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Polymorphisms of Nucleotide Excision Repair Genes Predict Melanoma Survival

  • Chunying Li
    Correspondence
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China

    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Ming Yin
    Affiliations
    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Li-E Wang
    Affiliations
    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Author Footnotes
    5 Current address: Christopher I. Amos and Dakai Zhu, Department of Community and Family Medicine, Geisel College of Medicine, Dartmouth College, Hanover, New Hampshire 03755, USA
    Christopher I. Amos
    Footnotes
    5 Current address: Christopher I. Amos and Dakai Zhu, Department of Community and Family Medicine, Geisel College of Medicine, Dartmouth College, Hanover, New Hampshire 03755, USA
    Affiliations
    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Author Footnotes
    5 Current address: Christopher I. Amos and Dakai Zhu, Department of Community and Family Medicine, Geisel College of Medicine, Dartmouth College, Hanover, New Hampshire 03755, USA
    Dakai Zhu
    Footnotes
    5 Current address: Christopher I. Amos and Dakai Zhu, Department of Community and Family Medicine, Geisel College of Medicine, Dartmouth College, Hanover, New Hampshire 03755, USA
    Affiliations
    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Jeffrey E. Lee
    Affiliations
    Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Jeffrey E. Gershenwald
    Affiliations
    Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Elizabeth A. Grimm
    Affiliations
    Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Qingyi Wei
    Correspondence
    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
    Affiliations
    Department of Epidemiology, Unit 1365, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
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  • Author Footnotes
    5 Current address: Christopher I. Amos and Dakai Zhu, Department of Community and Family Medicine, Geisel College of Medicine, Dartmouth College, Hanover, New Hampshire 03755, USA
      Melanoma is the most highly malignant skin cancer, and nucleotide excision repair (NER) is involved in melanoma susceptibility. In this analysis of 1,042 melanoma patients, we evaluated whether genetic variants of NER genes may predict survival outcome of melanoma patients. We used genotyping data of 74 tagging single-nucleotide polymorphisms (tagSNPs) in eight core NER genes from our genome-wide association study (including two in XPA, 14 in XPC, three in XPE, four in ERCC1, 10 in ERCC2, eight in ERCC3, 14 in ERCC4, and 19 in ERCC5) and evaluated their associations with prognosis of melanoma patients. Using the Cox proportional hazards model and Kaplan–Meier analysis, we found a predictive role of XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871 SNPs in the prognosis of melanoma patients (rs28720291: AG vs. GG, adjusted hazard ratio (adjHR)=11.2, 95% confidence interval (CI) 3.04–40.9, P=0.0003; rs4150314: AG vs. GG, adjHR=4.76, 95% CI 1.09–20.8, P=0.038; rs2470458: AA vs. AG/GG, adjHR=2.11, 95% CI 1.03–4.33, P=0.040; and rs50871: AA vs. AC/CC adjHR=2.27, 95% CI 1.18–4.35, P=0.015). Patients with an increasing number of unfavorable genotypes had markedly increased death risk. Genetic variants of NER genes, particularly XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871, may independently or jointly modulate survival outcome of melanoma patients. Because our results were based on a median follow-up of 3 years without multiple test corrections, additional large prospective studies are needed to confirm our findings.

      Abbreviations

      adjHR
      adjusted hazard ratio
      AJCC
      American Joint Committee on Cancer
      CI
      confidence interval
      HR
      hazard ratio
      NER
      nucleotide excision repair
      OS
      overall survival
      tagSNPs
      tagging single-nucleotide polymorphisms

      Introduction

      Melanoma is the most lethal skin cancer, ranking the sixth most common cancer in the United States. There were estimated 76,250 new melanoma cases, in addition to 55,560 melanoma in situ, in 2012 (
      • Siegel R.
      • Naishadham D.
      • Jemal A.
      Cancer statistics, 2012.
      ). Although surgery remains the mainstay treatment, biochemotherapy and radiotherapy are also considered in an attempt to improve local control and overall survival (OS). Despite aggressive treatment, patients’ prognosis varies substantially between individuals, with a 5-year survival rate ranging from over 80% in early stages to <10% in patients with distant metastasis (
      • Buettner P.G.
      • Leiter U.
      • Eigentler T.K.
      • et al.
      Development of prognostic factors and survival in cutaneous melanoma over 25 years: an analysis of the Central Malignant Melanoma Registry of the German Dermatological Society.
      ).
      Some important tumor morphological and biological characteristics are known to be associated with patients’ survival, including primary tumor thickness, ulceration, mitotic activity, lymph node infiltration, and distant metastasis (
      • Spatz A.
      • Batist G.
      • Eggermont A.M.
      The biology behind prognostic factors of cutaneous melanoma.
      ). However, these histopathological features of primary tumors do not provide sufficient information for assessing tumor malignancy. For example, a subset of “thin” melanoma (tumor thickness<0.76mm) can be lethal because of undetected metastasis (
      • Woods J.E.
      • Soule E.H.
      • Creagan E.T.
      Metastasis and death in patients with thin melanomas (less than 0.76 mm).
      ). Although the underlying mechanisms are unclear, tumor genetic heterogeneity and interactions among the host and tumor factors may be responsible for rapid evolution and development of malignancies in these patients. Some somatic mutations (e.g., BRAF and p16) are commonly implicated in melanoma progression (
      • Chin L.
      • Garraway L.A.
      • Fisher D.E.
      • et al.
      Malignant melanoma: genetics and therapeutics in the genomic era.
      ), whereas an enhanced host’s immune system can efficiently suppress cancer cell spreading, contributing to prolonged survival (
      • DiFronzo L.A.
      • Gupta R.K.
      • Essner R.
      • et al.
      Enhanced humoral immune response correlates with improved disease-free and overall survival in American Joint Committee on Cancer stage II melanoma patients receiving adjuvant polyvalent vaccine.
      ). Nevertheless, it is possible that some other unknown genetic factors, by interacting with the known clinicopathological factors, may modulate survival outcomes of melanoma patients, thus uncovering biomarker for patients’ long-term survival.
      Previous epidemiologic studies have supported the notion that DNA-damaging UV irradiation causes cutaneous melanoma by inducing genetic abnormality (
      • von Thaler A.K.
      • Kamenisch Y.
      • Berneburg M.
      The role of ultraviolet radiation in melanomagenesis.
      ). The well-studied nucleotide excision repair (NER) pathway consists of at least 23 genes/proteins that act to remove UV-induced DNA lesions. Several single-nucleotide polymorphisms (SNPs) of the NER genes have been shown to be associated with melanoma susceptibility (
      • Li C.
      • Hu Z.
      • Liu Z.
      • et al.
      Polymorphisms in the DNA repair genes XPC, XPD, and XPG and risk of cutaneous melanoma: a case-control analysis.
      ). However, their influence on patients’ survival has not been thoroughly investigated. In a recent study of eight nonsynonymous SNPs of DNA repair genes (i.e., XPC p.Ala499Val, XPC p.Lys939Gln, ERCC2 p.Lys751Gln, and ERCC5 p.Asp1104His of NER; APEX1 p.Asp148Glu, XRCC1 p.Arg399Gln of base excision repair; and XRCC3 p.Thr241Met and NBS1 p.Glu185Gln of the homologous recombination repair), only ERCC5 p.Asp1104His (rs17655) and ERCC2 p.Lys751Gln (rs13181) were found to have an effect on the prognosis of melanoma (
      • Schrama D.
      • Scherer D.
      • Schneider M.
      • et al.
      ERCC5 p.Asp1104His and ERCC2 p.Lys751Gln polymorphisms are independent prognostic factors for the clinical course of melanoma.
      ), suggesting that the NER genes may be involved in melanoma outcomes, although genes involved in cell cycle checkpoint are also found to be important (
      • Kauffmann A.
      • Rosselli F.
      • Lazar V.
      • et al.
      High expression of DNA repair pathways is associated with metastasis in melanoma patients.
      ).
      Here we report our results of an analysis of prognosis of 1,042 melanoma patients in association with 74 tagging SNPs of the NER genes available to us in a previously published genome-wide association study of melanoma (
      • Amos C.I.
      • Wang L.E.
      • Lee J.E.
      • et al.
      Genome-wide association study identifies novel loci predisposing to cutaneous melanoma.
      ). In the present analysis, we evaluated the association between these SNPs and survival and explored their interactions with clinicopathological risk factors in determining melanoma patients’ prognosis.

