ERCC5 p.asp1104his and ERCC2 p.lys751gln Polymorphisms Are Independent Prognostic Factors for the Clinical Course of Melanoma

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1 ORIGINAL ARTICLE ER5 p.asp1104his ad ER2 p.lys751gl Polymorphisms Are Idepedet Progostic Factors for the Cliical Course of Melaoma David Schrama 1,2, Domiique Scherer 3, Michael Scheider 1, Marc Zapatka 4, Eva-Bettia Bröcker 1, Dirk Schadedorf 5, Selma Ugurel 1,2, Rajiv Kumar 3 ad Jürge C. Becker 2 Geetic variats i DNA repair ezymes cotribute to the susceptibility to cutaeous melaoma; cosequetly, we aalyzed whether commo osyoymous sigle-ucleotide polymorphisms i DNA repair ezyme gees might also ifluece the course of disease. To this ed, we determied eight polymorphisms of seve differet DNA repair ezymes i 742 patiets with cutaeous melaoma, ad correlated these with overall survival. Uivariate Cox proportioal hazards model aalyses revealed that ER5 (XPG) 1104 His/His was sigificatly associated with impaired survival. Ideed, the uivariate hazard ratio (HR) was 2.8 times higher for patiets with ER His/His (Po0.001) compared with ER Asp/Asp. Accordigly, the rate was 55% (95% cofidece iterval 43 71) for patiets with ER His/His, whereas 82% (95% cofidece iterval 78 86) of patiets with ER Asp/Asp were still alive at this time. Importatly, adjusted Cox regressio aalysis ot oly cofirmed ER His/His as a idepedet progostic factor (multivariate HR ¼ 4.5; Po0.001), but also revealed the sigificat impact of ER2 (XPD) 751 Gl/Gl o progosis, with a 2.2-fold icreased HR compared with ER2 751 Lys/Lys (P ¼ 0.009). Thus, ER5 codo 1104 ad ER2 codo 751 polymorphisms are idepedet progostic factors i patiets with cutaeous melaoma. Joural of Ivestigative Dermatology (2011) 131, ; doi: /jid ; published olie 10 March 2011 INTRODUCTION Maligat melaoma is a aggressive cacer with a high mortality rate oce metastasized. Oe of the major risk factors for melaoma is su exposure (Halper ad Altma, 1999). Notably, UV radiatio causes various kids of DNA damage. UVB, i.e., m, provokes DNA damage by cyclobutae pyrimidie dimers ad pyrimidie photoproducts, whereas wavelegths of m, i.e., UVA, cause sigle-strad breaks, DNA protei crosslikig, ad geeratio of free oxidative radicals (Pfeifer et al., 2005). As urepaired DNA damage ca either result i apoptosis or 1 Departmet of Dermatology, Uiversity Hospital Würzburg, Würzburg, Germay; 2 Divisio of Geeral Dermatology, Medical Uiversity Graz, Graz, Austria; 3 Departmet of Molecular Geetic Epidemiology, Germa Cacer Research Ceter, Heidelberg, Germay; 4 Divisio of Molecular Geetics, Germa Cacer Research Ceter, Heidelberg, Germay ad 5 Departmet of Dermatology, Uiversity Hospital Esse, Esse, Germay Correspodece: David Schrama, Departmet of Dermatology, Medical Uiversity of Graz, Auebruggerplatz 8, 8010 Graz, Austria. david.schrama@meduigraz.at Abbreviatios: BER, base excisio repair; ER5, excisio repair crosscomplemetig rodet repair deficiecy, complemetatio group 5; HR, hazard ratio; NER, ucleotide excisio repair; NBN, Nijmege break sydrome mutated gee; SNP, sigle-ucleotide polymorphism; XP, xeroderma pigmetosum complemetatio group; XR, X-ray repair complemetig defective repair i Chiese hamster cells Received 21 July 2010; revised 17 December 2010; accepted 19 Jauary 2011; published olie 10 March 2011 DNA aberratios leadig to uregulated cell growth ad cacer, cells are edued with various DNA repair pathways that are activated upo DNA damage. I order to maitai the itegrity of the geome, at least four differet pathways of DNA repair operate o specific types of damaged DNA. For example, base excisio repair (BER) corrects small DNA lesios such as oxidized or reduced bases, as well as fragmeted or obulky adducts. The ucleotide excisio repair (NER) pathway repairs bulky lesios such as pyrimidie dimers, larger chemical adducts, or DNA crossliks. I additio, at least two pathways to repair double-strad breaks exist, i.e., the homologous recombiatio pathway ad the ohomologous ed-joiig repair pathway (reviewed i Goode et al., 2002). As maiteace of DNA itegrity is importat to prevet carciogeesis, gees ecodig DNA repair molecules are prime cadidates for cacer-susceptibility gees (Bartsch et al., 2007). Ideed, several sigle-ucleotide polymorphisms (SNPs) i these gees have bee implicated i icreased cacer susceptibility (Kiyohara ad Yoshimasu, 2007; Naccarati et al., 2007): e.g., ER2 (XPD) 751 Gl ad XR3 241 Met are both associated with a icreased risk to develop cutaeous melaoma (Wisey et al., 2000; Li et al., 2006a). However, geetic variatio i DNA repair ezymes might also affect the cliical course of cacer by, e.g., affectig geetic stability (Umar ad Kukel, 1996; Kloor et al., 2010). Ideed, it has bee recetly reported that 1280 Joural of Ivestigative Dermatology (2011), Volume 131 & 2011 The Society for Ivestigative Dermatology

