| BackgroundLiver cancer is a lethal disease, hepatocellular carcinoma is the fifth most common cancer in the world and the third most prevalent cause of tumor-related death. An estimated 782,500 new liver cancer cases and 745,500 deaths occurred worldwide during 2012, with China alone accounting for about 50% of the total number of cases and deaths. The disease morbidity and mortality is increasing year after year. Hepatocellular carcinoma accounts for 80-90% of liver cancer.The development of HCC depends on accumulation of risk factors and genetic predisposition which lead to a wide array of molecular and cellular abnormalities within hepatic cells. The main pathogenic factors include chronic B viral hepatitis, chronic C viral hepatitis, aflatoxin B1 exposure, drinking and so on. But it is still a huge challenge to elucidate the patho genesis and to identify the subjects at risk. Epidermiological studies have shown that genetic factors play an important role in hepatocarcinogenesis. The growth of tumor needs new blood vessels to supply nutrients. HCC is a highly vascular tumor, and the importance of the angiogenic process in HCC progression and the correlation between tumor blood vessel density and clinical (or preclinical) outcome has already been described in the literature. Vascular endothelial growth factors (VEGF) and their receptors are believed to be important for the process of regulating angiogenesis. The kinase insert domain receptor (KDR) is the principal receptor that promotes the proangiogenic action of VEGF and has been the principal target of antiangiogenic therapies. KDR gene located in the fourth chromosome q11-q12, and the main biological function of KDR protein included regulating the endothelial cell proliferation, migration, permeability and survival. As the third generation of genetic marker, SNPs are DNA sequence polymorphisms at the genomic level which were caused by a single nucleotide mutation, and the rate of variation is greater than 1%. The genetic marker is relatively stable and SNPs can affect the function of genes through a variety of ways. Most studies about single nucleotide polymorphisms (SNPs) and hepatocellular carcinoma are concentrated on disease susceptibility, while studies about SNPs and HCC prognosis are rare. Given the role played by angiogenesis in the pathogenesis of HCC, there is increasing interest in the role of angiogenesis factors as a predictor of outcome in HCC. We therefore decided to investigate whether genetic variants in the KDR gene could act as biomarkers for TACE treatment outcome. In this study, we examined the SNPs in KDR gene and assessed the associations of the SNPs with progression-free survival in a Chinese cohort of unresectable HCC patients treated by TACE. Correlations of encountered genetic variants with clinicopathological features, and progression-free survival of these patients were analyzed to assess their potential biological and clinical implications. We intended to look for some meaningful genetic markers, and provide the basis for individualized treatment of hepatocellular carcinoma.MethodsPatients with unresectable HCC who underwent TACE as an initial treatment modality at the Department of Interventional Radiology in our hospital between 2011 and 2013 were selected from a prospectively maintained hepatobiliary tumor database. All patients satisfied the diagnostic criteria for HCC based on radiologic or histo logic grounds according to the American Association for the Study of the Liver guidelines. Four milliliters of venous blood sample was collected from each patient before any treatment, and the blood samples was anticoagulant using EDTA at-80℃. Leukocyte genomic DNA was extracted from each blood sample using Qiagen DNA Blood mini kits, following the manufacturer’s instructions. Concentration and purity index of each sample were evaluated by UV spectrophotometry as the ratio absorbance 260/280 nm.The KDR SNPs selected for genotyping were accessed from the public databases:dbSNP (http://www.ncbi.nih.gov) and HapMap (http://www.hapmap.org). Tag SNPs were selected using the Haploview software and SNP tagger approach. Only common SNPs were selected as tagSNPs by analysis of HapMap genotyping data with minor allele frequency of 0.1 or higher and a minimum correlation coefficient of the frequencies (r2) of 0.8. In addition, four SNPs (rs2305948, rs1870377, rs34231037 andrs34038364) were selected because they coded for a nonsynonymous amino acid change in the KDR gene and a functional SNP (rs2071559) in the KDR promoter previously reported to affect transcriptional activity of the KDR promoter, which were included by documentary search. SNPs genotyping was carried out using the iPLEX GoldTN assay on the Mass ARRAY Platform (Sequenom, CA)Tumor response was assessed according to modified response evaluation criteria in solid tumors.We defined CR+PR+SD as response group, and PD as progressive group.Data analysis was performed