- Email: [email protected]
Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation
Radiotherapy and Oncology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Original article
Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation Jian Zhang a,b,c, Baosheng Li a,b,⇑, Xiuping Ding a,b, Mingping Sun a,b, Hongsheng Li a,b, Ming Yang d, Changchun Zhou a,b, Haiying Yu e, Hong Liu f, Gongqi Yu f a
Department of Radiation Oncology, Shandong Cancer Hospital, Shandong Academy of Medical Sciences, Jinan; b Shandong’s Key Laboratory of Radiation Oncology, Jinan; Department of Radiation Oncology, Cancer Hospital, Tianjin Medical University; d College of Life Science and Technology, Beijing University of Chemical Technology; e Department of Radiology, Shandong Cancer Hospital, Jinan; and f Shandong Provincial Institute of Dermatology and Venereology, Jinan, PR China
c
a r t i c l e
i n f o
Article history: Received 18 October 2012 Received in revised form 2 February 2014 Accepted 9 March 2014 Available online xxxx Keywords: Single nucleotide polymorphism Inducible nitric oxide synthase Radiation-induced lung injury Lung cancer Radiotherapy
a b s t r a c t Background and purpose: Nitric oxide (NO), mainly synthesized by inducible nitric oxide synthase (NOS2) in pathological conditions, plays an important role in cytotoxicity, inflammation and fibrosis. Elevations in exhaled NO after thoracic radiation have been reported to predict radiation-induced lung injury (RILI). This study examined whether genetic variations in NOS2 gene is associated with the risk of RILI. Material and methods: A cohort of 301 patients between 2009 and 2011 were genotyped for 21 single nucleotide polymorphisms (SNPs) in the NOS2 gene by the Sequenom MassArray system. Kaplan–Meier cumulative probability was used to assess RILI risk and Cox proportional hazards analyses were performed to evaluate the effect of NOS2 genotypes on RILI. Results: Multivariate analysis found that three SNPs (rs2297518, rs1137933 and rs16949) in NOS2 were significantly associated with risk of RILI P 2 (P value = 0.001, 0.000092, 0.001, respectively) after adjusting for other covariates. Their associations were independent of radiation dose and mean lung dose. Further haplotype analysis indicated that the ATC haplotype of three SNPs is associated with reducing the risk of developing RILI. Conclusion: Our results demonstrate that genetic variants of NOS2 may serve as a reliable predictor of RILI in lung cancer patients treated with thoracic radiation. Ó 2014 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2014) xxx–xxx
Currently, radiotherapy is one of the main treatment modalities in lung cancer, contributing to both its cure and palliation. However, radiation-induced lung injury (RILI), one of the most common dose-limiting toxicities of thoracic radiotherapy, might compromise the success of current efforts regarding lung cancer treatment intensification [1]. Recently, many studies have investigated and identified clinical and dosimetric factors that may influence RILI risk, including mean lung dose (MLD) [2], percent lung volume receiving more than a threshold radiation dose (VDose) [3], performance status and pulmonary function [4], chronic obstructive pulmonary disease [5], concurrent chemotherapy [6], etc. However, only some patients in whom normal lung is exposed to a certain dose and volume of irradiation develop RILI even after stratifying
⇑ Corresponding author at: Department of Radiation Oncology, Shandong Cancer Hospital, 440, Jiyan Road, Jinan, PR China. E-mail address: [email protected] (B. Li).