2 primary melaomas overexpressig DNA repair gees are characterized by a impaired progosis (Kauffma et al., 2008). The authors hypothesized that tumor cells i the process of metastasis try to replicate i a fast ad error-free mode to maitai those geetic aberratios associated with a growth advatage. To further extet this otio, we scrutiized the progostic impact of polymorphic variatio i DNA repair gees o the course of melaoma by aalyzig eight SNPs i a cohort of 742 melaoma patiets. All of the addressed SNPs are osyoymous. The icluded SNPs were xeroderma pigmetosum complemetatio group C (XPC) p.ala499val ad XPC p.lys939gl, excisio repair cross-complemetig rodet repair deficiecy, complemetatio group 2 (ER2; alias XPD) p.lys751gl ad ER5 (XPG) p.asp1104his all ivolved i NER, APEX1 (APEX uclease (multifuctioal DNA repair ezyme) 1) p.asp148glu ad X-ray repair complemetig defective repair i Chiese hamster cells 1 (XR1) p.arg399gl both participatig i BER ad XR3 p.thr241met ad Nijmege break sydrome mutated gee (NBN; NBS1) p.glu185gl, i.e., molecules of the homologous recombiatio repair pathway. This aalysis revealed that ER5 p.asp1104his ad ER2 p.lys751gl have a sigificat ad idepedet impact o the cliical course of melaoma. RESULTS Patiet characteristics Of the 742 patiets, 328 were female (44. 2%) ad 414 male (55.8%). Superficial spreadig melaoma (346 patiets) ad odular melaoma (182 patiets) were the most commo histological diagoses. The media age at diagosis of the patiet cohort was 54.8 years ad the media follow-up time was 74.2 moths. A total of 226 patiets died durig the follow-up period. The detailed patiet ad tumor characteristics are preseted i Table 1. Geotypig frequecies of polymorphisms ad survival The allelic ad geotypic frequecies of SNPs are give i Table 2. Notably, from the majority of samples the geotype could be determied; oly 4 to 12.9%, with a average of B7.6%, of samples did ot give a distict result. The observed geotype frequecies were i the rage of the frequecies reported o the respective SNP database websites of the Natioal Ceter for Biotechology Iformatio. From the aalyzed SNPs oly XPC p.lys939gl ad XPC p.ala499val were statistically sigificat i likage disequilibrium after Boferroi Holmes adjustmet (Po0.001; D ¼ 0.906; r 2 ¼ 0.19). This likage disequilibrium for the two XPC SNPs has bee published previously (Huag et al., 2006). The effect of each polymorphism o overall survival was estimated by the Kapla Meier method, revealig that ER5 p.asp1104his has a sigificat impact o overall survival (Po0.001; log-rak test): ER His/His was associated with a highly impaired progosis compared with ER His/Asp or Asp/Asp (Figure 1). This observatio was cofirmed by the rate (Table 3): 153 patiets died durig the first 5 years after diagosis, ad 469 patiets had a follow-up time of 460 moths. Validated progostic factors such as age, geder, tumor classificatio, ad cliical stage at diagosis iflueced the sigificatly, thereby demostratig that our cohort is represetative for the geeral melaoma populatio (Balch et al., 2001b). Notably, from the aalyzed DNA repair gee SNPs, oly ER polymorphisms sigificatly iflueced the rate. Ideed, the survival impact was obvious i both the whole patiet populatio ad the geder subgroups. I this regard, the rate was 82% (95% cofidece iterval 78 86) for ER Asp/Asp versus 56% (95% cofidece iterval 43 71) for His/ His (Po0.001; log-rak test). Hazard ratio (HR) for DNA repair polymorphisms Next, we calculated the HRs for DNA repair SNPs by uivariate ad multivariate Cox regressio aalyses. The uivariate aalyses cofirmed that geder, age, tumor classificatio, ad stage at diagosis as well as ER5 p.asp1104his polymorphisms iflueced the risk to die from melaoma (Figure 2). The HR for ER His/His was 2.8 times higher tha for ER Asp/Asp (Po0.001). Additioally, the ER2 p.lys751gl polymorphism, for which we observed a tred i the aalysis, sigificatly iflueced progosis: patiets with ER2 751 Gl/Gl had a 1.7 times icreased risk to die compared with ER2 751 Lys/Lys patiets (P ¼ 0.035). To test whether ER5 p.asp1104his ad ER2 p.lys751gl are idepedet progostic factors, we performed multivariate