using the computer software Statistical Package for Social Sciences (SPSS) v.13 for Windows (SPSS Inc., Chicago, IL). Categorical variables were compared using the chi-square test, and continuous variables were compared using t test or the Mann-Whitney U test. The correlation between disorderly classification variables were analyzed using the chi-square test. Survival probability over time was estimated by Kaplan-Meier method. All tests were two-tailed, and p<0.05 was considered statistically significant.ResultsThe majority of patients (159,82.8%) were men, and 79.2%(152) of all patients were HBsAg-positive. Anti-viral treatments were offered in 139 HBV-infected patients (with entecavir, lamivudine or telbivudine). The median patient age was 53 years, and 126 patients were elder than 50 years. About 51.0% of patients had cirrhosis, and 64.6%(124) with significantly increased serum alpha-fetoprotein (AFP) (≥400μg/L). The range of tumor diameter was 3.4-13cm, and 76.0%(146) of patients had a tumor size larger than 5 cm. The range of tumor lesions was 1-8, and 57.8%(111) had multiple lesions (≥2).71.8% had a Child-Pugh score A and 28.1%had a child-pugh score B.31.8%(61) with portal vein thrombus, BCLC stage status was as follows-110 (57.3%) patients at stage B and 82 (42.7%) at stage C. Of the 192 patients evaluated, no complete responders, 42 patients (21.9%) had a partial response,36 (18.7%) had stable disease, and 114 (59.4%) had progressive disease, respectively.The distribution of patient age(<50years vs≥50 years), gender, lesion number (1 vs≥2), AFP level (<400ng/ml vs≥400ng/ml), liver cirrhosis, lymph node metastasis in response group and progressive group was similar (all P> 0.05). The difference of the distribution of BCLC stage, tumor diameter and portal vein tumor thrombus (PVTT) between two groups was statistically significant.The tumor BCLC C stage, tumor diameter more than 5 centimeters and presence of PVTT indicated shorter PFS. Age, gender, serum AFP level, lesion number, and Child-Pugh score were not associated with progression-free survival. The median PFS of patients with tumor diameter more than 5 centimeter was 11.5 months compared with 18 months for patients with tumor diameter less than 5 centimeter (P< 0.001). The median PFS of patients with PVTT was 10 months compared with 13 months for patients without PVTT (P< 0.001). The median PFS of patients with BCLC B stage was 13.5 months compared with 11 months for patients with BCLC C stage (P=0.011)The distribution of the tested genotypes did not deviate from Hardy-Weinberg equilibrium (P> 0.05).There were 3 genotypes in KDR gene rs2071559 loci, CC,TT and CT. The distribution frequency of the CC genotype in the response group and progressive group was 20.5% and 36.0%, respectively. The distribution frequency of the TT genotype in the response group and progressive group was 26.9% and 46.5%, respectively. The distribution frequency of the CT genotype in the response group and progressive group was 41.0% and 26.3%, respectively. The frequency of three genotypes in the response and progressive group were significant different, P=0.018.There were 3 genotypes in KDR gene rs1870377 loci, TT,TA and AA. The distribution frequency of the TT genotype in the response group and progressive group was 20.5% and 36.0%, respectively. The distribution frequency of the TA genotype in the response group and progressive group was 34.6% and 36.0%, respectively. The distribution frequency of the TT genotype in the response group and progressive group was 44.9% and 28.1%, respectively. The frequency of three genotypes in the response and progressive group were significant different, P=0.024. The frequency of the rest SNP loci between two groups was not significant different, P>0.05. The genotypes of rs2071559 and rs1870377 loci had no correlation with the baseline tumor diameter, PVTT, lymph node metastasis, performance status and child-puge stage.The PFS of CC genotype and CT+TT genotype in rs 2071559 locus was 15.0 months and 13.0 months, P= 0.006. The PFS of AA genotype and TT+TA genotype in rs 1870377 locus was 15.0 months and 11.5 months, P= 0.006. No other significant difference of PFS in the different genotypes of the rest SNPs loci was detected.Unconditioned logistic regression analysis showed that the CC genotypes of rs 2071559 and AA genotypes of rs1870377 would decrease the risk of disease progression in HCC patients (OR= 0.347,95% CI= 0.136-0.875, P=0.028; OR= 0.131,95% CI= 0.032-0.451, P=0.034). Multivariate Logistic regression analysis showed that BCLC stage and the polymorphism of rs2071559 were independent prognostic factors for TACE treated HCC (OR= 0.133,95% CI= 0.021-0.513, P= 0.025; OR= 0.415,95% CI= 0.218-0.677, P= 0.031).ConclusionsThe rate of disease progression is comparatively lower in HCC patients with CC genotypes of KDR gene rs2071559, which may be a prognostic factor for HCC. |
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