for smoking status [7], suggesting that genetic variants may play a critical role in RILI development. Radiation pneumonitis and pulmonary fibrosis represent acute and late phases in the development of RILI. Recent studies suggest that endogenous nitric oxide (NO) formation by the inducible nitric oxide synthase (iNOS or Nos2) after irradiation may be closely associated with the occurrence and development of RILI [8–11]. Nitric oxide, a reactive radical and proinflammatory mediator, mainly synthesized from iNOS in pathological conditions, has been reported to display diverse biological activities in lung tissues, including cytotoxicity [12], inflammation [13], and fibrosis [14,15]. NOS2 gene is present on the human chromosome 17q11.2–12. The gene product iNOS is commonly absent in resting cells, but it is capable of being rapidly expressed in response to inflammatory stimuli, such as cytokines [16]. In addition, elevations in exhaled NO at the end of thoracic radiation were found in symptomatic RILI in esophageal or lung cancer patients. Recently, several studies have reported that genetic susceptibility
http://dx.doi.org/10.1016/j.radonc.2014.03.001 0167-8140/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
2
Variants in NOS2 gene are associated with RILI
factors, such as TGF-b [17], ATM [18], P53 [19], DNA double-strand breaks repair gene [20], and inflammation-related genes [21] play a role in RILI development. However, to our knowledge, the influences of NOS2 variants on RILI have not been reported so far. In this study, we hypothesized that SNPs of the NOS2 gene are biomarkers for predicting susceptibility to RILI among patients with lung cancer who receive thoracic radiation. To test this hypothesis, 21 SNPs of NOS2 were performed in this study. The aim is to figure out the associations between RILI risk and common variants of NOS2 in lung cancer patients, and to predict RILI susceptibility before the initiation of radiation therapy.
2006), as follows: Grade 0, no change; Grade 1, asymptomatic radiographic findings only; Grade 2, symptomatic, not interfering with activities of daily living; Grade 3, symptomatic, interfering with activities of daily living, oxygen required; Grade 4, life-threatening ventilatory support required; and Grade 5 if the patient died from RILI. The time to the endpoint development was based on the duration from the beginning of radiation treatment to occurrence of toxicity, and patients who did not experience the endpoint were censored at the last follow-up.
SNP selection and genotyping Materials and methods Patient population and clinical data collection In this study, a cohort of 301 newly diagnosed lung cancer patients were treated with definitive radiation between December 2009 and January 2011 at Shandong Cancer Hospital (Jinan). The eligible patients were those with histologically or cytologically confirmed lung cancer, Karnofsky performance status (KPS) P60, and an expected survival >6 months. Each patient signed an informed consent before starting therapy. Detailed clinical information and dosimetric factors (MLD, V20) were collected. This study was approved by the Shandong Cancer Hospital institutional review board. We complied with Health Insurance Portability and Accountability Act regulations. Follow-up and evaluation of RILI All patients included in this study were examined and evaluated by their radiation oncologists weekly during radiotherapy and 4–6 weeks after completion of treatment. Patients were then followed up every 3 months for the first 3 years unless they had symptoms that required immediate examination or intervention. Radiographic examination by chest X-ray or computerized tomography was performed at each follow-up visit after completion of treatment. RILI was assessed by three radiation oncologists according to the symptoms and imaging information of each patient, and graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 3.0 (August 9,
Table 1 Characteristics of 21 SNPs in the NOS2 gene. SNP ID
Chromosome
Position
Allele
Function class
rs2297515 rs2297518
17 17
23117460 23120724
A/C A/G
rs2779248 rs3794763 rs4795067 rs8072199 rs2314809 rs7208775 rs16949 rs3730013 rs9906835 rs11080344 rs944722 rs944725 rs1137933
17 17 17 17 17 17 17 17 17 17 17 17 17
23151959 23135353 23130802 23140975 23119505 23109235 23148826 23150045 23113501 23128638 23116164 23133698 23130059
C/T A/G A/G C/T C/T C/G C/T C/T A/G C/T C/T C/T C/T
rs2072324 rs2248814 rs2255929 rs2314810 rs10459953 rs17722851
17 17 17 17 17 17
23141023 23124448 23112094 23128237 23151645 23134963
A/C A/G A/T C/G C/G A/T
Intron CDSnonsynonymous Promoter Intron Intron Intron Intron Intron Intron Intron Intron Intron Intron Intron CDSsynonymous Intron Intron Intron Intron 50 UTR Intron
0
0
Abbreviations: 5 UTR, 5 untranslational region; CDS, Coding Sequence.
tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP
We chose tag SNP through the tagger software included in the Haploview software 4.2 and the common SNPs based on previous literature in NOS2. The tag SNP was selected based on ability to tag surrounding variants (NOS2, chr17) in the Han Chinese panel (Beijing, China) of the International HapMap project, NCBI build B36 assembly HapMap phase II + III (http://www.hapmap.org). The tag SNP was obtained with a minor allele frequency (MAF) of
Table 2 Baseline clinical characteristics of patients (n = 301). Characteristic Sex Female Male Age, years Median Range KPS 90–100 80 <80 Histopathology Squamous cell Adenocarcinoma NSCLC, NOS SCLC Tumor location Peripheral Central Clinical stage I–II III IV Smoking status Nonsmoker CSI P 600 CSI < 600 COPD Yes No Treatment RT C + RT CRT C + CRT Radiation technique 2-Dimensional RT 3D-CRT IMRT Radiation dose, Gy Median Range MLD, Gy (n = 259) Median Range V20 of both lungs, % Median Range
No.
%
69 232
22.9 77.1 60 25–87
152 111 38
50.5 36.9 12.6
105 70 69 57
34.9 23.3 22.9 18.9
169 132
56.1 43.9
28 151 122
9.3 50.2 40.5
77 132 92
25.6 43.9 30.6
42 259
14.0 86.0
24 126 24 127
8.0 41.9 8.0 42.2
42 163 96
14.0 54.0 32.0 59.6 40–80.6 14.6 3.2–35.6 26 2–75
Abbreviations: NOS, not otherwise specified; CSI, cigarette-smoke index; CRT, concurrent chemoradiation; 3D-CRT, three-dimensional conformal radiation therapy; IMRT, intensity modulated radiotherapy.
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
3
J. Zhang et al. / Radiotherapy and Oncology xxx (2014) xxx–xxx
0.05, a pairwise correlation coefficient r2 of 0.80. The characteristics of 21 selected SNPs were shown in Table 1. A 5 mL wholeblood sample was obtained from each patient before starting therapy, and genomic DNA was extracted from whole-blood cells using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen, USA). Genotyping analyses were conducted by using the Sequenom MassArray system (San Diego, USA). Statistical analysis The end point for this analysis was the development of RILI grade P2. Statistical analysis was performed using IBM SPSS 19.0. Student’s T-test or chi-square test was used to examine the differences in clinical characteristics. Hazard ratios (HRs) with 95% confidence intervals (CIs) of genotype for RILI were computed by the Cox proportional hazards model. In addition, further multivariate Cox regression analysis was performed to adjust other covariates. Kaplan–Meier analysis was performed to calculate the estimated cumulative RILI probability. Comparisons were made with the log-rank test. The Bonferroni method was used to adjust for multiple comparisons. A P value of 0.05 or less was considered statistically significant in two-tails of the T-test. Results Patient characteristics and association with RILI Table 2 lists the baseline clinical characteristics of the 301 patients, of whom 92.0% (N = 277) received combined platinumbased chemoradiotherapy. Of 277 patients, 57 (20.6%) patients received an etopside/cisplatin regimen and 220 (79.4%) patients received a gemcitabine/cisplatin or taxane/cisplatin or vinorelbine/ cisplatin regimen. The median radiation dose was 59.6 Gy (range, 40–80.6 Gy); 82.4% (N = 248) of patients received 50–70 Gy. By the time of the final analysis (January 2012), the median follow-up time had been 16 months (range, 3–25). After radiotherapy, 81 patients (26.9%) had PGrade 2 RILI (Grade 2, 3, and 5 were observed in 48, 28 and 5 patients, respectively). The median time in the development of RILI was 2 months. Table 3 shows the associations between patient-, tumor-, therapy-related characteristics and RILI grade P2 by univariate and multivariate analyses. Our results demonstrate that sex and radiation dosimetric factors (radiation dose, MLD, V20) are associated with RILI in univariate and multivariate analyses. Univariate and multivariate analyses Among the 21 SNPs studied, a total of four SNPs in NOS2 were found to be significantly associated with risk of RILI (grade P2)
Table 3 Associations Between patient-, tumor-, and therapy-related characteristics and RILI Grade P2. Parameter
Univariate analysis HR
