12,13]. And in this study, we supported that the G allele of rs17401966 in KIF1B may be a favorable prognostic biomarker for HCC. Our study was the first one to analyze the association between the polymorphism at KIF1B and the prognosis of intermediate or advanced HCC patients; however, a study conducted by Sopipong et al. [28] drawn a inconsistent conclusion that rs17401966 was not associated with the development of HBV-related hepatocellular carcinoma in Thai patients, which might be due to different genetic background. Additionally, we failed to collect enough and accurate information on viral factors which were also important in determining the patients’ survival, such as HBeAg levels, genotype and mutations of HBV DNA, HBV DNA levels, and antiviral therapy. Furthermore, as other association studies, the exact mechanism was still not elucidated in our study, though, it provided a clue for our future studies. In summary, our findings suggest that polymorphism at KIF1B may be a genetic biomarker for the prognosis of intermediate or advanced HCC patients. However, larger population based prospective studies are warranted to further elucidate the impact of rs17401996 on HCC prognosis and genetic mechanism underlying the association needs further comprehensive analyses as well.
Conflict of interest statement None. Authorship contribution Study concept and design: Juncheng Dai, Yao Liu, Hongbing Shen, and Hongxia Ma; acquisition of data: Jibin Liu, Yun Pan, Mingde Huang, and Fuzhen Qi; analysis and interpretation of data: Yun Pan, and Yao Liu; manuscript writing: Mingde Huang; critical revision of the manuscript for important intellectual content: Juncheng Dai, and Yun Pan; statistical analysis: Yun Pan, and Yao Liu; study supervision: Juncheng Dai. Acknowledgements This work was funded by the National Natural Science Foundation of China (81372606 and 81072344), Project supported by the National Key Basic Research Program Grant (2013CB911400), the project supported by the National Science Foundation for Distinguished Young Scholars of China (81225020), Foundation of Jiangsu Province for Distinguished Young Scholars (BK2012042), Foundation for the Program for New Century Excellent Talents in University (NCET-10-0178), the Fok YingTong Education Foundation for Young Teachers in the Higher Education Institutions (122031), Young tip-top talents support program by the Organization Department of the CPC Central Committee, the Author of National Excellent Doctoral Dissertation (201081), Collaborative Innovation Center For Cancer Personalized Medicine, Jiangsu Province Clinical Science and Technology Projects (BL2012008) and the Priority Academic Program for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canep.2014.07.012. References [1] Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61:69–90.
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[2] Perz JF, Armstrong GL, Farrington LA, et al. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol 2006;45:529–38. [3] Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–917. [4] Kung JW, Macdougall M, Madhavan KK, et al. Predicting survival in patients with hepatocellular carcinoma: a UK perspective. Eur J Surg Oncol 2007;33:188–94. [5] Chen CH, Su WW, Yang SS, et al. Long-term trends and geographic variations in the survival of patients with hepatocellular carcinoma: analysis of 11,312 patients in Taiwan. J Gastroenterol Hepatol 2006;21:1561–6. [6] Fan ST, Ng IO, Poon RT, et al. Hepatectomy for hepatocellular carcinoma: the surgeon’s role in long-term survival. Arch Surg 1999;134:1124–30. [7] Llovet JM, Fuster J, Bruix J. Prognosis of hepatocellular carcinoma. Hepatogastroenterology 2002;49:7–11. [8] Yang H, Zhai G, Ji X, et al. LAPTM4B allele *2 is a marker of poor prognosis following hepatic tumor resection for hepatocellular carcinoma. PLoS ONE 2012;7:e34984. [9] Geng M, Cao YC, Chen YJ, et al. Loss of Wnt5a and Ror2 protein in hepatocellular carcinoma associated with poor prognosis. World J Gastroenterol 2012;18:1328–38. [10] Chan KC, Lai PB, Mok TS, et al. Quantitative analysis of circulating methylated DNA as a biomarker for hepatocellular carcinoma. Clin Chem 2008;54:1528–36. [11] Shen H, Jin G. Human genome epidemiology, progress and future. J Biomed Res 2013;27:167–9. [12] Zhang H, Zhai Y, Hu Z, et al. Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers. Nat Genet 2010;42:755–8. [13] Wang ZC, Gao Q, Shi JY, et al. Genetic polymorphism of the kinesin-like protein KIF1B gene and the risk of hepatocellular carcinoma. PLoS ONE 2013;8:e62571. [14] Stacey SN, Gudbjartsson DF, Sulem P, et al. Common variants on 1p36 and 1q42 are associated with cutaneous basal cell carcinoma but not with melanoma or pigmentation traits. Nat Genet 2008;40:1313–8. [15] Dong Z, Xu X, Du L, et al. Leptin-mediated regulation of MT1-MMP localization is KIF1B dependent and enhances gastric cancer cell invasion. Carcinogenesis 2013;34:974–83.