      Results

      Patient characteristics

      The analysis consisted of 1,042 patients with primary cutaneous melanoma (Table 1), who had available data from questionnaire, genotyping, and survival. The patients were aged between 18 and 84 years at diagnosis with a mean of 50.8 years and standard deviation of 13.1 years. There were slightly more women than men (58.8 vs. 41.2%); 83.1% of the patients had early-stage melanoma (in situ and stages I and II), and 16.9% had later-stage melanoma (stages III and IV). We also collected complete information on tumor morphology, including primary tumor thickness, ulceration, metastasis to local lymph nodes, mitotic rate (mitoses per mm2) of tumor cells (because there was no association with mitotic rate by the American Joint Committee on Cancer (AJCC) staging system of mitoses ≥1/mm2 vs. <1/mm2, we used 3/mm2 as the cutoff as shown in Table 1), anatomic site of the tumor, and patient biological characteristics, including colors of the skin, hair, and eyes, tanning ability after sun exposure, lifetime sunburns with blistering, moles, and family history of skin cancer. The median follow-up time was 35.7 months, during which 52 (5.0%) of the 1,042 patients had died at the last follow-up.
      Table 1Associations of patient demographics and tumor-related characteristics with overall survival
      Parameter
      The numbers of subjects in some of the strata were less than the total number of subjects included in our study, because some subjects did not provide complete information in their screening questionnaires.
      PatientDeathUnivariate analysisMultivariate analysis
      Multivariate Cox regression analyses were adjusted for all factors listed in Table 1.
      No.%No.%HR95% CIP-valueHR95% CIP-value
      Age
       =5048146.21426.91.001.00
       >5056155.73873.12.651.43–4.890.0021.900.86–4.180.110
      Sex
       Female61358.83669.21.001.00
       Male42941.21630.80.560.31–1.010.0530.900.41–2.040.825
      Skin color
       Fair93890.1479.61.001.00
       Dark and brown1039.9590.40.980.39–2.460.9614.551.28–16.700.019
      Hair color
       Blond or red35534.11019.21.001.00
       Black or brown68665.94280.82.131.08–4.350.0300.860.37–2.000.735
      Eye color
       Not blue60458.02955.81.001.00
       Blue43742.02344.21.080.63–1.870.7810.620.30–1.270.192
      Tanning ability after prolonged sun exposure
       Good (high)65163.03669.21.001.00
       Poor (low)38237.01630.80.770.43–1.390.391.260.58–2.770.560
      Lifetime sunburns with blistering
       030229.01835.31.001.00
       =173871.03364.70.650.37–1.150.1400.970.47–2.010.927
      Freckling in the sun as a child
       No60057.61732.71.001.00
       Yes44142.43567.33.331.89–5.88<0.00012.901.33–6.330.008
      Moles
       No24223.21121.11.001.00
       Yes80076.84178.91.030.53–2.010.9251.220.53–2.830.639
      Dysplastic nevi
       No93990.15198.11.001.00
       Yes1039.911.90.180.02–1.270.0850.560.07–4.240.575
      Family history of skin cancer
       No38536.91936.51.001.00
       Yes65763.13363.51.100.62–1.930.7451.150.56–2.370.704
      AJCC stages
      In situ, I and II86683.12446.11.001.00
       III and IV17616.92853.96.873.97–11.9<0.00015.602.69–11.64<0.0001
      Primary tumor thickness
       <1mm42549.0817.01.001.00
       >0.99mm44351.03983.05.172.42–11.1<0.00012.140.72–6.350.169
      Ulceration
       No84687.22859.61.001.00
       Yes12412.81940.35.713.19–10.2<0.000012.721.28–5.780.0009
      SLNB
       No33332.11325.51.001.00
       Yes70567.93874.51.310.69–2.490.4170.440.19–1.030.058
      Mitotic rate (mitoses/mm
      Multivariate Cox regression analyses were adjusted for all factors listed in Table 1.
      )
       =358978.62047.61.001.00
       >316021.42252.44.702.56–8.63<0.00011.540.74–3.230.251
      Primary tumor anatomic site
       Face, head, and neck11711.5917.61.001.00
       Trunk, extremities, and others90288.54282.41.750.85–3.610.1520.520.17–1.570.244
      Abbreviations: CI, confidence interval; HR, hazard ratio; SLNB, sentinel lymph node biopsy.
      1 The numbers of subjects in some of the strata were less than the total number of subjects included in our study, because some subjects did not provide complete information in their screening questionnaires.
      2 Multivariate Cox regression analyses were adjusted for all factors listed in Table 1.
      To determine whether there was any confounding factor influencing patients’ death or survival time, we performed Cox proportional hazards regression analysis to assess the association between OS and clinicopathological characteristics. In the univariate analysis, older age, dark color of hair, freckling in the sun as a child, advanced tumor stages, thick tumor, presence of tumor ulceration, and increased primary tumor mitotic rate were significant predictors for poor survival. When all of these variables were included in a Cox proportional hazards regression model for adjustment to calculate hazard ratio (HR), only dark color of the skin (HR=4.55), freckling in the sun as a child (HR=2.90), advanced AJCC stage (HR=5.60), and presence of tumor ulceration (HR=2.72) remained statistically significant predictors for poor survival (Table 1).

