Radiotherapy and Oncology 99 (2011) 356–361

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Radiotherapy and Oncology

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Clinical radiobiology

Association between single nucleotide polymorphisms in the XRCC1and RAD51 genes and clinical radiosensitivity in head and neck cancer

Nicola Pratesi a, Monica Mangoni b,⇑, Irene Mancini a, Fabiola Paiar b, Lisa Simi a, Lorenzo Livi b, Sara Cassani b,Michela Buglione c, Salvatore Grisanti d, Camillo Almici e, Caterina Polli f, Calogero Saieva g,Stefano Maria Magrini c, Giampaolo Biti b, Mario Pazzagli a, Claudio Orlando a

a Clinical Biochemistry Unit; and b Radiotherapy Unit, Department of Clinical Physiopathology, University of Florence, Italy; c Department of Radiation Oncology, University of Brescia,Italy; d Department of Medical Oncology; and e Department of Hematology, Brescia, Italy; f Radiotherapy Unit, USL 4 Prato, Prato, Italy; g Molecular and Nutritional Epidemiology Unit,Cancer Research and Prevention Center, Scientific Institute of Tuscany, Florence, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 May 2011Received in revised form 21 May 2011Accepted 26 May 2011Available online 23 June 2011

Keywords:RadiotherapyToxicitiesSingle nucleotide polymorphismsHead and neck cancer

0167-8140/$ - see front matter � 2011 Elsevier Irelandoi:10.1016/j.radonc.2011.05.062

⇑ Corresponding author. Address: Radiotherapy UPhysiopathology, Viale Morgagni 85, 50139 Florence,

E-mail address: m.mangoni@dfc.unifi.it (M. Mango

Purpose: Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymor-phisms (SNPs) in genes involved in DNA repair and in protection against reactive oxygen species (ROS)could be responsible for such cases of radiosensitivity. We investigated the association between theoccurrence of acute reactions in 101 patients with squamous cell carcinoma of the head and neck(SCCHN) after radiotherapy (RT) and five genetic polymorphisms: XRCC1 c.1196A > G, XRCC3c.722C > T, RAD51 (c.-3429G > C, c.-3392G > T), and GSTP1 c.313A > G.Materials and methods: Genetic polymorphisms were detected by high resolution melting analysis(HRMA). The development of acute reactions (oral mucositis, skin erythema and dysphagia) associatedwith genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologicallyeffective dose (BED).Results: Development of grade P2 mucositis was increased in all patients (chemo-radiotherapy andradiotherapy alone) with XRCC1-399Gln allele (HR = 1.72). The likelihood of developing grade P2 dys-phagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR = 4.00). The presence of at leastone SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G > C status were asso-ciated to higher likelihood of occurrence of acute toxicities (HR = 2.03).Conclusions: Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A > Gand RAD51 c.-3429 G > C, and the development of radiation-induced toxicities in SCCHN patients.

� 2011 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 99 (2011) 356–361

Squamous cell carcinoma of the head and neck (SCCHN) is a rel-atively common type of cancer, with a worldwide incidence of500,000 cases each year [1]. Multiple treatments, including sur-gery, radiotherapy (RT) and chemotherapy (CT) have been devel-oped for the anatomically different subtypes. Improvements inRT techniques with the introduction of three-dimensional confor-mal radiotherapy (3-DCRT) and intensity-modulated radiotherapy(IMRT) have increased the possibility of dose escalation and radio-curability [2].

Despite these improvements, the radiation dose necessary toachieve local tumor control is limited by the tolerance dose of nor-mal tissues in the irradiation field. Treatment of SCCHN patientswith RT can be significantly compromised by acute reactions andlate damages occurring in the normal tissue exposed to radiation.Advances in radiation technology and dose-fractionation have

d Ltd. All rights reserved.

nit, Department of ClinicalItaly.ni).

changed the pattern of side effects, but there is considerable evi-dence that both treatment – and patient – related factors influenceseverity of damage to normal tissue after therapeutic RT [3]. Intrin-sic factors of individual radiosensitivity can influence the variabil-ity of side effects observed [4–6]. In about 70% of SCCHN patients,clinical radiosensitivity should be considered as a direct effect of acellular response in the irradiation field caused by a loss of effi-ciency in the DNA repair mechanism. Single nucleotide polymor-phisms (SNPs) in DNA repair genes may alter protein functionand individual’s capacity to repair damaged DNA. The presenceof variants in specific genes, the products of which play a role inradiation response, could be associated to the development ofthe adverse radiation effects [7–14].