Sex Female 1.000 Male 1.935 Age, years <65 1.000 P65 1.048 KPS 90–100 1.000 80 0.698 <80 0.839 Stage IV 1.000 I, II, III 1.037 COPD No 1.000 Yes 1.141 Tumor location Central 1.000 Peripheral 1.459 Histology Squamous cell 1.000 Adenocarcinoma 1.142 SCLC 1.082 NSCLC, NOS 1.190 Tobacco use Never 1.000 CSI < 600 0.571 CSI P 600 1.114 CRT No 1.000 Yes 0.867 Radiation dose, Gy <60 1.000 P60 1.774 MLD, Gy* (n = 259) <17.7 1.000 P17.7 2.051 V20 (n = 259) < 30% <26% 1.000 26–37% 1.147 P37% 2.568
Multivariate analysis
95% CI
P
1.023–3.659
HR
95% CI
P
1.000 0.042 2.099
1.101–4.003
0.024
0.663–1.656
1.000 0.840 1.140
0.687–1.889
0.612
0.425–1.146 0.423–1.665
1.000 0.155 0.661 0.616 0.759
0.386–1.132 0.361–1.594
0.131 0.466
0.662–1.625
1.000 0.875 0.981
0.597–1.612
0.939
0.617–2.110
1.000 0.674 1.370
0.660–2.841
0.398
0.920–2.314
1.000 0.108 1.484
0.866–2.543
0.151
0.629–2.074 0.570–2.052 0.661–2.144
1.000 0.662 1.478 0.810 1.514 0.561 1.063
0.743–2.941 0.748–3.065 0.575–1.966
0.266 0.249 0.846
0.203–1.606 0.497–2.499
1.000 0.288 0.642 0.793 1.110
0.319–1.293 0.629–1.959
0.215 0.719
0.557–1.349
1.000 0.527 1.064
0.658–1.720
0.802
1.126 to 2.795
1.000 0.013 1.717
1.033–2.852
0.037
1.275–3.298
1.000 0.003 1.829
1.072–3.119
0.027
0.709–1.854 1.318–5.004
1.000 0.577 1.1519 0.690–1.918 0.006 2.475 1.193–5.136
0.590 0.015
NOTE: Multivariate analyses were adjusted for all factors listed in this table. Abbreviations: NOS, not otherwise specified; CSI, cigarette-smoke index; CRT, concurrent chemoradiation. * Either total radiation dose, MLD or V20 was used in multivariate analyses, but not together.
in the univariate Cox regression analyses (Table 4). In multivariate analysis, this effect is virtually unchanged after adjustment for other covariates (P value = 0.001, 0.000092, 0.024, 0.001, respectively). After further Bonferroni corrections (P value = 0.019,
Table 4 Associations between NOS2 genotypes and Grade P2 RILI. Polymorphisms and genotypes
rs2297518 AA or AG GG rs1137933 CC CT or TT rs3794763 AA AG or GG rs16949 CC or CT TT
No. of patients
300 68 232 300 231 69 301 33 268 300 73 227
Events
Univariate analysis (Crude)
Multivariate analysis (Adjusted)
HR
95% CI
P
HR
95% CI
P
10 70
1 2.551
1.274–5.107
.008
1 3.585
1.711–7.510
72 8
1 0.321
0.154–0.666
.002
1 0.209
0.095–0.458
14 66
1 0.516
0.300–0.919
.025
1 0.488
0.262–0.909
.024
10 69
1 2.480
1.277–4.814
.007
1 3.481
1.707–7.099
.001
.001
9.2E05
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
4
Variants in NOS2 gene are associated with RILI
In addition, Table 4 lists the Kaplan–Meier incidences of grade P2 in patients with different genotypes of 4 SNPs at 12 months from starting radiation. In general, RILI developed more often in patients exhibiting GG in rs2297518, CC in rs1137933, AA in rs3794763, or TT in rs16949 genotypes, with RILI rates of 30.2%, 31.2%, 42.4%, and 30.3%, respectively, than in those having AG/AA in rs2297518, CT/TT in rs1137933, AG/GG in rs3794763, or CC/CT in rs16949 genotypes, for which the incidences were 14.7%, 11.6%, 24.6%, and 13.7%, respectively. Cumulative probability of RILI grade P2 as a function of time from the beginning of radiotherapy by genotypes in rs2297518, rs1137933, and rs16949 was shown in Fig. 1. Subgroup analysis for dosimetric factors Since dosimetric factors were also independent risk factors for RILI, we then investigated the subgroup analysis results by radiation dose and MLD. The cumulative RILI grade P2 probability as a function of time was analyzed according to genotypes and radiation dose (Supplementary files-Fig. 1A–C), MLD (Supplementary files-Fig. 1D–F). The results suggest that the effect of the genetic variants on RILI grade P2 was independent on dosimetric parameters. Haplotype analysis Since rs2297518, rs1137933, and rs16949 are in strong linkage disequilibrium (r2 = 0.57–0.73), we further constructed three-marker haplotypes (Supplemental file-Table 1). Compared to the most common GCT haplotype, only the ATC haplotype is associated with reducing risk of developing RILI grade P2, with the adjusted OR being 0.157 (95% CI, 0.047–0.522; P < 0.001). Discussion
Fig. 1. Cumulative probability of RILI grade P2 as a function of time from the beginning of radiotherapy by genotypes. (A) rs2297518; (B) rs1137933; (C) rs16949. The rs2297518:GG, rs1137933:CC and rs16949:TT genotype was associated with a statistically significant higher incidence of RILI compared with other genotypes, respectively.