      Determination of melanoma survival prediction model

      We performed the stepwise multivariate Cox proportional hazards regression analysis to further screen for optimal predictors of survival in melanoma patients, using covariates listed in Table 1 and the 74 selected SNPs of the eight NER core genes (i.e., two SNPs for XPA, 14 SNPs for XPC, three SNPs for XPE/DDB1, four SNPs for ERCC1, 10 SNPs for ERCC2/XD, eight SNPs for ERCC3/XPB, 14 SNPs for ERCC4/XPF, and 19 SNPs for ERCC5/XPG). As shown in Table 2, clinicopathological factors of age (≤50 vs. >50), stage (in situ/I/II vs. III/IV), ulceration (no vs. yes) and mitotic rate (≤3 vs. >3/mm2), and SNPs of rs28720291 (GG vs. AG), rs4150314 (GG vs. AG), rs2470458 (AG+GG vs. AA), and rs50871 (AC+CC vs. AA) were selected as the most significant predictors of survival, of which covariates of late stages (III/IV) (HR=6.34; 95% confidence interval (CI) 3.11–11.9), rs28720291 GG genotype (HR=6.69; 95% CI 1.83–23.7), and rs4150314 GG genotype (HR=6.15; 95% CI 1.46–28.5), were among the strongest predictors. Older age, ulceration, increased mitotic rate, rs2470458 AG/GG genotypes, and rs50871 AC/CC genotypes were of low or moderate risk factors (1< HR <3).
      Table 2Predictors of overall survival in melanoma patients obtained from stepwise multivariate cox regression analysis of selected variables
      Age, sex, tumor Breslow thickness, tumor stages, ulceration of the tumor, tumor cell mitotic rate, involvement of lymph nodes, primary tumor anatomic site, and the 74 selected SNPs of the eight NER core genes (i.e., XPA (rs1800975 and rs2808667), XPC (rs1350344, rs2227999, rs2228000, rs2228001, rs2470458, rs2607772, rs2733533, rs2733537, rs3731062, rs3731125, rs3731127, rs3731146, rs3731149, and rs3731151), XPE (rs2230356, rs4939513, and rs28720291), ERCC1 (rs11615, rs1007616, rs2298881, and rs3212955), ERCC2 (rs13181, rs50871, rs171140, rs238406, rs238416, rs1052555, rs1618536, rs1799786, rs1799787, and rs1799793), ERCC3 (rs1566823, rs1803541, rs4150403, rs4150436, rs4150496, rs4150523, rs4662718, and rs9282675), ERCC4 (rs254942, rs1799801, rs1800067, rs1800124, rs2276464, rs2276465, rs2276466, rs3136146, rs3136166, rs3136187, rs3136189, rs3136195, rs3743538, and rs16963255), and ERCC5 (rs17655, rs751402, rs873601, rs1047768, rs1047769, rs2227869, rs2296147, rs2296148, rs4150260, rs4150275, rs4150314, rs4150330, rs4150339, rs4150342, rs4150355, rs4150383, rs4771436, rs8002276, and rs11069498)) genotypes were included in the stepwise multivariate Cox proportional hazards regression analysis.
      Selected variablesP-valueHR95% CI
      Age (≤50 vs. >50)0.0031.051.01–1.07
      Stage (in situ, I, II vs. III, IV)<0.00016.343.11–11.9
      Ulceration (no vs. yes)0.0132.521.25–5.35
      Mitotic rate (≤3 vs. >3)0.0122.551.06–4.61
      rs28720291 (GG vs. AG)0.0046.691.83–23.7
      rs4150314 (GG vs. AG)0.0176.151.46–28.5
      rs2470458 (AG+GG vs. AA)0.0252.421.08–4.76
      rs50871 (AC+CC vs. AA)0.0202.231.18–4.50
      Abbreviations: CI, confidence interval; HR, hazard ratio.
      1 Age, sex, tumor Breslow thickness, tumor stages, ulceration of the tumor, tumor cell mitotic rate, involvement of lymph nodes, primary tumor anatomic site, and the 74 selected SNPs of the eight NER core genes (i.e., XPA (rs1800975 and rs2808667), XPC (rs1350344, rs2227999, rs2228000, rs2228001, rs2470458, rs2607772, rs2733533, rs2733537, rs3731062, rs3731125, rs3731127, rs3731146, rs3731149, and rs3731151), XPE (rs2230356, rs4939513, and rs28720291), ERCC1 (rs11615, rs1007616, rs2298881, and rs3212955), ERCC2 (rs13181, rs50871, rs171140, rs238406, rs238416, rs1052555, rs1618536, rs1799786, rs1799787, and rs1799793), ERCC3 (rs1566823, rs1803541, rs4150403, rs4150436, rs4150496, rs4150523, rs4662718, and rs9282675), ERCC4 (rs254942, rs1799801, rs1800067, rs1800124, rs2276464, rs2276465, rs2276466, rs3136146, rs3136166, rs3136187, rs3136189, rs3136195, rs3743538, and rs16963255), and ERCC5 (rs17655, rs751402, rs873601, rs1047768, rs1047769, rs2227869, rs2296147, rs2296148, rs4150260, rs4150275, rs4150314, rs4150330, rs4150339, rs4150342, rs4150355, rs4150383, rs4771436, rs8002276, and rs11069498)) genotypes were included in the stepwise multivariate Cox proportional hazards regression analysis.