Various studies examined the effects of polymorphisms in DNArepair genes on RT response in terms of toxicity, but very few stud-ies have examined the correlation between polymorphisms andradiation-induced toxicities in SCCHN [15,16].

Radiosensitivity candidate genes that have been linked to en-hanced radiation responses include RAD51, XRCC1, XRCC3 and

N. Pratesi et al. / Radiotherapy and Oncology 99 (2011) 356–361 357

GSTP1. XRCC1 (X-ray repair cross-complementing 1) protein is anick sensor and through its action is able to detect and to repairthree of the most common forms of DNA damage induced byionizing radiation: single strand breaks (SSB) repair, doublestrand breaks (DSB) repair and base-excision repair (BER) mech-anisms [17–21]. A specific polymorphism of XRCC1 gene,c.1196A > G p.Gln399Arg, is a relatively common variant alleleresulting in a protein with altered fidelity and repair capacitywhich consequently alters the radiosensitivity risk [22]. More-over, subjects with XRCC1 AA genotypes have higher levels ofchromosomal breaks per cell than those with other genotypes[23].

RAD51 (RecA homolog, Escherichia coli) and RAD51 paralogXRCC3 (X-ray repair complementary 3) proteins are componentsof the homologous recombinational (HR) repair of DNA double-strand breaks and interstrand cross-links [24]. SNPs in these DSBrepair genes could interfere with their functions making them suit-able candidate genes to understand the genetic impact in acutenormal tissue reactions after RT [25].

RT exerts its cytotoxic effects by producing reactive oxygenspecies (ROS), thus, it is plausible that genetic variants in genesinvolved in the protection from oxidative stress could influencesusceptibility to acute side effects in subjects receiving RT. Glu-tathione-associated metabolism is a major mechanism for cellu-lar protection against ROS and their toxic products, andglutathione S-transferases (GSTs) a, l, p, and h are active indetoxifying reactive bases and lipid peroxides produced by reac-tive oxidant damage to DNA and lipids, respectively [26]. GSTP1(Glutathione S-transferase pi 1) are the GSTs isoforms mainlyexpressed in oral cavity and in pharynx. Single nucleotide sub-stitutions A > G at base pair 313 of the GSTP1 locus, a memberof the GST family, result in an aminoacid substitution of valineto isoleucine at codon 105, which decreases the enzyme activity[27].

On this basis, we evaluated the impact of XRCC1 c.1196A > Gp.Gln399Arg, XRCC3 c.722C > T p.Thr241Met, RAD51 (c.-3429G > C, c.-3392 G > T), and GSTP1 c.313A > G p.Ile105Val polymor-phisms and the occurrence of acute radiation-induced toxicity in101 SCCHN patients after RT.

Table 1Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribut

SNPs/genotypes All patients (radiotherapy and chemother

Mucositis grade p

<2 P2

XRCC1 c.1196 A > G GG 20 23 0.011AG 7 31 0.018AA 6 14 0.776AA + AG 13 45 0.011

XRCC3 c.722 C > T CC 12 19 0.389CT 12 31 0.379TT 9 18 0.932TT + CT 21 49 0.389

RAD51 c.-3429 G > C GG 28 54 0.512GC 4 12 0.476CC 1 2 0.699CC + GC 5 14 0.512

RAD51 c.-3392 G > T GG 7 26 0.087GT 19 29 0.159TT 7 13 0.804TT + GT 26 42 0.087

GSTP1 c.313 A > G AA 20 39 0.756AG 9 23 0.507GG 4 6 0.603GG + AG 13 29 0.756

Abbreviations: SNPs, single-nucleotide polymorphisms; <2 and P2, understood respect

Materials and methods

Study patients and clinical data

The study protocol was approved by the Local Ethics Commit-tee. The entry criteria were the following: histologically provenHNSCC; no distant metastases when first diagnosed; no previousRT and/or CT; patients aged P18 years; availability for correct fol-low-up; informed consent obtained from each patient.

