Journal name: Cancer Management and Research Article Designation: Original Research Year: 2018 Volume: 10 Cancer Management and Research Dovepress Running head verso: Yang et al Running head recto: Diagnostic and prognostic values of ERCC in HBV-related HCC open access to scientific and medical research DOI: http://dx.doi.org/10.2147/CMAR.S179043

Open Access Full Text Article Original Research Diagnostic and prognostic values of the mRNA expression of excision repair cross- complementation enzymes in hepatitis B virus- related hepatocellular carcinoma

Lu Yang1,* Background: The current study aims at using the whole genome expression profile chips for Ming Xu2,* systematically investigating the diagnostic and prognostic values of excision repair cross-com- Chuan-Bao Cui1 plementation (ERCC) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Peng-Hai Wei1 Materials and methods: Whole genome expression profile chips were obtained from the Shu-Zhi Wu1 GSE14520. The receiver-operating characteristic (ROC) curve, survival analysis, and nomogram were used to investigate the diagnostic and prognostic values of ERCC genes. Investigation of Zuo-Jie Cen1 the potential function of ERCC8 was carried out by set enrichment analysis (GSEA) and Xing-Xing Meng1 genome-wide coexpression analysis. Qiong-Guang Huang1 Results: ROC analysis suggests that six ERCC genes (ERCC1, ERCC2, ERCC3, ERCC4, 1 Zhi-Chun Xie ERCC5, and ERCC8) were dysregulated and may have potential to distinguish between HBV- 1Department of Epidemiology, related HCC tumor and paracancerous tissues (area under the curve of ROC ranged from 0.623 Guangxi Medical University, Nanning to 0.744). Survival analysis demonstrated that high ERCC8 expression was associated with a 530021, Guangxi Zhuang Autonomous Region, People’s Republic of China; significantly decreased risk of recurrence (adjusted P=0.021; HR=0.643; 95% CI=0.442–0.937) 2Department of Human Anatomy and death (adjusted P=0.049; HR=0.631; 95% CI=0.399–0.998) in HBV-related HCC. Then, and Histology and Embryology, Qilu we also developed two nomograms for the HBV-related HCC individualized prognosis predic- Medical University, Zibo 255213, Shandong Province, People’s Republic tions. GSEA suggests that the high expression of ERCC8 may have involvement in the energy of China metabolism biological processes. As the genome-wide coexpression analysis and functional *These authors contributed equally to assessment of ERCC8 suggest, those coexpressed genes were significantly enriched in multiple this work biological processes of DNA damage and repair. Conclusion: The present study indicates that six ERCC genes (ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, and ERCC8) were dysregulated between HBV-related HCC tumor and para- cancerous tissues and that the mRNA expression of ERCC8 may serve as a potential biomarker for the HBV-related HCC prognosis. Keywords: HBV, hepatocellular carcinoma, diagnosis, prognosis, ERCC

Introduction Liver cancer is one of the most frequent malignant tumors occurring in China; besides, 1 Correspondence: Zhi-Chun Xie it has emerged as the third leading cause of cancer death in China. According to cancer Department of Epidemiology, Guangxi statistics in China in 2015, liver cancer is most frequently observed in males as well Medical University, Shuang Yong Road as in rural areas. Moreover, the temporal tendency of incidence rates in liver cancer 22#, Nanning 530021, Guangxi Zhuang Autonomous Region, People’s Republic is decreased.1 The histological type of most primary liver cancers is hepatocellular of China carcinoma (HCC).2 Hepatitis B virus (HBV) infection is identified as a risk factor for Tel +86 771 535 8847 Email [email protected] HCC, especially in China, where the infection rate of HBV is quite high. HBV is a submit your manuscript | www.dovepress.com Cancer Management and Research 2018:10 5313–5328 5313 Dovepress © 2018 Yang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://dx.doi.org/10.2147/CMAR.S179043 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Yang et al Dovepress single-stranded DNA virus, which can be integrated into the use of the match probes package in the R platform; subse- host genome, subsequently may activate the cellular proto- quent to that, the robust multi-array average (RMA) method oncogenes or suppress growth-regulating genes in cis,3 and in the R affy package was used to obtain probe set expres- induce chromosomal instability or insertional .4 sion summaries.16,17 Details of the experiment protocols and HBV-induced DNA damage is one of the potential mecha- data processing are available at https://www.ncbi.nlm.nih. nisms that contribute to the hepatocarcinogenesis of HCC.5 gov/geo/query/acc.cgi?acc=GSM362992. Moreover, just The excision repair cross-complementation (ERCC) the data set of the Affymetrix HT U133A genes include ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, Array (platform GPL3921) was included in the current study ERCC6, and ERCC8, constituting a set of that are to avoid the batch effect. These patients having a clearly involved in DNA repair. Nucleotide excision repair (NER) HBV infection were included in the current study, and those is considered as an extensively pivotal excision mechanism, patients without complete prognostic information and other which removes DNA damage caused by various kinds fac- clinical features were excluded. Owing to the fact that the tors, and a key DNA repair pathway in eukaryotic cells, population included in the current study originates from whereas the ERCC genes constitute the key genes in the the public databases and the present study does not contain NER pathway.6,7 As revealed by the investigations, the any studies performed by any of the authors with human genetic variation or of the NER pathway genes participants or animals, there is no need for approval by is likely to impact the cancer risk by affecting the DNA additional ethics committees. repair efficacy, and some nonsynonymous coding single- nucleotide polymorphisms or mutations in DNA repair Expression distribution and survival genes or their regulatory sequences induce the phenotypic analysis of ERCC genes changes and are involved in cancer development.8 Previous Investigation of the expression distribution of ERCC genes reports of the ERCC genes, suggesting they were associated was carried out with the help of the independent samples with multiple cancer prognoses,9–12 and polymorphisms t-test and the Metabolic gEne RApid Visualizer (MERAV, of the ERCC genes also have significant association with http://merav.wi.mit.edu/, accessed May 13, 2018) online the HCC development, progression, survival, and cancer tool.18 Receiver-operating characteristic (ROC) curves were susceptibility.5,13–15 However, the systematic investigation of put to use for the investigation of the potential application the diagnostic and prognostic values of the expression level values in distinct HBV-related HCC tumors and adjacent of ERCC genes in HCC continue to be unclear. Therefore, normal liver tissues. The Kaplan–Meier method, with the aim of the present study was to use the whole genome a log-rank test, and the univariate and multivariate Cox expression data set from the Omnibus proportional hazards regression models were employed (GEO; https://www.ncbi.nlm.nih.gov/geo/) public data- for the purpose of comparing the prognosis between dif- base for the purpose of systematically investigating the ferent groups. Stratified analysis was employed for the diagnostic and prognostic values of ERCC genes in HBV- assessment of the prognostic values of prognostic ERCC related HCC. genes in different strata of HBV-related HCCs. Joint effect survival analyses between prognostic ERCC genes and Materials and methods serum alpha-fetoprotein (AFP), which is a recognized Data processing prognostic biomarker in HCC, have also been carried out In order to perform the present study, the inclusion criteria for investigating the combined effect on HCC prognosis of public data set were set as follows: 1) the patients were prediction. The high- and low-expression groups of the HBV-related HCC; 2) the information of clinical outcome ERCC family genes were cutoff in accordance with the and features should be available; and 3) the data are the median values.19–22 whole genome expression profile data. Through the retrieval of the public database, only the data set of GSE14520 caters Nomogram construction to the inclusion criteria. GSE14520 (https://www.ncbi.nlm. To investigate the individualized prognostic prediction and nih.gov/geo/query/acc.cgi?acc=GSE14520; accessed May evaluation system, we also developed a nomogram for the 13, 2018), a data set from the GEO public database, was evaluation of the prognostic risk of each patient, which was included in the present study. Calculation of the expression based on the prognostic clinical indicators and prognostic- profile chip data set of GSE14520 was carried out with the related ERCC genes of Cox regression model. Besides that,

