DNA Repair 31 (2015) 64–72

Contents lists available at ScienceDirect

DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

DNA repair genes XPC, XPD, XRCC1, and XRCC3 are associated with risk and survival of squamous cell carcinoma of the head and neck Lovisa Farnebo a,1 , Annika Stjernström b,1 , Mats Fredrikson c , Anna Ansell d , Stina Garvin e , Lena K. Thunell b,∗ a

Department of Otorhinolaryngology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden Division of Cell Biology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden c Division of Environmental and Occupational Medicine, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden d Division of Otorhinolaryngology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden e Department of Clinical Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden b

a r t i c l e

i n f o

Article history: Received 16 October 2014 Received in revised form 3 May 2015 Accepted 5 May 2015 Available online 8 May 2015 Keywords: Head and neck squamous cell carcinoma XPC XPD HPV p53 Overall survival

a b s t r a c t Head and neck squamous cell carcinomas (HNSCC) are a heterogenous group of tumors with a high rate of early recurrences, second primary tumors, and mortality. Despite advances in diagnosis and treatment over the past decades, the overall 5-year survival rate remains around 50%. Since the head-and neck-region is continuously exposed to potentially DNA-damaging exogenous and endogenous factors, it is reasonable to expect that the DNA repair genes play a part in the development, progression, and outcome of HNSCC. The aim of this study was to investigate the SNPs XPC A499V, XPD K751Q, XRCC1 R399Q, and XRCC3 T241M as potential risk factors and indicators of survival among Caucasian patients. One-hundred-sixty-nine patients as well as 344 healthy controls were included and genotyped with PCR–RFLP. We showed that XPC A499V was associated with increased risk of HNSCC, especially laryngeal carcinoma. Among women, XPD K751Q was associated with increased risk of oral SCC. Furthermore, XPD homozygous mutant individuals had the shortest survival time, a survival time that increased however after full dose radiotherapy. Wild-type individuals of XRCC3 T241M demonstrated an earlier age of onset. HPV-positive never smokers had lower frequencies of p53 mutation. Among HNSCC patients, HPV-positivity was significantly associated with XRCC1 R399Q homozygous mutant genotype. Moreover, combinations of putative risk alleles seemed to act synergistically, increasing the risk of HNSCC. In conclusion, our results suggest that SNPs of the DNA repair genes XPC, XPD, XRCC1, and XRCC3 may affect risk and survival of HNSCC. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Head and neck squamous cell carcinoma (HNSCC) constitutes the sixth most frequent cancer worldwide [1]. More men than

Abbreviations: HNSCC, head and neck squamous cell carcinoma; HPV, human Papilloma Virus; BER, base-excision repair; NER, nucleotide-excision repair; DSB, double-strand break repair; MMR, mismatch repair; ROS, reactive oxygen species; SNP, single-nucleotide polymorphisms; XPC, xeroderma pigmentosum complement group C; XPD, xeroderma pigmentosum complement group D; XRCC1, X-ray repair cross-complementing group 1; XRCC3, X-ray repair cross-complementing group 3; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; RR, relative risk; OR, odds ratio; CI, confidence intervals; Gy, gray. ∗ Corresponding author at: Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE−581 85 Linköping, Sweden. Tel.: +46 10 103 2679; fax: +46 10 103 4273. E-mail address: [email protected] (L.K. Thunell). 1 L. F and A. S contributed equally to this work. http://dx.doi.org/10.1016/j.dnarep.2015.05.003 1568-7864/© 2015 Elsevier B.V. All rights reserved.

women suffer from HNSCC, but recent findings show that the incidence is increasing in women and decreasing in men [2]. Despite advances in detection and diagnosis, the overall 5-year survival rate for patients with HNSCC is among the lowest of the major cancers [3]. It is known that exogenous mutagens like tobacco smoke and alcohol constitute major risk factors for HNSCC [4] along with infections with Human Papilloma Virus (HPV)[5]. The DNA repair system plays a pivotal role in the protection of cells against carcinogenesis due to environmental factors such as the above mentioned [6]. Besides protection against carcinogenesis, the DNA repair capacity may also affect therapy outcome as well as serve as a prognostic indicator of patient survival [7,8]. Hence, changes in DNA repair genes might not only influence an individual’s susceptibility to HNSCC but also affect response to therapy and prognosis. The four major DNA repair mechanisms operate on specific types of damaged DNA [6,9]. The nucleotide-excision repair (NER) pathway repairs bulky lesions often caused by exogenous sources

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

such as tobacco smoke and UVB irradiation while the base-excision repair (BER) pathway is the main protector against damage due to endogenous factors such as reactive oxygen species (ROS). Double-strand breaks (DSB), caused by ROS, ionising radiation, and replication, are repaired by DSB repair. Mismatch repair (MMR) is the main defender against replication errors caused by DNA polymerase. Variations such as single-nucleotide polymorphisms (SNPs) in several DNA repair genes have been found to be associated with many forms of cancer [9]. Xeroderma pigmentosum complement group C and D (XPC and XPD, also called ERCC2) are both members of the NER pathway. The SNP A499V in XPC has been studied extensively and correlated with increased cancer risk in general [10] as well as for HNSCC [11]. A SNP in XPD, K751Q, has been widely studied in HNSCC [7,11–22], and although a majority of the authors report no risk association, the results are still considered inconsistent. Contradictory results have also been reported for the BER gene X-ray repair cross-complementing group 1 (XRCC1) R399Q, where some studies point towards no change in risk [7,16,23–28] while others suggest an increased [17,21,29] or decreased [12,13,15,30,31] risk of HNSCC. The polymorphism T241M in the DSB repair gene XRCC3 has also been studied in HNSCC [13,15,16,32–35] with similarly conflicting results. Low penetrance variants (like SNPs) often affect the risk for cancer development, however, the potency of these variants often lies within their combined effect which is often more pronounced [36]. Candidate genes harbouring low penetrance variants have been found to encode proteins such as carcinogen metabolizing enzymes, methylation enzymes, DNA repair proteins, microenvironmental modifiers, oncoproteins, and tumor suppressors. To our knowledge, the combined effects of the four mentioned SNPs in HNSCC have not been studied previously. Therefore, we aimed to investigate the SNPs XPC A499V (rs2228000), XPD K751Q (rs13181), XRCC1 R399Q (rs25487) and XRCC3 T241M (rs861539) as potential risk factors for HNSCC and indicators of survival among Caucasian HNSCC patients. 2. Material and methods

65

Table 1 Clinical characteristics of HNSCC cases and healthy controls. Cases (n = 169) n (%)

Controls (n = 344) n (%)

Gender Male Female

112 (66) 57 (34)

224 (65) 120 (35)

Age of onset 65

16 (10) 67 (40) 86 (50)

201 (58) 88 (26) 55 (16)

Cigarette smoking Current Former Never No data

76 (45) 31 (18) 26 (16) 36 (21)

55 (16) 106 (31) 183 (53) 0 (0)

Tumour site Oral cavity Oropharynx Larynx Other

71 (42) 40 (24) 49 (29) 9 (5)

TNM-stage T1–T2 T3–T4

80 (47) 89 (53)

Variable

Treatment Radiotherapy, full dose (66 Gy) Pre- or postoperative radiotherapy (46 Gy) Surgery alone Othera

21 (12) 14 (8)

p53 Mutationb Mutation Wildtype

23 (33) 46 (67)

HPV statusb Positive Negative

95 (58) 68 (42)

82 (49) 52 (31)

Abbreviations: n (number of individuals). a Other: due to disrupted treatment or palliative radiotherapy. b Number of samples not analyzed; 100 for p53, 6 for HPV.