Cox regressio aalysis. For this aalysis, we adjusted each SNP to each other as well as to the kow progostic parameters geder, age, ad stage at diagosis. I order to decrease the umber of patiet subgroups, stage IIB ad IIC as well as stage IIIB ad IIIC patiets were grouped. These aalyses cofirmed ER His/His (HR ¼ 4.5; Po0.001) ad ER2 751 Lys/Lys (HR ¼ 2.2; P ¼ 0.009) as idepedet progostic factors for overall survival of cutaeous melaoma patiets (Figure 3). All results of the multivariate aalyses were cofirmed by bootstrappig, i.e., aalysis of radomly selected patiet cohorts. DISCUSSION DNA repair is essetial to esure itegrity ad stability of the geome. Geetic istability is resposible for the cellular chages that cofer progressive trasformatio o cacerous cells; thus, defects i DNA repair ca be expected to promote cacer. Ideed, patiets with XP, a autosomal recessive disease associated with defective NER, have a 1,000-fold higher susceptibility for ski cacer (Kraemer et al., 2007). The ability to repair DNA damages, however, also varies to a cosiderable extet withi the ormal huma populatio. These variatios could be a cosequece of commo polymorphic amio-acid substitutios i DNA repair gees (Mohreweiser et al., 2003). Cosequetly, may studies have bee performed to reveal possible associatios betwee SNPs i DNA repair gees ad susceptibility to cacer (Berwick ad Vieis, 2000; Goode et al., 2002). Most studies, however, do ot address whether such variatios i DNA repair impact o the cliical course of cacer. Several lies of

3 Table 1. Patiet characteristics Variables Total Wome Me Media (IQR) age at diagosis 54.8 ( ) (=742) 53.7 ( ) (=328) 55.8 ( ) (=414) Media (IQR) age at death 61.0 ( ) (=226) 58.4 ( ) (=94) 61.5 ( ) (=132) Media (IQR) follow-up time (moths) 74.2 ( ) (=742) 80.8 ( ) (=328) 70.7 ( ) (=414) Death 226 (30.1%) 94 (28.7%) 132 (31.9%) Therapy 1 Adjuvat IFN 154 (20.8%) 73 (22.3%) 81 (19.6%) Chemotherapy 213 (28.7%) 89 (27.1%) 124 (30%) Radiatio 84 (11.3%) 37 (11.3) 47 (11.4%) Immuotherapy 160 (21.6%) 67 (20.4%) 93 (22.5%) T-classificatio 2 T1a 202 (28.7%) 90 (28.7%) 112 (28.7%) T1b 13 (1.8%) 4 (1.3%) 9 (2.3%) T2a 187 (26.6%) 84 (26.8%) 103 (26.4%) T2b 27 (3.8%) 9 (2.9%) 18 (4.6%) T3a 126 (17.9%) 66 (21.0%) 60 (15.4%) T3b 51 (7.2%) 22 (7.0%) 29 (7.4%) T4a 71 (10.1%) 30 (9.6%) 41 (10.5%) T4b 27 (3.8%) 9 (2.9%) 18 (4.6%) Stage at diagosis 2 IA 198 (27.7%) 87 (27.6%) 111 (27.8%) IB 183 (25.6%) 77 (24.4%) 106 (26.6%) IIA 134 (18.8%) 65 (20.6%) 69 (17.3%) IIB 96 (13.4%) 45 (14.3%) 51 (12.8%) IIC 13 (1.8%) 2 (0.6%) 11 (2.8%) IIIA 32 (4.5%) 15 (4.8%) 17 (4.3%) IIIB 44 (6.2%) 18 (5.7%) 26 (6.5%) IIIC 5 (0.7%) 3 (1.0%) 2 (0.5%) IV 9 (1.3%) 3 (1.0%) 6 (1.5%) Histological type ALM 44 (6.8%) 19 (6.8%) 25 (6.8%) LMM 37 (5.7%) 15 (5.3%) 22 (6.0%) NM 182 (28.2%) 77 (27.4%) 105 (28.8%) SSM 346 (53.6%) 154 (54.8%) 192 (52.6%) Other 12 (1.9%) 6 (2.1%) 6 (1.6%) Noclassifiable 25 (3.9%) 10 (3.6%) 15 (4.1%) Abbreviatios: ALM, acral letigious melaoma; IFN, iterfero; IQR, iterquartile rage; LMM, letigo maliga melaoma; NM, odular melaoma; SSM, superficial spreadig melaoma. 1 Number of patiets receivig the respective therapies. 2 T-classificatio of primary tumor as well as pathological stagig at diagosis was performed accordig to the America Joit Committee o Cacer (AJ) Classificatio from 2002 (Balch et al., 2001a) Joural of Ivestigative Dermatology (2011), Volume 131

4 Table 2. Results of SNP determiatio SNP Allele (AA) Allele frequecy Geotypes Patiets (frequecy) Wome (frequecy) Me (frequecy) APEX1 p.asp148glu T (Asp) 0.52 TT 189 (28.0) 70 (23.4) 119 (31.5) (rs ) G (Glu) 0.48 TG 324 (47.9) 155 (51.8) 169 (44.7) GG 164 (24.2) 74 (24.7) 90 (23.8) ER2 p.lys751gl A (Lys) 0.63 AA 285 (40.2) 122 (38.4) 163 (41.4) (rs13181) C (Gl) 0.37 AC 329 (46.4) 148 (46.5) 181 (45.9) 98 (13.8) 48 (15.1) 50 (12.7) ER5 p.asp1104his G (Asp) 0.78 GG 449 (63.1) 204 (63.9) 245 (62.3) (rs17655) C (His) 0.22 GC 206 (28.9) 91 (28.5) 115 (29.3) 57 (8.0) 24 (7.5) 33 (8.4) NBN p.glu185gl G (Glu) 0.69 GG 329 (48.5) 146 (48.0) 183 (48.8) (rs ) C (Gl) 0.31 GC 282 (41.5) 120 (39.5) 162 (43.2) 68 (10.0) 38 (12.5) 30 (8.0) XPC p.ala499val C (Ala) (57.1) 168 (55.3) 221 (58.6) (rs ) T (Val) 0.26 CT 236 (34.7) 104 (34.2) 132 (35.0) TT 56 (8.2) 32 (10.5) 24 (6.4) XPC p.lys939gl A (Lys) 0.6 AA 238 (36.8) 108 (37.4) 130 (36.4) (rs ) C (Gl) 0.4 AC 295 (45.7) 129 (44.6) 166 (46.5) 113 (17.5) 52 (18.0) 61 (17.1) XR1 p.arg399gl G (Arg) 0.66 GG 303 (44.1) 139 (44.8) 164 (43.5) (rs25487) A (Gl) 0.34 GA 302 (44.0) 133 (42.9) 169 (44.8) AA 82 (11.9) 38 (12.3) 44 (11.7) XR3 p.thr241met C (Thr) (40.7) 118 (37.6) 165 (43.2) (rs861539) T (Met) 0.38 CT 300 (43.1) 142 (45.2) 158 (41.4) TT 113 (16.2) 54 (17.2) 59 (15.4) Abbreviatios: AA, amio acid; APEX1, APEX uclease (multifuctioal DNA repair ezyme) 1; ER5, excisio repair cross-complemetig rodet repair deficiecy, complemetatio group 5; NBN, Nijmege break sydrome mutated gee; SNP, sigle-ucleotide polymorphism; XPC, xeroderma pigmetosum complemetatio group C; XR, X-ray repair complemetig defective repair i Chiese hamster cells. evidece suggest that this may be the case: for melaoma, geomic istability is ot oly icreased i primary tumors whe compared with evi, but also i metastases whe compared with primary tumors (Chi et al., 2006); similarly, hypoxia, which is associated with a aggressive pheotype of cacer, mediates amog other chages geetic istability (Bristow ad Hill, 2008). Thus, we scrutiized whether commo osyoomous SNPs i DNA repair gees are related to the progressio of melaoma i a cohort of 4700 melaoma patiets for whom detailed iformatio ad a close follow-up was available. The majority of the aalyzed SNPs were selected as they are amog the most studied DNA repair gee SNPs for melaoma susceptibility (Li et al., 2006a, b; Mocelli et al., 2009). I additio, the NBN SNP was icluded as germlie mutatios have bee described for melaoma (Steffe et al., 2004). Our study revealed that ER5 p.asp1104his ad ER2 p.lys751gl are idepedet progostic factors for the cliical course of melaoma. Iterestigly, although ER2 SNP has bee described as melaoma susceptibility SNP i oe study (Li et al., 2006a), a large study of almost 1,200 melaoma patiets as well as a review of the literature could ot reveal a impact of these two SNPs o the susceptibility for melaoma (Figl et al., 2010). The umber of studies addressig the associatio betwee SNPs i DNA repair gees ad the course of eoplastic diseases is surprisigly low, particularly if compared with those addressig cacer susceptibility. This is probably because of the fact that the diagosis of cacer itself is more widely available tha the detailed medical history. Cosequetly, studies addressig SNPs as progostic factors are largely characterized by a low patiet umber. For example, i a recet study scrutiizig three DNA repair SNPs, i.e., XR1 p.arg399gl, ER2 p.lys751gl, ad ER1 p.thr118cys, ad their associatio with survival i stage IV melaoma patiets receivig biochemotherapy, oly 90 patiets were icluded (Liu et al., 2005). This aalysis, however, revealed that the ER1 polymorphism was weakly associated with overall survival, ad the Gl allele of the ER2 polymorphism was ufavorable without reachig sigificace. I cotrast to our study, Liu et al. (2005) searched for a predictive marker, whereas we addressed

5 Pts. at risk: Probability of survival (%) Asp/Asp Asp/His His/His 0 Asp/Asp Asp/His His/His Moths Figure 1. ER5 (excisio repair cross-complemetig rodet repair deficiecy, complemetatio group 5) p.asp1104his polymorphism iflueces progosis. Kapla Meier survival estimatio for 712 melaoma patiets stratified to ER5 p.asp1104his polymorphism (Po0.0001; lograk test). The patiets (Pts.) at risk are depicted i 20-moth itervals. progostic markers for melaoma. Moreover, the study of Liu et al. (2005) is likely to be uderpowered to reach a sigificat result for ER2. Iterestigly, ER2 p.lys751gl was suggested to be a progostic factor for melaoma progressio, as i 244 Swedish patiets the Lys/ Gl pheotype was more frequet i patiets with advaced melaoma (Kertat et al., 2008). I a previous study of 400 melaoma patiets, Figl et al. (2009) reported XR1 p.arg399gl to be associated with progosis: patiets with XR1 Gl/Gl demostrated a media overall survival of 24.4 years compared with 11.5 years for the two other geotypes. I the same study, however, this associatio was ot detectable i a idepedet Spaish patiet cohort selected by the same criteria, i.e., oly patiets first diagosed at stage I or II. The authors cocluded that this may have bee because of the fact that oly a few deaths were recorded for this cohort. I our study, although the idepedet patiet cohort reflects the whole variety of tumor stages at diagosis traslatig ito a worse survival rate, we were ot able to cofirm the previous results. This observatio demostrates the importace of coductig multiple studies o potetial biomarkers. Alteratively, supportive evidece might come from more frequet cacer etities aalyzig the impact of variatios i DNA repair o