5314 submit your manuscript | www.dovepress.com Cancer Management and Research 2018:10 Dovepress Dovepress Diagnostic and prognostic values of ERCC in HBV-related HCC evaluation of the nomogram was using the concordance index Program (CLIP) stage were significantly associated with the (c-index). The nomogram was carried out by an rms package recurrence-free survival (RFS) of the HBV-related HCC, (https://CRAN.R-project.org/package=rms), together with whereas the clinical features including cirrhosis, tumor size, its dependent packages in the R platform, with visualization BCLC stage, TNM stage, CLIP stage, and serum AFP were using gplots.23,24 significantly correlated with overall survival (OS).

Gene set enrichment analysis (GSEA) Expression distribution and survival In a bid to figure out the potential mechanism of prognos- analysis of ERCC genes tic-related ERCC genes participating in the HBV-related Through the comparison of the expression of ERCC genes HCC prognosis, we further performed a GSEA (http:// in the HBV-related HCC tumor tissues and adjacent normal software.broadinstitute.org/gsea/index.jsp) to discover liver tissues, we observed that ERCC1, ERCC2, ERCC3, the potential biological processes and pathways involved and ERCC8 were significantly upregulated in the HBV- in the different expression levels of ERCC genes.25,26 The related HCC tumor tissues, whereas ERCC4 and ERCC5 Molecular Signatures Database of GSEA used the c2 (c2. were significantly downregulated in the HBV-related HCC all.v6.1.symbols.gmt) and c5 (c5.all.v6.1.symbols.gmt) tumor tissues (Figure 1). These dysregulated genes are also reference gene sets.27 Results with a P-value below 0.05 observable in liver cancer using the MERAV online tool and false discovery rate (FDR) below 0.25 were considered (Figure 2A–G). Thereafter, we performed the investigation statistically significant. of the potential application values of the ERCC genes in distinguishing the HBV-related HCC tumor tissues and Coexpression analysis adjacent normal liver tissues using the ROC curve. As the To further evaluate the function of ERCC8 in HBV-related ROC analysis suggests, six ERCC genes (ERCC1, ERCC2, HCC, we further carried out a genome-wide coexpres- ERCC3, ERCC4, ERCC5, and ERCC8) could be a biomarker sion analysis of ERCC8 in the HBV-related HCC of the used to distinguish between HBV-related HCC tumor tis- GSE14520 cohort. The Pearson’s correlation coefficient was sues and the adjacent normal liver tissues; the area under used to evaluate the coexpression genes, and the Database the curve of the ROC was in the range between 0.623 and for Annotation, Visualization and Integrated Discovery 0.744 (Figure 3A–G). (DAVID; https://david.ncifcrf.gov/home.jsp, accessed May Survival analysis of ERCC family genes has been pre- 13, 2018) v6.828,29 was used for the purpose of functional sented in Figures 4A–G and 5A–G; these findings provide assessment. the demonstration that the high ERCC8 expression sig- nificantly reduced the risk of recurrence (adjusted P=0.021; Statistical analysis HR=0.643; 95% CI=0.442–0.937, Table 2, Figure 4G) and FDR in the GSEA was used to estimate the probability death (adjusted P=0.049; HR=0.631; 95% CI=0.399–0.998, that the normalized enrichment score represents a false- Table 2, Figure 5G) in the HBV-related HCC. Stratification positive finding, which was calculated with the help of analysis was also used to assess the prognostic values of the Benjamini and Hochberg method. A P-value <0.05 ERCC8 in the HBV-related HCC. The high expression of was regarded as statistically significant. All the statisti- ERCC8 significantly reduced the risk of recurrence in the cal analyses were performed using SPSS v20.0 (IBM patients having cirrhosis, serum AFP >300 ng/mL, single Corporation, Armonk, NY, USA) and R3.3.1 (https:// nodules, and in the male patients (Table 3), whereas the www.r-project.org/). high expression of ERCC8 significantly reduced the risk of death in the patients having single nodules, together Results with the male patients (Table 3). The joint effect survival Study population analysis of ERCC8 and serum AFP suggests that the patients A total of 212 HBV-related HCC patients were included in with high ERCC8 expression and a serum AFP ≤300 ng/ the present study, coupled with 204 adjacent normal liver mL (adjusted P=0.035; HR=0.583; 95% CI=0.354–0.962, tissue specimens. Both the demographic and clinical fea- Table 4, Figure 6A) or >300 ng/mL (adjusted P=0.004; tures have been presented in Table 1. The clinical features HR=0.426; 95% CI=0.237–0.768, Table 4, Figure 6A) including gender, cirrhosis, Barcelona Clinic Liver Cancer showed a significantly decreased risk of recurrence in the (BCLC) stage, TNM stage, and Cancer of the Liver Italian HBV-related HCC compared with patients having AFP >300

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Table 1 Distribution of HCC patients’ characteristics and prognosis analysis Variables RFS OS Events/ MRT HR (95% CI) Log-rank Events/ MST HR (95% CI) Log- total (months) P total (months) rank P Age (years) 0.916 0.629 ≤60 96/175 46 1 69/175 NA 1 >60 20/37 48 0.974 (0.602–1.578) 13/37 NA 0.864 (0.478–1.564) Gender 0.018 0.148 Male 106/183 40 1 74/183 NA 1 Female 10/29 NA 0.467 (0.244–0.893) 8/29 NA 0.587 (0.283–1.218) Cirrhosis 0.029 0.025 No 5/17 1 2/17 NA 1 Yes 111/195 38 2.612 (1.066–6.402) 80/195 NA 4.335 (1.065–17.638) Tumor sizea 0.073 0.002 ≤5 cm 73/137 51 1 46/137 NA 1 >5 cm 43/74 28 1.409 (0.966–2.056) 36/74 53 1.975 (1.274–3.060) Multinodular 0.381 0.052 Single 90/167 49 1 59/167 NA 1 Multiple 26/45 28 1.216 (0.785–1.883) 23/45 48 1.607 (0.992–2.604) BCLC stage <0.001 <0.001 0 6/20 NA 1 2/20 NA 1 A 74/143 51 2.050 (0.892–4.711) 48/143 NA 4.119 (1.001–16.951) B 15/22 27 4.019 (1.550– 12/22 46 8.992 (2.005–40.320) 10.421) C 21/27 9 6.163 (2.477–15.333) 20/27 13 18.993 (4.419–81.632) TNM stage <0.001 <0.001 I 35/89 NA 1 20/89 NA 1 II 48/76 28 1.995 (1.289–3.088) 32/76 NA 2.214 (1.265–3.873) III 33/47 18 3.220 (1.993–5.204) 30/47 18 5.197 (2.930–9.218) CLIP stage 0.001 <0.001 0 46/94 57 1 26/94 NA 1 1 37/71 40 1.197 (0.776–1.846) 25/71 NA 1.414 (0.816–2.449) 2/3/4/5 33/47 19 2.233 (1.424–3.500) 31/47 27 3.638 (2.153–6.149) Serum AFPb 0.327 0.047 ≤300 ng/mL 62/115 48 1 39/115 NA 1 >300 ng/mL 54/94 35 1.200 (0.833–1.728) 43/94 NA 1.546 (1.002–2.385) Notes: aInformation of tumor size was unavailable in one patient. bInformation of serum AFP was unavailable in three patients. Abbreviations: AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; HCC, hepatocellular carcinoma; MRT, median recurrence time; MST, median survival time; OS, overall survival; RFS, recurrence-free survival.