Blood samples were collected before starting radiotherapy.Radiation-induced toxicities (oral mucositis, skin erythema anddysphagia) were documented during the treatment, and severityof acute side effects was assessed using the Common Toxicity Cri-teria for Adverse Events (CTCAE) of the U.S. NIH. [28]. Developmentof acute side effects of Grade 2 was considered to indicate in-creased sensitivity for acute effects in our study. Each toxicitywas analyzed accounting to biologically effective radiation dose(BED) considering the time of the higher grade of toxicity develop-ment. BED was calculated to account for difference in fractionationand overall treatment time, using the formula:

BED ¼ ndð1þ da=bÞ � c

aðT� T0Þ;

given the number of fractions n, the fraction size of d, an a/b ratio of10 Gy for acute reaction, a time factor c/a of 0,7 Gy/day, the overalltreatment time T and a starting time for compensatory proliferationT0 of 21 days [29]. Overall 101 patients affected with HNSSC treatedwith RT at the Radiotherapy Units of Florence University, BresciaUniversity and ASL4 Prato were enrolled in the study between June2009 and September 2010. All patients underwent radiotherapy.

The mean tumor dose was 62 Gy, (range 54–70 Gy). Irradiationwas performed with photon beams from 6-MeV linear acceleratorsusing 3D conformal radiation therapy or IMRT (Intensity Modu-lated Radiation Therapy) with SIB (simultaneous integrated boost).Fifty-six patients out of 101 received both radiotherapy plus che-motherapy. Sequential or concomitant chemotherapy scheduleswere based on derivatives of platinum, 5-fluorouracile, taxanesand/or cetuximab.

ion of acute mucositis after radiotherapy in SCCHN patients.

apy) Patients treated with radiotherapy alone

OR Mucositis grade p OR

<2 P2

0.33 15 7 0.025 0.253.11 6 11 0.098 2.831.17 2 4 0.311 2.333.01 8 15 0.025 4.020.68 10 6 0.256 0.491.47 6 11 0.098 2.830.96 7 5 0.559 0.671.47 13 16 0.256 2.050.68 18 17 0.936 0.941.55 4 5 0.47 1.40.97 1 / 0.511 0.961.45 5 5 0.609 1.062.3 6 7 0.672 1.320.55 13 13 0.862 1.110.88 4 2 0.354 0.470.43 17 15 0.672 0.760.87 14 13 0.903 0.931.36 7 6 0.815 0.860.7 2 3 0.478 1.661.14 9 9 0.903 1.08

to mucositis CTCAE score.

358 SNPs and radiosensitivity in head & neck cancer

High resolution melting analysis

For DNA exctraction, 3 ml of blood was collected in EDTA tubesfrom each patient. DNA was purified using the GENTRA DNABLOOD Kit (Qiagen, Hilden, Germany). For the rapid and cost-lim-ited detection of polymorphisms in SCCHN patients we developedaccurate protocols based on high resolution melting analysis(HRMA). As initial step, 100 ng DNA were amplified in a final vol-ume of 10 ll using: 1.25 ll of PCR Buffer (Life Technology, Italy),1.5 mM of MgCl2 (Life Technology, Italy), 300 nM of each primer,1.5 lM of SYTO�9 Dye (Invitrogen Corp., Carlsbad, CA), 1.25 U ofTaq Gold Polymerase (Life Technology, Italy). PCR protocol was de-fined by an initial denaturation step at 95 �C for 10 min, followedby 35 cycles of 60 s at 95 �C, 60 s at 60 �C and 60 s at 72 �C, witha final extension at 72 �C for 15 min.