the course of disease. For example, i a large study of lug cacer patiets, the authors detected 15 osyoomous SNPs associated with progosis (Matakidou et al., 2007); these primarily mapped to the NER ad BER repair pathways, ad ER5 p.asp1104his was amog the most sigificat oes. Thus, ER5 p.asp1104his seems to be associated with progosis i differet cacer etities. Notably, i cotrast to a previous report by Di Lucca et al. (2010), we did ot fid a associatio betwee ER5 p.asp1104his or ay other of our aalyzed geotypes ad Breslow thickess. Ideed, i multivariate aalysis with tumor class istead of tumor stage, we obtaied the same results (data ot show). To date, all SNPs associated with progosis belog to the NER ad BER pathways. This otio is further corroborated by the observatio that i early small cell lug cacer patiets a higher expressio of DNA sythesis ad repair ezymes RRM1 ad ER1 i the tumors was associated with a better overall survival (Zheg et al., 2007). However, the picture is gettig more complex as it has recetly bee demostrated that primary melaomas with poor progosis overexpress DNA repair gees (Kauffma et al., 2008). Ideed, the majority of these overexpressed gees code for proteis ivolved i rescuig stalled DNA replicatio forks, DNA double-strad break/iterstrad crosslik repair, ad telomere maiteace. Similar results were obtaied i a larger study, i which overexpressio of DNA repair gees ad gees ivolved i cell cycle progressio was observed i samples of relapsig compared with orelapsig patiets (Jewell et al., 2010). Thus, it seems that a defective or impaired DNA repair system ca cotribute to the iitiatio of tumors, whereas i the tumor stage patiets whose tumors express larger amouts of DNA repair ezymes preset with a faster relapse. This overexpressio of DNA repair gees might allow the fast-growig cacer cells to replicate more correctly ad thereby to prevet severe, i.e., deleterious, DNA damage. Somehow surprisigly, however, metastatic sites ofte demostrate higher levels of geetic istability (Balazs et al., 2001), ad i mice the metastatic potetial of tumor cells is associated with icreasig geetic istability (Cifoe ad Fidler, 1981). As it is kow that E2F itegrates cell cycle progressio with DNA repair (Re et al., 2002), overexpressio of DNA repair gees ca at least i part be ascribed to icreased proliferatio. The precise mechaism of how the ER5 ad ER2 SNPs ifluece overall survival i cutaeous melaoma patiets remais elusive; as always for disease-associated SNPs, the cacer pheotype could be altered i two ways: either directly by alterig the DNA repair capacity of the tumor cell or idirectly by beig i likage disequilibrium with other disease-modulatig alleles. ER5 ad ER2 are core proteis of the NER pathway ad therefore play crucial roles i the correctio of cyclobutae pyrimidie dimers, 6 4 photoproducts, ad bulky adducts iduced by chemical agets (Costa et al., 2003). The potetial of amio-acid substitutios ecoded by SNPs to impact protei structure ad activity ca be predicted by two differet algorithms: i.e., SIFT (Sortig Itolerat from Tolerat) ad PolyPhe (Polymorphism Pheotypig). These algorithms are based o sequece coservatio over evolutioary time, the physical ad chemical properties of the exchaged residues, ad/or protei structural domai iformatio. Several bechmarkig studies have demostrated that these algorithms predict protei fuctio very correctly (reviewed i Xi et al., 2004). Both the SIFT ad the PolyPhe algorithms estimate the His substitutio of Asp at codo 1104 of ER5 as itolerat or possible damagig, whereas the Lys substitutio by Gl at codo 751 of ER2 was predicted by both programs as beig. However, fuctioal aalysis of the respective 1284 Joural of Ivestigative Dermatology (2011), Volume 131

6 Table 3. The rates Variables Geder (at risk/evets) 1 Me 414 (246/98) 76 (72 80) Total Wome Me rate (95% CI) 2 P-value 3 (at risk/evets) 1 Wome 328 (223/55) 82 (78 87) rate (95% CI) 2 P-value 3 (at risk/evets) 1 rate (95% CI) 2 P-value 3 Age at oset 4 r (155/36) 84 (79 89) 113 (79/17) 85 (78 92) 106 (76/19) 83 (75 90) (158/44) 81 (76 86) 94 (69/14) 85 (78 93) 139 (89/30) 78 (71 85) (156/73) 73 (68 78) (75/24) 78 (70 86) (81/49) 70 (62 77) Tumor classificatio T1a 202 (147/14) 92 (89 96) 90 (65/4) 95 (90 100) 112 (82/10) 90 (85 96) T1b 13 (9/1) 92 (78 100) 4 (4/0) 100 (NA) 9 (5/1) 89 (68 100) T2a 187 (134/18) 90 (86 95) 84 (64/8) 90 (83 96) 103 (70/10) 91 (85 96) T2b 27 (13/12) 56 (37 76) 9 (5/2) 71 (38 100) 18 (8/10) 50 (27 73) T3a 126 (84/23) 81 (74 