Adjacent normal tissue Tumor tissue ng/mL and low ERCC8 expression. In contrast, the patients 10 **** **** **** *** **** **** with high ERCC8 expression and AFP ≤300 ng/mL showed 8 a significantly reduced risk of death in the HBV-related HCC 6 (adjusted P=0.028; HR=0.499; 95% CI=0.268–0.927, Table

4 4, Figure 6B) compared with patients having AFP >300 ng/ mL and low ERCC8 expression. 2 Thereafter, we also performed the verification of these Relative mRNA expression 0 joint effect survival analyses between ERCC8 and AFP mRNA expression level and demonstrated that the AFP ERCC1ERCC1ERCC2ERCC2ERCC3ERCC3ERCC4ERCC4ERCC5ERCC5ERCC6ERCC6ERCC8ERCC8 mRNA expression also performed well in the HCC prognosis Figure 1 Gene expression distribution of ERCC genes in GSE14520 HBV-related HCC cohort. prediction. Patients with high ERCC8 expression and low Notes: ***P<0.001; ****P<0.0001. (adjusted P=0.003; HR=0.443; 95% CI=0.259–0.757, Table Abbreviations: ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma. 4, Figure 6C) or high (adjusted P=0.002; HR=0.418; 95%

5316 submit your manuscript | www.dovepress.com Cancer Management and Research 2018:10 Dovepress Dovepress Diagnostic and prognostic values of ERCC in HBV-related HCC

A BCD

ERCC1 ERCC2 ERCC3 ERCC4 9.5 8.5 8.0

9.0 8.0 6.4

7.5

Tissue Tissue 7.5 Tissue Tissue 8.5 Normal tissue Normal tissue Normal tissue 6.0 Normal tissue Primary tumor Primary tumor Primary tumor Primary tumor Expression Expression 7.0 Expression Expression 7.0

8.0 5.6

6.5 6.5

7.5 Liver Liver Liver Liver

E F G

ERCC5 ERCC6 ERCC8 9.0 5.75 5.75 8.5 5.50 Tissue Tissue Tissue Normal tissue Normal tissue Normal tissue Primary tumor Primary tumor 5.50 Primary tumor 8.0 5.25 Expression Expression Expression

5.00 7.5 5.25

4.75 Liver Liver Liver

Figure 2 Gene expression distribution of ERCC gene in liver cancer using the MERAV. Note: The order of box plots of ERCC genes were as follows: ERCC1 (A), ERCC2 (B), ERCC3 (C), ERCC4 (D), ERCC5 (E), ERCC6 (F), and ERCC8 (G). Abbreviations: ERCC, excision repair cross-complementation; MERAV, Metabolic gEne RApid Visualizer.

AB100 100 CD100 100

80 80 80 80 ) ) 60 60 60 60

40 40 40 40

ERCC1 ERCC2 Sensitivity (% ERCC3 Sensitivity (% ERCC4 Sensitivity (100%) Sensitivity (100%) 20 20 20 20 P<0.0001 P<0.0001 P<0.0001 P<0.0001 AUC (95% CI)=0.710 (0.660–0.760) AUC (95% CI)=0.627 (0.574–0.681) AUC (95% CI)=0.744 (0.696–0.792) AUC (95% CI)=0.623 (0.569–0.676) 0 0 0 0 020406080 100 020406080 100 020406080 100 020406080 100 100(%)-Specificity (%) 100(%)-Specificity (%) 100(%)-Specificity (%) 100(%)-Specificity (%)

E 100 F 100 G 100

80 80 80 ) 60 60 60

40 40 40

Sensitivity (%) ERCC5 Sensitivity (%) ERCC6 Sensitivity (% ERCC8 20 20 20 P<0.0001 P=0.777 P<0.0001 AUC (95% CI)=0.703 (0.652–0.753) AUC (95% CI)=0.508 (0.452–0.564) AUC (95% CI)=0.676 (0.624–0.728) 0 0 0 020406080 100 020406080 100 020406080 100 100(%)-Specificity (%) 100(%)-Specificity (%) 100(%)-Specificity (%)

Figure 3 ROC curves of ERCC genes in distinguish HBV-related HCC tissue from adjacent normal liver tissue. Note: The order of ROC curves of ERCC genes were as follows: ERCC1 (A), ERCC2 (B), ERCC3 (C), ERCC4 (D), ERCC5 (E), ERCC6 (F), and ERCC8 (G). Abbreviations: AUC, area under the curve of ROC; ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; ROC, receiver- operating characteristic.

CI=0.238–0.734, Table 4, Figure 6C) AFP level showed a we observed that the patients with high ERCC8 expression significantly decreased risk of recurrence in the HBV-related and low AFP expression showed a significantly reduced HCC compared with patients who had high AFP and low risk of death in the HBV-related HCC (adjusted P=0.035; ERCC8 expression, as well as in patients with low AFP and HR=0.487; 95% CI=0.249–0.952, Table 4, Figure 6D) ERCC8 expression (adjusted P=0.004; HR=0.425; 95% compared with patients who had high AFP and low ERCC8 CI=0.238–0.757, Table 4, Figure 6C). With respect to OS, expression.

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AB100 100 C 100 D 100 Low ERCC1 Low ERCC2 Low ERCC3 Low ERCC4

e High ERCC1 e e High ERCC3 e High ERCC4 80 80 High ERCC2 80 80

60 60 60 60

40 40 40 40

20 Percent recurrenc Percent recurrenc 20 Percent recurrenc 20 Percent recurrenc 20 Log-rank P=0.583 Log-rank P=0.395 Log-rank P=0.759 Log-rank P= 0.061 0 0 0 0 0122436486072 0122436486072 0122436486072 0122436486072 Recurrence-free survival (months) Recurrence-free survival (months) Recurrence-free survival (months) Recurrence-free survival (months)

E 100 F 100 G 100 Low ERCC5 Low ERCC6 Low ERCC8

e High ERCC8 80 High ERCC5 80 High ERCC6 80

60 60 60

40 40 40

Percent recurrence 20 Percent recurrenc Percent recurrence 20 20 Log-rank P=0.731 Log-rank P=0.059 Log-rank P=0.009 0 0 0 0122436486072 0122436486072 0122436486072 Recurrence-free survival (months) Recurrence-free survival (months) Recurrence-free survival (months)

Figure 4 Kaplan–Meier curves of ERCC genes in HBV-related HCC RFS. Note: The order of Kaplan–Meier curves of ERCC genes were as follows: ERCC1 (A), ERCC2 (B), ERCC3 (C), ERCC4 (D), ERCC5 (E), ERCC6 (F), and ERCC8 (G). Abbreviations: ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; RFS, recurrence-free survival.

