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Association between single nucleotide polymorphisms in the XRCC1 and RAD51 genes and clinical radiosensitivity in head and neck cancer
Radiotherapy and Oncology 99 (2011) 356–361
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Clinical radiobiology
Association between single nucleotide polymorphisms in the XRCC1 and 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
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Article history: Received 1 May 2011 Received in revised form 21 May 2011 Accepted 26 May 2011 Available online 23 June 2011 Keywords: Radiotherapy Toxicities Single nucleotide polymorphisms Head and neck cancer
a b s t r a c t Purpose: Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (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 the occurrence 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, XRCC3 c.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) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologically effective dose (BED). Results: Development of grade P2 mucositis was increased in all patients (chemo-radiotherapy and radiotherapy alone) with XRCC1-399Gln allele (HR = 1.72). The likelihood of developing grade P2 dysphagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR = 4.00). The presence of at least one SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G > C status were associated to higher likelihood of occurrence of acute toxicities (HR = 2.03). Conclusions: Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A > G and RAD51 c.-3429 G > C, and the development of radiation-induced toxicities in SCCHN patients. Ó 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 relatively common type of cancer, with a worldwide incidence of 500,000 cases each year [1]. Multiple treatments, including surgery, radiotherapy (RT) and chemotherapy (CT) have been developed for the anatomically different subtypes. Improvements in RT techniques with the introduction of three-dimensional conformal radiotherapy (3-DCRT) and intensity-modulated radiotherapy (IMRT) have increased the possibility of dose escalation and radiocurability [2]. Despite these improvements, the radiation dose necessary to achieve local tumor control is limited by the tolerance dose of normal tissues in the irradiation field. Treatment of SCCHN patients with RT can be significantly compromised by acute reactions and late damages occurring in the normal tissue exposed to radiation. Advances in radiation technology and dose-fractionation have ⇑ Corresponding author. Address: Radiotherapy Unit, Department of Clinical Physiopathology, Viale Morgagni 85, 50139 Florence, Italy. E-mail address: [email protected]fi.it (M. Mangoni). 0167-8140/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2011.05.062
changed the pattern of side effects, but there is considerable evidence that both treatment – and patient – related factors influence severity of damage to normal tissue after therapeutic RT [3]. Intrinsic factors of individual radiosensitivity can influence the variability of side effects observed [4–6]. In about 70% of SCCHN patients, clinical radiosensitivity should be considered as a direct effect of a cellular response in the irradiation field caused by a loss of efficiency in the DNA repair mechanism. Single nucleotide polymorphisms (SNPs) in DNA repair genes may alter protein function and individual’s capacity to repair damaged DNA. The presence of variants in specific genes, the products of which play a role in radiation response, could be associated to the development of the adverse radiation effects [7–14]. Various studies examined the effects of polymorphisms in DNA repair genes on RT response in terms of toxicity, but very few studies have examined the correlation between polymorphisms and radiation-induced toxicities in SCCHN [15,16]. Radiosensitivity candidate genes that have been linked to enhanced radiation responses include RAD51, XRCC1, XRCC3 and
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GSTP1. XRCC1 (X-ray repair cross-complementing 1) protein is a nick sensor and through its action is able to detect and to repair three of the most common forms of DNA damage induced by ionizing radiation: single strand breaks (SSB) repair, double strand breaks (DSB) repair and base-excision repair (BER) mechanisms [17–21]. A specific polymorphism of XRCC1 gene, c.1196A > G p.Gln399Arg, is a relatively common variant allele resulting in a protein with altered fidelity and repair capacity which consequently alters the radiosensitivity risk [22]. Moreover, subjects with XRCC1 AA genotypes have higher levels of chromosomal breaks per cell than those with other genotypes [23]. RAD51 (RecA homolog, Escherichia coli) and RAD51 paralog XRCC3 (X-ray repair complementary 3) proteins are components of the homologous recombinational (HR) repair of DNA doublestrand breaks and interstrand cross-links [24]. SNPs in these DSB repair genes could interfere with their functions making them suitable candidate genes to understand the genetic impact in acute normal tissue reactions after RT [25]. RT exerts its cytotoxic effects by producing reactive oxygen species (ROS), thus, it is plausible that genetic variants in genes involved in the protection from oxidative stress could influence susceptibility to acute side effects in subjects receiving RT. Glutathione-associated metabolism is a major mechanism for cellular protection against ROS and their toxic products, and glutathione S-transferases (GSTs) a, l, p, and h are active in detoxifying reactive bases and lipid peroxides produced by reactive oxidant damage to DNA and lipids, respectively [26]. GSTP1 (Glutathione S-transferase pi 1) are the GSTs isoforms mainly expressed in oral cavity and in pharynx. Single nucleotide substitutions A > G at base pair 313 of the GSTP1 locus, a member of the GST family, result in an aminoacid substitution of valine to isoleucine at codon 105, which decreases the enzyme activity [27]. On this basis, we evaluated the impact of XRCC1 c.1196A > G p.Gln399Arg, XRCC3 c.722C > T p.Thr241Met, RAD51 (c.-3429 G > C, c.-3392 G > T), and GSTP1 c.313A > G p.Ile105Val polymorphisms and the occurrence of acute radiation-induced toxicity in 101 SCCHN patients after RT.