      NER genetic polymorphisms as independent survival risk factors

      The initial stepwise Cox proportional hazards regression analysis suggested four SNPs (XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871) as important and independent predictors for survival of melanoma patients. We further performed univariate and multivariate Cox proportional hazards regression analyses to evaluate their effects on risk of death or in the presence of other clinicopathological covariates. In the univariate analysis, XPE rs28720291AG and ERCC2 rs50871AA genotypes were associated with increased hazards of early death (AG vs. GG: HR=4.92, 95% CI 1.77–13.70, P=0.002; and AA vs. AC+CC: HR=2.18, 95% CI 1.26–3.77, P=0.005, respectively). In the multivariate analyses performed with adjustment for age, sex, tumor Breslow thickness, tumor stage, ulceration, tumor cell mitotic rate, involvement of lymph nodes, and tumor anatomic site, the four SNPs remained significantly associated with survival outcome of melanoma patients (i.e., rs28720291: AG (no AA was observed) vs. GG 11.2, 95% CI 3.04–40.9, P=0.0003; rs4150314: AG (no AA was observed) vs. GG 4.76, 95% CI 1.09–20.8, P=0.038; rs2470458: AA vs. AG+GG 2.11, 95% CI 1.03–4.33, P=0.040; and rs50871: AA vs. AC+CC 2.27, 95% CI 1.18–4.35, P=0.015) (Table 3).
      Table 3Association between selected NER genetic variants
      Only listed the four SNPs from the stepwise multivariate Cox proportional hazards regression analysis model shown in Table 2.
      and overall survival of melanoma patients
      Genotypes
      Only listed the four SNPs from the stepwise multivariate Cox proportional hazards regression analysis model shown in Table 2.
      PatientDeathUnivariate analysisMultivariate analysis
      Adjusted by age, sex, tumor Breslow thickness, tumor stage, ulceration of the tumor, tumor cell mitotic rate, involvement of lymph nodes, and primary tumor anatomic site.
      No.%No.%HR95% CIP-valueHR95% CIP-value
      XPE
       rs28720291
        GG1,01997.84892.31.001.00
        AG232.247.74.921.77–13.70.00211.23.04–40.90.0003
      ERCC5
       rs4150314
        GG1,02198.25096.11.001.00
        AG191.823.92.390.58–9.830.2274.761.09–20.80.038
      XPC
       rs2470458
        AA64662.13771.11.001.00
        AG34533.11223.10.600.31–1.150.1260.420.19–0.920.031
        GG504.835.81.060.33–3.430.9270.890.21–3.780.879
        AG+GG39537.91528.90.660.36–1.200.1750.470.23–0.970.040
        AG+GG39537.91528.91.001.00
        AA64662.13771.11.520.83–2.770.1722.111.03–4.330.040
      ERCC2
       rs50871
        AA28627.42344.21.001.00
        AC52950.81834.60.410.22–0.760.0050.450.22–0.900.024
        CC22721.81121.20.570.28–1.170.1250.440.17–1.150.093
        AC+CC75672.62955.80.460.27–0.790.0050.440.23–0.850.015
        AC+CC75672.62955.81.001.00
        AA28627.42344.22.181.26–3.770.0052.271.18–4.350.015
      Abbreviations: CI, confidence interval; HR, hazard ratio; NER, nucleotide excision repair.
      1 Only listed the four SNPs from the stepwise multivariate Cox proportional hazards regression analysis model shown in Table 2.
      2 Adjusted by age, sex, tumor Breslow thickness, tumor stage, ulceration of the tumor, tumor cell mitotic rate, involvement of lymph nodes, and primary tumor anatomic site.