CD AB100 100 100 100 Low ERCC1 Low ERCC2 Low ERCC3 Low ERCC4 High ERCC1 High ERCC2 High ERCC3 High ERCC4

l 80 l 80 l 80

l 80

60 60 60 60

40 40 40 40 Percent surviva Percent surviva Percent surviva 20 20 20 Percent surviva 20 Log-rank P=0.509 Log-rank P=0.327 Log-rank P=0.231 Log-rank P=0.043 0 0 0 0 0122436486072 0122436486072 0122436486072 0122436486072 Overall survival (months) Overall survival (months) Overall survival (months) Overall survival (months)

FG E100 100 100 Low ERCC5 Low ERCC6 Low ERCC8 High ERCC5 High ERCC6 High ERCC8 l l 80 80 l 80

60 60 60

40 40 40 Percent surviva Percent surviva 20 20 Percent surviva 20 Log-rank P=0.770 Log-rank P=0.057 Log-rank P=0.008 0 0 0 0122436486072 0122436486072 0122436486072 Overall survival (months) Overall survival (months) Overall survival (months)

Figure 5 Kaplan–Meier curves of ERCC genes in HBV-related HCC OS. Note: The order of Kaplan–Meier curves of ERCC genes were as follows: ERCC1 (A), ERCC2 (B), ERCC3 (C), ERCC4 (D), ERCC5 (E), ERCC6 (F), and ERCC8 (G). Abbreviations: ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; OS, overall survival.

Nomogram construction for RFS and OS, we observed that the contribution of ERCC8 Thereafter, we performed the investigation of the potential expression in the HBV-related HCC prognosis prediction was application of ERCC8 mRNA expression in the individual- lower compared with some conventional clinical indicators; ized prognosis prediction. Those clinical features that were however, the expression of ERCC8 contributed much more significantly correlated to RFS or OS were included in the than the serum AFP in the nomogram of the HBV-related development of nomogram for RFS and OS, correspond- HCC OS (Figure 7B). ingly. The nomogram of the HBV-related HCC RFS was developed in accordance with the gender, cirrhosis, TMN GSEA of ERCC8 stage, BCLC stage, CLIP stage, and ERCC8 expression Investigation by GSEA using the c2 reference gene set indi- (c-index [95% CI]=0.68 [0.63–0.74], Figure 7A), whereas cates that the high expression of ERCC8 was significantly the nomogram of HBV-related HCC OS was developed enriched in the NF-kappa-B and Wnt signaling pathway, by tumor size, cirrhosis, TMN stage, BCLC stage, CLIP as well as the P53 independent G1-S DNA damage check- stage, AFP, and ERCC8 expression (c-index [95% CI]=0.72 point, TCA cycle and respiratory electron transport, and [0.66–0.79], Figure 7B). In accordance with the nomogram oxidative phosphorylation (Figure 8A–F, Table S1). GSEA

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Group a AB100 100 Group A Group b Group B Group c 80 80 Group d

e Group C l Group D 60 60

40 40 Percent surviva Percent recurrenc 20 20 Log-rank P=0.0159 Log-rank P=0.0072 0 0 0122436486072 0122436486072 Recurrence-free survival (months) Overall survival (months)

Group i CDC 100 100 Group I Group ii Group II Group iii 80 80 Group iv

e Group III l Group IV 60 60

40 40 Percent surviva Percent recurrenc 20 20 Log-rank P=0.001 Log-rank P=0.01 0 0 0122436486072 0122436486072 Recurrence-free survival (months) Overall survival (months)

Figure 6 Joint effect survival analysis of ERCC8 and AFP in HBV-related HCC prognosis. Notes: (A) RFS stratified by ERCC8 and serum AFP level; (B) OS stratified by ERCC8 and serum AFP level; (C) RFS stratified by ERCC8 and AFP mRNA level; and (D) OS stratified byERCC8 and serum AFP mRNA level. Abbreviations: AFP, alpha-fetoprotein; ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; OS, overall survival; RFS, recurrence-free survival.

with c5 indicates that the high expression of ERCC8 was analysis. A total of 273 genes with |Pearson’s correlation significantly correlated to metabolically related biological coefficient|>0.4 and P<0.05 were taken into consideration processes, for instance, nucleoside triphosphate and cel- as coexpressed with ERCC8 in the HBV-related HCC lular amine metabolic processes, and the generation of (Figure 9, Table S3). Thereafter, the functional assess- precursor metabolites and energy (Figure 8G–I, Table S2). ment of the ERCC8-coexpressed genes was performed In comparison, using the results of the c2 and c5 reference using DAVID v6.8. (GO) term analysis gene set, we observed that the high expression of ERCC8 suggested that ERCC8 and its coexpressed genes together may be involved in energy metabolism biological processes, are significantly enriched in the MAPK cascade, damaged which may have an impact on the prognosis of the HBV- DNA binding, DNA synthesis involved in DNA repair, related HCC. and DNA damage checkpoints (Figure 10). In contrast, the Kyoto Encyclopedia of Genes and Genomes suggested Genome-wide coexpression analysis and that ERCC8 and its coexpressed genes were significantly function assessment of ERCC8 enriched in the ribosome-signaling pathway (hsa03010, For the purpose of further exploring the function of P=0.003, gene count=8). ERCC8 in the HBV-related HCC, a genome-wide coex- pression analysis was carried out. With respect to the Discussion multiple probes corresponding to one gene, we used the Through the review of the literature, we observed that exten- mean values of multiple probes to present the expres- sive previous studies have focused on the investigation of the sion values of this gene in a genome-wide coexpression association between the ERCC gene polymorphisms and the