2.1. Tissue samples and DNA isolation

2.2. Treatment with radiotherapy

This study was based on 169 cases of HNSCC from which tumor biopsies were collected at the University Hospital in Linköping between the years 2004 and 2009. The patients consisted of 112 (66%) men and 57 (34%) women with a mean age of 65 (range 34–94; Table 1). Cases were stratified according to age (65 years), cigarette smoking (current, former, or never smokers), tumor site (oral cavity, oropharynx, or larynx), TNM-stage (T1–T2 or T3–T4), as well as treatment (full dose radiotherapy, pre- or postoperative radiotherapy, or surgery; Table 1). For controls, blood samples were collected from 344 healthy participants from the southeast region of Sweden during the years 2000–2001, of which 224 (65%) were men and 120 (35%) were women; mean age 46 years (range 23–78; Table 1). The population-based controls also answered a questionnaire about biological origin (immigration, adoption, etc), life-style (dietary habits, smoking, medication, and medical history), and occupational exposure. The rate of participation was 97% and since none of the controls reported a current or former tumor, no individuals were excluded. The study was approved by the Linköping University ethical committee, and all participants provided informed consent. DNA isolation from the tumor biopsies was performed by standard proteinase K treatment and phenol–chloroform extractions or MaxwellTM 16 DNA Purification Kit (Promega, Madison, WI, USA). DNA from blood samples was isolated with QIAamp® DNA Blood Maxi Kit (Qiagen, Solna, Sweden).

Patients who were offered surgery were given pre- or postoperative radiotherapy up to 46 Gray (Gy). Non-surgical patients received full dose radiotherapy up to 66 Gy, depending on the tumor site. Radiotherapy was delivered with 4MeV photons generated by a linear accelerator (Clinac 4/100, Varian, Palo Alto, CA, USA), delivering a dose-rate of 2.0 Gy/min. Two Gy were given daily, five days a week, and interruptions were avoided if possible. In total, 134 (80%) tumor patients were given either full dose or pre- or postoperative radiotherapy (Table 1). 2.3. Determination of genotypes employing PCR–RFLP The polymerase chain reaction (PCR) was carried out on a Mastercycler® ep (Eppendorf, Horsholm, Denmark) in a total volume of 20 ␮l containing the following: 50 ng of genomic DNA; 200 ␮M of each dNTP; 1.0 ␮M of each primer; 2 mM MgCl2 ; 1× PCR buffer (containing 200 mM (NH4) 2SO4, 750 mM Tris pH 9.0 and 0.1% Tween 20); and 0.5 units Thermo White Taq DNA polymerase (Saveen & Werner AB, Limhamn, Sweden). All primers were custom-made by Invitrogen, (Paisley, UK) and their sequences are available upon request. The amplifications were performed by annealing at 55–62 ◦ C for 35 cycles. Prior to restriction fragment length polymorphism (RFLP), samples were loaded on a 1.5% agarose gel (Invitrogen) and stained with ethidium bromide for detection of PCR products in UV light. For XPC A499V, XPD K751Q,

66

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

XRCC1 R399Q, and XRCC3 T241M the following digestion enzymes were utilized according to the manufacturer’s instructions: MspA1I, PstI, HpaII, and NlaIII respectively. For detection of restriction fragments, the products for XPC and XPD were loaded on a 3% NuSieve (Cambrex Bio Science Rockland Inc Rockland, ME, USA): 1% agarose (Invitrogen) gel, XRCC1 on a 2% agarose gel, and XRCC3 on a 1.5% agarose gel. The gels were stained with ethidium bromide and the bands were visualised in UV light. The following banding patterns were characterized: for XPC A499 V Ala/Ala 146 and 188 bp, Ala/Val 146, 188, and 334 bp and Val/Val 334 bp; for XPD K751Q Lys/Lys 100 and 224 bp, Lys/Gln 66 100, 158, and 224 bp, and Gln/Gln 66, 100, and 158 bp; for XRCC1 R399Q Arg/Arg 80 and 161 bp, Arg/Gln 80, 161, and 241 bp, and Gln/Gln 241 bp; and for XRCC3 T241M Thr/Thr 305 bp, Thr/Met 72, 233, and 305 bp and Met/Met 72 and 233 bp.

As a methodological control, we also analyzed 10% of randomly selected samples, representing all three genotypes of the SNPs, by Sanger sequencing (3500 Genetic Analyzer, Applied Biosystems). BigDye Terminator v3.1Cycle sequencing kit (Applied Biosystems) was used to label samples according to the manufacturer’s protocols. 2.4. HPV analysis Analysis of the presence of HPV in DNA from the tumors was performed by an Abbott RealTime High-Risk HPV method. Briefly, 200 ng genomic DNA was used as template in the PCR reaction with three forward and two reverse primers to amplify HPV DNA from the high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66

Table 2 Predisposition and risk of HNSCC in relation to the alleles of the DNA repair genes XPC, XPD, XRCC1 and XRCC3. Gene SNP

Genotype

HNSCC n (%)

Control n(%)

OR

(95% CI)

CC CT TT T-allele CC CT TT T-allele CC CT TT T-allele

89 (53) 63 (37) 17 (10) 97 (29) 63 (57) 34 (30) 15 (13) 64 (29) 26 (45) 29 (51) 2 (4) 33 (29)

219 (64) 105 (30) 20 (6) 145 (21) 148 (66) 62 (28) 14 (6) 90 (20) 71 (59) 43 (36) 6 (5) 55 (23)

1.00 1.48 (0.99–2.20) 2.09 (1.05–4.18) 1.51 (1.10–2.05) 1.00 1.29 (0.77–2.15) 2.52 (1.15–5.52) 1.59 (1.08–2.34) 1.00 1.84 (0.96–3.53) 0.91 (0.17–4.80) 1.37 (0.80–2.33)

AA AC CC C-allele AA AC CC C-allele AA AC CC C-allele

59 (35) 81 (48) 29 (17) 139 (41) 40 (36) 50 (45) 22 (19) 94 (42) 19 (33) 31 (54) 7 (12) 45 (39)