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Contents lists available at ScienceDirect
Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net
A genetic variant at KIF1B predicts clinical outcome of HBV-related hepatocellular carcinoma in Chinese Mingde Huang a,1, Yun Pan b,1, Jibin Liu c, Fuzhen Qi d, Juan Wen b, Kaipeng Xie b, Hongxia Ma b,e, Hongbing Shen b,e, Yao Liu f,**, Juncheng Dai b,e,* a
Department of Oncology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, Jiangsu 223300, China Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166, China c Department of Hepatobiliary Surgery, Nantong Tumor Hospital, Nantong, Jiangsu 226361, China d Department of Hepatopancreatobiliary Surgery, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, Jiangsu 223300, China e State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu 211166, China f Pathology Center and Department of Pathology, Soochow University, Suzhou, Jiangsu 215123, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 April 2014 Received in revised form 22 July 2014 Accepted 28 July 2014 Available online 18 August 2014
Background: Recently, a genome-wide association study conducted in Chinese reported a single nucleotide polymorphism at KIF1B, rs17401966, associated with the susceptibility of hepatitis B virusrelated hepatocellular carcinoma. In this study, we aim to investigate the effect of rs17401966 on the prognosis of hepatitis B virus-related hepatocellular carcinoma patients at intermediate or advanced stages. Methods: The SNP rs17401966 was genotyped using the TaqMan allelic discrimination assay in 414 intermediate or advanced hepatocellular carcinoma patients. Log-rank test and Cox proportional hazard models were used for survival analyses. Results: Previous studies have identified that the G allele of rs17401966 demonstrated protective effect for the susceptibility of hepatitis B virus-related hepatocellular carcinoma. Here we found that subjects carrying the G allele of rs17401966 was significantly associated with a better survival compared with those carrying the A allele (adjusted hazard ratio = 0.82, 95% confidence intervals = 0.68–0.99, P = 0.044 in an additive genetic model). Conclusion: The variant G allele of rs17401966 may be a favorable biomarker for the prognosis of intermediate or advanced hepatitis B virus-related hepatocellular carcinoma patients in this Chinese population. ß 2014 Elsevier Ltd. All rights reserved.
Keywords: KIF1B Single nucleotide polymorphism Hepatocellular carcinoma Outcome
1. Introduction Primary liver cancer is the sixth most common cancer worldwide. An estimated 748,300 new liver cancer cases and 695,900 cancer deaths occurred worldwide in 2008 [1]. Half of these cases and deaths were estimated to occur in China [2]. Hepatocellular carcinoma (HCC) accounts for 70–85% of primary malignancies in liver [3]. Given its non-obvious symptoms at early
* Corresponding author at: Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, 211166, Jiangsu, China. Tel.: +86 25 8686 8437; fax: +86 25 8686 8439. ** Corresponding author at: Pathology Center and Department of Pathology, Soochow University, Suzhou 215123, Jiangsu, China. Tel.: +86 13605184096. E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Dai). 1 These authors contributed equally in this work. http://dx.doi.org/10.1016/j.canep.2014.07.012 1877-7821/ß 2014 Elsevier Ltd. All rights reserved.