0.0017, 0.019, respectively), three SNPs (rs2297518, rs1137933 and rs16949) remained significant for its association with RILI (grade P2).
The human NOS2 gene comprises 27 exons, exon 1–13 code for the oxygenase domain and exon 14–27 encode for the reductase domain of the protein. Previous studies have indicated that even single amino acid change in iNOS may have dramatic effects on enzymatic activity [22,23]. The rs2297518 (Ser608Leu, missense), located in exon 16 of the NOS2 gene, has been associated with risk of silicosis [24] and asthma [25], and the C > T substitution may lead to a more activated iNOS in the target cells, finally elevating NO to a higher level [26]. In spite of being a silent mutation, rs1137933 (exon 10, D385D) has also been implicated with Crohns disease [27], asthma [25] and multiple sclerosis [28]. In our study, we used symptomatic RILI as the end point and found that rs2297518 (Ser608Leu), rs1137933 (D385D), and rs16949 (intron) were significantly associated with risk of developing RILI. And their associations were independent of radiation dose and MLD. Our observations are consistent with data reported by Salam et al. [25], stating that genetic variations, mainly located in coding and downstream region of the NOS2 gene, but not in NOS1 and NOS3 genes, were the determinants of exhaled nitric oxide(FeNO) levels in children. Compared to eNOS encoded by NOS3, iNOS can produce much higher levels of NO and thus the resulting cytotoxicity [29]. In addition, Leu/Leu homozygotes of rs2297518 have been reported to confer higher enzymatic activity and NOS2 expression [22,30], resulting in increased NO production and inflammation. Our results that patients with rs2297518:CC has a higher risk of developing RILI, suggested that this functional SNP may influence RILI through inducing the expression of NOS2. However, although rs2297518 is of functional significance and most likely to be the susceptibility site, the observed association with RILI might be by linkage disequilibrium with other SNPs rather than in a single effect manner. Further haplotype analysis of three
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
J. Zhang et al. / Radiotherapy and Oncology xxx (2014) xxx–xxx
SNPs evidenced an association of haplotype ATC with decreased RILI susceptibility, may support the hypothesis that rs1137933 and rs16949 may have a role in RILI, perhaps not as a single etiologic polymorphism but because of strong LD with rs2297518. Although there are many possible mechanistic explanations for these findings, it is clear that these results should be validated in future study. Despite these positive findings and relatively large sample size, some limitations of the study need to be mentioned. Firstly, there is no further validation or replication in the current study. Secondly, we used a candidate polymorphism approach, combinations of tag SNPs and common SNPs reported in the literatures, but did not cover all SNPs in the entire gene. Some important SNPs may have been missed or the observed association may result from genetic linkages with other unidentified SNPs. Thirdly, because some genetic markers are ethnic specific, our results also should be validated among different ethnic populations in the future. Fourthly, since CTCAE scoring was utilized, data presented do not distinguish between acute radiation pneumonitis and early lung fibrosis. Such a ‘mixed phenotype’ may overestimate the incidence of radiation pneumonitis. Fifthly, recurrent disease may mimic RILI, so we assume that patients are censored in case of a recurrence. However, this may underestimate the incidence of radiation-induced lung injury. In conclusion, to our knowledge, this report is the first study to demonstrate that polymorphisms of the NOS2 gene have a significant effect on the risk of developing RILI in lung cancer patients who received definitive radiotherapy. Our results suggest that in addition to measures of clinical and radiation dosimetric factors, the naturally occurring genetic variants in the NOS2 gene might be useful to predict the risk of RILI. However, our conclusion should be interpreted with caution in clinical practice because of some of the problems previously encountered in the field [31–33]. Furthermore, we need to validate and replicate our results, and we are planning to do so more recently at our institution. Conflict of interest The authors declare that they have no competing interests. Acknowledgements This work was supported, in part, by the National Nature Science Foundation (Grant No. 30970861) and by Science and Technology Project of the Shandong Province (2009GG10002011 and 2011GGC03054).