142 (41) 153 (45) 49 (14) 251 (36) 86 (39) 106 (47) 32 (14) 170 (38) 56 (47) 47 (39) 17 (14) 81 (34)

1.00 1.27 (0.85–1.91) 1.42 (0.82–2.47) 1.22 (0.92–1.60) 1.00 1.01 (0.61–1.68) 1.48 (0.76–2.86) 1.18 (0.84–1.66) 1.00 1.94 (0.97–3.88) 1.21 (0.44–3.37) 1.28 (0.78–2.08)

GG GA AA A-allele GG GA AA A-allele GG GA AA A-allele

73 (43) 72 (43) 24 (14) 120 (36) 48 (43) 46 (41) 18 (16) 82 (37) 25 (44) 26 (46) 6 (10) 38 (33)

135 (39) 160 (47) 49 (14) 258 (38) 97 (43) 100 (45) 27 (12) 154 (34) 38 (32) 60 (50) 22 (18) 104 (43)

1.00 0.83 (0.56–1.24) 0.91 (0.51–1.59) 0.92 (0.69–1.21) 1.00 0.93 (0.57–1.52) 1.35 (0.68–2.68) 1.10 (0.77–1.56) 1.00 0.66 (0.33–1.30) 0.41 (0.15–1.17) 0.65 (0.40–1.07)

CC CT TT T-allele CC CT TT T-allele CC CT TT T-allele

77 (46) 70 (41) 22 (13) 114 (34) 47 (42) 48 (43) 17 (15) 82 (37) 30 (53) 22 (39) 5 (9) 32 (28)

158 (46) 144 (42) 42 (12) 228 (33) 108 (48) 90 (40) 26 (12) 142 (32) 50 (42) 54 (45) 16 (13) 86 (36)

1.00 1.00 (0.67–1.48) 1.07 (0.60–1.93) 1.03 (0.77–1.36) 1.00 1.23 (0.75–2.00) 1.50 (0.75–3.03) 1.24 (0.87–1.77) 1.00 0.68 (0.35–1.33) 0.52 (0.17–1.57) 0.70 (0.41–1.16)

P-value

OR (95% CI) P-value Adjusted for age and smoking

XPC A499V Total

Men

Women

XPD K751Q Total

Men

Women

XRCC1 R399Q Total

Men

Women

XRCC3 T241M Total

Men

Women

0.05 0.04 0.007 0.33 0.02 0.014 0.07 0.91 0.22

0.24 0.21 0.15 0.96 0.25 0.31 0.06 0.71 0.29

0.37 0.73 0.53 0.77 0.40 0.57 0.23 0.09 0.07

0.99 0.81 0.85 0.42 0.26 0.20 0.26 0.25 0.15

1.00 1.43 (0.83–2.49) 1.81 (0.71–4.60)

0.20 0.21

1.00 1.32 (0.66–2.64) 1.91 (0.67–5.45)

0.44 0.23

1.00 1.81 (0.71–4.58) 0.69 (0.43–11.07)

0.21 0.79

1.00 0.79 (0.45–1.39) 0.97 (0.47–2.01) 1.00 0.73 (0.36–1.47) 1.15 (0.47–2.83) 1.00 0.98 (0.37–2.63)

0.41

0.38 0.76

0.97 0.71

0.78 (0.22–2.80) 1.00 1.14 (0.66–1.99) 1.25 (0.58–2.68)

0.63 0.57

1.00 1.60 (0.80–3.20) 2.46 (0.90–6.72)

0.18 0.07

1.00 0.65 (0.24–1.74) 0.44 (0.12–1.66)

0.39 0.23

1.00 1.10 (0.64–1.90) 1.23 (0.54–2.81)

0.71 0.62

1.00 1.38 (0.70–2.71) 1.77 (0.64–4.86)

0.35 0.27

1.00 0.66 (0.25–1.73) 0.62 (0.15–2.54)

0.40 0.50

Abbreviations: CI (confidence interval), n (number of individuals), OR (odds ratio). The relation between amino acid and nucleotide is as follows: XPCA499V, C/T; XPD K751Q, A/C; XRCC1 R399Q, G/A; and XRCC3 T241M, C/T.

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

67

Table 3 Risk of HNSCC in relation to interaction of the polymorphisms XPC A499V, XPD K751Q, XRCC1 R399Q, and XRCC3 T241M. Gender

Women

Men

Genes Genotypes

OR (95% CI)

XPC A499V

XPD K751Q

XRCC1 R399Q

XRCC3 T241M

CC CT/TT CT/TT CT/TT – – – CT/TT CC

AA CA/CC – – CA/CC CA/CC – CA/CC AA

GA/AA – GG – GG – GG GA/AA GG

CT/TT – – CC – CC CC CT/TT CC

P-value

OR (95% CI)

P-value

Adjusted for age and smoking 1.00 3.39 (1.25–9.21) 2.41 (1.01–5.73) 2.49 (0.97–6.36) 3.06 (1.12–8.35) 3.91 (1.27–12.05) 2.52 (1.02–6.23) 1.00 10.00 (1.10–90.78)

0.02 0.04 0.06 0.03 0.02 0.04 0.04

1.00 1.93 (0.48–7.72) 2.87 (0.82–10.02) 2.75 (0.77–9.84) 1.73 (0.48–6.21) 1.65 (0.40–6.89) 2.80 (0.75–10.42) 1.00 3.77 (0.33–43.00)

0.35 0.09 0.12 0.40 0.49 0.12 0.29

Abbreviations: CI (confidence interval), OR (odds ratio). The relation between amino acid and nucleotide is as follows: XPC A499V, C/T; XPD K751Q, A/C; XRCC1 R399Q, G/A; and XRCC3 T241M, C/T.

and 68) and endogenous human beta-globin was used as an internal control. The signal was detected using fluorescently labelled probes, and the cut off value was set to 32 cycles of amplification.

Hardy–Weinberg equilibrium for all four polymorphisms. Genotyping of 10% of the samples by Sanger sequencing showed 100% concordance with the initial genotyping method.