88) 66 (46/12) 82 (72 92) 60 (38/11) 80 (70 91) T3b 51 (26/13) 72 (60 85) 22 (15/4) 80 (62 98) 29 (11/9) 67 (49 85) T4a 71 (29/37) 50 (38 61) 30 (12/16) 49 (31 67) 41 (17/21) 50 (34 66) T4b 27 (8/16) 36 (17 55) o (3/4) 47 (10 83) o (5/12) 32 (10 54) o Pathological stagig IA 198 (146/11) 94 (90 97) 87 (64/2) 97 (94 100) 111 (82/9) 91 (86 97) IB 183 (132/16) 91 (87 95) 77 (61/6) 91 (85 98) 106 (71/10) 91 (85 97) IIA 134 (87/26) 79 (72 86) 65 (46/9) 85 (75 94) 69 (41/17) 74 (64 85) IIB 96 (51/30) 69 (60 79) 45 (26/14) 70 (56 84) 51 (25/16) 69 (56 82) IIC 13 (6/5) 57 (28 86) 2 (1/0) (5/5) 52 (21 83) IIIA 32 (16/16) 55 (38 73) 15 (9/6) 67 (43 91) 17 (7/10) 44 (20 68) IIIB 44 (11/28) 33 (19 48) 18 (6/10) 40 (16 64) 26 (5/18) 28 (9 44) IIIC 5 (0/4) 0 (NA) 3 (0/2) 0 (NA) 2 (0/2) 0 (NA) IV 9 (1/8) 11 (0 32) o (1/2) 33 (0 87) o (0/6) 0 (NA) o ER5 p.asp1104his GG 449 (296/77) 82 (79 86) 204 (146/27) 86 (81 91) 244 (150/48) 79 (74 85) GC 206 (130/46) 77 (72 83) 91 (59/16) 81 (72 89) 115 (71/28) 75 (67 83) 57 (26/23) 56 (42 70) o (12/9) 57 (38 80) (14/14) 53 (35 72) ER2 p.lys751gl AA 285 (190/52) 82 (77 86) 122 (81/19) 84 (77 91) 163 (109/33) 80 (74 86) AC 329 (203/67) 79 (74 83) 148 (104/22) 85 (79 91) 181 (99/45) 74 (68 81) 98 (59/27) 72 (62 81) (32/11) 76 (64 89) (27/16) 67 (54 80) XPC p.lys939gl AA 238 (150/48) 79 (74 85) 108 (73/19) 82 (75 90) 130 (77/29) 77 (69 84) CA 295 (186/64) 78 (73 83) 129 (88/22) 82 (76 89) 166 (98/42) 74 (67 81) 113 (64/29) 73 (65 82) (30/11) 73 (61 87) (34/18) 71 (60 83) Table 3 cotiued o the followig page

7 Table 3. Cotiued Variables (at risk/evets) 1 Total Wome Me rate (95% CI) 2 P-value 3 (at risk/evets) 1 rate (95% CI) 2 P-value 3 (at risk/evets) 1 rate (95% CI) 2 P-value 3 XPC p.ala499val 389 (238/83) 78 (73 82) 168 (109/28) 82 (76 88) 221 (129/55) 75 (69 80) CT 236 (148/50) 78 (73 84) 104 (75/15) 85 (78 92) 132 (73/35) 73 (65 81) TT 56 (38/11) 81 (71 92) (22/7) 80 (66 94) (16/4) 83 (67 98) XR1 p.arg399gl GG 303 (188/59) 80 (75 84) 139 (87/25) 80 (73 87) 164 (101/34) 79 (73 85) GA 302 (188/67) 77 (73 82) 133 (98/21) 85 (79 91) 169 (90/46) 71 (64 78) AA 82 (56/13) 83 (74 91) (27/5) 86 (74 97) (29/8) 81 (69 93) XR3 p.thr241met 283 (178/64) 77 (72 82) 118 (79/24) 79 (71 87) 165 (99/40) 75 (69 82) CT 300 (191/61) 79 (74 83) 142 (96/24) 82 (76 89) 158 (95/37) 75 (68 82) TT 113 (71/21) 82 (74 89) (40/5) 90 (81 98) (31/16) 75 (63 86) APEX1 p.asp148glu TT 189 (117/35) 80 (74 86) 70 (43/10) 83 (73 93) 119 (74/25) 78 (70 86) TG 324 (209/75) 77 (73 82) 155 (111/29) 82 (75 88) 169 (98/46) 73 (67 80) GG 164 (100/31) 80 (74 86) (49/9) 87 (79 95) (51/22) 74 (65 83) NBN p.glu185gl GG 329 (204/70) 78 (73 83) 146 (96/27) 80 (74 87) 183 (108/43) 76 (70 82) CG 282 (178/57) 79 (74 84) 120 (80/19) 83 (76 90) 162 (98/38) 76 (69 83) 68 (44/16) 79 (69 89) (29/5) 89 (78 99) (15/11) 66 (49 83) Abbreviatios: AA, amio acid; APEX1, APEX uclease (multifuctioal DNA repair ezyme) 1; CI, cofidece iterval; ER5, excisio repair crosscomplemetig rodet repair deficiecy, complemetatio group 5; NBN, Nijmege break sydrome mutated gee; SNP, sigle-ucleotide polymorphism; XPC, xeroderma pigmetosum complemetatio group C; XR, X-ray repair complemetig defective repair i Chiese hamster cells. 1 Give is the total umber of patiets (N) as well as those who were observed for 45 years (at risk) ad those who died withi 5 years after diagosis (evets). 2 The rates were determied by Kapla Meier estimatios that comprise also those patiets cesored withi 5 years. 3 P-values were calculated by the log-rak test. 4 The patiet cohort was divided ito three groups accordig to age at oset i such a way that almost equipollet groups resulted. amio-acid substitutios is still the gold stadard. To this ed, we recetly demostrated for XPG Asp1104His that TT ¼ T dimer repair was osigificatly lower i heterozygotes compared with the homozygous wild type (Kumar et al., 2003); furthermore, we demostrated a associatio betwee this SNP ad the level of sigle-strad breaks (Vodicka et al., 2004). I cotrast, for ER2 751 variat proteis o differece i NER activity was detectable (Laie et al., 2007). This observatio, o oe had, cofirms the results of the predictio algorithms ad, o the other had, argues that there is o causal relatioship betwee ER2 p.lys751gl polymorphism ad reduced DNA repair (Clarkso ad Wood, 2005). However, Wolfe et al. (2007) recetly demostrated that the mior allele of ER2 751 is associated with decreased costitutive ER2 mrna levels caused by a altered mrna secodary structure. This