Materials and methods Study patients and clinical data The study protocol was approved by the Local Ethics Committee. The entry criteria were the following: histologically proven HNSCC; no distant metastases when first diagnosed; no previous RT and/or CT; patients aged P18 years; availability for correct follow-up; informed consent obtained from each patient. Blood samples were collected before starting radiotherapy. Radiation-induced toxicities (oral mucositis, skin erythema and dysphagia) were documented during the treatment, and severity of acute side effects was assessed using the Common Toxicity Criteria for Adverse Events (CTCAE) of the U.S. NIH. [28]. Development of acute side effects of Grade 2 was considered to indicate increased sensitivity for acute effects in our study. Each toxicity was analyzed accounting to biologically effective radiation dose (BED) considering the time of the higher grade of toxicity development. BED was calculated to account for difference in fractionation and overall treatment time, using the formula:
BED ¼ ndð1 þ
d
c a
Þ ðT T0Þ;
a=b
given the number of fractions n, the fraction size of d, an a/b ratio of 10 Gy for acute reaction, a time factor c/a of 0,7 Gy/day, the overall treatment time T and a starting time for compensatory proliferation T0 of 21 days [29]. Overall 101 patients affected with HNSSC treated with RT at the Radiotherapy Units of Florence University, Brescia University and ASL4 Prato were enrolled in the study between June 2009 and September 2010. All patients underwent radiotherapy. The mean tumor dose was 62 Gy, (range 54–70 Gy). Irradiation was performed with photon beams from 6-MeV linear accelerators using 3D conformal radiation therapy or IMRT (Intensity Modulated Radiation Therapy) with SIB (simultaneous integrated boost). Fifty-six patients out of 101 received both radiotherapy plus chemotherapy. Sequential or concomitant chemotherapy schedules were based on derivatives of platinum, 5-fluorouracile, taxanes and/or cetuximab.
Table 1 Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribution of acute mucositis after radiotherapy in SCCHN patients. SNPs/genotypes
All patients (radiotherapy and chemotherapy) Mucositis grade
XRCC1 c.1196 A > G
XRCC3 c.722 C > T
RAD51 c.-3429 G > C
RAD51 c.-3392 G > T
GSTP1 c.313 A > G
GG AG AA AA + AG CC CT TT TT + CT GG GC CC CC + GC GG GT TT TT + GT AA AG GG GG + AG
<2
P2
20 7 6 13 12 12 9 21 28 4 1 5 7 19 7 26 20 9 4 13
23 31 14 45 19 31 18 49 54 12 2 14 26 29 13 42 39 23 6 29
p
0.011 0.018 0.776 0.011 0.389 0.379 0.932 0.389 0.512 0.476 0.699 0.512 0.087 0.159 0.804 0.087 0.756 0.507 0.603 0.756
Patients treated with radiotherapy alone OR
0.33 3.11 1.17 3.01 0.68 1.47 0.96 1.47 0.68 1.55 0.97 1.45 2.3 0.55 0.88 0.43 0.87 1.36 0.7 1.14
Mucositis grade <2
P2
15 6 2 8 10 6 7 13 18 4 1 5 6 13 4 17 14 7 2 9
7 11 4 15 6 11 5 16 17 5 / 5 7 13 2 15 13 6 3 9
Abbreviations: SNPs, single-nucleotide polymorphisms; <2 and P2, understood respect to mucositis CTCAE score.
p
OR
0.025 0.098 0.311 0.025 0.256 0.098 0.559 0.256 0.936 0.47 0.511 0.609 0.672 0.862 0.354 0.672 0.903 0.815 0.478 0.903
0.25 2.83 2.33 4.02 0.49 2.83 0.67 2.05 0.94 1.4 0.96 1.06 1.32 1.11 0.47 0.76 0.93 0.86 1.66 1.08
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SNPs and radiosensitivity in head & neck cancer
High resolution melting analysis For DNA exctraction, 3 ml of blood was collected in EDTA tubes from each patient. DNA was purified using the GENTRA DNA BLOOD Kit (Qiagen, Hilden, Germany). For the rapid and cost-limited detection of polymorphisms in SCCHN patients we developed accurate protocols based on high resolution melting analysis (HRMA). As initial step, 100 ng DNA were amplified in a final volume 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 SYTOÒ9 Dye (Invitrogen Corp., Carlsbad, CA), 1.25 U of Taq Gold Polymerase (Life Technology, Italy). PCR protocol was defined by an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of 60 s at 95 °C, 60 s at 60 °C and 60 s at 72 °C, with a final extension at 72 °C for 15 min. Amplification and HRMA were performed on RotorGene™6000 (Corbett Research, Australia). Primers were: Xrcc1 – 50 -CCCCAAGTACAGCCAGGTC-3’ (Forw) and 5’-CAGTCTGACTCCCCTCCAGA-30 (Rev); Xrcc3 – 50 -CCATTCCGCTGTGAATTTG-30 (Forw) and 50 CCGCATCCTGGCTAAAAATA-30 (Rev); Rad51 – 50 -GCTGGGAACTGCAACTCATCT-30 (Forw) and 50 - GCAGCGCTCCTCTCTCCAGC-30 (Rev); Gstp1 – 50 -CTCTATGGGAAGGACCAGCA-30 (Forw) and 50 GAAGCCCCTTTCTTTGTTCA-30 (Rev). HRMA protocol was defined by an initial denaturation step at 95 °C for 5 min, followed by reanneling 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 expected genotype frequencies using the v2 test. The effect of chemotherapy treatment on the development of adverse radiation reactions was evaluated by subdividing patients by each clinical end-point according to presence or absence of chemotherapy treatment using the v2 test. The crude association between clinical toxicity endpoints and SNPs genotypes was measured by odds ratio (OR) and its 95% confidence interval. The degree of significance was calculated using the v2 test, except in case of small sample size (<5), where Fisher0 s Exact test was used. The relation between dose parameter, derived from mean BED (BEDmean), and adverse
radiation effects was tested with the Mann–Whitney U test. To minimize difference due to fractionation and timing of radiotherapy we account for BED at the time of toxicity appearance. The probability of not developing higher scores of acute reactions radiation (CTC P 2) was analyzed in relation to BED, using the Kaplan– Meier method [30]. Comparison of BED curves was made using the log-rank test. Hazard ratios (HRs) and their 95% confidence interval were determined using the Cox proportional hazard model [8,31]. A probability level of less than 0.05 was used as the criterion of significance. All the analyses were carried out using the SPSS 17.0 software package (SPSS INC, Chicago, IL).