2.5. Statistical analysis

3.1. SNPs in XPC, XPD, XRCC1, and XRCC3 were associated with risk for HNSCC

Relative risk (RR), odds ratio (OR), 95% confidence intervals (CI) and P-values were calculated using STATA 13.1. Hardy–Weinberg equilibrium was calculated using 2-test. Results were considered as statistically significant when P < 0.05 (two-sided). Overall survival was estimated with the Kaplan–Meier method and the significance of difference between survival rates for patients with different genotypes was assessed by log rank test (SPSS 22.0 for Windows). 3. Results The clinical characteristics of the cases and controls are presented in Table 1. We successfully genotyped 169HNSCC cases and 344 healthy controls, from the same geographical area, for the four polymorphisms XPC A499V, XPD K751Q, XRCC1 R399Q and XRCC3 T241M. The genotype frequencies of the control group were in

Statistical analysis revealed an increased risk of HNSCC in the presence of the XPCT-allele. There was more than a 2-fold increase in risk for the overall group as well as for men homozygous XPC499V, OR = 2.09 (CI 95% 1.05–4.18, P = 0.04) and OR = 2.52 (CI 95% 1.15–5.52, P = 0.02; Table 2), respectively. After adjusting for age and smoking, the odds ratio was stable, although no longer statistically significant, implying no negative confounding effect. In order to identify possible synergistic effects of variations in the different DNA repair genes, we compared the four risk alleles against the non-risk alleles among both women and men. According to our results, the risk alleles for men were regarded as T for XPC, C for XPD, A for XRCC1, and T for XRCC3. For men, the combination of all four putative risk alleles (n = 12) was associated with a tenfold risk of HNSCC (OR = 10.00, CI 95% 1.10–90.78, P = 0.04; Table 3), although adjustment for age and smoking yielded a non-significant

Table 4 Oral SCC and laryngeal SCC in relation to XPC A499V and XPD K751Q. Tumor site Genes

Genotype

HNSCC cases

Controls

OR (95% CI)

P-value

10 28 34 70 8 25 25 59 8 10

20 219 145 543 14 148 90 358 55 185

3.13 (1.26–7.77)

0.014

1.82 (1.13–2.91)

0.008

2.96 (1.09–8.06)

0.034

1.69 (0.97–2.93)

0.05

XPC A499V Women

TT vs CC T-allele vs C-allele TT vs CC T-allele vs C-allele T-allele vs C-allele

2.69 (0.91–7.85)

0.04

Oral cavity XPC A499V Total XPD K751Q Total

CT vs CC CA vs AA

31 40 41 22

105 219 153 142

1.69 (0.99–2.89)

0.054

1.81 (1.01–3.25)

0.047

CA vs AA XPC CT/TT & XPD CA/CC vs XPC CC & XPD AA

24 10 24 11

47 56 70 87

3.43 (1.40–8.37)

0.007

2.98 (1.34–6.65)

0.008

Larynx XPC A499V Total

XPC A499V Men

XPD K751Q Women XPC A499V & XPD K751Q Total

Abbreviations: CI (confidence interval), OR (odds ratio). The relation between amino acid and nucleotide is as follows: XPC A499V, C/T; XPD K751Q, A/C.

68

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

Table 5 Association between age of HNSCC onset and XRCC3 T241M. Selected population characteristicsGenes

Genotype

HNSCC cases

Age of onset XRCC3 T241M Total XRCC3 T241M Men XRCC3 T241M Women

CC vs TT CC vs TT CC vs CT

65

46 5 27 4 19 6

31 17 20 13 11 16

RR (95% CI)

P-value

5.05 (1.69–15.10) 4.39 (1.24–15.48) 4.61 (1.39–15.24)

0.004 0.022 0.012

The relation between amino acid and nucleotide is XRCC3 T241 M, C/T.

OR of 3.77, warranting cautious interpretation. For women, the risk alleles for XPC and XPD were regarded the same as for men whereas for XRCC1 and XRCC3 they appeared to be G and C, respectively. Only one woman showed this combination however, and no significant association was found (data not shown). Since few men and women possessed all four risk alleles we were interested in studying the possible effects of having two risk alleles. The risk alleles were thus combined two-and-two and thereafter statistically analyzed. For men, no statistically significant associations were seen (data not shown). For women on the other hand, nearly all combinations of polymorphisms were associated with an increased risk of HNSCC (Table 3). Again after adjusting for age and smoking, the outcomes were non-significant, probably due to small sample size. However, our interpretation was that the most interesting combinations were XPC 499V and XRCC1 399Q as well as XRCC1 399R and XRCC3 241T, showing an approximate 2.5-fold increased risk of HNSCC.

3.2. SNPs in XPC A499V and XPD K751Q were associated with tumor localization When analyzing genotype in relation to tumor localization, homozygous XPC 499 V individuals showed a higher risk of laryngeal SCC (OR = 3.13, CI 95% 1.26–7.77, P = 0.014; Table 4). Subgrouping for gender revealed similar risks for both men and women. Looking at SCC in the oral cavity, there was a trend towards increased risk among heterozygotes for XPC A499 V (OR = 1.69, CI 95% 0.99–2.89, P = 0.054). Being heterozygous for XPD K751Q was associated with higher risk of oral SCC (OR = 1.81, CI 95% 1.01–3.25, P = 0.047). For women, the risk was even more pronounced (OR = 3.43, CI 95% 1.40–8.37, P = 0.007). Interestingly, the risk of oral SCC was increased among individuals homozygous mutant or heterozygous for the combination of both XPC and XPD (OR = 2.98, CI 95% 1.34–6.65, P–= 0.008).

Table 6 HPV, p53 mutation, cigarette smoking, and XRCC1 R399Q among HNSCC cases. Tumor site HPV Negative Positive P53 Wildtype

Mutated

Former smokers Current smokers

Larynx 24 23 (49%)

16 10 (38%)

13 5 (28%)

14 7 (33%)

GG GA AA

4

HPV Negative Positive

Never smokers

Oropharynx 5 34 (87%)

Age of onset ≤65 24 57 Smoking Current/former 48 55 p53 status p53 wildtype 21 21 1 3 17 14 3 4 HPV Negative 36 28

HPV Negative Positive

All cases

Oral 34 35 (51%)

HPV Negative Positive Negative Positive Negative Positive Negative Positive XRCC1 R399Q

Adjusted for age and smoking Abbreviations: CI (confidence interval), RR (relative risk). The relation between amino acid and nucleotide is XRCC1 R399Q.

Chi2

P-value

>65 44 39

9.23

0.002

Never 6 20

4.72

0.03

Chi2

P-value

2.62

0.10

3.94

0.047

1.14

0.29

0.69

0.41

RR (95% CI)

P-value

p53 mutation 15 6 3 0 12 5 0 1 Positive 36 41 18

1.00 1.46 (0.75–2.85) 4.50 (1.39–14.61) 4.32 (1.31–14.28)

0.26 0.01 0.016

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

69

3.3. Association between age of onset and XRCC3 T241M

4. Discussion

Being wild-type for XRCC3 241T was associated with an earlier age of onset (below 65 years) compared to homozygous 241M individuals (RR = 5.05, CI 95% 1.69–15.10, P = 0.004; Table 5). Stratification according to gender revealed an association between XRCC3 and age of onset among both men and women.