      Survival of melanoma patients and combined genetic risk factors

      To assess the joint effect of the four SNPs on patients’ prognosis, we combined their unfavorable genotypes (i.e., XPE rs28720291AG, ERCC5 rs4150314AG, XPC rs2470458AA, and ERCC2 rs50871AA genotypes). The frequencies of patients with 0, 1, 2, and 3 unfavorable genotypes were 276, 566, 188, and 10, respectively; no patient had all four unfavorable genotypes. Patients with an increasing number of unfavorable genotypes had markedly increased risk of death by over 30-fold (HR=34.3; 95% CI 7.48–157.2; P<0.0001) in patients with any three unfavorable genotypes, compared with those without any unfavorable genotypes (Table 4 and Figure 1). As there were only 10 patients carrying three unfavorable genotypes, we next grouped all patients into a low-risk group (patients with ≤1 unfavorable genotypes) and a high-risk group (patients with two or three unfavorable genotypes) for further stratified analysis (Table 4).
      Table 4Association between combined NER variants and overall survival of melanoma patients
      No. of variant genotypes
      rs28720291AG, rs4150314AG, rs2470458AA, and rs50871AA.
      PatientDeathUnivariate analysisMultivariate analysis
      Multivariate Cox proportional Hazards regression analysis with adjustment for age, sex, tumor Breslow thickness, tumor stage, ulceration of the tumor, tumor cell mitotic rate, involvement of lymph nodes, and primary tumor anatomic site.
      No.%No.%HR95% CIP-valueHR95% CIP-value
      No. of variant genotypes
      rs28720291AG, rs4150314AG, rs2470458AA, and rs50871AA.
      0.0005<0.00001
       027626.5917.31.001.00
       156654.42344.21.280.59–2.780.5251.260.51–3.100.061
       218818.11732.72.941.31–6.590.0093.901.50–10.10.005
       3101.035.717.84.77–66.3<0.000134.37.48–157.2<0.0001
      Ptrend <0.0001
      Combined group
       0–184481.03261.51.001.00
       2–319819.02038.52.101.42–3.120.00024..012.04–7.86<0.0001
      Abbreviations: CI, confidence interval; HR, hazard ratio.
      1 rs28720291AG, rs4150314AG, rs2470458AA, and rs50871AA.
      2 Multivariate Cox proportional Hazards regression analysis with adjustment for age, sex, tumor Breslow thickness, tumor stage, ulceration of the tumor, tumor cell mitotic rate, involvement of lymph nodes, and primary tumor anatomic site.
      Figure thumbnail gr1
      Figure 1Kaplan–Meier overall survival analysis for patients with primary melanoma by combined nucleotide excision repair (NER) genotypes (i.e., rs28720291 GG, rs4150314 GG, rs2470458 AG+GG, and rs50871 AC+CC). (a) By 0, 1, 2, and 3 NER variant genotypes (P<0.0001); and (b) by 0–1 and 2–3 NER variant genotypes (P=0.0001).

      Stratification analysis between the unfavorable genotypes and melanoma survival

      We further performed stratified analysis to investigate whether the combined effect of unfavorable genotypes on survival was modified by some important clinicopathological factors in Table 1. We found that only patients in the high-risk genotype group, but not the low-risk genotype group, showed substantially increased risk of death in the presence or absence of concomitant clinicopathological risk factors (e.g., thick tumor, involvement of lymph nodes, increased mitotic rate, advanced AJCC tumor stages, presence of tumor ulceration, and tumor site in face, head and neck), except for the subgroups of thin tumor and without lymph node involvement (Figure 2).
      Figure thumbnail gr2
      Figure 2Kaplan–Meier overall survival curves for patients with primary melanoma of 0–1 and 2–3 nucleotide excision repair (NER) variant genotypes (i.e., rs28720291 GG, rs4150314 GG, rs2470458 AG+GG, and rs50871 AC+CC) by tumor-related characters. (a, b) By tumor Breslow thickness (P<0.0001); (c, d) by sentinel lymph node biopsy (SLNB) (P<0.0001); (e, f) by mitotic rate (P<0.0001); (g, h) by American Joint Committee on Cancer stages (P<0.0001); (i, j) by ulceration (P<0.0001); and (k, l) by primary tumor anatomic site (P<0.0001).