Results Study patients Oral mucositis, dysphagia, and skin erythema were considered the clinical endpoints of this study. Study population consisted in 81.2% male and 18.2% female. The primary tumor site was nasopharynx in 9.9%, oropharynx in 28.7%, oral cavity in 18.8%, hypopharynx in 12.9%, larynx in 26.7%, sinuses in 2% and salivary glands in 1% of patients. The 55% of patients underwent also chemotherapy. Chemotherapy schedules were based on platinum derivatives, taxanes, 5-fluorouracile and cetuximab alone or associated. Sixty-eight experienced grade P2 mucositis, 39 severe skin erythema, 12 high grade dysphagia. Because a significant proportion of the study patients underwent CT, the effect of this treatment on the development of radiation-induced acute toxicities was investigated by sorting the patients 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 were 0.39 (XRCC11 c.1196A > G p.Gln399Arg), 0.48 (XRCC3 c.722C > T p.Thr241Met), 0.25 (GSTP1 c.313A > G p.Ile105Val), 0.11 (RAD51 c.-3429 G > C) and 0.44 (RAD51 c.-3392 G > T), consistently with previously reports in Caucasian populations [8,31,32].
Fig. 1. (a) Cumulative incidence of acute mucositis (CTCAE score P2) after radiotherapy among carriers of the wild-type genotype (G/G) and of the variant genotypes (G/A or A/A), genetic polymorphism XRCC1 c.1196A > G p.Gln399Arg both (p log rank = 0.035). SCCHN patients treated with radiotherapy or chemo-radiotherapy (n = 101). BED = biologically effective radiation dose. (b) Cumulative incidence of acute mucositis (CTCAE score P 2) after radiotherapy among carriers of the wild-type genotype (G/G) and of the variant genotypes (G/A or A/A), genetic polymorphism XRCC1 c.1196A > G p.Gln399Arg (p log rank = 0.049). SCCHN patients treated with radiotherapy alone (n = 45).
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Association between polymorphisms and the development of clinical radiosensitivity Mucositis XRCC1-399Gln 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 patients who experienced grade P2 and <2 mucositis, suggesting that the different severity of toxicity observed in this group was not related to BED. Patients receiving CT in addition to radiotherapy showed a significantly higher mucositis score compared with subjects treated with radiotherapy treatment only (p = 0.001). Based on this result, this parameter was accounted in further analysis with regard to mucositis. The statistically significant association between XRCC1-399Gln allele and higher score mucositis was confirmed also 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 significantly increased in patients with XRCC1-399Gln allele genotypes both 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. Erythema Patients receiving chemo-radiotherapy showed a significantly higher erythema grade compared with subjects treated with radiotherapy alone (p = 0.027). Based on these result, this parameter was accounted in further analysis. XRCC1-399Gln allele showed an association with skin toxicity in patients treated with chemoradiotherapy, but it was not statistically significant (p = 0.057, OR = 2.25, CI = 0.97–5.23). The trend was not confirmed in patients treated with radiotherapy alone (p = 0.208) (Table 2). Dysphagia The 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. No statistical difference between patients receiving chemotherapy in addition to radiotherapy and patients receiving radiotherapy alone
Table 3 Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribution of dysphagia after radiotherapy in SCCHN patients. SNPs/genotypes
Dysphagia Grade
XRCC1 c.1196 A > G
GG AG AA AA + AG CC CT TT TT + CT GG GC CC CC + GC GG GT TT TT + GT AA AG GG GG + AG
XRCC3 c.722 C > T
RAD51 c.-3429 G > C
RAD51 c.-3392 G > T
GSTP1 c.313 A > G
<2
P2
39 33 17 50 28 36 25 61 75 12 2 14 31 42 16 58 52 27 10 37
4 5 3 8 3 7 2 9 7 4 1 5 2 6 4 10 7 5 / 5
p
OR
0.490 0.758 0.630 0.490 0.649 0.240 0.401 0.649 0.031 0.077 0.244 0.031 0.208 0.855 0.210 0.208 0.995 0.428 0.221 0.995
0.64 1.21 1.41 1.56 0.73 2.06 0.51 1.38 0.26 3.21 3.95 3.83 0.37 1.12 2.28 2.67 0.99 1.64 0.88 1.00
was found for the dysphagia (p = 0.306). Based on these results, this parameter was not included in further analysis. As shown in Fig. 2, RAD51 -3429 CC/CG genotypes were associated to a higher risk 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 status and the development of toxicities We evaluated the significance of XRCC1 c.1196A > G/RAD51 c.-3429 G > C polymorphisms status in the risk of grade P2 toxicities. All the SCCHN patients were subdivided according to the grade CTCAE of radiation-induced toxicity in a manner irrespective of specific damage. Patients receiving chemotherapy in addition to radiotherapy showed a significantly higher grade toxicity compared with subjects treated with radiotherapy treatment only
Table 2 Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribution of skin erythema after radiotherapy in SCCHN patients. SNPs/genotypes
All patients (radiotherapy and chemotherapy) Erythema grade
XRCC1 c.1196 A > G
XRCC3 c.722 C > T
RAD51 c.-3429 G > C
RAD51 c.-3392 G > T
GSTP1 c.313 A > G
GG AG AA AA + AG CC CT TT TT + CT GG GC CC CC + GC GG GT TT TT + GT AA AG GG GG + AG
<2
P2
31 20 11 31 21 26 15 41 49 11 2 13 20 31 11 42 39 16 7 23
12 18 9 27 10 17 12 29 33 5 1 6 13 17 9 26 20 16 3 19
p
0.057 0.160 0.512 0.057 0.383 0.870 0.467 0.383 0.485 0.510 0.669 0.485 0.911 0.530 0.512 0.911 0.249 0.109 0.411 0.249
Patients treated with radiotherapy alone OR
0.44 1.80 1.39 2.25 0.67 1.07 1.39 1.48 1.46 0.68 0.79 0.68 1.05 0.77 1.39 0.95 0.62 2.00 0.65 1.61
Erythema grade <2
P2
18 12 3 15 13 12 8 20 26 6 1 7 10 20 3 23 20 9 4 13
4 5 3 8 3 5 4 9 9 3 / 3 3 6 3 9 7 4 1 5
Abbreviations: SNPs, single-nucleotide polymorphisms; <2 and P2, understood respect to erythema CTCAE score.