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Table 2 Survival analysis of ERCC genes in HBV-related HCC prognosis Gene Patients MST/MRT Crude HR (95% CI) Crude P Adjusted HR (95% CI) Adjusted Pa expression (n=212) (months) RFS MRT ERCC1 Low 106 40 1 1 High 106 49 0.903 (0.627–1.300) 0.583 1.061 (0.723–1.556) 0.762 ERCC2 Low 106 35 1 1 High 106 49 0.854 (0.593–1.229) 0.396 0.934 (0.646–1.351) 0.717 ERCC3 Low 106 46 1 1 High 106 46 1.059 (0.735–1.524) 0.759 1.173 (0.805–1.710) 0.405 ERCC4 Low 106 36 1 1 High 106 53 0.706 (0.489–1.019) 0.063 0.818 (0.560–1.194) 0.298 ERCC5 Low 106 41 1 1 High 106 46 1.066 (0.740–1.535) 0.731 0.952 (0.651–1.392) 0.798 ERCC6 Low 106 30 1 1 High 106 54 0.704 (0.448–1.016) 0.061 0.768 (0.527–1.118) 0.168 ERCC8 Low 106 30 1 1 High 106 57 0.613 (0.424–0.887) 0.009 0.643 (0.442–0.937) 0.021 OS MST ERCC1 Low 106 NA 1 1 High 106 NA 0.864 (0.560–1.334) 0.509 1.040 (0.663–1.630) 0.865 ERCC2 Low 106 NA 1 1 High 106 NA 0.805 (0.522–1.243) 0.328 0.865 (0.558–1.343) 0.518 ERCC3 Low 106 NA 1 1 High 106 NA 1.303 (0.844–2.012) 0.233 1.511 (0.961–2.375) 0.074 ERCC4 Low 106 NA 1 1 High 106 NA 0.637 (0.410–0.989) 0.044 0.729 (0.465–1.142) 0.167 ERCC5 Low 106 NA 1 1 High 106 NA 1.067 (0.691–1.647) 0.77 0.899 (0.572–1.412) 0.643 ERCC6 Low 106 58 1 1 High 106 NA 0.654 (0.421–1.015) 0.059 0.723 (0.462–1.133) 0.157 ERCC8 Low 106 54 1 1 High 106 NA 0.549 (0.351–0.858) 0.008 0.631 (0.399–0.998) 0.049 Note: aAdjustment for age, gender, cirrhosis, tumor size, tumor number, BCLC stage, and serum AFP level. Abbreviations: AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; MRT, median recurrence time; MST, median survival time; OS, overall survival; RFS, recurrence-free survival. risk of cancers30,31 or on the ERCC gene polymorphisms and Previous studies substantiated that polymorphisms in cancer prognosis.32,33 Besides that, the ERCC gene polymor- ERCC2 and ERCC5 confer susceptibility to the acute myeloid phisms have also been reported for having correlation with leukemia (AML)40 and that polymorphisms in ERCC2 and the prognosis of patients treated with chemotherapy.34–37 XPC were significantly associated with the AML survival.41 However, there are rare reports of the systematic investiga- Polymorphisms in ERCC5 also make contribution toward tions of the expression of ERCC in cancer diagnosis and the susceptibility of lung cancer and squamous cell carcino- prognosis.9,38,39 mas of the oropharynx, larynx, and esophagus;42 head and

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Table 3 Stratified survival analysis ofERCC8 in HBV-related HCC prognosis Variables Patients Low High RFS OS (n=218) ERCC8 ERCC8 Adjusted HR Adjusted Pa Adjusted HR Adjusted (95% CI) (95% CI) Pa Age (years) ≤60 175 88 87 0.694 (0.456–1.055) 0.087 0.659 (0.396–1.098) 0.11 >60 37 18 19 0.498 (0.172–1.442) 0.199 0.506 (0.133–1.926) 0.318 Gender Male 183 91 92 0.664 (0.447–0.984) 0.041 0.647 (0.398–1.053) 0.08 Female 29 15 14 0.099 (0.007–1.319) 0.08 0.140 (0.011–1.844) 0.135 Cirrhosis No 17 6 11 1×10e-6 (1.8×10e-196– 0.952 4.9×10e-5 0.57 3.7×10e183) (6.9×10e-20–3.7×10e10) Yes 195 100 95 0.673 (0.458–0.988) 0.043 0.676 (0.425–1.075) 0.098 Tumor sizeb ≤5 cm 137 63 74 0.705 (0.437–1.136) 0.151 0.575 (0.306–1.081) 0.086 >5 cm 74 42 32 0.570 (0.292–1.114) 0.1 0.757 (0.363–1.579) 0.458 Multinodular Single 167 81 86 0.618 (0.406–0.939) 0.024 0.524 (0.309–0.888) 0.016 Multiple 45 25 20 0.837 (0.305–2.298) 0.73 1.575 (0.533–4.659) 0.411 BCLC stage 0 20 9 11 0.592 (0.101–3.457) 0.561 7×10e-6 0.968 (2.64×10e-264–1.7×10e245) A 143 65 78 0.755 (0.475–1.199) 0.234 0.684 (0.385–1.216) 0.196 B 22 14 8 0.473 (0.123–1.815) 0.275 1.006 (0.238–4.263) 0.993 C 27 18 9 0.893 (0.291–2.734) 0.842 0.981 (0.312–3.083) 0.973 TNM stage I 89 37 52 0.533 (0.266–1.068) 0.076 0.553 (0.223–1.372) 0.201 II 76 39 37 0.807 (0.438–1.488) 0.492 0.987 (0.472–2.064) 0.973 III 47 30 17 1.057 (0.459–2.435) 0.897 1.081 (0.440–2.655) 0.866 CLIP stage 0 94 40 54 0.827 (0.455–1.503) 0.532 0.735 (0.330–1.639) 0.452 1 71 35 36 0.681 (0.340–1.364) 0.278 0.509 (0.218–1.189) 0.119 2/3/4/5 47 31 16 0.411 (0.159–1.061) 0.066 0.978 (0.391–2.448) 0.962 Serum AFPc ≤300 ng/mL 115 52 63 0.860 (0.516–1.432) 0.562 0.737 (0.386–1.405) 0.354 >300 ng/mL 94 53 41 0.422 (0.232–0.767) 0.005 0.512 (0.259–1.016) 0.055 Notes: aAdjustment for age, gender, cirrhosis, tumor size, tumor number, BCLC stage, and serum AFP level. bInformation of tumor size was unavailable in one patient. cInformation of serum AFP was unavailable in three patients. Abbreviations: AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; OS, overall survival; RFS, recurrence-free survival.

neck;43 prostate cancer;44 HCC;15 and bladder cancer (BC).31 significantly correlated with a better clinical outcome.47 Pre- A study performed by Sakano et al also found that the genetic vious studies demonstrated that genetic variants of ERCC2 variants of ERCC5 contribute to the tumor invasiveness in and ERCC5 may be the independent prognostic factors BC.45 In addition, the polymorphisms ERCC2-rs1799793 and in the patients with melanoma32,48 and osteosarcoma.49 In ERCC5-rs17655 may also contribute to the recurrence risk addition, polymorphisms of ERCC5 were also correlated of squamous cell carcinoma of the oropharynx.37 with the response to chemotherapy and the prognosis of There have also been reports of the prognostic values of osteosarcoma.50,51 Furthermore, polymorphisms of ERCC5 ERCC genetic variants in multiple cancers. An investigation have also been reported to have an influence in multiple carried out by Liu et al revealed the fact that ERCC1-rs11615 cancer prognosis, such as lung cancer,33,47 and colorectal is likely to be a useful genetic marker for the prediction of cancer (CRC).52,53 the osteosarcoma prognosis and that the CC genotype is cor- The interindividual differences in the patients treated with related with a better clinical outcome.46 The work performed chemotherapy mainly affected the efficiency of the DNA by Liu et al indicated that the XRCC1-399A/A genotype was damage–repair system in the cells and then affected the clini-