Since the head and neck region is continuously exposed to both exogenous and endogenous DNA-damaging factors, it is reasonable to expect that DNA repair genes may play a part in development, progression, and outcome of HNSCC. We found that homozygous carriers of XPC 499V had more than a 2-fold increased risk of HNSCC and a 3-fold increased risk of laryngeal SCC. This is in line with findings that XPC 499V is linked to an overall increased risk of cancer [10]. Furthermore a large case-control study [11] and a meta-analysis on various SNPs in XPC (including Caucasian and Asian populations) [37], both showed an increased risk of HNSCC. In line with this, our results indicate that the SNP XPC 499V increased the risk of developing tumours of primarily the larynx, but possibly also the oral cavity. Many authors have not been able to find associations between XPD K751Q and risk of HNSCC [11,13–16,19,22] whereas others have found correlations to overall risk and/or a gene-environment interaction [12,17,18]. To our knowledge, our study is the first to show a relationship between the SNP XPD 751Q and an increased risk of a specific tumor site in the head and neck region, the oral cavity, but not the larynx. Interestingly, when pooling the risk genotypes of XPC 499V and XPD 751Q, the risk of oral SCC increased, suggesting a synergistic effect. A meta-analysis on XPD 751Q found an increased risk for premalignant oral leukoplakia, but not for oral cancer [22]. In our study, a novel finding is that XRCC3 241M correlated significantly to age of onset in both men and women. A recent publication on HNSCC patients under the age of 45 indicated a correlation of the T-allele in younger individuals, although it was displayed in heterozygotes alone [38]. The results of other previous studies on XRCC3 T241M are in conflict; some authors report no association with overall risk of HNSCC [13,16] whereas others report an increased risk [15,34,39], an association specific to gender [15,33], or only when combined with SNPs of other genes [35]. Furthermore, associations have also been reported between XRCC3 T241M and supraglottic [32] and pharyngeal cancer [34]. The variant allele of XRCC3 241M which results in an amino acid change, Thr to Met is stipulated to have biological effects on the function of the enzyme and/or interaction with other proteins involved in DNA damage and repair [40]. It has been shown that healthy homozygous carriers of XRCC3 241M possess higher levels of DNA adducts [40], which may affect age of onset. HPV is one of the major risk factors for HNSCC which is why we also analyzed the material for HPV status using a real time PCR method. We found that 58% of the tumors were HPV-positive, which is in accordance with the literature (12.8–59.9%) [41–44]. As expected, HPV-positivity was more common in oropharyngeal tumors (87%), followed by 51% in oral cavity, and 49% in the larynx. This is in accordance with a review of 60 published studies which demonstrated a higher prevalence of HPV-positivity in oropharyngeal SCC vs oral or laryngeal SCCs (35.6% vs 23.5% vs 24.0%) [41]. In addition, more recent studies have shown HPV-positivity in up to 72% of oropharyngeal SCC [45–47]. The fact that our overall detection rate was high could be due to the method which detects numerous high-risk HPV genotypes (12 types) and/or a reflection of the steady increase in incidence of HPV-positive HNSCC [48–50]. Controversy remains about whether or not smoking is associated with an increased number of HPV-positive HNSCC [51,52] or if these risk factors are independent [53]. Statistical analysis of HPV and smoking in our material showed that never smokers were significantly more often HPV-positive, as compared to current/former smokers (P = 0.03). The mechanisms by which smoking and HPV may interact to cause HNSCC are unknown. One hypothesis is that smoking alters the immune response to HPV and prolongs the clearance of the virus from the cells. Gillison et al. recently showed that

3.4. HPV, p53 mutation, smoking, and XRCC1 R399Q were associated with risk for HNSCC Since HPV is an important risk factor for HNSCC, we analyzed the 169 tumors for HPV-positivity using a PCR based method. One hundred sixty-three samples were successfully analyzed, and 59% were positive for high-risk HPV. The distribution of HPVpositivity according to tumor site was 51% for oral cavity, 87% for oropharynx, and 49% for larynx. HPV-positivity was more frequent among cases under the age of 65 (P = 0.002). Mutation analysis of p53 was performed on the first 69 consecutive cases, of which 33% were mutated. Specified by tumor site, p53-mutated tumors represented 38% of those in the oral cavity, 28% of those in the oropharynx, and 33% of those in the larynx. To evaluate the associations between HPV, p53-mutation, and smoking, we performed Chi2 analysis as summarized in Table 6. The frequency of HPVpositive never smokers was higher compared to current/former smokers (P = 0.03). Moreover, HPV-positive never smokers had lower frequencies of p53 mutation (P = 0.047). We performed statistical analysis between HPV-status and genotypes of all four DNA repair genes. Our results showed a significant association between HPV-positivity and the homozygous mutant genotype XRCC1399Q (RR = 4.5, CI 1.39–14.61, P = 0.01). After adjusting for age and smoking, the relative risk was 4.32 (P = 0.016).

3.5. Relationship between survival and XPD K751Q Looking at XPD K751Q, there was a statistically significant difference in overall survival between wild-type 751K as compared to heterozygotes K751Q and homozygous 751Q mutants (P = 0.04 respectively, log rank test) with a median survival time of 57 months for wild-type as compared to 25 and 23 months for heterozygotes and homozygous mutants, respectively (Fig. 1A). When stratifying according to gender, a significant difference was seen exclusively in women, both when comparing wild-type individuals with heterozygous or with heterozygous pooled with homozygous mutant individuals (P = 0.02 for both, log rank test; Fig. 1B). The median survival time observed for men wild-type for XPD 751K was 57 months, for heterozygous K751Q 42 months, and for homozygous 751Q mutant 23 months in contrast to women, where median survival time was 53 months for wild-type, but only 17 and 8 months for heterozygous K751Q and homozygous 751Q mutant, respectively. When stratifying for radiotherapy, a significant difference in overall survival time was observed. The median survival time for individuals receiving full dose radiotherapy (66 Gy) was more than 74 months among wild-type for XPD 751K, 53 months for heterozygous K751Q, and 32 months for homozygous 751Q mutant (751K vs. 751Q P = 0.02; 751K vs. K751Q pooled with 751Q P = 0.03, log rank test; Fig. 1C). Looking at patients who received pre- or postoperative radiotherapy (46 Gy), wild-type 751K had a median survival time of 40 months; heterozygous K751Q and homozygous 751Q mutant individuals survived only 16 and 18 months, respectively (no significant differences).

70

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

Fig. 1. Kaplan–Meier analysis of SNP in XPD K751Q and survival of HNSCC patients. (A) Illustrates a statistically significant difference in survival of HNSCC depending on genotype: homozygous wild-type (AA), heterozygous (AC), or homozygous mutant (CC). (P = 0.04 for both, log rank test). (B) Stratification by gender shows a significant difference among women when comparing wild-type against heterozygous or heterozygous pooled with homozygous mutant individuals (P = 0.02 for both, log rank test). (C) Stratification by radiotherapy regimes displays a significant difference in survival time among HNSCC patients receiving full dose radiotherapy when comparing wild-type individuals against homozygous mutants or heterozygous pooled with homozygous mutants (P = 0.02 and P = 0.03, respectively, log rank test). Overall survival was measured in months.