[5]
[6]
[7] [8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
[21]
[22] [23]
[24]
[25]
Appendix A. Supplementary data
[26]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.radonc. 2014.03.001.
[27]
[28]
References [29] [1] Mehta V. Radiation pneumonitis and pulmonary fibrosis in non-small-cell lung cancer: pulmonary function, prediction, and prevention. Int J Radiat Oncol Biol Phys 2005;63:5–24. [2] Kim M, Lee J, Ha B, Lee R, Lee KJ, Suh HS. Factors predicting radiation pneumonitis in locally advanced non-small cell lung cancer. Radiat Oncol J 2011;29:181–90. [3] Claude L, Perol D, Ginestet C, et al. A prospective study on radiation pneumonitis following conformal radiation therapy in non-small-cell lung cancer: clinical and dosimetric factors analysis. Radiother Oncol 2004;71:175–81. [4] Robnett TJ, Machtay M, Vines EF, McKenna MG, Algazy KM, McKenna WG. Factors predicting severe radiation pneumonitis in patients receiving
[30]
[31] [32]
[33]
5
definitive chemoradiation for lung cancer. Int J Radiat Oncol Biol Phys 2000;48:89–94. Rancati T, Ceresoli GL, Gagliardi G, Schipani S, Cattaneo GM. Factors predicting radiation pneumonitis in lung cancer patients: a retrospective study. Radiother Oncol 2003;67:275–83. Yamada M, Kudoh S, Hirata K, Nakajima T, Yoshikawa J. Risk factors of pneumonitis following chemoradiotherapy for lung cancer. Eur J Cancer 1998;34:71–5. Tucker SL, Liu HH, Liao Z, et al. Analysis of radiation pneumonitis risk using a generalized Lyman model. Int J Radiat Oncol Biol Phys 2008;72:568–74. Nozaki Y, Hasegawa Y, Takeuchi A, et al. Nitric oxide as an inflammatory mediator of radiation pneumonitis in rats. Am J Physiol 1997;272:L651–8. Giaid A, Lehnert SM, Chehayeb B, Chehayeb D, Kaplan I, Shenouda G. Inducible nitric oxide synthase and nitrotyrosine in mice with radiation-induced lung damage. Am J Clin Oncol 2003;26:e67–72. Zhang H, Han G, Liu H, et al. The development of classically and alternatively activated macrophages has different effects on the varied stages of radiationinduced pulmonary injury in mice. J Radiat Res 2011;52:717–26. McCurdy MR, Wazni MW, Martinez J, McAleer MF, Guerrero T. Exhaled nitric oxide predicts radiation pneumonitis in esophageal and lung cancer patients receiving thoracic radiation. Radiother Oncol 2011;101:443–8. Morgan DL, Shines CJ. Alveolar macrophage cytotoxicity for normal lung fibroblasts is mediated by nitric oxide release. Toxicol In Vitro 2004;18:139–46. Kang JL, Park W, Pack IS, et al. Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation. J Appl Physiol 2002;92:795–801. Giri SN, Biring I, Nguyen T, Wang Q, Hyde DM. Abrogation of bleomycininduced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice. Nitric Oxide 2002;7:109–18. Yung GL, Kriett JM, Jamieson SW, et al. Outpatient inhaled nitric oxide in a patient with idiopathic pulmonary fibrosis: a bridge to lung transplantation. J Heart Lung Transpl 2001;20:1224–7. Geller DA, Nussler AK, Di Silvio M, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A 