      Discussion

      In this relatively large melanoma patient cohort, we found that some variants of the NER genes (e.g., XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871) may independently or jointly modulate survival of melanoma patients. These genetic variants, in combination with clinicopathological factors, effectively predicted survival in subgroups of melanoma patients.
      Previous studies demonstrated that some clinicopathological characteristics were associated with the prognosis of melanoma patients, such as hair color, history of childhood freckling in the sun, tumor stage, and ulceration status (
      • Buettner P.G.
      • Leiter U.
      • Eigentler T.K.
      • et al.
      Development of prognostic factors and survival in cutaneous melanoma over 25 years: an analysis of the Central Malignant Melanoma Registry of the German Dermatological Society.
      ). These results were also confirmed in the current analysis. However, we were interested in finding out some genetic variants of the NER genes that may have a role in modulating patient survival. This is because NER is an essential DNA repair mechanism that ensures genomic integrity. There is evidence that genomic instability increases not only in primary melanoma, compared with nevi, but also in metastases, compared with primary tumors (
      • Chin L.
      • Garraway L.A.
      • Fisher D.E.
      • et al.
      Malignant melanoma: genetics and therapeutics in the genomic era.
      ). Hence, an increased NER capacity may reduce DNA mutations that may stimulate malignant progression and metastasis. More interestingly, previously reported two NER SNPs [ERCC5 p.Asp1104His (rs17655) and ERCC2 p.Lys751Gln (rs13181)] (
      • Schrama D.
      • Scherer D.
      • Schneider M.
      • et al.
      ERCC5 p.Asp1104His and ERCC2 p.Lys751Gln polymorphisms are independent prognostic factors for the clinical course of melanoma.
      ) were replicated in our current analysis, because they are tagged by our selected tagging SNPs (ERCC5 rs4150314 and ERCC2 rs50871). However, we found that additional two NER SNPs (XPE rs28720291 and XPC rs2470458) also independently predicted the prognosis of melanoma. Therefore, our data further support the notion that genetic variants in the NER pathway may modulate clinical outcome of melanoma patients.
      NER uses a relatively small number of essential repair proteins, including XPA, XPC, XPE/DDB1, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, and ERCC5/XPG, to correct bulky DNA damage induced by chemical carcinogens or UV exposure. In brief, repair of the damaged DNA strand includes making an incision at the 5′ and 3′ of the lesion, removing a 30-nucleotide section containing the damage, and ligating the gap by pairing DNA synthesis (
      • Sancar A.
      DNA repair in humans.
      ). XPC-hHR23B is an NER factor that detects DNA damage and recruits TFIIH to the damaged site (
      • Yokoi M.
      • Masutani C.
      • Maekawa T.
      • et al.
      The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.
      ); then, proteins encoded by XPA-G genes, the ERCC1-hHR23B-RPA trimmers, and TFIIH are involved in the excision step (
      • de Boer J.
      • Hoeijmakers J.H.
      Cancer from the outside, aging from the inside: mouse models to study the consequences of defective nucleotide excision repair.
      ). Two helicase subunits of TFIIH (i.e., XPB and XPD), together with XPA, RPA, and XPG, form a 30 base-pair bubble around the lesion for damage verification and correct positioning of two endonucleases (i.e., XPG and XPF-ERCC1) before incision (
      • Missura M.
      • Buterin T.
      • Hindges R.
      • et al.
      Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair.
      ). The incisions made by XPG and XPF-ERCC1 are at the double- and single-stranded DNA border in the incision complex (
      • Sijbers A.M.
      • de Laat W.L.
      • Ariza R.R.
      • et al.
      Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.
      ). Observation of the mobility of various NER proteins in living cells suggests that NER proceeds by the sequential assembly of individual factors involved, rather than through the action of a preassembled repairosome (
      • Houtsmuller A.B.
      • Rademakers S.
      • Nigg A.L.
      • et al.
      Action of DNA repair endonuclease ERCC1/XPF in living cells.
      ).
      Previous studies have extensively explored associations between NER and melanoma susceptibility, but few investigated the effects of NER on clinical outcomes of melanoma patients. In a study of 90 stage IV melanoma patients, an ERCC1-rs11615 SNP was found to be weakly associated with OS (
      • Liu D.
      • O'Day S.J.
      • Yang D.
      • et al.
      Impact of gene polymorphisms on clinical outcome for stage IV melanoma patients treated with biochemotherapy: an exploratory study.
      ). In another study of 244 melanoma patients in Sweden, an ERCC2-rs13181 SNP was suggested to be a prognostic factor for melanoma progression (
      • Kertat K.
      • Rosdahl I.
      • Sun X.F.
      • et al.
      The Gln/Gln genotype of XPD codon 751 as a genetic marker for melanoma risk and Lys/Gln as an important predictor for melanoma progression: a case control study in the Swedish population.
      ). More recently, both ERCC5-rs17655 and ERCC2-rs13181 SNPs were found to be independent prognostic factors in 742 melanoma patients (
      • Schrama D.
      • Scherer D.
      • Schneider M.
      • et al.
      ERCC5 p.Asp1104His and ERCC2 p.Lys751Gln polymorphisms are independent prognostic factors for the clinical course of melanoma.
      ). However, none of the above SNPs were replicated or in linkage with the four positive SNPs (i.e., XPE-rs28720291, ERCC5-rs4150314, XPC-rs2470458, and ERCC2-rs50871) investigated in the present study. Several possible reasons may explain the discrepancies: first, the studies by Liu et al. and Kertat et al. were small sample-sized (90 cases and 244 cases, respectively), which could lead to chance findings or miss some SNPs with mild effects because of a limited study power; second, the majority of melanoma patients in these two studies had late-stage tumors (stages III/IV), whereas a large proportion of patients in our analysis and the study by Schrama et al. had early-stage tumors (866/1044 and 652/742, respectively).
      Despite these discrepancies, there was some consistency among these published studies and ours. If excluding the most small-sized study by Liu et al., it appears that there are increasing levels of significance as well as increasing number of genes as the study patient population size increases, and the significant SNPs/genes identified in previous small-sized study could be confirmed by later larger-sized studies (ERCC2 in 244 patients, ERCC2 and ERCC5 in 742 patients, and ERCC2, ERCC5, XPC, and XPE in 1,042 patients). As SNPs may alter the related gene’s function, our analysis, together with previous studies, suggested that the four genes, ERCC2/XPD, ERCC5/XPG, XPC, and XPE/ERCC3, may have important roles in modulating melanoma patients’ survival.
      Although the effect of a single SNP on cancer risk or clinical outcomes, if any, may be limited, the combined effect of several SNPs in the same or different genes could be more significant. In the present analysis, we were also interested in whether there was an additive/synergistic effect in the association of the four SNPs (XPE-rs28720291, ERCC5-rs4150314, XPC-rs2470458, and ERCC2-rs50871) with melanoma survival. Indeed, patients with two or three unfavorable genotypes showed markedly increased risk of death, compared with those with none or one unfavorable genotype. This is biologically plausible, because multiple variants may be more likely to have a substantial joint effect on the DNA repair capacity phenotype.
      Through stratified analyses, we found that the genotype–survival association was the most pronounced in the presence of clinicopathological risk factors, suggesting that suboptimal repair of DNA damage induced externally (UV exposure) or internally (free radicals from metabolism) could aggregate the existing genomic instability of a fast-growing melanoma, promoting melanoma development and progression in the high-risk populations. As these high-risk patients comprised 20.0% (198 out of 1,042) of all the study subjects, our analysis identified a significant proportion of melanoma patients (such as those with unfavorable genotypes) that may require close clinical surveillance or alternative treatment to improve their survival.
      The current analysis has some limitations. First, as we used a tagging SNP approach, we were not able to explore the mechanism by which the studied genetic polymorphisms influence melanoma patients’ survival. Although the four identified SNPs and their tagged SNPs (LD ≥0.8) may have potential biological functions as predicted by software tools (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm), none of them have been reported or investigated as functional SNPs in the literature. Only four SNPs tagged by XPC-rs2470458 were found to be associated with the risk of bladder cancer (rs2228000, rs2470352, and rs2470458) and lung cancer (rs2229090) in previous association studies (
      • Shen M.
      • Berndt S.I.
      • Rothman N.
      • et al.
      Polymorphisms in the DNA nucleotide excision repair genes and lung cancer risk in Xuan Wei, China.
      ;
      • Stern M.C.
      • Lin J.
      • Figueroa J.D.
      • et al.
      Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the international consortium of bladder cancer.
      ). Further functional studies of these SNPs are required. Second, there were only 52 deaths out of 1,042 patients at our last follow-up at a median of nearly 3 years. Therefore, the current study is, to a large extent, an interim survival analysis. We will report updated results after we have a longer follow-up time. Third, we did not adjust for multiple tests, simply because this was an exploratory study with a limited study power. We plan to confirm current findings in our ongoing prospective expansion studies in more stringent conditions with a larger study population. Fourth, as treatment of melanoma, advanced melanoma in particular, has not been standardized, patients included in the current analysis who developed advanced melanoma may have received a wide variety of systemic therapies, often sequentially. The systemic therapies available for the cohort of patients included in our analysis would only have been expected to be modestly effective in a minority of melanoma patients (the study period ended in 2008; vemurafenib was approved by the FDA for the treatment of advanced melanoma in 2011). Because of the variety of treatments administered (often multiple types of treatments to the same patient) and their very modest anticipated effect on OS, we did not evaluate the potential role of these therapies in the outcomes of the patients, or their potential relationship to the polymorphisms identified. Although evaluation of the association between the polymorphisms investigated and response to a variety of melanoma systemic therapies is important, such an evaluation is beyond the scope of the current analysis.
      In summary, we identified four SNPs of the NER genes (i.e., XPE rs28720291, ERCC5 rs4150314, XPC rs2470458, and ERCC2 rs50871) that may have independent or joint effects on survival of melanoma patients. These findings, once validated in future prospective studies with large sample sizes and better study designs, will provide some promising guidance for clinical management and tailored or personalized therapeutics in treating melanoma patients.