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

PCR detection of HPV from oral rinse was positively associated with the current number of cigarettes smoked [54,55]. Another theory is that individuals prone to risk behavior might have an additive effect from exposure to several risk factors for HNSCC, such as unprotected sex, smoking, and alcohol. p53 mutation analysis was carried out in order to identify associations with HPV and smoking. Our results showed that HPVpositive never smokers had lower frequencies of p53 mutation. (P = 0.047). It has previously been shown that HPV-positive tumors have less p53 mutations in HNSCC [56]. Furthermore, it was shown that p53 mutations found in HPV-positive tumors did not affect the transactivation site of the P53 protein, suggesting that carcinogenesis mediated by HPV is independent of p53 mutation, even in cases where a mutation exists [56]. Conversely, smoking increases the risk of p53 mutations [57] and is associated with a greater number of genetic alterations than in tumors from non-smoking HPV-positive patients. Interestingly, it was recently shown that among patients with HPV-positive HNSCC, the worst outcome was related to smoking [58–60]. An association between HPV positivity and XRCC1 399Q was noted in our material. This finding suggests that the role of XRCC1 in base excision repair may influence the risk of HNSCC when infected with HPV, although this hypothesis needs further investigation. Of the SNPs analyzed in this study, XPD 751Q appeared to be most closely linked to overall survival, with homozygous mutant women demonstrating the shortest median survival time. Furthermore, we found a significant difference in survival time between the two presented radiotherapy regimes, where genotype was associated to survival after full dose radiotherapy. These findings support those of our previous study on 40HNSCC patients, where we showed that patients with XPD 751Q treated with radiotherapy had a significantly shorter overall survival time (P = 0.048) compared to 751K [61]. Another study examining the relationship between polymorphisms in XPD and outcome of HNSCC [7] did not find a correlation with survival or time to progression. However, a more recent study on an Indian population has shown an increase in relapse-free survival, but not disease-specific survival, in C-allele carriers [62]. In agreement with our results, no associations were found between XRCC1 R399Q and HNSCC survival in work by Carles et al., [7]. To our knowledge, no previous studies have analyzed the combined effect of XPC A499V, XPD K751Q, XRCC1 R399Q and XRCC3 T241M. In our analyses of combined genotypes, putative risk genotypes were those with ORs > 1.0. When combining all four putative risk alleles for men we found a 10-fold increase in risk of developing HNSCC. The four putative risk alleles among women did not yield any significant differences in risk of HNSCC, probably due to few cases. However, when the risk alleles for women were combined two and two, five of six possible combinations yielded an almost 3-fold increase in risk of HNSCC. Altogether, our findings suggest a synergistic interaction among the susceptibility genotypes. Another study combined XRCC1 R194W, XRCC3 T241M,and XPD exon 6 and found up to a 9-fold increase in risk of HNSCC [15]. Interestingly, this study, as well as ours, combined genes which encode enzymes belonging to different DNA repair systems and found a synergistic effect. This is consistent with the pathogenesis of HNSCC, where multiple DNA lesions are induced by cigarette smoking and other environmental agents, requiring different pathways for repair. The increased effect seen when combining several susceptibility genes is in line with the polygenic model in which a large number of alleles each conferring to a small genotypic risk are combined to confer susceptibility in the population [36]. The SNPs analyzed in our study might very well be in linkage disequilibrium with a more functional variant, even though functional studies of XPD K751Q, XRCC1 A399G and XRCC3 T241M indicate a direct effect on the func-

71

tion [40,63–66]. Many of the above mentioned studies have not yielded reproducible results, which can be explained by the selection of tumors from different sites in the head and neck region, small sample size yielding low study power, ethnic differences in genotype distribution and exposure to DNA damaging agents, as well as interactions with other susceptibility genes. Thus, further larger studies are required before the role of the four selected SNPs in this study can be elucidated. 5. Conclusion In our study, XPC 499V was associated with the overall risk of HNSCC and especially risk of laryngeal SCC. In women, XPD 751Q was associated with risk of oral SCC and prolonged overall survival. XPD 751Q was associated with significantly prolonged overall survival following full dose radiotherapy. Wild-type individuals of XRCC3 241T showed an earlier age of onset. Taken together, our results suggest that polymorphisms of the DNA repair genes XPC, XPD, XRCC1, and XRCC3 may affect both risk for and survival of HNSCC and may act synergistically in HNSCC pathogenesis. Conflict of interest statement The authors declare that there are no conflicts of interest. Funding This work was supported by the Swedish Cancer Society and Laryngfonden. Acknowledgments The authors wish to thank Linda Vainikka for excellent technical support. This work was supported by the Swedish Cancer Society and Laryngfonden. References [1] D.M. Parkin, F. Bray, J. Ferlay, P. Pisani, Global cancer statistics: 2002, CA: Cancer J. Clin. 55 (2) (2005) 74–108. [2] M.P. Curado, M. Hashibe, Recent changes in the epidemiology of head and neck cancer, Curr. Opin. Oncol. 21 (3) (2009) 194–200. [3] D.L. Crowe, J.G. Hacia, C.L. Hsieh, U.K. Sinha, H. Rice, Molecular pathology of head and neck cancer, Histol. Histopathol. 17 (3) (2002) 909–914. [4] J.M. Elwood, J.C. Pearson, D.H. Skippen, S.M. Jackson, Alcohol, smoking, social and occupational factors in the aetiology of cancer of the oral cavity, pharynx and larynx, Int. J. Cancer J. Int. Cancer 34 (5) (1984) 603–612. [5] G.C. Blitzer, M.A. Smith, S.L. Harris, R.J. Kimple, Review of the clinical and biologic aspects of human papillomavirus-positive squamous cell carcinomas of the head and neck, Int. J. Radiat. Oncol. Phys. 88 (4) (2014) 761–770. [6] J.H. Hoeijmakers, Genome maintenance mechanisms for preventing cancer, Nature 411 (6835) (2001) 366–374. [7] J. Carles, M. Monzo, M. Amat, et al., Single-nucleotide polymorphisms in base excision repair: nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer, Int. J. Radiat. Oncol. Biol. Phys. 66 (4) (2006) 1022–1030. [8] L.L. Gleich, F.N. Salamone, Molecular genetics of head and neck cancer, Cancer Control 9 (5) (2002) 369–378. [9] E.L. Goode, C.M. Ulrich, J.D. Potter, Polymorphisms in DNA repair genes and associations with cancer risk, Cancer Epidemiol. Biomarkers Prev. 11 (12) (2002) 1513–1530. [10] G. Francisco, P.R. Menezes, J. Eluf-Neto, R. Chammas, XPC polymorphisms play a role in tissue-specific carcinogenesis: a meta-analysis, Eur. J. Hum. Genet. 16 (6) (2008) 724–734. [11] J. An, Z. Liu, Z. Hu, et al., Potentially functional single nucleotide polymorphisms in the core nucleotide excision repair genes and risk of squamous cell carcinoma of the head and neck, Cancer Epidemiol. Biomarkers Prev. 16 (8) (2007) 1633–1638. [12] V. Harth, M. Schafer, J. Abel, et al., Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair, J. Toxicol. Environ. Health Part A 71 (13–14) (2008) 887–897.