stage, most HCC patients are diagnosed at advanced stage, when they were not suitable for operation and most patients were in poor prognosis [4]. However, patients at the same stage may present different prognosis [5], because many factors can affect the long-term outcome, including tumor characteristics [6,7] and emerging biomarkers [8–11]. Here we explore the genetic factors affecting the long-term survival outcome in intermediate or advanced HCC patients. Recently, a single nucleotide polymorphism (SNP), rs17401966, locates at intron 24 of KIF1B, was identified associated with the susceptibility of hepatitis B virus (HBV)-related HCC from a genome-wide association study (GWAS) [12]. Several studies further confirmed that KIF1B was involved in the progression from hepatitis B virus (HBV) infection to HCC [12,13]. Moreover, frequent chromosomal loss at this locus was linked to several types of cancer, including basal cell carcinoma [14], gastric cancer [15], gastrointestinal stromal tumor [16], and neural and
M. Huang et al. / Cancer Epidemiology 38 (2014) 608–612
nonneural tumors [17]. However, no comprehensive analysis has been performed to explore this genetic variant on the prognosis of HCC patients. Here we hypothesize that rs17401966 at KIF1B may be associated with the prognosis of HCC patients, and we performed a case-only study to investigate the association between rs17401966 at KIF1B and the clinical outcome of HCC patients at intermediate or advanced stages. 2. Materials and methods 2.1. Participants The study was approved by the institutional review board of Nanjing Medical University. Written informed consent was obtained. The participants’ enrollment was described previously [18,19]. All the patients were treatment-naı¨ve newly diagnosis in consideration of prognostic modeling in HCC patients has a high complexity and should consider several tightly related aspects, including tumor stage, degree of liver function impairment, patient’s general condition, and treatment efficacy. In this study, we use the Barcelona Clinic Liver Cancer (BCLC) Stage System to evaluate the prognosis of HCC [20,21]. To construct a relatively homogenous population with similar treatment, our current study subjects were restricted to HCC patients at either intermediate (B) or advanced stage (C) without surgery and treatment-naive newly diagnosed. We recruited 414 HCC patients at intermediate or advanced stages with HBV positive plus hepatitis C virus (HCV) negative from Nantong Tumor Hospital and the first affiliated hospital of Nanjing Medical University, Jiangsu, China. All patients were followed up by personal or family contacts every 3 months from the time of enrollment until death or the last time of follow-up (last follow-up: January 2013). As a result, a total of 331 HCC patients at intermediate (B) or advanced stage (C) who had complete follow-ups and clinical information were enrolled in our study with a response rate of 80.0%. The maximum follow-up time (MFT) for the 331 patients was 60.7 months and the median survival time (MST) was 14.5 months. Demographic data, exposure information and clinical data of each patient were collected by a standard questionnaire. Individuals who smoked one cigarette per day for over one year were defined as smokers (ever-smokers were included) and those who consumed one or more alcohol drinks a week for over six months were categorized as alcohol drinkers (ever-drinkers were included). All the subjects included in the current study were not blood related.
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2.4. Statistical methods Survival time was calculated from the date of HCC diagnosis to the date of patients dead or the last time of follow-up. Hardy– Weinberg equilibrium was assessed within patients by using a goodness-of-fit x2 test. Median survival time (MST) was calculated, and mean survival time was presented when the MST could not be calculated. Log-rank test was used to compare the survival time in different subgroups categorized by patient characteristics, clinical features and genotypes. The significance level was set at P < 0.05 and P values are given for two-sided testing. Univariate and multivariate Cox proportional hazard regression analysis were performed to estimate the crude or adjusted hazard ratio (HR) and their 95% confidence intervals (CIs), with adjustment of age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE (transcatheter hepatic arterial chemoembolization) status. The Chi-square-based Q test was applied to test the heterogeneity of associations between subgroups [22]. Analyses were carried out using Statistical Analysis System software (version 9.1.3; SAS Institute, Cary, NC). 3. Results 3.1. Patient characteristics and clinical features The demographic characteristics and clinical information for the 331 HCC patients in stage B or C recruited in the study were described in Table 1 and Supplementary Table 1. The median age was 53 years old. Of the 331 patients, 284 (85.8%) were males. 211(63.7%) and 204 (61.6%) were smokers or drinkers, respectively. 304 (91.8%) patients were in intermediate stage (B), 27 (8.2%) patients were in advanced stage (C). Among all patients, 240 (72.5%) received chemotherapy or TACE and 91 (27.5%) not. For the 331 HCC patients in stage B or C retained in our survival analysis, 258 died from HCC, and 2 died from other causes during a period of up to 60.7 months of follow-up. Drinking status and chemotherapy or TACE were significantly associated with the survival time (logrank P for drinking status = 0.006, for chemotherapy or TACE status <0.001). The drinkers had a higher risk of death than those who never drink (HR = 1.43, 95% CI = 1.11–1.84) and compared with those received neither chemotherapy nor TACE (MST = 3.4 months), patients received chemotherapy or TACE (MST = 16.8 months) had a 61% significantly decreased risk of death (HR = 0.39, 95% CI = 0.29–0.51).