p
OR
0.208 0.746 0.183 0.208 0.300 0.746 0.542 0.372 0.787 0.450 0.733 0.539 0.520 0.524 0.183 0.729 0.891 0.48 0.596 0.891
0.42 1.25 3.33 2.4 0.51 1.25 1.56 1.95 0.81 1.5 0.97 1.24 0.77 0.65 3.33 1.1 0.91 1.3 0.66 1.3
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SNPs and radiosensitivity in head & neck cancer
Discussion
Fig. 2. Cumulative incidence of severe dysphagia (CTCAE score P2) after radiotherapy among the wild-type genotype (G/G) and of the variant genotypes (G/C or C/C), genetic polymorphism RAD51 c.-3429 G > C (p log rank = 0.011). SCCHN patients treated with radiotherapy or chemo-radiotherapy (n = 101). BED = biologically effective radiation dose.
(p = 0.001). Based on these results, this parameter was accounted in further analysis. The association between grade P2 clinical toxicity end-points and the presence of at least one SNP or the co-presence of SNPs in both XRCC1-399Gln or RAD51 c.-3429 G > C was demonstrated in our patients (p = 0.001, OR = 4.77, CI = 1.77–12.84) and was weakly confirmed in patients treated with radiotherapy alone (p = 0.079, OR = 3.02, CI = 0.86–10.52). No statistical difference was found for the BEDmean. The presence of at least one SNP or the co-presence of both SNPs in XRCC1 c.1196A > G/RAD51 c.-3429 G > C status were associated to higher likelihood of occurrence of toxicities both in chemo-radiotherapy and 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).
In this study we evaluated whether SNPs in RAD51, XRCC1, XRCC3 and GSTP1 genes, related to DNA repair and cellular protection against oxidative stress mechanisms, could be responsible for the biological variability in the clinical radiosensitivity in 101 SCCHN patients. For this analysis we considered grade P2 toxicities as indicator of clinical radiosensitivity. To minimize difference due to fractionation and timing of radiotherapy we account for BED at the time of toxicity appearance. Our findings demonstrated that SCCHN patients with the AA/AG genotypes in XRCC1 c.1196A > G have a higher probability of mucositis than those with GG genotype. Analysis of BED at the appearance of mucositis shows that patients with the XRCC1399Gln allele experienced early mucosal toxicity compared with those with the GG genotype. The association between highest grade mucositis and XRCC1 c.1196-A allele was confirmed both in chemo-radiotherapy patients and in those treated with radiotherapy alone, suggesting that this SNP may have a strong impact in radiosensitivity. Our hypothesis is that the XRCC1-399Gln allele results in lower levels of DNA repair capacity, thus leading to increased toxicity. Our data provided evidence that RAD51 c.-3429 G > C could play a role to increase risk of dysphagia after RT. Patients with the RAD51 c.-3429 C allele (CC/CG genotypes) experienced a grade P2 toxicity at lower BED compared to patients with the GG genotype. Likewise to XRCC1 c.1196 A > G, we hypothesized that RAD51 c.-3429 G > C leads to a loss of efficiency in DNA repair process in SCCHN patients and to an increased toxicity in terms of dysphagia. To our knowledge, this is the first report that demonstrates in SCCHN patients an association between XRCC1-399Gln and the occurrence of mucositis and between RAD51 c.-3429 G > C and the development of acute dysphagia after RT. Thus, different SNPs correlate with different categories of acute toxicity; it seems reasonable since XRCC1 and RAD51 genes take part in two different DNA repair mechanisms, the Base Excision Repair and the Homologous Recombinational process respectively, and could be responsible for two distinct categories of clinical toxicity. Controversial data still exist about the association between dysphagia and SNPs:
Fig. 3. (a) Cumulative incidence of toxicities grade P2 after radiotherapy among carriers of neither SNP and carriers of one or both polymorphisms, XRCC1 c.1196A > G/ RAD51 c.-3429G > C polymorphisms status (p log rank = 0.004). SCCHN patients treated with radiotherapy chemo-radiotherapy (n = 101). BED = biologically effective radiation dose. (b) Cumulative incidence of toxicities grade P2 after radiotherapy among carriers of neither SNP and carriers of one or both polymorphisms, XRCC1 c.1196A > G/RAD51 c.-3429 G > C polymorphisms status (p log rank = 0.011). SCCHN patients treated with radiotherapy alone (n = 45). BED = biologically effective radiation dose.
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as reported in Werbrouck et al. [16], an association between the development of this clinical reaction and XRCC3 p.Thr241Met has been found, but we did not confirm this association. Moreover the study of Werbrouck, that involved IMRT-treated SCCHN patients, failed to find an association between RAD51 and acute reactions. Further studies in a higher number of SCCHN patients are necessary to clarify this critical issue. After all, apart from specific acute toxicity considered, the determination of XRCC1 c.1196A > G/RAD51 c.-3429 G > C status in SCCHN patients identifies two categories of subjects with different capacity to tolerate the genotoxic damage induced by ionizing radiation. Thus, our findings showed that SCCHN patients with at least one SNP or with both the SNPs in XRCC1 c.1196A > G or RAD51 c.-3429 G > C have a higher occurrence of acute toxicities compared to patients with no SNPs. Several studies have shown the functional relevance of these SNPs as markers of DNA damage [23,25,33–39]. Our findings could support the strong value of these SNPs in the risk of development radiosensitivity. Because of the relatively small size of analyzed patient population (n = 101), our findings require further validation in subsequent studies. However, our data provided relevant information 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 individualizing of tailored treatment strategies.