Amplification and HRMA were performed on RotorGene™6000(Corbett Research, Australia). Primers were: Xrcc1 – 50-CCCCAAG-TACAGCCAGGTC-3’ (Forw) and 5’-CAGTCTGACTCCCCTCCAGA-30

(Rev); Xrcc3 – 50-CCATTCCGCTGTGAATTTG-30 (Forw) and 50-CCGCATCCTGGCTAAAAATA-30 (Rev); Rad51 – 50-GCTGGGAACTG-CAACTCATCT-30 (Forw) and 50- GCAGCGCTCCTCTCTCCAGC-30

(Rev); Gstp1 – 50-CTCTATGGGAAGGACCAGCA-30 (Forw) and 50-GAAGCCCCTTTCTTTGTTCA-30 (Rev). HRMA protocol was definedby an initial denaturation step at 95 �C for 5 min, followed by rean-neling step of 60 s at 40 �C with a HRM step from 75 to 90 �C.

Statistical analysis

Each polymorphism was tested for deviation from Hardy–Weinberg equilibrium by comparing the observed and expectedgenotype frequencies using the v2 test. The effect of chemotherapytreatment on the development of adverse radiation reactions wasevaluated by subdividing patients by each clinical end-pointaccording to presence or absence of chemotherapy treatment usingthe v2 test. The crude association between clinical toxicity end-points and SNPs genotypes was measured by odds ratio (OR) andits 95% confidence interval. The degree of significance was calcu-lated using the v2 test, except in case of small sample size (<5),where Fisher0s Exact test was used. The relation between doseparameter, derived from mean BED (BEDmean), and adverse

Fig. 1. (a) Cumulative incidence of acute mucositis (CTCAE score P2) after radiotherapyA/A), genetic polymorphism XRCC1 c.1196A > G p.Gln399Arg both (p log rank = 0.03BED = biologically effective radiation dose. (b) Cumulative incidence of acute mucositis (Cand of the variant genotypes (G/A or A/A), genetic polymorphism XRCC1 c.1196A > G p(n = 45).

radiation effects was tested with the Mann–Whitney U test. Tominimize difference due to fractionation and timing of radiother-apy we account for BED at the time of toxicity appearance. Theprobability of not developing higher scores of acute reactions radi-ation (CTC P 2) was analyzed in relation to BED, using the Kaplan–Meier method [30]. Comparison of BED curves was made using thelog-rank test. Hazard ratios (HRs) and their 95% confidence intervalwere determined using the Cox proportional hazard model [8,31].A probability level of less than 0.05 was used as the criterion of sig-nificance. All the analyses were carried out using the SPSS 17.0software package (SPSS INC, Chicago, IL).

Results

Study patients

Oral mucositis, dysphagia, and skin erythema were consideredthe clinical endpoints of this study. Study population consisted in81.2% male and 18.2% female. The primary tumor site was naso-pharynx in 9.9%, oropharynx in 28.7%, oral cavity in 18.8%, hypo-pharynx in 12.9%, larynx in 26.7%, sinuses in 2% and salivaryglands in 1% of patients. The 55% of patients underwent also che-motherapy. Chemotherapy schedules were based on platinumderivatives, taxanes, 5-fluorouracile and cetuximab alone or asso-ciated. Sixty-eight experienced grade P2 mucositis, 39 severe skinerythema, 12 high grade dysphagia.

Because a significant proportion of the study patients under-went CT, the effect of this treatment on the development of radia-tion-induced acute toxicities was investigated by sorting thepatients by each acute effect according to treatment.

Genotypes and alleles frequencies

All the genotype distributions were in equilibrium with Hardy–Weinberg law (data not shown). Frequencies of variant alleles were0.39 (XRCC11 c.1196A > G p.Gln399Arg), 0.48 (XRCC3 c.722C > Tp.Thr241Met), 0.25 (GSTP1 c.313A > G p.Ile105Val), 0.11 (RAD51c.-3429 G > C) and 0.44 (RAD51 c.-3392 G > T), consistently withpreviously reports in Caucasian populations [8,31,32].

among carriers of the wild-type genotype (G/G) and of the variant genotypes (G/A or5). SCCHN patients treated with radiotherapy or chemo-radiotherapy (n = 101).TCAE score P 2) after radiotherapy among carriers of the wild-type genotype (G/G).Gln399Arg (p log rank = 0.049). SCCHN patients treated with radiotherapy alone

Table 3Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequencydistribution of dysphagia after radiotherapy in SCCHN patients.