      Materials and Methods

      Study populations

      Patients were accrued for an ongoing, hospital-based, case–control study of epidemiologic and genetic risk factors for melanoma. A total of 1,042 histologically confirmed patients with melanoma in situ and stage I to stage IV were enrolled between January 2000 and September 2008. Patients were enrolled into the study regardless of age, sex, or disease stage. On entry into the study, each patient had a personal interview to elicit lifestyle factors, using a standardized questionnaire. Each patient also had a 30-ml sample of blood drawn for various biomarker studies, including genotyping. All patients were enrolled and diagnosed with staging system defined by the AJCC at The University of Texas MD Anderson Cancer Center, Houston, TX. Specifically, melanoma patients with melanoma in situ, stage I/II (primary tumor without evidence of regional or distant metastasis at diagnosis), stage III (locoregional disease, including in transit, satellite, and/or regional lymph node metastasis at diagnosis), and stage IV (distant metastasis at diagnosis) were classified according to the AJCC melanoma staging system (
      • Balch C.M.
      • Gershenwald J.E.
      • Soong S.J.
      • et al.
      Final version of 2009 AJCC melanoma staging and classification.
      ). For patients with stage I/II disease, staging elements included Breslow primary tumor thickness, presence or absence of primary tumor ulceration, and mitotic rate (i.e., number of mitoses per square millimeter using dermal hotspot approach). All patients gave a written informed consent, and the protocol was approved by the MD. Anderson Cancer Center Institutional Review Board. Patients were evaluated, staged, treated, and followed using the standard guidelines, including the use of sentinel lymph node biopsy for high-risk primary melanoma (
      • Gershenwald J.E.
      • Ross M.I.
      Sentinel-lymph-node biopsy for cutaneous melanoma.
      ). Patients with high-risk local-regional, and those with recurrent and metastatic melanoma, received a variety of protocol-based and off-protocol systemic therapies, on the basis of standard guidelines, physician recommendations, and patient preferences. The study protocol and informed consent were in compliance with Declaration of Helsinki Principles.

      Polymorphism selection and genotyping

      Genomic DNA was extracted from the buffy coat fraction of each blood sample by using a Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 and 280nm by a UV spectrophotometer (Nano Drop Technologies, Wilmington, DE). The SNPs were genotyped using the Illumina HumanOmni1-Quad_v1-0_B array and were called using the BeadStudio algorithm, at the John Hopkins University Center for Inherited Disease Research. In this analysis, we selected the available 74 tagging SNPs in eight core NER genes, including XPA (rs1800975 and rs2808667), XPC (rs1350344, rs2227999, rs2228000, rs2228001, rs2470458, rs2607772, rs2733533, rs2733537, rs3731062, rs3731125, rs3731127, rs3731146, rs3731149, and rs3731151), XPE (rs2230356, rs4939513, and rs28720291), ERCC1 (rs11615, rs1007616, rs2298881, and rs3212955), ERCC2 (rs13181, rs50871, rs171140, rs238406, rs238416, rs1052555, rs1618536, rs1799786, rs1799787, and rs1799793), ERCC3 (rs1566823, rs1803541, rs4150403, rs4150436, rs4150496, rs4150523, rs4662718, and rs9282675), ERCC4 (rs254942, rs1799801, rs1800067, rs1800124, rs2276464, rs2276465, rs2276466, rs3136146, rs3136166, rs3136187, rs3136189, rs3136195, rs3743538, and rs16963255), and ERCC5 (rs17655, rs751402, rs873601, rs1047768, rs1047769, rs2227869, rs2296147, rs2296148, rs4150260, rs4150275, rs4150314, rs4150330, rs4150339, rs4150342, rs4150355, rs4150383, rs4771436, rs8002276, and rs11069498). Any SNP with a call rate lower than 95% was excluded from further analysis.

      Statistical analysis

      We used the Cox proportional hazards regression model to evaluate the effect of genotypes and clinicopathological variables on OS, calculated as hazard ratios (HRs) with their corresponding 95% CIs. We performed a stepwise conditional logistic regression analysis to explore the best model to predict the survival outcome. The survival time was calculated from the first day of diagnosis until the date of event or the last-known follow-up. All HRs were adjusted for age, sex, tumor stage, tumor Breslow thickness, ulceration of tumor, tumor cell mitotic rate, involvement of lymph node, and primary tumor anatomic site. Kaplan–Meier analysis was used to evaluate the effects of clinicopathological and genetic variables on the cumulative probability of OS. All reported P-values were two-sided, and P<0.05 was considered to indicate statistical significance. All analyses were performed using SAS software (version 9.1; SAS Institute, Cary, NC).