Conflict of interest notification No conflict of interest.
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Clinical radiobiology
Association between single nucleotide polymorphisms in the XRCC1 and 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
Article history: Received 1 May 2011 Received in revised form 21 May 2011 Accepted 26 May 2011 Available online 23 June 2011 Keywords: Radiotherapy Toxicities Single nucleotide polymorphisms Head and neck cancer
a b s t r a c t Purpose: Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (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 the occurrence 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, XRCC3 c.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) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologically effective dose (BED). Results: Development of grade P2 mucositis was increased in all patients (chemo-radiotherapy and radiotherapy alone) with XRCC1-399Gln allele (HR = 1.72). The likelihood of developing grade P2 dysphagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR = 4.00). The presence of at least one SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G > C status were associated to higher likelihood of occurrence of acute toxicities (HR = 2.03). Conclusions: Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A > G and RAD51 c.-3429 G > C, and the development of radiation-induced toxicities in SCCHN patients. Ó 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 relatively common type of cancer, with a worldwide incidence of 500,000 cases each year [1]. Multiple treatments, including surgery, radiotherapy (RT) and chemotherapy (CT) have been developed for the anatomically different subtypes. Improvements in RT techniques with the introduction of three-dimensional conformal radiotherapy (3-DCRT) and intensity-modulated radiotherapy (IMRT) have increased the possibility of dose escalation and radiocurability [2]. Despite these improvements, the radiation dose necessary to achieve local tumor control is limited by the tolerance dose of normal tissues in the irradiation field. Treatment of SCCHN patients with RT can be significantly compromised by acute reactions and late damages occurring in the normal tissue exposed to radiation. Advances in radiation technology and dose-fractionation have ⇑ Corresponding author. Address: Radiotherapy Unit, Department of Clinical Physiopathology, Viale Morgagni 85, 50139 Florence, Italy. E-mail address: [email protected]fi.it (M. Mangoni). 0167-8140/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2011.05.062
changed the pattern of side effects, but there is considerable evidence that both treatment – and patient – related factors influence severity of damage to normal tissue after therapeutic RT [3]. Intrinsic factors of individual radiosensitivity can influence the variability of side effects observed [4–6]. In about 70% of SCCHN patients, clinical radiosensitivity should be considered as a direct effect of a cellular response in the irradiation field caused by a loss of efficiency in the DNA repair mechanism. Single nucleotide polymorphisms (SNPs) in DNA repair genes may alter protein function and individual’s capacity to repair damaged DNA. The presence of variants in specific genes, the products of which play a role in radiation response, could be associated to the development of the adverse radiation effects [7–14]. Various studies examined the effects of polymorphisms in DNA repair genes on RT response in terms of toxicity, but very few studies have examined the correlation between polymorphisms and radiation-induced toxicities in SCCHN [15,16]. Radiosensitivity candidate genes that have been linked to enhanced radiation responses include RAD51, XRCC1, XRCC3 and
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GSTP1. XRCC1 (X-ray repair cross-complementing 1) protein is a nick sensor and through its action is able to detect and to repair three of the most common forms of DNA damage induced by ionizing radiation: single strand breaks (SSB) repair, double strand breaks (DSB) repair and base-excision repair (BER) mechanisms [17–21]. A specific polymorphism of XRCC1 gene, c.1196A > G p.Gln399Arg, is a relatively common variant allele resulting in a protein with altered fidelity and repair capacity which consequently alters the radiosensitivity risk [22]. Moreover, subjects with XRCC1 AA genotypes have higher levels of chromosomal breaks per cell than those with other genotypes [23]. RAD51 (RecA homolog, Escherichia coli) and RAD51 paralog XRCC3 (X-ray repair complementary 3) proteins are components of the homologous recombinational (HR) repair of DNA doublestrand breaks and interstrand cross-links [24]. SNPs in these DSB repair genes could interfere with their functions making them suitable candidate genes to understand the genetic impact in acute normal tissue reactions after RT [25]. RT exerts its cytotoxic effects by producing reactive oxygen species (ROS), thus, it is plausible that genetic variants in genes involved in the protection from oxidative stress could influence susceptibility to acute side effects in subjects receiving RT. Glutathione-associated metabolism is a major mechanism for cellular protection against ROS and their toxic products, and glutathione S-transferases (GSTs) a, l, p, and h are active in detoxifying reactive bases and lipid peroxides produced by reactive oxidant damage to DNA and lipids, respectively [26]. GSTP1 (Glutathione S-transferase pi 1) are the GSTs isoforms mainly expressed in oral cavity and in pharynx. Single nucleotide substitutions A > G at base pair 313 of the GSTP1 locus, a member of the GST family, result in an aminoacid substitution of valine to isoleucine at codon 105, which decreases the enzyme activity [27]. On this basis, we evaluated the impact of XRCC1 c.1196A > G p.Gln399Arg, XRCC3 c.722C > T p.Thr241Met, RAD51 (c.-3429 G > C, c.-3392 G > T), and GSTP1 c.313A > G p.Ile105Val polymorphisms and the occurrence of acute radiation-induced toxicity in 101 SCCHN patients after RT.