Cancer Management and Research 2018:10 submit your manuscript | www.dovepress.com 5321 Dovepress Yang et al Dovepress b P 0.035 0.161 0.074 0.003 0.004 0.002 0.028 0.165 0.057 0.035 0.098 0.004 Adjusted 0.487 (0.249–0.952) 0.612 (0.308–1.215) 0.533 (0.267–1.063) 1 0.443 (0.259–0.757) 0.425 (0.238–0.757) 0.418 (0.238–0.734) 1 0.499 (0.268–0.927) 0.661 (0.369–1.185) 0.522 (0.268–1.018) 1 0.583 (0.354–0.962) 0.656 (0.398–1.081) 0.426 (0.237–0.768) Adjusted HR (95% CI) 1 0.01 0.083 0.007 0.006 0.011 0.001 0.002 0.052 0.021 0.012 0.104 0.006 Crude P A FP level. 0.478 (0.272–0.841) 0.602 (0.339–1.069) 1 0.400 (0.206–0.775) 0.524 (0.331–0.830) 0.519 (0.313–0.859) 1 0.389 (0.224–0.673) 0.401 (0.223–0.719) 0.575 (0.329–1.005) 1 0.463 (0.242–0.889) 0.546 (0.340–0.877) 0.671 (0.414–1.086) 1 0.448 (0.252–0.795) Crude HR (95% CI) (95% Crude HR NA NA 47 NA MST 53 51 21 NA MRT NA NA 36 NA MST 51 48 23 NA MRT/MST (months) MRT H BV, hepatitis B virus; CC, hepatocellular carcinoma; MRT, median recurrence time; M S T, survival O , overall survival; RF S , 60 46 60 46 60 46 60 46 63 52 53 41 63 52 53 41 Patients (n = 209) djustment for age, gender, cirrhosis, tumor size, number, BC L C stage, and serum A djustment for age, b and serum A FP in H BV-related CC prognosis H igh L ow L ow H igh H igh L ow L ow H igh H igh L ow L ow H igh H igh L ow L ow H igh ERCC8 expression ERCC8 a L ow L ow H igh H igh L ow L ow mRNA H igh H igh < 300 < 300 > 300 > 300 < 300 < 300 AFP level Serum (ng/mL ) > 300 > 300 A FP, alpha-fetoprotein; E RCC, excision repair cross-complementation; Joint effect survival analysis of I nformation of serum A FP was unavailable in three patients. a G roup iv G roup iii G roup i G roup ii G roup I V G roup III G roup I G roup II G roup d G roup c G roup a G roup b G roup D G roup C G roup A G roup B OS RFS OS Group RFS Abbreviations: recurrence-free survival. Table 4 Notes:

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AB

0102030405060708090 100 0102030405060708090 100 Points Points Male ≤ 5 cm Gender Tumor_size > 5 cm Female Yes Yes Cirrhosis Cirrhosis No No II II TNM_stage TNM_stage I III I III AC ACBCLC_stage BCLC_stage 0 B 0B 1 CLIP_stage 1 0 3/4/5 CLIP_stage 3/4/5 0 >300 ng/mL AFP Low expression ≤ 300 ng/mL ERCC8 Low expression High expression ERCC8 High expression Total points 050 100 150 200 250 300 350 Total points 020406080 100 140 180 220 260 1-year survival 1-year survival 0.9 0.8 0.7 0.6 0.5 0.4 0.99 0.9 0.8 0.7 0.6 0.5 3-year survival 3-year survival 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 5-year survival 5-year survival 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

Figure 7 Nomogram for predicting the 1-, 3-, and 5-year event with ERCC8 expression and clinical indicators. Notes: (A) Nomogram for ERCC8 expression in HBV-related HCC RFS. (B) Nomogram for ERCC8 expression in HBV-related HCC OS. Abbreviations: AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; OS, overall survival; RFS, recurrence-free survival.

cal outcomes. Extensive studies have demonstrated that the susceptibility and therapeutic outcomes to the colorectal ERCC genes were involved in the responses to chemotherapy cancer patients, who received oxaliplatin chemotherapy.36,54–56 and the prognosis of the patients with CRC36,54–56 as well By reviewing the above literature, we found that a consid- as advanced non-small cell lung cancer (NSCLC).57,58 The erable amount of literature has been published on the genetic ERCC5 polymorphism His46His was reported to be associ- variation of ERCC genes in cancer susceptibility, prognosis, ated with the susceptibility of platinum-based chemotherapy and therapeutic outcomes; however, the expression of ERCC in the advanced NSCLC35 and significantly affected clinical genes in cancer diagnostic and prognostic values were rarely outcomes.58 The ERCC5 promoter polymorphism rs751402 mentioned. Several investigations verified the fact that the was significantly associated with the chemotherapy response low expression of ERCC1 was significantly correlated with in the advanced NSCLC, and the AA genotype may contrib- a better clinical outcome in patients with breast cancer,38 ute to increase the effect of chemotherapy response.59 Hu et NSCLC,61 and ovarian cancer,9 as well as in advanced NSCLC al substantiated that the rs2296147 T allele and rs873601 treated with platinum-based chemotherapy.10 A systematic G allele were associated with the better progression-free investigation of the ERCC genes in the ovarian cancer prog- survival (PFS) and OS in the advanced NSCLC patients nosis was carried out by Zhao et al using the Kaplan–Meier undergoing the platinum-based chemotherapy57; further- Plotter online tool and revealed that the high expression of more, the results of the rs2296147 T allele in OS of the ERCC8 and ERCC1 had correlations with a poor OS, whereas advanced NSCLC patients undergoing platinum-based che- the high expression of ERCC4 was associated with a better motherapy are also verifiable in another cohort.60 However, OS.9 In our current study, we observed that the high expres- the rs1047768 TT genotype, which showed a significantly sion of ERCC8 was significantly correlated with a better RFS shortened median PFS and OS in the advanced NSCLC and OS in the HBV-related HCC after hepatectomy, and our patients undergoing platinum-based chemotherapy in the current study was the first to investigate the potential prog- study of Zhang et al,60 is unable to be verified in the cohort nostic application values of ERCC8 in the HBV-related HCC. collected by Hu et al.57 With respect to the ERCC genetic Comprehensive survival analysis of stratification analysis and variation in CRC response to platinum-based chemotherapy joint effect survival analysis indicates that ERCC8 may be and therapeutic outcomes, multiple studies demonstrated served as an independent prognostic indicator for the HBV- that the ERCC5 polymorphisms may have an impact on the related HCC; the nomograms indicate that the combination

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A B C Enrichment plot: Enrichment plot: Reactome_signaling_by WNT Enrichment plot: 0.6 Reactome_actlvatlon_of _NF _kappab_in_B_cells Reactome_p53_independent_G1_S_DNA 0.5 NES=1.966 Nominal P<0.0001 _damage_checkpoint 0.6 0.4 0.6 NES=1.923 FDR=0.097 NES=1.862 0.5 0.5 Nominal P=0.006 0.3 Nominal P=0.004 0.4 0.4 FDR=0.086 0.2 FDR=0.101 0.3 0.3 0.1 0.2

