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Authors: Neung Hwa Park, Seok Won Jung, Eun‐Soon Shin, Young‐Hwa Chung
Journal: Hepatology (2014)

We appreciate Liu et al. for their interest in our study regarding the prognostic impact of telomere maintenance gene polymorphisms in hepatocellular carcinoma (HCC) patients with chronic hepatitis B.1 In our study, genotype identification was performed with the GenomeLab SNPstream ultrahigh-throughput system, which uses multiplexed polymerase chain reaction in conjunction with tag array single-base extension genotyping (Beckman Coulter, Fullerton, CA).2 The 4 × 4 arrays of oligonucleotides are attached to the glass surface in each well in 384-well format. Three positive controls and one negative control are included in each 4 × 4 array. The top-left location is a heterozygous control which has an equimolar mixture of two probes hybridizing to self-extending oligonucleotides that incorporate two dye-labeled terminators. The top-right location has probes that specifically hybridize to self-extending oligonucleotides that incorporate blue dye-labeled terminators. The bottom-left location has probes that specifically hybridize to self-extending oligonucleotides that incorporate green dye-labeled terminators. The bottom-right location has probes that are not self-extending and lack complementarity to any DNA in the reaction. These probes serve as negative controls in each well; 10% of randomly selected samples were repeated to verify genotyping results. As you suggested, we further provided other factors, such as alcohol consumption, smoking, and family history of HCC, in both HCC patients and controls. Patients with alcoholic liver diseases, defined as male patients whose daily alcohol intake was 40 g or more for 10 years and female patients whose daily alcohol intake was 20 g or more for 10 years or more, were excluded from this study. In addition, the amount of alcohol consumption was similar in both groups (P = 0.14). The prevalence of cigarette smoking status was similar between HCC cases (32%) and controls (29%, P = 0.135). The rate of family history of HCC was also similar in the HCC cases and controls (11% versus 9%, P = 0.210). We investigated the association between selective single nucleotide polymorphisms (SNPs) of telomere maintenance genes and the risk of the development of HCC and its clinical outcomes. In univariate analysis, five (TEP1 rs1713449, TEP1 rs872072, POT1 rs7784168, TERT rs13167280, and TERF1 rs2306494) of the 20 SNPs showed a significant effect on overall survival. Furthermore, the combined genotypic effect of five SNPs on the overall survival of HCC was strongest in the pooled analysis of the two cohorts. We also analyzed survival analysis according to significant genotypes stratified by tumor staging and tumor postoperative recurrence, as you suggested. The combined effect of TEP1 rs1713449, TEP1 rs872072, POT1 rs7784168, TERT rs13167280, and TERF1 rs2306494 in overall tumor staging were associated with overall survival (P < 0.001); they are represented in Fig. 1. Especially, the Kaplan-Meier curves showed that HCC patients with no high-risk genotype, one high-risk genotype, or two high-risk genotypes showed significantly better survival in comparison with patients with three or more high-risk genotypes in the very early (0), early (A), and intermediate (B) stages. Furthermore, in the recurrence and no recurrence of HCC groups after surgery, having three or more putatively high-risk genotypes tended to reduce survival compared with HCC patients with no high-risk genotype, one high-risk genotype, or two high-risk genotypes (P = 0.08). Finally, we conducted a multivariate analysis of the effects of genotype on survival with Cox proportional hazards models adjusted for well-known clinicopathological factors. The multivariate survival analysis showed that tumor staging and postoperative recurrence were the most important confounding factors. Fortunately, these five SNPs remained significant independent predictors of survival among HCC patients after adjustments for clinical factors. Furthermore, having three or more putatively high-risk genotypes also remained a significant independent predictor for survival. In conclusion, these results suggest that the polymorphisms of telomere maintenance genes may act as prognostic factors for predicting clinical outcomes of HCC patients with chronic hepatitis B virus (HBV) infections, regardless of well-known prognostic factors such as advanced cirrhosis, a diffuse tumor, advanced tumor staging, the presence of major vessel invasion and distant metastasis, an unresected tumor, and postoperative recurrence. Neung Hwa Park, M.D.1 Seok Won Jung, M.D.1 Eun-Soon Shin, Ph.D.2 Young-Hwa Chung, M.D.3 1Department of Internal Medicine University of Ulsan College of Medicine Ulsan University Hospital Ulsan, Korea 2DNA Link, Inc. Seoul, Korea 3Department of Internal Medicine University of Ulsan College of Medicine Asan Medical Center Seoul, Korea