2.2. SNPs genotyping 3.2. Genotypes of KIF1B polymorphisms and HCC patients’ survival Genomic DNA was extracted from a leukocyte pellet by traditional proteinase K digestion, phenol–chloroform extraction and ethanol precipitation. The SNP, rs17401966 A>G was genotyped using the TaqMan allelic discrimination assay on a 7900 system (Applied Biosystems). The primers and probes for rs17401966 were as follows. Primers: sense, 50 -TTTCCAGCACTTAATGAAAACACATAG, antisense, 50 -CAAAGTTAAATTTCCCTGCTTTGAA; Probes: allele A, FAM-TCTATGAGTCCGTATTGAGT-MGB, allele G, HEX-CTCTATGAGTCCATATTGAGT-MGB. The genotyping was performed blindly without knowing the subjects’ case or control status. Two blank (water) controls in each 384-well plate were used for quality control and more than 5% samples were randomly selected and repeated, yielding a 100% concordant.
The success rate of genotyping for rs17401966 was above 99%. We adopted Kaplan–Meier method and log-rank test were to examine the associations of KIF1B rs17401966 with HCC survival in different genetic models. Log-rank tests showed that patients carrying GG genotype of rs17401966 had an improved MST compared to those with AA genotype, although not reaching statistical significance (16.0 vs. 14.3 months, Table 2). In the Cox proportional hazard regression analyses, the G allele of rs17401966 was significantly associated with a better survival of intermediate or advanced HCC after adjusting for age, gender, smoking status, drinking status, BCLC stage, and chemotherapy or TACE status (adjusted HR = 0.82, 95% CI = 0.68–0.99, P = 0.044 in the additive model.
2.3. Serological testing 3.3. Stratified analysis HBsAg, anti-HBs, anti-HBc and anti-HCV were detected by the enzyme-linked immunosorbent assay (Kehua Bio-engineering Co., Ltd., Shanghai, China) following the manufacturer’s instructions as described previously [18].
The associations between KIF1B rs17401966 polymorphism and HCC survival were further investigated by stratified analysis of age, gender, smoking status, drinking status, BCLC stage, and
M. Huang et al. / Cancer Epidemiology 38 (2014) 608–612
610 Table 1 Patient characteristics and clinical features. Variables Age 53 >53 Gender Male Female Smoking status No Yes Drinking status No Yes BCLC stage stage B stage C Chemotherapy or TACE None Yes Ascitesa No Yes Hepatic encephalopathyb No Yes Vascular invasionc No Yes ALPd Normal (35–125 U/L) Abnormal (>125 U/L) PTe Normal (11–14 s) Abnormal (>14 s) GGTf Normal (0–50 U/L) Abnormal (>50 U/L) TBILIg Normal (4–20 mmol/L) Abnormal (>20 mmol/L) ALBh Normal (40–55 g/L) Abnormal (>55 g/L)
Patients (N = 331) (%)
Deaths (N = 258)
MST (months)
173 (52.3) 158 (47.7)
138 120
14.3 15.9
284 (85.8) 47 (14.2)
221 37
14.7 12.7
120 (36.3) 211 (63.7)
90 168
16 13.5
127 (38.4) 204 (61.6)
94 164
18 12.6
304 (91.8) 27 (8.2)
236 22
14.5 13
91 (27.5) 240 (72.5)
77 181
3.4 16.8
176 (53.2) 127 (38.4)
135 99
14.7 12.1
184 (55.6) 119 (40.0)
137 97
12.1 14.7
298 (90.0) 21 (6.3)
230 16
14.0 17.4
97 (29.3) 206 (62.2)
72 162
15.0 13.0
136 (41.1) 159 (48.4)
102 125
14.9 12.8
50 (15.1) 253 (76.4)
34 200
14.1 13.4
11 (3.3) 292 (88.2)
7 227
19.3 13.5
176 (53.2) 119 (36.0)
131 96
14.5 12.4
Log-rank P
HR (95% CI)
0.257 1 0.87 (0.68–1.11) 0.755 1 1.06 (0.75–1.50) 0.192 1 1.19 (0.92–1.53) 0.006 1 1.43 (1.12–1.84) 0.389 1 1.21 (0.78–1.88) <0.001 1 0.39 (0.29–0.51) 0.517 1 1.09 (0.84–1.41) 0.543 1 1.09 (0.83–1.42) 0.708 1 1.10 (0.66–1.83) 0.135 1 1.24 (0.94–1.63) 0.150 1 1.21 (0.93–1.58) 0.295 1 1.21 (0.84–1.75) 0.289 1 1.50 (0.71–3.19) 0.292 1 1.15 (0.86–1.50)