SNPs/genotypes Dysphagia

Grade p OR

<2 P2

XRCC1 c.1196 A > G GG 39 4 0.490 0.64AG 33 5 0.758 1.21AA 17 3 0.630 1.41AA + AG 50 8 0.490 1.56

XRCC3 c.722 C > T CC 28 3 0.649 0.73CT 36 7 0.240 2.06TT 25 2 0.401 0.51TT + CT 61 9 0.649 1.38

RAD51 c.-3429 G > C GG 75 7 0.031 0.26GC 12 4 0.077 3.21CC 2 1 0.244 3.95CC + GC 14 5 0.031 3.83

RAD51 c.-3392 G > T GG 31 2 0.208 0.37GT 42 6 0.855 1.12TT 16 4 0.210 2.28TT + GT 58 10 0.208 2.67

GSTP1 c.313 A > G AA 52 7 0.995 0.99AG 27 5 0.428 1.64

N. Pratesi et al. / Radiotherapy and Oncology 99 (2011) 356–361 359

Association between polymorphisms and the development of clinicalradiosensitivity

MucositisXRCC1-399Gln allele were significantly associated with higher

risk of mucositis (p = 0.011, OR = 3.01, CI = 1.27–7.11) (Table 1).The BEDmean was not significantly different between patientswho experienced grade P2 and <2 mucositis, suggesting that thedifferent severity of toxicity observed in this group was not relatedto BED. Patients receiving CT in addition to radiotherapy showed asignificantly higher mucositis score compared with subjects trea-ted with radiotherapy treatment only (p = 0.001). Based on this re-sult, this parameter was accounted in further analysis with regardto mucositis. The statistically significant association betweenXRCC1-399Gln allele and higher score mucositis was confirmedalso in a subgroup of patients treated with radiotherapy alone(p = 0.025, OR = 4.02, CI = 1.16–13.90).

As shown in Fig. 1 (a and b), the risk of mucositis was signifi-cantly increased in patients with XRCC1-399Gln allele genotypesboth in chemo-radiotherapy (p = 0.035, HR = 1.72, CI = 1.03–2.86)and in radiotherapy alone (p = 0.049, HR = 2.50, CI = 0.97–6.47)groups.

GG 10 / 0.221 0.88GG + AG 37 5 0.995 1.00

ErythemaPatients receiving chemo-radiotherapy showed a significantly

higher erythema grade compared with subjects treated with radio-therapy alone (p = 0.027). Based on these result, this parameterwas accounted in further analysis. XRCC1-399Gln allele showedan association with skin toxicity in patients treated with chemo-radiotherapy, but it was not statistically significant (p = 0.057,OR = 2.25, CI = 0.97–5.23). The trend was not confirmed in patientstreated with radiotherapy alone (p = 0.208) (Table 2).

DysphagiaThe risk of higher grade dysphagia was significantly increased

in RAD51 -3429 CC/CG patients (p = 0.031, OR = 3.83, CI = 1.06–13.79) (Table 3). No statistical difference was found for the BEDmean

between patients who experienced dysphagia grade <2 or P2. Nostatistical difference between patients receiving chemotherapy inaddition to radiotherapy and patients receiving radiotherapy alone