Materials and methods Study patients and clinical data The study protocol was approved by the Local Ethics Committee. The entry criteria were the following: histologically proven HNSCC; no distant metastases when first diagnosed; no previous RT and/or CT; patients aged P18 years; availability for correct follow-up; informed consent obtained from each patient. Blood samples were collected before starting radiotherapy. Radiation-induced toxicities (oral mucositis, skin erythema and dysphagia) were documented during the treatment, and severity of acute side effects was assessed using the Common Toxicity Criteria for Adverse Events (CTCAE) of the U.S. NIH. [28]. Development of acute side effects of Grade 2 was considered to indicate increased sensitivity for acute effects in our study. Each toxicity was analyzed accounting to biologically effective radiation dose (BED) considering the time of the higher grade of toxicity development. BED was calculated to account for difference in fractionation and overall treatment time, using the formula:
BED ¼ ndð1 þ
d
c a
Þ ðT T0Þ;
a=b
given the number of fractions n, the fraction size of d, an a/b ratio of 10 Gy for acute reaction, a time factor c/a of 0,7 Gy/day, the overall treatment time T and a starting time for compensatory proliferation T0 of 21 days [29]. Overall 101 patients affected with HNSSC treated with RT at the Radiotherapy Units of Florence University, Brescia University and ASL4 Prato were enrolled in the study between June 2009 and September 2010. All patients underwent radiotherapy. The mean tumor dose was 62 Gy, (range 54–70 Gy). Irradiation was performed with photon beams from 6-MeV linear accelerators using 3D conformal radiation therapy or IMRT (Intensity Modulated Radiation Therapy) with SIB (simultaneous integrated boost). Fifty-six patients out of 101 received both radiotherapy plus chemotherapy. Sequential or concomitant chemotherapy schedules were based on derivatives of platinum, 5-fluorouracile, taxanes and/or cetuximab.
Table 1 Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribution of acute mucositis after radiotherapy in SCCHN patients. SNPs/genotypes
All patients (radiotherapy and chemotherapy) Mucositis grade
XRCC1 c.1196 A > G
XRCC3 c.722 C > T
RAD51 c.-3429 G > C
RAD51 c.-3392 G > T
GSTP1 c.313 A > G
GG AG AA AA + AG CC CT TT TT + CT GG GC CC CC + GC GG GT TT TT + GT AA AG GG GG + AG
<2
P2
20 7 6 13 12 12 9 21 28 4 1 5 7 19 7 26 20 9 4 13
23 31 14 45 19 31 18 49 54 12 2 14 26 29 13 42 39 23 6 29
p
0.011 0.018 0.776 0.011 0.389 0.379 0.932 0.389 0.512 0.476 0.699 0.512 0.087 0.159 0.804 0.087 0.756 0.507 0.603 0.756
Patients treated with radiotherapy alone OR
0.33 3.11 1.17 3.01 0.68 1.47 0.96 1.47 0.68 1.55 0.97 1.45 2.3 0.55 0.88 0.43 0.87 1.36 0.7 1.14
Mucositis grade <2
P2
15 6 2 8 10 6 7 13 18 4 1 5 6 13 4 17 14 7 2 9
7 11 4 15 6 11 5 16 17 5 / 5 7 13 2 15 13 6 3 9
Abbreviations: SNPs, single-nucleotide polymorphisms; <2 and P2, understood respect to mucositis CTCAE score.
p
OR
0.025 0.098 0.311 0.025 0.256 0.098 0.559 0.256 0.936 0.47 0.511 0.609 0.672 0.862 0.354 0.672 0.903 0.815 0.478 0.903
0.25 2.83 2.33 4.02 0.49 2.83 0.67 2.05 0.94 1.4 0.96 1.06 1.32 1.11 0.47 0.76 0.93 0.86 1.66 1.08
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High resolution melting analysis For DNA exctraction, 3 ml of blood was collected in EDTA tubes from each patient. DNA was purified using the GENTRA DNA BLOOD Kit (Qiagen, Hilden, Germany). For the rapid and cost-limited detection of polymorphisms in SCCHN patients we developed accurate protocols based on high resolution melting analysis (HRMA). As initial step, 100 ng DNA were amplified in a final volume 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 SYTOÒ9 Dye (Invitrogen Corp., Carlsbad, CA), 1.25 U of Taq Gold Polymerase (Life Technology, Italy). PCR protocol was defined by an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of 60 s at 95 °C, 60 s at 60 °C and 60 s at 72 °C, with a final extension at 72 °C for 15 min. Amplification and HRMA were performed on RotorGene™6000 (Corbett Research, Australia). Primers were: Xrcc1 – 50 -CCCCAAGTACAGCCAGGTC-3’ (Forw) and 5’-CAGTCTGACTCCCCTCCAGA-30 (Rev); Xrcc3 – 50 -CCATTCCGCTGTGAATTTG-30 (Forw) and 50 CCGCATCCTGGCTAAAAATA-30 (Rev); Rad51 – 50 -GCTGGGAACTGCAACTCATCT-30 (Forw) and 50 - GCAGCGCTCCTCTCTCCAGC-30 (Rev); Gstp1 – 50 -CTCTATGGGAAGGACCAGCA-30 (Forw) and 50 GAAGCCCCTTTCTTTGTTCA-30 (Rev). HRMA protocol was defined by an initial denaturation step at 95 °C for 5 min, followed by reanneling 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 expected genotype frequencies using the v2 test. The effect of chemotherapy treatment on the development of adverse radiation reactions was evaluated by subdividing patients by each clinical end-point according to presence or absence of chemotherapy treatment using the v2 test. The crude association between clinical toxicity endpoints and SNPs genotypes was measured by odds ratio (OR) and its 95% confidence interval. The degree of significance was calculated using the v2 test, except in case of small sample size (<5), where Fisher0 s Exact test was used. The relation between dose parameter, derived from mean BED (BEDmean), and adverse
radiation effects was tested with the Mann–Whitney U test. To minimize difference due to fractionation and timing of radiotherapy we account for BED at the time of toxicity appearance. The probability of not developing higher scores of acute reactions radiation (CTC P 2) was analyzed in relation to BED, using the Kaplan– Meier method [30]. Comparison of BED curves was made using the log-rank test. Hazard ratios (HRs) and their 95% confidence interval were determined using the Cox proportional hazard model [8,31]. A probability level of less than 0.05 was used as the criterion of significance. All the analyses were carried out using the SPSS 17.0 software package (SPSS INC, Chicago, IL).