      ACKNOWLEDGMENTS

      We thank Margaret Lung and Jessica Fiske for assistance in recruiting the subjects, and Yawei Qiao, Jianzhong He, and Kejing Xu for laboratory assistance. This study was supported by the National Institutes of Health, National Cancer Institute grants R01 CA 100264 and CA 131274 (QW) and P50 CA 093459 (EAG), and in part by the National Institute of Environmental Health Sciences grants R01 ES11740 (QW) and P30 CA16672 (MD Anderson Cancer Center).
      Author Contributions
      Conception and design: CL, MY, L-EW, CIA, JEL, JEG, EAG, and QW. Financial support: CIA, JEL, EAG, and QW. Administrative support: CIA, JEL, EAG, and QW. Provision of study materials or patients: CL, MY, L-EW, CIA, JEL, JEG, EAG, and QW. Collection and assembly of data: CL, MY, L-EW, CIA, DZ, JEL, JEG, EAG, and QW. Data analysis and interpretation: CL, MY, L-EW, CIA, DZ, JEL, JEG, EAG, and QW. Manuscript writing: CL, MY, L-EW, CIA, DZ, JEL, JEG, EAG, and QW. Final approval of manuscript: CL, MY, L-EW, CIA, DZ, JEL, JEG, EAG, and QW.

      REFERENCES

        • Amos C.I.
        • Wang L.E.
        • Lee J.E.
        • et al.
        Genome-wide association study identifies novel loci predisposing to cutaneous melanoma.
        Hum Mol Genet. 2011; 20: 5012-5023
        • Balch C.M.
        • Gershenwald J.E.
        • Soong S.J.
        • et al.
        Final version of 2009 AJCC melanoma staging and classification.
        J Clin Oncol. 2009; 27: 6199-6206
        • Buettner P.G.
        • Leiter U.
        • Eigentler T.K.
        • et al.
        Development of prognostic factors and survival in cutaneous melanoma over 25 years: an analysis of the Central Malignant Melanoma Registry of the German Dermatological Society.
        Cancer. 2005; 103: 616-624
        • Chin L.
        • Garraway L.A.
        • Fisher D.E.
        • et al.
        Malignant melanoma: genetics and therapeutics in the genomic era.
        Genes Dev. 2006; 20: 2149-2182
        • de Boer J.
        • Hoeijmakers J.H.
        Cancer from the outside, aging from the inside: mouse models to study the consequences of defective nucleotide excision repair.
        Biochimie. 1999; 81: 127-137
        • DiFronzo L.A.
        • Gupta R.K.
        • Essner R.
        • et al.
        Enhanced humoral immune response correlates with improved disease-free and overall survival in American Joint Committee on Cancer stage II melanoma patients receiving adjuvant polyvalent vaccine.
        J Clin Oncol. 2002; 20: 3242-3248
        • Gershenwald J.E.
        • Ross M.I.
        Sentinel-lymph-node biopsy for cutaneous melanoma.
        N Engl J Med. 2011; 364: 1738-1745
        • Houtsmuller A.B.
        • Rademakers S.
        • Nigg A.L.
        • et al.
        Action of DNA repair endonuclease ERCC1/XPF in living cells.
        Science. 1999; 284: 958-961
        • Siegel R.
        • Naishadham D.
        • Jemal A.
        Cancer statistics, 2012.
        CA Cancer J Clin. 2012; 62: 10-29
        • Kauffmann A.
        • Rosselli F.
        • Lazar V.
        • et al.
        High expression of DNA repair pathways is associated with metastasis in melanoma patients.
        Oncogene. 2008; 27: 565-573
        • Kertat K.
        • Rosdahl I.
        • Sun X.F.
        • et al.
        The Gln/Gln genotype of XPD codon 751 as a genetic marker for melanoma risk and Lys/Gln as an important predictor for melanoma progression: a case control study in the Swedish population.
        Oncol Rep. 2008; 20: 179-183
        • Li C.
        • Hu Z.
        • Liu Z.
        • et al.
        Polymorphisms in the DNA repair genes XPC, XPD, and XPG and risk of cutaneous melanoma: a case-control analysis.
        Cancer Epidemiol Biomarkers Prev. 2006; 15: 2526-2532
        • Liu D.
        • O'Day S.J.
        • Yang D.
        • et al.
        Impact of gene polymorphisms on clinical outcome for stage IV melanoma patients treated with biochemotherapy: an exploratory study.
        Clin Cancer Res. 2005; 11: 1237-1246
        • Missura M.
        • Buterin T.
        • Hindges R.
        • et al.
        Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair.
        EMBO J. 2001; 20: 3554-3564
        • Sancar A.
        DNA repair in humans.
        Annu Rev Genet. 1995; 29: 69-105
        • Schrama D.
        • Scherer D.
        • Schneider M.
        • et al.
        ERCC5 p.Asp1104His and ERCC2 p.Lys751Gln polymorphisms are independent prognostic factors for the clinical course of melanoma.
        J Invest Dermatol. 2011; 131: 1280-1290
        • Shen M.
        • Berndt S.I.
        • Rothman N.
        • et al.
        Polymorphisms in the DNA nucleotide excision repair genes and lung cancer risk in Xuan Wei, China.
        Int J Cancer. 2005; 116: 768-773
        • Sijbers A.M.
        • de Laat W.L.
        • Ariza R.R.
        • et al.
        Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.
        Cell. 1996; 86: 811-822
        • Spatz A.
        • Batist G.
        • Eggermont A.M.
        The biology behind prognostic factors of cutaneous melanoma.
        Curr Opin Oncol. 2010; 22: 163-168
        • Stern M.C.
        • Lin J.
        • Figueroa J.D.
        • et al.
        Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the international consortium of bladder cancer.
        Cancer Res. 2009; 69: 6857-6864
        • von Thaler A.K.
        • Kamenisch Y.
        • Berneburg M.
        The role of ultraviolet radiation in melanomagenesis.
        Exp Dermatol. 2010; 19: 81-88
        • Woods J.E.
        • Soule E.H.
        • Creagan E.T.
        Metastasis and death in patients with thin melanomas (less than 0.76 mm).
        Ann Surg. 1983; 198: 63-64
        • Yokoi M.
        • Masutani C.
        • Maekawa T.
        • et al.
        The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.
        J Biol Chem. 2000; 275: 9870-9875