reduced expressio of the mrna may explai the icreased umber of DNA adducts i idividuals carryig Gl alleles suggestig lower DNA repair capacity i these idividuals (reviewed i Behamou ad Sarasi, 2005). It should be further oted that besides their fuctio i NER, ER2 is ivolved i trascriptioal regulatio (Schaeffer et al., 1993), ad ER5 acts as a cofactor for a DNA glycosylase; a ezyme that removes oxidized pyrimidies from DNA. Moreover, ER5 has also bee ivolved i both trascriptio-coupled repair ad RNA trascriptio itself (Clarkso, 2003). The SNPs i these gees may therefore affect ot oly NER, but also ay of these fuctios Joural of Ivestigative Dermatology (2011), Volume 131

8 Geder Female Male Age to 60 > 60 Tumor classificatio T1a T1b T2a T2b T3a T3b T4a T4b Stage at diagosis IA IB IIA IIB IIC IIIA IIIB IIIC IV ER5 p.asp1104his GG GC ER2 p.lys751gl AA AC XR3 p.thr241met CT TT APEX p.asp148glu TT TG GG NBN p.glu185gl GG GC XPC p.ala499val CT TT XR1 p.arg399gl AA GA GG XPC p.lys939gl AA AC Uivariate HR ad 95% CI (7 28.6) 20.6 ( ) 76 ( ) 72 ( ) P-value Figure 2. Uivariate hazard ratios (HRs) for overall survival. All factors aalyzed are depicted. Dots represet the HRs, lies the 95% cofidece itervals (CIs). I cases where the HRs ad/or the CIs are beyod the depicted rage, the values are give. A polymorphism is relevat whe the respective CI does ot cross the value 1. 1 Patiet umbers for each aalysis are give i Table 1 ad Table 2, respectively. I coclusio, i this study we idetified ER5 p.asp1104his ad ER2 p.lys751gl as idepedet progostic factors for the cliical course of melaoma. Whether these SNPs impact the progosis of melaoma directly via alteratio of DNA repair efficacy or by other, possibly idirect, mechaisms has to be addressed by future studies. I ay case, idetificatio of progostic factors will help to idividualize ad optimize medical care of patiets; i.e., high-risk patiets should receive a closer follow-up ad should be cosidered for adjuvat therapy. PATIENTS AND METHODS Patiets ad sera Serum samples were selected from froze serum baks hosted by Ski Cacer Uit, Maheim ad the Departmet of Dermatology, Wuerzburg. Serum samples to be icluded i the aalysis were chose radomly but selected to meet the followig criteria: (1) histological cofirmatio of primary cutaeous melaoma, (2) patiet of Caucasia origi, ad (3) availability of exteded iformatio of the medical history, primary tumor characteristics, as well as patiet follow-up. The oly exclusio criteria were sera from patiets with secodary maligacies or i situ melaoma. The detailed patiet characteristics are give i Table 1. All serum samples were obtaied ad processed followig a stadardized protocol. Briefly, blood was draw ito gel-coated serum tubes (Sarstedt, Nuembrecht, Germay) ad allowed to clot at room temperature for miutes. After cetrifugatio, the serum phase was harvested ad subsequetly froze without ay additives at 20 1C. All measures were performed with the approval of the istitutioal review board after patiets iformed coset ad i adherece to the Declaratio of Helsiki Priciples. Geotypig DNA was extracted from 200 ml serum of each cryopreserved sample with the QIAamp DNA mii kit (Qiage, Hilde, Germay) as described by the maufacturer ad geotyped for eight differet

9 Multivariate HR ad 95% CI 1 P-value Geder Female Male Age to 60 >60 Stage at diagosis IA IB IIA IIB/C IIIA IIIB/C IV ER5 p. Asp1104His GG GC ER2 p.lys751gl TT TG GG XR3 p.thr241met CT TT APEX1 p.asp148glu TT TG GG NBN p.glu185gl GG GC XPC p.ala499val CT TT XR1 Arg399Gl AA GA GG XPC p.lys939gl AA AC ( ) ( ) < Figure 3. Multivariate hazard ratios (HRs) for overall survival. Besides the eight DNA repair sigle-ucleotide polymorphisms (SNPs), geder, categorized age, ad stage at diagosis were icluded. Dots represet the HRs, lies the 95% cofidece itervals (CIs). I cases where the HRs ad/or the CIs are beyod the depicted rage, the values are give. A polymorphism is relevat whe the respective CI does ot cross the value 1. 