0.2 Enrichment score (ES) 0.0 0.1 0.1 Enrichment score (ES) Enrichment score (ES) 0.0 0.0 )

‘High’ (positively correlated) ‘High’ (positively correlated) 0.25 ‘High’ (positively correlated) 0.25 0.25 Zero cross at 5,900 Zero cross at 5,900 0.00 Zero cross at 5,900 0.00 0.00

–0.25 –0.25 –0.25 ‘Low’ (negatively correlated) ‘Low’ (negatively correlated) ‘Low’ (negatively correlated) 0 2,500 5,000 7,500 10,000 12,500 0 2,500 5,000 7,500 10,000 12,500 0 2,500 5,000 7,500 10,000 12,500 Rank in ordered dataset Rank in ordered dataset Rank in ordered dataset Ranked list metric (signal-to-noise) Ranked list metric (signal-to-noise) Ranked list metric (signal-to-noise Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores

D E F Enrichment plot: Enrichment plot: Enrichment plot: Reactome_TCA_cycle_and respiratory electron KEGG_oxidatlve_phosphorylation Reactome_regulatlon_of_apoptosis _transport 0.5 0.4 NES=1.824 NES=1.758 0.5 NES=1.863 Nominal P=0.037 0.4 Nominal P=0.033 0.4 Nominal P=0.016 0.3 FDR=0.127 0.3 FDR=0.202 0.3 FDR=0.109 0.2 0.2 0.2 0.1 0.1 Enrichment score (ES) Enrichment score (ES) 0.1 Enrichment score (ES) 0.0 0.0 0.0 )

‘High’ (positively correlated) ‘High’ (positively correlated) ‘High’ (positively correlated) 0.25 0.25 0.25

Zero cross at 5,900 0.00 Zero cross at 5,900 0.00 0.00 Zero cross at 5,900

–0.25 –0.25 –0.25 ‘Low’ (negatively correlated) ‘Low’ (negatively correlated) ‘Low’ (negatively correlated) 0 2,500 5,000 7,500 10,000 12,500 0 2,500 5,000 7,500 10,000 12,500 0 2,500 5,000 7,500 10,000 12,500

Ranked list metric (signal-to-noise Rank in ordered dataset Rank in ordered dataset Rank in ordered dataset Ranked list metric (signal-to-noise) Ranked list metric (signal-to-noise)

Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores

G H I Enrichment plot: Enrichment plot: Enrichment plot: Go_generation_of_precursor_metabolites Go regulatlon_of_cellular_amine_metabolic Go_nucleoside_triphosphate_metabolic_process _and_energy _process

NES=1.713 NES=1.700 0.4 NES=1.704 0.4 0.4 Nominal P=0.012 Nominal P=0.010 0.3 Nominal P=0.018 0.3 FDR=0.245 0.3 FDR=0.247 FDR=0.248 0.2 0.2 0.2 0.1 0.1 0.1 Enrichment score (ES) Enrichment score (ES) Enrichment score (ES) 0.0 0.0 0.0 ) ) )

‘High’ (positively correlated) ‘High’ (positively correlated) ‘High’ (positively correlated) 0.25 0.25 0.25

Zero cross at 5,900 0.00 0.00 Zero cross at 5,900 0.00 Zero cross at 5,900

–0.25 –0.25 –0.25 ‘Low’ (negatively correlated) ‘Low’ (negatively correlated) ‘Low’ (negatively correlated) 0 2,500 5,000 7,500 10,000 12,500 0 2,500 5,000 7,500 10,000 12,500 0 2,500 5,000 7,500 10,000 12,500 Rank in ordered dataset Rank in ordered dataset Rank in ordered dataset Ranked list metric (signal-to-noise Ranked list metric (signal-to-noise Ranked list metric (signal-to-noise Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores

Figure 8 GSEA results of ERCC8. Notes: (A)–(F) GSEA results of c2 reference gene sets for high ERCC8 expression. (G)–(I) GSEA results of c5 reference gene sets for high ERCC8 expression. Abbreviations: ERCC, excision repair cross-complementation; GSEA, gene set enrichment analysis; FDR, false discovery rate; ES, Enrichment Score; NES, Normalized Enrichment Score. of ERCC8 and traditional prognostic indicators may have an was derived from the Kaplan–Meier method, which was a application value in the individualized prognosis prediction. univariate survival analysis and that the results obtained from It is worth noting that the results of ERCC8 obtained in our such kinds of statistical methods may be unreliable. However, current study were contradictory to the results of the study the key advantage of the results of the current study was carried out by Zhao et al in the ovarian cancer prognosis.9 assessed by a multivariate Cox proportional hazards regres- The possible causes of the above inconsistency results sug- sion model, and the statistical method was more reliable than gested that the survival analysis in the study by Zhao et al the Kaplan–Meier method in survival analysis. Another pos-

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P2RXL1 OPHN1 PAX2 NACAP1 PGF MYEF2 PRR11 MTCH2 LOC732095 OSGEPL1 MRPL46 RPL23 LOC441258

RPL39 PIGO MEN1 LOC390612 KIAA1659KIAA0754 IKZF1 LOC152719 HTN1 SCN11A LOC23117 HNRPD LDHAL6B LOC643287 H2AFX PLD2 MLH1 LRRC36 KLK3 ITFG1 SDC3 MAG GYPA KRT84 NRBF2 KIFAP3

MNT PRY KIAA2002 GPR23 OLAH NUP62 SEC14L5 PLEKHO1 NFIX KERA MYH2 RAB40C POLH MAP3K3 KIAA0372 GPC6 PNPO GALNT10 POU4F2 MCCC2 MAN2A1 LOC389906 SERPINA2 MGAT4B JTV1 JARID1A NAP1L4 RAB4A RERE KIRREL IKZF4 GMIP MRPS12 INSL4 RPE65 LOC647070 HOXC4 SLC14A1 OTOF RARG NIT1 MGC5590 KHDRBS1 HSPC171 ELK1 FLJ14327 ITGA9