Results Study patients Oral mucositis, dysphagia, and skin erythema were considered the clinical endpoints of this study. Study population consisted in 81.2% male and 18.2% female. The primary tumor site was nasopharynx in 9.9%, oropharynx in 28.7%, oral cavity in 18.8%, hypopharynx in 12.9%, larynx in 26.7%, sinuses in 2% and salivary glands in 1% of patients. The 55% of patients underwent also chemotherapy. Chemotherapy schedules were based on platinum derivatives, taxanes, 5-fluorouracile and cetuximab alone or associated. Sixty-eight experienced grade P2 mucositis, 39 severe skin erythema, 12 high grade dysphagia. Because a significant proportion of the study patients underwent CT, the effect of this treatment on the development of radiation-induced acute toxicities was investigated by sorting the patients 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 were 0.39 (XRCC11 c.1196A > G p.Gln399Arg), 0.48 (XRCC3 c.722C > T p.Thr241Met), 0.25 (GSTP1 c.313A > G p.Ile105Val), 0.11 (RAD51 c.-3429 G > C) and 0.44 (RAD51 c.-3392 G > T), consistently with previously reports in Caucasian populations [8,31,32].
Fig. 1. (a) Cumulative incidence of acute mucositis (CTCAE score P2) after radiotherapy among carriers of the wild-type genotype (G/G) and of the variant genotypes (G/A or A/A), genetic polymorphism XRCC1 c.1196A > G p.Gln399Arg both (p log rank = 0.035). SCCHN patients treated with radiotherapy or chemo-radiotherapy (n = 101). BED = biologically effective radiation dose. (b) Cumulative incidence of acute mucositis (CTCAE score P 2) after radiotherapy among carriers of the wild-type genotype (G/G) and of the variant genotypes (G/A or A/A), genetic polymorphism XRCC1 c.1196A > G p.Gln399Arg (p log rank = 0.049). SCCHN patients treated with radiotherapy alone (n = 45).
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Association between polymorphisms and the development of clinical radiosensitivity Mucositis XRCC1-399Gln 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 patients who experienced grade P2 and <2 mucositis, suggesting that the different severity of toxicity observed in this group was not related to BED. Patients receiving CT in addition to radiotherapy showed a significantly higher mucositis score compared with subjects treated with radiotherapy treatment only (p = 0.001). Based on this result, this parameter was accounted in further analysis with regard to mucositis. The statistically significant association between XRCC1-399Gln allele and higher score mucositis was confirmed also 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 significantly increased in patients with XRCC1-399Gln allele genotypes both 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. Erythema Patients receiving chemo-radiotherapy showed a significantly higher erythema grade compared with subjects treated with radiotherapy alone (p = 0.027). Based on these result, this parameter was accounted in further analysis. XRCC1-399Gln allele showed an association with skin toxicity in patients treated with chemoradiotherapy, but it was not statistically significant (p = 0.057, OR = 2.25, CI = 0.97–5.23). The trend was not confirmed in patients treated with radiotherapy alone (p = 0.208) (Table 2). Dysphagia The 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. No statistical difference between patients receiving chemotherapy in addition to radiotherapy and patients receiving radiotherapy alone
Table 3 Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribution of dysphagia after radiotherapy in SCCHN patients. SNPs/genotypes
Dysphagia Grade
XRCC1 c.1196 A > G
GG AG AA AA + AG CC CT TT TT + CT GG GC CC CC + GC GG GT TT TT + GT AA AG GG GG + AG
XRCC3 c.722 C > T
RAD51 c.-3429 G > C
RAD51 c.-3392 G > T
GSTP1 c.313 A > G
<2
P2
39 33 17 50 28 36 25 61 75 12 2 14 31 42 16 58 52 27 10 37
4 5 3 8 3 7 2 9 7 4 1 5 2 6 4 10 7 5 / 5
p
OR
0.490 0.758 0.630 0.490 0.649 0.240 0.401 0.649 0.031 0.077 0.244 0.031 0.208 0.855 0.210 0.208 0.995 0.428 0.221 0.995
0.64 1.21 1.41 1.56 0.73 2.06 0.51 1.38 0.26 3.21 3.95 3.83 0.37 1.12 2.28 2.67 0.99 1.64 0.88 1.00
was found for the dysphagia (p = 0.306). Based on these results, this parameter was not included in further analysis. As shown in Fig. 2, RAD51 -3429 CC/CG genotypes were associated to a higher risk 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 status and the development of toxicities We evaluated the significance of XRCC1 c.1196A > G/RAD51 c.-3429 G > C polymorphisms status in the risk of grade P2 toxicities. All the SCCHN patients were subdivided according to the grade CTCAE of radiation-induced toxicity in a manner irrespective of specific damage. Patients receiving chemotherapy in addition to radiotherapy showed a significantly higher grade toxicity compared with subjects treated with radiotherapy treatment only
Table 2 Association of XRCC1, XRCC3, RAD51 and GSTP1 polymorphisms with frequency distribution of skin erythema after radiotherapy in SCCHN patients. SNPs/genotypes
All patients (radiotherapy and chemotherapy) Erythema grade
XRCC1 c.1196 A > G
XRCC3 c.722 C > T
RAD51 c.-3429 G > C
RAD51 c.-3392 G > T
GSTP1 c.313 A > G
GG AG AA AA + AG CC CT TT TT + CT GG GC CC CC + GC GG GT TT TT + GT AA AG GG GG + AG
<2
P2
31 20 11 31 21 26 15 41 49 11 2 13 20 31 11 42 39 16 7 23
12 18 9 27 10 17 12 29 33 5 1 6 13 17 9 26 20 16 3 19
p
0.057 0.160 0.512 0.057 0.383 0.870 0.467 0.383 0.485 0.510 0.669 0.485 0.911 0.530 0.512 0.911 0.249 0.109 0.411 0.249
Patients treated with radiotherapy alone OR
0.44 1.80 1.39 2.25 0.67 1.07 1.39 1.48 1.46 0.68 0.79 0.68 1.05 0.77 1.39 0.95 0.62 2.00 0.65 1.61
Erythema grade <2
P2
18 12 3 15 13 12 8 20 26 6 1 7 10 20 3 23 20 9 4 13
4 5 3 8 3 5 4 9 9 3 / 3 3 6 3 9 7 4 1 5
Abbreviations: SNPs, single-nucleotide polymorphisms; <2 and P2, understood respect to erythema CTCAE score.