72

L. Farnebo et al. / DNA Repair 31 (2015) 64–72

[13] W.Y. Huang, A.F. Olshan, S.M. Schwartz, et al., Selected genetic polymorphisms in MGMT, XRCC1, XPD, and XRCC3 and risk of head and neck cancer: a pooled analysis, Cancer Epidemiol. Biomarkers Prev. 14 (7) (2005) 1747–1753. [14] Y.B. Ji, K. Tae, Y.S. Lee, et al., XPD polymorphisms and risk of squamous cell carcinoma of the head and neck in a Korean sample, Clin. Exp. Otorhinolaryngol. 3 (1) (2015) 42–47. [15] S. Kietthubthew, H. Sriplung, W.W. Au, T. Ishida, Polymorphism in DNA repair genes and oral squamous cell carcinoma in Thailand, Int. J. Hyg. Environ. Health 209 (1) (2006) 21–29. [16] G. Matullo, A.M. Dunning, S. Guarrera, et al., DNA repair polymorphisms and cancer risk in non-smokers in a cohort study, Carcinogenesis 27 (5) (2006) 997–1007. [17] S. Ramachandran, K. Ramadas, R. Hariharan, R. Rejnish Kumar, M. Radhakrishna Pillai, Single nucleotide polymorphisms of DNA repair genes XRCC1 and XPD and its molecular mapping in Indian oral cancer, Oral Oncol. 42 (4) (2006) 350–362. [18] E.M. Sturgis, R. Zheng, L. Li, et al., XPD/ERCC2 polymorphisms and risk of head and neck cancer: a case-control analysis, Carcinogenesis 21 (12) (2000) 2219–2223. [19] J. Cui, D. Li, L. Shen, W. Zhang, X. Xu, XPD Lys751Gln polymorphism is not associated with oral cancer risk: evidence from a meta-analysis, Tumour Biol. 35 (7) (2014) 6335–6341. [20] M. Gugatschka, D. Dehchamani, T.C. Wascher, G. Friedrich, W. Renner, DNA repair gene ERCC2 polymorphisms and risk of squamous cell carcinoma of the head and neck, Experiment. Mol. Pathol. 91 (1) (2011) 331–334. [21] R. Khlifi, I. Kallel, B. Hammami, A. Hamza-Chaffai, A. Rebai, DNA repair gene polymorphisms and risk of head and neck cancer in the Tunisian population, J. Oral Pathol. Med.: Off. Publ. Int. Assoc. Oral Pathol. Am. Acad. Oral Pathol. 43 (3) (2014) 217–224. [22] E. Zhang, Z. Cui, Z. Xu, et al., Association between polymorphisms in ERCC2 gene and oral cancer risk: evidence from a meta-analysis, BMC Cancer 13 (2013) 594. [23] S. Demokan, D. Demir, Y. Suoglu, E. Kiyak, U. Akar, N. Dalay, Polymorphisms of the XRCC1 DNA repair gene in head and neck cancer, Pathol. Oncol. Res. 11 (1) (2005) 22–25. [24] K.M. Applebaum, M.D. McClean, H.H. Nelson, C.J. Marsit, B.C. Christensen, K.T. Kelsey, Smoking modifies the relationship between XRCC1 haplotypes and HPV16-negative head and neck squamous cell carcinoma, Int. J. Cancer J. Int. Cancer 124 (11) (2009) 2690–2696. [25] M. Kowalski, K. Przybylowska, P. Rusin, et al., Genetic polymorphisms in DNA base excision repair gene XRCC1 and the risk of squamous cell carcinoma of the head and neck, J. Exp. Clin. Cancer Res. 28 (2009) 37. [26] C. Li, Z. Hu, J. Lu, et al., Genetic polymorphisms in DNA base-excision repair genes ADPRT: XRCC1, and APE1 and the risk of squamous cell carcinoma of the head and neck, Cancer 110 (4) (2007) 867–875. [27] K. Tae, H.S. Lee, B.J. Park, et al., Association of DNA repair gene XRCC1 polymorphisms with head and neck cancer in Korean population, Int. J. Cancer J. Int. Cancer 111 (5) (2004) 805–808. [28] B. Liu, T. Shen, XRCC1 Arg399Gln polymorphism is not associated with oral cancer risk: evidence from a meta-analysis, Tumour Biol.: J. Int. Soc. Oncodev. Biol. Med. 35 (1) (2014) 507–512. [29] E.M. Sturgis, E.J. Castillo, L. Li, et al., Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck, Carcinogenesis 20 (11) (1999) 2125–2129. [30] A.F. Olshan, M.A. Watson, M.C. Weissler, D.A. Bell, XRCC1 polymorphisms and head and neck cancer, Cancer Lett. 178 (2) (2002) 181–186. [31] R.J. Hung, J. Hall, P. Brennan, P. Boffetta, Genetic polymorphisms in the base excision repair pathway and cancer risk: a HuGE review, Am. J. Epidemiol. 162 (10) (2005) 925–942. [32] S. Benhamou, J. Tuimala, C. Bouchardy, P. Dayer, A. Sarasin, A. Hirvonen, DNA repair gene XRCC2 and XRCC3 polymorphisms and susceptibility to cancers of the upper aerodigestive tract, Int. J. Cancer J. Int. Cancer 112 (5) (2004) 901–904. [33] H. Shen, E.M. Sturgis, K.R. Dahlstrom, Y. Zheng, M.R. Spitz, Q. Wei, A variant of the DNA repair gene XRCC3 and risk of squamous cell carcinoma of the head and neck: a case-control analysis, Int. J. Cancer J. Int. Cancer 99 (6) (2002) 869–872. [34] J. Werbrouck, K. De Ruyck, F. Duprez, et al., Single-nucleotide polymorphisms in DNA double-strand break repair genes: association with head and neck cancer and interaction with tobacco use and alcohol consumption, Mutat. Res. 656 (1–2) (2008) 74–81. [35] C.Y. Yen, S.Y. Liu, C.H. Chen, et al., Combinational polymorphisms of four DNA repair genes XRCC1: XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan, J. Oral Pathol. Med.: Off. Publ. Int. Assoc. Oral Pathol. Am. Acad. Oral Pathol. 37 (5) (2008) 271–277. [36] R.S. Houlston, J. Peto, The search for low-penetrance cancer susceptibility alleles, Oncogene 23 (38) (2004) 6471–6476. [37] Y. Zhang, Z. Li, Q. Zhong, et al., Polymorphisms of the XPC gene may contribute to the risk of head and neck cancer: a meta-analysis, Tumour Biol.: J. Int. Soc. Oncodev. Biol. Med. 35 (4) (2014) 3917–3931. [38] M. Kostrzewska-Poczekaj, W. Gawecki, J. Illmer, et al., Polymorphisms of DNA repair genes and risk of squamous cell carcinoma of the head and neck in young adults, Eur. Arch. Oto-rhino-laryngol.: Off. J. Eur. Fed. Oto-Rhino-Laryngolog. Soc. 270 (1) (2013) 271–276.