1993;90:522–6. Yuan X, Liao Z, Liu Z, et al. Single nucleotide polymorphism at rs1982073:T869C of the TGFbeta 1 gene is associated with the risk of radiation pneumonitis in patients with non-small-cell lung cancer treated with definitive radiotherapy. J Clin Oncol 2009;27:3370–8. Zhang L, Yang M, Bi N, et al. ATM polymorphisms are associated with risk of radiation-induced pneumonitis. Int J Radiat Oncol Biol Phys 2010;77:1360–8. Yang M, Zhang L, Bi N, et al. Association of P53 and ATM polymorphisms with risk of radiation-induced pneumonitis in lung cancer patients treated with radiotherapy. Int J Radiat Oncol Biol Phys 2011;79:1402–7. Yin M, Liao Z, Liu Z, et al. Genetic variants of the nonhomologous end joining gene LIG4 and severe radiation pneumonitis in nonsmall cell lung cancer patients treated with definitive radiotherapy. Cancer 2012;118:528–35. Hildebrandt MA, Komaki R, Liao Z, et al. Genetic variants in inflammationrelated genes are associated with radiation-induced toxicity following treatment for non-small cell lung cancer. PLoS One 2010;5:e12402. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999;1411:217–30. Johannesen J, Pie A, Pociot F, Kristiansen OP, Karlsen AE, Nerup J. Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes. J Clin Endocrinol Metab 2001;86:2792–6. Qu Y, Tang Y, Cao D, et al. Genetic polymorphisms in alveolar macrophage response-related genes, and risk of silicosis and pulmonary tuberculosis in Chinese iron miners. Int J Hyg Environ Health 2007;210:679–89. Salam MT, Bastain TM, Rappaport EB, et al. Genetic variations in nitric oxide synthase and arginase influence exhaled nitric oxide levels in children. Allergy 2011;66:412–9. Holla LI, Stejskalova A, Znojil V, Vasku A. Analysis of the inducible nitric oxide synthase gene polymorphisms in Czech patients with atopic diseases. Clin Exp Allergy 2006;36:1592–601. Martin MC, Martinez A, Mendoza JL, et al. Influence of the inducible nitric oxide synthase gene (NOS2A) on inflammatory bowel disease susceptibility. Immunogenetics 2007;59:833–7. Barcellos LF, Begovich AB, Reynolds RL, et al. Linkage and association with the NOS2A locus on chromosome 17q11 in multiple sclerosis. Ann Neurol 2004;55:793–800. Marletta MA. Nitric oxide synthase: function and mechanism. Adv Exp Med Biol 1993;338:281–4. Shen J, Wang RT, Wang LW, Xu YC, Wang XR. A novel genetic polymorphism of inducible nitric oxide synthase is associated with an increased risk of gastric cancer. World J Gastroenterol 2004;10:3278–83. Andreassen CN. Searching for genetic determinants of normal tissue radiosensitivity – are we on the right track? Radiother Oncol 2010;97:1–8. Kerns SL, Ruysscher DD, Andreassen CN, et al. STROGAR – STrengthening the Reporting Of Genetic Association studies in Radiogenomics. Radiother Oncol 2013. Andreassen CN, Barnett GC, Langendijk JA, et al. Conducting radiogenomic research – do not forget careful consideration of the clinical data. Radiother Oncol 2012;105:337–40.