p
OR
0.208 0.746 0.183 0.208 0.300 0.746 0.542 0.372 0.787 0.450 0.733 0.539 0.520 0.524 0.183 0.729 0.891 0.48 0.596 0.891
0.42 1.25 3.33 2.4 0.51 1.25 1.56 1.95 0.81 1.5 0.97 1.24 0.77 0.65 3.33 1.1 0.91 1.3 0.66 1.3
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SNPs and radiosensitivity in head & neck cancer
Discussion
Fig. 2. Cumulative incidence of severe dysphagia (CTCAE score P2) after radiotherapy among the wild-type genotype (G/G) and of the variant genotypes (G/C or C/C), genetic polymorphism RAD51 c.-3429 G > C (p log rank = 0.011). SCCHN patients treated with radiotherapy or chemo-radiotherapy (n = 101). BED = biologically effective radiation dose.
(p = 0.001). Based on these results, this parameter was accounted in further analysis. The association between grade P2 clinical toxicity end-points and the presence of at least one SNP or the co-presence of SNPs in both XRCC1-399Gln or RAD51 c.-3429 G > C was demonstrated in our patients (p = 0.001, OR = 4.77, CI = 1.77–12.84) and was weakly confirmed in patients treated with radiotherapy alone (p = 0.079, OR = 3.02, CI = 0.86–10.52). No statistical difference was found for the BEDmean. The presence of at least one SNP or the co-presence of both SNPs in XRCC1 c.1196A > G/RAD51 c.-3429 G > C status were associated to higher likelihood of occurrence of toxicities both in chemo-radiotherapy and 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).
In this study we evaluated whether SNPs in RAD51, XRCC1, XRCC3 and GSTP1 genes, related to DNA repair and cellular protection against oxidative stress mechanisms, could be responsible for the biological variability in the clinical radiosensitivity in 101 SCCHN patients. For this analysis we considered grade P2 toxicities as indicator of clinical radiosensitivity. To minimize difference due to fractionation and timing of radiotherapy we account for BED at the time of toxicity appearance. Our findings demonstrated that SCCHN patients with the AA/AG genotypes in XRCC1 c.1196A > G have a higher probability of mucositis than those with GG genotype. Analysis of BED at the appearance of mucositis shows that patients with the XRCC1399Gln allele experienced early mucosal toxicity compared with those with the GG genotype. The association between highest grade mucositis and XRCC1 c.1196-A allele was confirmed both in chemo-radiotherapy patients and in those treated with radiotherapy alone, suggesting that this SNP may have a strong impact in radiosensitivity. Our hypothesis is that the XRCC1-399Gln allele results in lower levels of DNA repair capacity, thus leading to increased toxicity. Our data provided evidence that RAD51 c.-3429 G > C could play a role to increase risk of dysphagia after RT. Patients with the RAD51 c.-3429 C allele (CC/CG genotypes) experienced a grade P2 toxicity at lower BED compared to patients with the GG genotype. Likewise to XRCC1 c.1196 A > G, we hypothesized that RAD51 c.-3429 G > C leads to a loss of efficiency in DNA repair process in SCCHN patients and to an increased toxicity in terms of dysphagia. To our knowledge, this is the first report that demonstrates in SCCHN patients an association between XRCC1-399Gln and the occurrence of mucositis and between RAD51 c.-3429 G > C and the development of acute dysphagia after RT. Thus, different SNPs correlate with different categories of acute toxicity; it seems reasonable since XRCC1 and RAD51 genes take part in two different DNA repair mechanisms, the Base Excision Repair and the Homologous Recombinational process respectively, and could be responsible for two distinct categories of clinical toxicity. Controversial data still exist about the association between dysphagia and SNPs:
Fig. 3. (a) Cumulative incidence of toxicities grade P2 after radiotherapy among carriers of neither SNP and carriers of one or both polymorphisms, XRCC1 c.1196A > G/ RAD51 c.-3429G > C polymorphisms status (p log rank = 0.004). SCCHN patients treated with radiotherapy chemo-radiotherapy (n = 101). BED = biologically effective radiation dose. (b) Cumulative incidence of toxicities grade P2 after radiotherapy among carriers of neither SNP and carriers of one or both polymorphisms, XRCC1 c.1196A > G/RAD51 c.-3429 G > C polymorphisms status (p log rank = 0.011). SCCHN patients treated with radiotherapy alone (n = 45). BED = biologically effective radiation dose.
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as reported in Werbrouck et al. [16], an association between the development of this clinical reaction and XRCC3 p.Thr241Met has been found, but we did not confirm this association. Moreover the study of Werbrouck, that involved IMRT-treated SCCHN patients, failed to find an association between RAD51 and acute reactions. Further studies in a higher number of SCCHN patients are necessary to clarify this critical issue. After all, apart from specific acute toxicity considered, the determination of XRCC1 c.1196A > G/RAD51 c.-3429 G > C status in SCCHN patients identifies two categories of subjects with different capacity to tolerate the genotoxic damage induced by ionizing radiation. Thus, our findings showed that SCCHN patients with at least one SNP or with both the SNPs in XRCC1 c.1196A > G or RAD51 c.-3429 G > C have a higher occurrence of acute toxicities compared to patients with no SNPs. Several studies have shown the functional relevance of these SNPs as markers of DNA damage [23,25,33–39]. Our findings could support the strong value of these SNPs in the risk of development radiosensitivity. Because of the relatively small size of analyzed patient population (n = 101), our findings require further validation in subsequent studies. However, our data provided relevant information 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 individualizing of tailored treatment strategies.
Conflict of interest notification No conflict of interest.
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