[39] P. Gresner, J. Gromadzinska, K. Polanska, E. Twardowska, J. Jurewicz, W. Wasowicz, Genetic variability of Xrcc3 and Rad51 modulates the risk of head and neck cancer, Gene 504 (2) (2012) 166–174. [40] G. Matullo, D. Palli, M. Peluso, et al., XRCC1: XRCC3, XPD gene polymorphisms, smoking and (32)P-DNA adducts in a sample of healthy subjects, Carcinogenesis 22 (9) (2001) 1437–1445. [41] A.R. Kreimer, G.M. Clifford, P. Boyle, S. Franceschi, Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review, Cancer Epidemiol. Biomarkers Prev. 14 (2) (2005) 467–475. [42] F. Dayyani, C.J. Etzel, M. Liu, C.H. Ho, S.M. Lippman, A.S. Tsao, Meta-analysis of the impact of human papillomavirus (HPV) on cancer risk and overall survival in head and neck squamous cell carcinomas (HNSCC), Head Neck Oncol. 2 (2010) 15. [43] H. Mehanna, T. Beech, T. Nicholson, et al., Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and neck cancer–systematic review and meta-analysis of trends by time and region, Head Neck 35 (5) (2013) 747–755. [44] N. Termine, V. Panzarella, S. Falaschini, et al., HPV in oral squamous cell carcinoma vs head and neck squamous cell carcinoma biopsies: a meta-analysis (1988–2007), Ann. Oncol. 19 (10) (2008) 1681–1690. [45] N. Stransky, A.M. Egloff, A.D. Tward, et al., The mutational landscape of head and neck squamous cell carcinoma, Science 333 (6046) (2011) 1157–1160. [46] E.M. Sturgis, K.K. Ang, The epidemic of HPV-associated oropharyngeal cancer is here: is it time to change our treatment paradigms? J. Natl. Compr. Cancer Network 9 (6) (2011) 665–673. [47] H.S. van Monsjou, A.J. Balm, M.M. van den Brekel, V.B. Wreesmann, Oropharyngeal squamous cell carcinoma: a unique disease on the rise? Oral Onco.l 46 (11) (2010) 780–785. [48] T.Z. Hwang, J.R. Hsiao, C.R. Tsai, J.S. Chang, Incidence trends of human papillomavirus-related head and neck cancer in Taiwan, 1995–2009, Int. J. Cancer (2014). [49] A.P. Stein, S. Saha, M. Yu, R.J. Kimple, P.F. Lambert, Prevalence of human papillomavirus in oropharyngeal squamous cell carcinoma in the United States across time, Chem. Res. Toxicol. 27 (4) (2014) 462–469. [50] C.H. Shiboski, B.L. Schmidt, R.C. Jordan, Tongue and tonsil carcinoma: increasing trends in the U.S. population ages 20–44 years, Cancer 103 (9) (2005) 1843–1849. [51] R. Herrero, X. Castellsague, M. Pawlita, et al., Human papillomavirus and oral cancer: the international agency for research on cancer multicenter study, J. Natl. Cancer Inst. 95 (23) (2003) 1772–1783. [52] S.M. Schwartz, J.R. Daling, D.R. Doody, et al., Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection, J. Natl. Cancer Inst. 90 (21) (1998) 1626–1636. [53] K.M. Applebaum, C.S. Furniss, A. Zeka, et al., Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer, J. Natl. Cancer Inst. 99 (23) (2007) 1801–1810. [54] M.L. Gillison, T. Broutian, R.K. Pickard, et al., Prevalence of oral HPV infection in the United States: 2009–2010, Jama 307 (7) (2012) 693–703. [55] C. Fakhry, M.L. Gillison, G. D’Souza, Tobacco use and oral HPV-16 infection, Jama 312 (14) (2014) 1465–1467. [56] W.H. Westra, J.M. Taube, M.L. Poeta, S. Begum, D. Sidransky, W.M. Koch, Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck, Clin. Cancer Res. 14 (2) (2008) 366–369. [57] M. Urashima, T. Hama, T. Suda, et al., Distinct effects of alcohol consumption and smoking on genetic alterations in head and neck carcinoma, PLoS One 8 (11) (2013) e80828. [58] K.K. Ang, J. Harris, R. Wheeler, et al., Human papillomavirus and survival of patients with oropharyngeal cancer, N. Engl. J. Med. 363 (1) (2010) 24–35. [59] C. Fakhry, W.H. Westra, S. Li, et al., Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial, J. Natl. Cancer Inst. 100 (4) (2008) 261–269. [60] P. Sinha, H.L. Logan, W.M. Mendenhall, Human papillomavirus, smoking, and head and neck cancer, Am. J. Otolaryngol. 33 (1) (2012) 130–136. [61] L. Farnebo, K. Tiefenbock, A. Ansell, L.K. Thunell, S. Garvin, K. Roberg, Strong expression of survivin is associated with positive response to radiotherapy and improved overall survival in head and neck squamous cell carcinoma patients, Int. J. Cancer J. Int. Cancer 133 (8) (2013) 1994–2003. [62] M.B. Mahimkar, T.A. Samant, S. Kannan, J. Tulsulkar, P.S. Pai, D. Anantharaman, Polymorphisms in GSTM1 and XPD genes predict clinical outcome in advanced oral cancer patients treated with postoperative radiotherapy, Mol. Carcinog. 51 (Suppl. 1) (2012) E94–103. [63] Y. Qiao, M.R. Spitz, H. Shen, et al., Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes, Carcinogenesis 23 (2) (2002) 295–299. [64] R.M. Lunn, R.G. Langlois, L.L. Hsieh, C.L. Thompson, D.A. Bell, XRCC1 polymorphisms: effects on aflatoxin B1-DNA adducts and glycophorin A variant frequency, Cancer Res. 59 (11) (1999) 2557–2561. [65] E.J. Duell, J.K. Wiencke, T.J. Cheng, et al., Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells, Carcinogenesis 21 (5) (2000) 965–971. [66] Y.C. Lei, S.J. Hwang, C.C. Chang, et al., Effects on sister chromatid exchange frequency of polymorphisms in DNA repair gene XRCC1 in smokers, Mutat. Res. 519 (1–2) (2002) 93–101.