MST: median survival time; HR: hazard ratio; CI: confidence interval; HCC: hepatocellular carcinoma; BCLC stage: Barcelona Clinic Liver Cancer stage; TACE: transcatheter hepatic arterial chemoembolization; ALP: alkaline phosphatase; PT: prothrombintime; GGT: glutamyl transpeptidase; TBILI: total bilirubin; ALB: albumin. a Ascites information are unavailable in 28 patients. b Hepatic encephalopathy information are unavailable in 28 patients. c Vascular invasion information are unavailable in 12 patients. d ALP information are unavailable in 28 patients. e PT information are unavailable in 36 patients. f GGT information are unavailable in 28 patients. g TBILI information are unavailable in 28 patients. h ALB information are unavailable in 36 patients.
chemotherapy or TACE status. As shown in Table 3, the G allele of rs17401966 was still significantly associated with HCC survival in patients older than 53 years (adjusted HR = 0.72, 95% CI = 0.55– 0.95) and in males (adjusted HR = 0.79, 95% CI = 0.64–0.97). However, no significant heterogeneity was observed between different subgroups.
4. Discussion Previous studies have identified that the G allele of rs17401966 demonstrated protective effect for the susceptibility of HBV-related HCC [12,13]. Here we found that the G allele of rs17401966 at KIF1B was significantly associated with a
Table 2 Genotypes of rs17401966 and hepatocellular carcinoma patients’ survival. Genotype
Patients
Deaths
MST (months)
1-year OS
3-year OS
Crude HR (95% CI)
Adjusted HR (95% CI)a
Pa
rs17401966 AA AG GG AG/GG Additive model
176 121 32 153
143 93 20 113
14.3 14.5 16.0 14.9
0.567 0.552 0.594 0.561
0.120 0.177 0.390 0.209
1 0.90 0.65 0.85 0.85
1 0.85 0.65 0.80 0.82
0.220 0.068 0.081 0.044
(0.70–1.17) (0.41–1.04) (0.66–1.08) (0.70–1.02)
(0.65–1.10) (0.40–1.03) (0.62–1.03) (0.68–0.99)
MST: median survival time; HR: hazard ratio; CI: confidence interval; TACE: transcatheter hepatic arterial chemoembolization; 1-year OS: 1-year cumulative overall survival rate; 3-year OS: 3-year cumulative overall survival rate. a Adjusted for age, gender, smoking and drinking status, BCLC stage, and chemotherapy or TACE status.
M. Huang et al. / Cancer Epidemiology 38 (2014) 608–612
611
Table 3 Stratified analysis of rs17401966 genotypes and hepatocellular carcinoma survival. Variables
Age 53 >53 Gender Male Female Smoking status No Yes Drinking status No Yes Chemotherapy or TACE None Chemotherapy TACE Both
rs17401966 (patients/deaths)
Adjusted HR (95% CI)a
P for heterogeneity
AA
AG
GG
90/77 86/66
63/49 58/44
18/10 14/10
0.72 (0.55–0.95) 0.92 (0.69–1.23)
0.227
147/121 29/22
110/85 11/8
25/13 7/7
0.79 (0.64–0.97) 1.23 (0.79–1.92)
0.077
66/50 110/93
38/29 83/64
16/11 16/9
0.95 (0.71–1.28) 0.79 (0.62–1.01)
0.345
68/53 108/90
47/34 74/59
12/7 20/13
0.80 (0.57–1.12) 0.89 (0.70–1.13)
0.614
47/41 50/32 57/52 22/18
38/31 30/18 35/30 18/14
6/5 10/4 12/8 4/3
0.85 0.69 0.79 1.14
0.554
(0.58–1.25) (0.45–1.07) (0.58–1.09) (0.66–1.97)
TACE: transcatheter hepatic arterial chemoembolization; HR: hazard ratio; CI: confidence interval. a Adjusted for age, gender, smoking and drinking status, BCLC stage, and chemotherapy or TACE status (excluded the stratified factor in each stratum).