1 This aalysis is based o 498 patiets sice 244 patiets had to be omitted due to absece of iformatio o primary tumor or missig geotype of at least oe of the aalyzed SNPs. SNPs i DNA repair gees icludig the NER gees XPC (A4C; p.lys939gl; rs ), XPC (C4T; p.ala499val; rs ), ER2 (A4C; p.lys751gl; rs13181), ad ER5 (G4C; p.asp1104his; rs17655); the BER gees APEX1 (T4G; p.asp148glu; rs ) ad XR1 (G4A; p.arg399gl; rs25487); ad the double-strad break repair gees XR3 (C4T; p.thr241met; rs861539) ad NBN (G4C; p.glu185gl; rs ). All these polymorphisms icluded i the study were osyoymous ad have mior allele frequecies 40.2 (Table 2). Geotypig was performed by the 5 0 uclease allelic discrimiatio assay i TaqMa techology (Applied Biosystems, Foster City, CA). TaqMa primers ad probes were purchased from Applied Biosystems as assays o demad (C_ _10 for rs ad C_622564_10 for rs861539) or by ow desig for all others (Supplemetary Table S1 olie). PCR was performed i 5 ml volume reactio usig 5 g DNA as template, master mix (Applied Biosystems), ad 0.5 probe/ primer mix. The iitial temperature coditios for PCR were set at 50 1C for 2 miutes ad 95 1C for 10 miutes followed by cycles at 92 1C for 15 secods ad 60 1C for 1 miute. Geotypig o amplified PCR products was scored by differeces i VIC ad 5-carboxyfluorescei, succiimidyl ester (FAM) fluorescet level i plate read operatio o ABI PRISM 7900HT sequece detectio system (Applied Biosystems) usig SDS 1.2 software (Applied Biosystems). Geotypig results from allelic discrimiatio assays were radomly verified by DNA sequecig. The sequecig reactios were performed usig BigDyeR Termiator Cycle Sequecig Kit (Applied Biosystems) i a 10 ml volume cotaiig PCR product pre-treated with ExoSapIT (USB Corporatio, Clevelad, OH), 5 Sequecig buffer (Applied Biosystems), ad a sequecig primer. The temperature coditios set for sequecig reactios were 96 1C for 2 miutes followed by 27 cycles at 96 1C for 30 secods, 54 1C for 10 secods, ad 60 1C for 4 miutes. Sequecig reactio products were precipitated with 2-propaol, washed with 75% ethaol, resuspeded i 25 ml water, ad loaded oto ABI prism 3100 Geetic aalyzer (Applied Biosystems). Primary sequecig data were aalyzed usig the accompaied sequece aalysis program (Applied Biosystems). Statistical methods Data were aalyzed by the statistic software R (versio 2.8) available at usig the libraries survival ad geetics. Age was categorized ito three groups (age p45, 46 60, ad 460 years). Each geotype-variable was dichotomized ito three idividual variables. For uivariate aalyses, the Kapla Meier method was used to compare survival times betwee groups. Differeces i rates ad survival times were assessed by the log-rak test. I additio, for uivariate as well as 1288 Joural of Ivestigative Dermatology (2011), Volume 131

10 multivariate aalyses, Cox s proportioal hazards regressio model was applied with dichotomized variables. As the majority of the melaoma-associated evets happe withi the first 5 years after diagosis, close follow-up accordig to the Germa guidelies is oly recommeded for this time period (Garbe et al., 2007). Cosequetly, we cesored our patiet cohort after this period. To scrutiize ad validate relevat variables, multiple aalyses icludig backward ad forward stepwise Cox regressios were performed. Likage disequilibrium was calculated with the fuctio likage disequilibrium of the library geetics. All preseted multivariate regressio models were evaluated by bootstrap methods accordig to the recommedatios of Altma ad Aderse (1989). CONFLICT OF INTEREST The authors state o coflict of iterest. ACKNOWLEDGMENTS We thak Eva-Maria Sarosi, Michelle Holterhus, ad Atje Sucker for excellet techical assistace. This work was supported by the Wilhelm Sader Stiftug ( ). 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11 Schaeffer L, Roy R, Humbert S et al. (1993) DNA repair helicase: a compoet of BTF2 (TFIIH) basic trascriptio factor. Sciece 260: Steffe J, Varo R, Mosor M et al. (2004) Icreased cacer risk of heterozygotes with NBS1 germlie mutatios i Polad. It J Cacer 111:67 71 Umar A, Kukel TA (1996) DNA-replicatio fidelity, mismatch repair ad geome istability i cacer cells. Eur J Biochem 238: Vodicka P, Kumar R, Stetia R et al. (2004) Geetic polymorphisms i DNA repair gees ad possible liks with DNA repair rates, chromosomal aberratios ad sigle-strad breaks i DNA. Carciogeesis 25: Wisey SL, Haldar NA, Marsh HP et al. (2000) A variat withi the DNA repair gee XR3 is associated with the developmet of melaoma ski cacer. Cacer Res 60: Wolfe KJ, Wickliffe JK, Hill CE et al. (2007) Sigle ucleotide polymorphisms of the DNA repair gee XPD/ER2 alter mrna expressio. Pharmacogeet Geomics 17: Xi T, Joes IM, Mohreweiser HW (2004) May amio acid substitutio variats idetified i DNA repair gees durig huma populatio screeigs are predicted to impact protei fuctio. Geomics 83:970 9 Zheg Z, Che T, Li X et al. (2007) DNA sythesis ad repair gees RRM1 ad ER1 i lug cacer. N Egl J Med 356: Joural of Ivestigative Dermatology (2011), Volume 131