Table 2Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribut

SNPs/genotypes All patients (radiotherapy and chemother

Erythema grade p

<2 P2

XRCC1 c.1196 A > G GG 31 12 0.057AG 20 18 0.160AA 11 9 0.512AA + AG 31 27 0.057

XRCC3 c.722 C > T CC 21 10 0.383CT 26 17 0.870TT 15 12 0.467TT + CT 41 29 0.383

RAD51 c.-3429 G > C GG 49 33 0.485GC 11 5 0.510CC 2 1 0.669CC + GC 13 6 0.485

RAD51 c.-3392 G > T GG 20 13 0.911GT 31 17 0.530TT 11 9 0.512TT + GT 42 26 0.911

GSTP1 c.313 A > G AA 39 20 0.249AG 16 16 0.109GG 7 3 0.411GG + AG 23 19 0.249

Abbreviations: SNPs, single-nucleotide polymorphisms; <2 and P2, understood respect

was found for the dysphagia (p = 0.306). Based on these results,this parameter was not included in further analysis. As shown inFig. 2, RAD51 -3429 CC/CG genotypes were associated to a higherrisk of occurrence of dysphagia of grade P2 (p = 0.011 HR = 4.00,CI = 1.27–12.65).

Association between Xrcc1 c.1196 A > G/Rad51 c.-3429 G > C statusand the development of toxicities

We evaluated the significance of XRCC1 c.1196A > G/RAD51c.-3429 G > C polymorphisms status in the risk of grade P2 toxic-ities. All the SCCHN patients were subdivided according to thegrade CTCAE of radiation-induced toxicity in a manner irrespectiveof specific damage. Patients receiving chemotherapy in addition toradiotherapy showed a significantly higher grade toxicity com-pared with subjects treated with radiotherapy treatment only

ion of skin erythema after radiotherapy in SCCHN patients.

apy) Patients treated with radiotherapy alone

OR Erythema grade p OR

<2 P2

0.44 18 4 0.208 0.421.80 12 5 0.746 1.251.39 3 3 0.183 3.332.25 15 8 0.208 2.40.67 13 3 0.300 0.511.07 12 5 0.746 1.251.39 8 4 0.542 1.561.48 20 9 0.372 1.951.46 26 9 0.787 0.810.68 6 3 0.450 1.50.79 1 / 0.733 0.970.68 7 3 0.539 1.241.05 10 3 0.520 0.770.77 20 6 0.524 0.651.39 3 3 0.183 3.330.95 23 9 0.729 1.10.62 20 7 0.891 0.912.00 9 4 0.48 1.30.65 4 1 0.596 0.661.61 13 5 0.891 1.3

to erythema CTCAE score.

Fig. 2. Cumulative incidence of severe dysphagia (CTCAE score P2) after radio-therapy among the wild-type genotype (G/G) and of the variant genotypes (G/C orC/C), genetic polymorphism RAD51 c.-3429 G > C (p log rank = 0.011). SCCHNpatients treated with radiotherapy or chemo-radiotherapy (n = 101). BED = biolog-ically effective radiation dose.

360 SNPs and radiosensitivity in head & neck cancer

(p = 0.001). Based on these results, this parameter was accountedin further analysis. The association between grade P2 clinicaltoxicity end-points and the presence of at least one SNP or theco-presence of SNPs in both XRCC1-399Gln or RAD51 c.-3429G > C was demonstrated in our patients (p = 0.001, OR = 4.77,CI = 1.77–12.84) and was weakly confirmed in patients treatedwith radiotherapy alone (p = 0.079, OR = 3.02, CI = 0.86–10.52).No statistical difference was found for the BEDmean. The presenceof at least one SNP or the co-presence of both SNPs in XRCC1c.1196A > G/RAD51 c.-3429 G > C status were associated to higherlikelihood of occurrence of toxicities both in chemo-radiotherapyand radiotherapy patients (p = 0.004, HR = 2.03, CI = 1.23–3.34;p = 0.011, HR = 2.96, CI = 1.22–7.15) (Fig. 3).

Fig. 3. (a) Cumulative incidence of toxicities grade P2 after radiotherapy among carrieRAD51 c.-3429G > C polymorphisms status (p log rank = 0.004). SCCHN patients trearadiation dose. (b) Cumulative incidence of toxicities grade P2 after radiotherapy amc.1196A > G/RAD51 c.-3429 G > C polymorphisms status (p log rank = 0.011). SCCHN patdose.