RPL7 NR5A1 IMPG1 HIST1H4C RAD17 PCDH8 OBFC2B GSK3A FLJ12151 FLJ13611 SLC25A42 REEP5 CREBL1 IGSF2 SLC24A2 PLEKHA4 GFRA3 FTS HOXD12 GYPE OSBP KLHL12 EXOSC5 GDF5 PDE4C FEZF2 MAPRE2 CXorf1 SLC27A6 RNF14 ST7L RPA4 HNRPL GRIK4 COQ3 HNRPU MCF2L2 IFNA6 PAFAH2 FMO4 PLGLB1 MDH2 CHRNB2 FBXW12 RAD50 SUHW1 GP6 FDX1 RPP38 RPL38 FAM106A CDK5 SLC35E1 ERCC8 HMHA1 PARP16 NNT C9orf156 FGF6 PTMA PTPN2 GTF2H3 EPAG TAS2R8 FHL3 SERPINB13 EWSR1 SCN4A PEX7 C1orf80 CBARA1 SOX30 RPL23AP13 PCAF CDRT4 FGF23 DUX5 GNRH1 TCF7 DNAI1 SLC30A5 C16orf80 SPON1 CLDN16 SLC4A5 PCDHGA9 YAF2 ARR3 FARS2 FAM82C RPL27A SNX13 ACBD4 C7orf26 DENND3 SUZ12P TCIRG1 DIP2A EMID2 TMEM51 CEP27 PDK2 DUX1 THRAP2 DACT1 RPL35A ZNF562 ARS2 DDX42 SOX15 ARHGAP28 C7orf25 TAX1BP3 PNO1 DSCR3 ELF4 TMEM113 CATR1 RPL37A DCLRE1B SMUG1 STARD3 PPP5C DHX8 BTRC DLEU1 TTTY1 QRSL1 DHX29 BTC TMSB4X CRADD DENND1C SLC4A7 UBQLN4 ARL6IP2 DAPP1 UBL3 XRCC2 ALMS1 BTD YOD1 ADAM8 AGRP TNIP3 SLITRK5 CUEDC1 CHSY1 YIPF3 AP3B1 SNX24 DHRS12 SPOCK2 ZBTB7B AP1G1 CSN2 TRA2A ZNHIT3 ADIPOR1 CHD8 TAS2R9 ADCK2 CDC42SE2 TMEM163 BRD3 TUG1 TPM2 BCL2 CHD4 TMEM135 ULBP1 ALF BAT2D1 COQ9 WHDC1 BTBD14B ZNF212 COMMD10 WRNIP1 CNOT1 ADAMTS9 XRCC6 ADAMTS12 ZC3H7B ADAM28 ZNF160 ACPP ADAM17

Figure 9 Coexpression genes of ERCC8 in HBV-related HCC. Notes: The green nodes represent negative correlation with ERCC8, and the red nodes represent positive correlation with ERCC8. Abbreviations: ERCC, excision repair cross-complementation; HBV, hepatitis B virus; HCC, hepatocellular carcinoma.

sible reason may be that ERCC8 plays different roles in both prognostic analysis; the incomplete clinical information, the ovarian cancer and HBV-related HCC. However, these such as postoperative treatment, may influence prognosis. results still require to be further verified in a larger sample, Second, the raw values of serum AFP level in GSE14520 and the hypotheses also need further experimental validation. were not provided as well, accordingly, we were unable to The current study has some limitations that need to clari- carry out a joint effect survival analysis in the serum with an fication. First, since the data set is from the public database, AFP cutoff 400 ng/mL, which is a recognized cutoff value. the clinical information in the GSE14520 was constrained. Third, through the retrieval of the public database, only the Consequently, we are incapable of attaining complete clini- GSE14520 data set caters to the requirements of the current cal information with which to carry out a comprehensive study. That is why the results of the present study are derived

Cancer Management and Research 2018:10 submit your manuscript | www.dovepress.com 5325 Dovepress Yang et al Dovepress ) 3. 5 3. 0 2. 5 2. 0 1. 5 −log10 ( P -value 0 08 06 04 02 t t x y s y y y n n n n g n g n g n in H BV-related CC. Nucleu Cell agin Ribosome ERCC8 Membrane Translatio Golgi lume Cell junction Nucleoplasm Mitochondrion MAPK cascade comple rRNA processing Visual perceptio Viral transcriptio Chromatin binding Beta-catenin binding Poly(A) RNA binding Phospholipid bindin Translational initiatio DNA duplex unwinding Damaged DNA binding DNA damage checkpoint Metalloendopeptidase activit Sequence-specific DNA binding ATP-dependent activity DNA-dependent ATPase activity Telomeric 3 ′ overhang formation Cytosolic large ribosomal subuni Condensed nuclear Structural constituent of ribosome Insulin receptor signaling pathwa Embryonic hindgut morphogenesis Sodium ion transmembrane transpor Cellular response to gamma radiatio Hormone-mediated signaling pathwa DNA synthesis involved in repair Sodium:bicarbonate symporter activity Nuclear chromosome, telomeric region Negative regulation of chondrocyte differentiatio E RCC, excision repair cross-complementation; G O, ene Ontology; H BV, hepatitis B virus; CC, hepatocellular carcinoma. Double−strand break repair via nonhomologous end joinin G O term enrichments of coexpression genes SRP-dependent cotranslational protein targeting to membrane Nuclear-transcribed mRNA catabolic process, nonsense-mediated deca Figure 10 Abbreviations:

5326 submit your manuscript | www.dovepress.com Cancer Management and Research 2018:10 Dovepress Dovepress Diagnostic and prognostic values of ERCC in HBV-related HCC from a single cohort, and additional cohorts to verify the 7. Hou SM, Fält S, Angelini S, et al. The XPD variant alleles are associ- results are needed. ated with increased aromatic DNA adduct level and lung cancer risk. Carcinogenesis. 2002;23(4):599–603. Despite these limitations, our current study is the first to 8. Karahalil B, Bohr VA, Wilson DM. Impact of DNA polymorphisms in systematically investigate the diagnostic and prognostic val- key DNA proteins on cancer risk. Hum Exp Toxicol. 2012;31(10):981–1005. ues of ERCC genes in the HBV-related HCC. As our findings 9. Zhao M, Li S, Zhou L, Shen Q, Zhu H, Zhu X. Prognostic values of suggest, the dysregulated ERCC genes may have a potential excision repair cross-complementing genes mRNA expression in ovarian application in the HBV-related HCC diagnosis and ERCC8 cancer patients. Life Sci. 2018;194:34–39. 10. Qin X, Yao W, Li W, et al. ERCC1 and BRCA1 mRNA expressions are may be a potential novel indicator for the HBV-related HCC associated with clinical outcome of non-small cell lung cancer treated prognosis prediction. We also developed two nomograms for with platinum-based chemotherapy. Tumour Biol. 2014;35(5):4697–4704. 11. Sun JM, Ahn MJ, Park MJ, et al. Expression of excision repair cross- the individualized prognosis prediction and investigated the complementation group 1 as predictive marker for nasopharyngeal potential mechanisms and functions of ERCC8 in the HBV- cancer treated with concurrent chemoradiotherapy. Int J Rad Oncol related HCC using the GSEA and genome-wide coexpression Biol Phys. 2011;80(3):655–660. 12. 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Yoon AJ, Kuo WH, Lin CW, Yang SF. Role of ERCC5 polymorphism in risk of hepatocellular carcinoma. Oncol Lett. 2011;2(5):911–914. 16. Roessler S, Long EL, Budhu A, et al. Integrative genomic identification Acknowledgments of genes on 8p associated with hepatocellular carcinoma progression This work was supported in part by the National Natural and patient survival. Gastroenterology. 2012;142(4):957–966. Science Foundation of China (Grant NO.81460512) and 17. Roessler S, Jia HL, Budhu A, et al. A unique metastasis gene signature enables prediction of tumor relapse in early-stage hepatocellular carci- Shandong Province Education Department Colleges and Uni- noma patients. Cancer Res. 2010;70(24):10202–10212. versities Science and Technology Plan Project (J17KB088). 18. Shaul YD, Yuan B, Thiru P, et al. MERAV: a tool for comparing gene The authors thank the contributors of the GSE14520 for expression across human tissues and cell types. 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