favorable prognosis in HCC patients at intermediate or advanced stage. KIF1B locates on chromosome 1p36, which encodes two spliced isoforms of kinesin protein, KIF1Ba and KIF1Bb, both of which involved in the transport of organelles and vesicles [23]. For many years, KIF1B gene has been elucidated to act as a tumor suppressor in multiple cancers by acting a variety of inhibitors of cell proliferation and/or activators of apoptotic cell death, such as aggressive neuroblastoma, pheochromocytoma, colon, liver, brain, breast, and other cancers [24]. Recent study also demonstrated that KIF1B was associated with gastric cancer (GC) invasion [25], which indicated that KIF1B may play a role in the progression of cancers. Rs17401966 locates at intron 24 of KIF1B. By using the database of RegulomeDB (http://regulomedb.org), we found that rs17401966 might has an effect on the binding of motif YY1 and the expression of KIF1B [26,27], which indicating that rs17401966 might function by interacting with YY1 and regulating KIF1B expression. Recent GWAS and the following replication studies have shown that the G allele of rs17401996 was associated with decreased risk for HCC [12,13]. And in this study, we supported that the G allele of rs17401966 in KIF1B may be a favorable prognostic biomarker for HCC. Our study was the first one to analyze the association between the polymorphism at KIF1B and the prognosis of intermediate or advanced HCC patients; however, a study conducted by Sopipong et al. [28] drawn a inconsistent conclusion that rs17401966 was not associated with the development of HBV-related hepatocellular carcinoma in Thai patients, which might be due to different genetic background. Additionally, we failed to collect enough and accurate information on viral factors which were also important in determining the patients’ survival, such as HBeAg levels, genotype and mutations of HBV DNA, HBV DNA levels, and antiviral therapy. Furthermore, as other association studies, the exact mechanism was still not elucidated in our study, though, it provided a clue for our future studies. In summary, our findings suggest that polymorphism at KIF1B may be a genetic biomarker for the prognosis of intermediate or advanced HCC patients. However, larger population based prospective studies are warranted to further elucidate the impact of rs17401996 on HCC prognosis and genetic mechanism underlying the association needs further comprehensive analyses as well.
Conflict of interest statement None. Authorship contribution Study concept and design: Juncheng Dai, Yao Liu, Hongbing Shen, and Hongxia Ma; acquisition of data: Jibin Liu, Yun Pan, Mingde Huang, and Fuzhen Qi; analysis and interpretation of data: Yun Pan, and Yao Liu; manuscript writing: Mingde Huang; critical revision of the manuscript for important intellectual content: Juncheng Dai, and Yun Pan; statistical analysis: Yun Pan, and Yao Liu; study supervision: Juncheng Dai. Acknowledgements This work was funded by the National Natural Science Foundation of China (81372606 and 81072344), Project supported by the National Key Basic Research Program Grant (2013CB911400), the project supported by the National Science Foundation for Distinguished Young Scholars of China (81225020), Foundation of Jiangsu Province for Distinguished Young Scholars (BK2012042), Foundation for the Program for New Century Excellent Talents in University (NCET-10-0178), the Fok YingTong Education Foundation for Young Teachers in the Higher Education Institutions (122031), Young tip-top talents support program by the Organization Department of the CPC Central Committee, the Author of National Excellent Doctoral Dissertation (201081), Collaborative Innovation Center For Cancer Personalized Medicine, Jiangsu Province Clinical Science and Technology Projects (BL2012008) and the Priority Academic Program for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canep.2014.07.012. References [1] Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61:69–90.
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