Discussion

In this study we evaluated whether SNPs in RAD51, XRCC1,XRCC3 and GSTP1 genes, related to DNA repair and cellular protec-tion against oxidative stress mechanisms, could be responsible forthe biological variability in the clinical radiosensitivity in 101SCCHN patients. For this analysis we considered grade P2 toxici-ties as indicator of clinical radiosensitivity. To minimize differencedue to fractionation and timing of radiotherapy we account for BEDat the time of toxicity appearance.

Our findings demonstrated that SCCHN patients with the AA/AGgenotypes in XRCC1 c.1196A > G have a higher probability ofmucositis than those with GG genotype. Analysis of BED at theappearance of mucositis shows that patients with the XRCC1-399Gln allele experienced early mucosal toxicity compared withthose with the GG genotype. The association between highestgrade mucositis and XRCC1 c.1196-A allele was confirmed bothin chemo-radiotherapy patients and in those treated with radio-therapy alone, suggesting that this SNP may have a strong impactin radiosensitivity. Our hypothesis is that the XRCC1-399Gln alleleresults in lower levels of DNA repair capacity, thus leading to in-creased toxicity.

Our data provided evidence that RAD51 c.-3429 G > C couldplay a role to increase risk of dysphagia after RT. Patients withthe RAD51 c.-3429 C allele (CC/CG genotypes) experienced a gradeP2 toxicity at lower BED compared to patients with the GG geno-type. Likewise to XRCC1 c.1196 A > G, we hypothesized that RAD51c.-3429 G > C leads to a loss of efficiency in DNA repair process inSCCHN patients and to an increased toxicity in terms of dysphagia.

To our knowledge, this is the first report that demonstrates inSCCHN patients an association between XRCC1-399Gln and theoccurrence of mucositis and between RAD51 c.-3429 G > C andthe development of acute dysphagia after RT. Thus, different SNPscorrelate with different categories of acute toxicity; it seems rea-sonable since XRCC1 and RAD51 genes take part in two differentDNA repair mechanisms, the Base Excision Repair and the Homol-ogous Recombinational process respectively, and could be respon-sible for two distinct categories of clinical toxicity. Controversialdata still exist about the association between dysphagia and SNPs:

rs of neither SNP and carriers of one or both polymorphisms, XRCC1 c.1196A > G/ted with radiotherapy chemo-radiotherapy (n = 101). BED = biologically effectiveong carriers of neither SNP and carriers of one or both polymorphisms, XRCC1

ients treated with radiotherapy alone (n = 45). BED = biologically effective radiation

N. Pratesi et al. / Radiotherapy and Oncology 99 (2011) 356–361 361

as reported in Werbrouck et al. [16], an association between thedevelopment of this clinical reaction and XRCC3 p.Thr241Met hasbeen found, but we did not confirm this association. Moreoverthe study of Werbrouck, that involved IMRT-treated SCCHN pa-tients, failed to find an association between RAD51 and acute reac-tions. Further studies in a higher number of SCCHN patients arenecessary to clarify this critical issue. After all, apart from specificacute toxicity considered, the determination of XRCC1c.1196A > G/RAD51 c.-3429 G > C status in SCCHN patients identi-fies two categories of subjects with different capacity to toleratethe genotoxic damage induced by ionizing radiation. Thus, ourfindings showed that SCCHN patients with at least one SNP or withboth the SNPs in XRCC1 c.1196A > G or RAD51 c.-3429 G > C have ahigher occurrence of acute toxicities compared to patients with noSNPs. Several studies have shown the functional relevance of theseSNPs as markers of DNA damage [23,25,33–39]. Our findings couldsupport the strong value of these SNPs in the risk of developmentradiosensitivity. Because of the relatively small size of analyzed pa-tient population (n = 101), our findings require further validationin subsequent studies. However, our data provided relevant infor-mation on the value of SNPs in XRCC1 c.1196A > G and RAD51 c.-3429 G > C in SCCHN patients and may be useful tools toward indi-vidualizing of tailored treatment strategies.

Conflict of interest notification

No conflict of interest.

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