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Original article
Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation Jian Zhang a,b,c, Baosheng Li a,b,⇑, Xiuping Ding a,b, Mingping Sun a,b, Hongsheng Li a,b, Ming Yang d, Changchun Zhou a,b, Haiying Yu e, Hong Liu f, Gongqi Yu f a
Department of Radiation Oncology, Shandong Cancer Hospital, Shandong Academy of Medical Sciences, Jinan; b Shandong’s Key Laboratory of Radiation Oncology, Jinan; Department of Radiation Oncology, Cancer Hospital, Tianjin Medical University; d College of Life Science and Technology, Beijing University of Chemical Technology; e Department of Radiology, Shandong Cancer Hospital, Jinan; and f Shandong Provincial Institute of Dermatology and Venereology, Jinan, PR China
c
a r t i c l e
i n f o
Article history: Received 18 October 2012 Received in revised form 2 February 2014 Accepted 9 March 2014 Available online xxxx Keywords: Single nucleotide polymorphism Inducible nitric oxide synthase Radiation-induced lung injury Lung cancer Radiotherapy
a b s t r a c t Background and purpose: Nitric oxide (NO), mainly synthesized by inducible nitric oxide synthase (NOS2) in pathological conditions, plays an important role in cytotoxicity, inflammation and fibrosis. Elevations in exhaled NO after thoracic radiation have been reported to predict radiation-induced lung injury (RILI). This study examined whether genetic variations in NOS2 gene is associated with the risk of RILI. Material and methods: A cohort of 301 patients between 2009 and 2011 were genotyped for 21 single nucleotide polymorphisms (SNPs) in the NOS2 gene by the Sequenom MassArray system. Kaplan–Meier cumulative probability was used to assess RILI risk and Cox proportional hazards analyses were performed to evaluate the effect of NOS2 genotypes on RILI. Results: Multivariate analysis found that three SNPs (rs2297518, rs1137933 and rs16949) in NOS2 were significantly associated with risk of RILI P 2 (P value = 0.001, 0.000092, 0.001, respectively) after adjusting for other covariates. Their associations were independent of radiation dose and mean lung dose. Further haplotype analysis indicated that the ATC haplotype of three SNPs is associated with reducing the risk of developing RILI. Conclusion: Our results demonstrate that genetic variants of NOS2 may serve as a reliable predictor of RILI in lung cancer patients treated with thoracic radiation. Ó 2014 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2014) xxx–xxx
Currently, radiotherapy is one of the main treatment modalities in lung cancer, contributing to both its cure and palliation. However, radiation-induced lung injury (RILI), one of the most common dose-limiting toxicities of thoracic radiotherapy, might compromise the success of current efforts regarding lung cancer treatment intensification [1]. Recently, many studies have investigated and identified clinical and dosimetric factors that may influence RILI risk, including mean lung dose (MLD) [2], percent lung volume receiving more than a threshold radiation dose (VDose) [3], performance status and pulmonary function [4], chronic obstructive pulmonary disease [5], concurrent chemotherapy [6], etc. However, only some patients in whom normal lung is exposed to a certain dose and volume of irradiation develop RILI even after stratifying
⇑ Corresponding author at: Department of Radiation Oncology, Shandong Cancer Hospital, 440, Jiyan Road, Jinan, PR China. E-mail address: [email protected] (B. Li).
for smoking status [7], suggesting that genetic variants may play a critical role in RILI development. Radiation pneumonitis and pulmonary fibrosis represent acute and late phases in the development of RILI. Recent studies suggest that endogenous nitric oxide (NO) formation by the inducible nitric oxide synthase (iNOS or Nos2) after irradiation may be closely associated with the occurrence and development of RILI [8–11]. Nitric oxide, a reactive radical and proinflammatory mediator, mainly synthesized from iNOS in pathological conditions, has been reported to display diverse biological activities in lung tissues, including cytotoxicity [12], inflammation [13], and fibrosis [14,15]. NOS2 gene is present on the human chromosome 17q11.2–12. The gene product iNOS is commonly absent in resting cells, but it is capable of being rapidly expressed in response to inflammatory stimuli, such as cytokines [16]. In addition, elevations in exhaled NO at the end of thoracic radiation were found in symptomatic RILI in esophageal or lung cancer patients. Recently, several studies have reported that genetic susceptibility
http://dx.doi.org/10.1016/j.radonc.2014.03.001 0167-8140/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
2
Variants in NOS2 gene are associated with RILI
factors, such as TGF-b [17], ATM [18], P53 [19], DNA double-strand breaks repair gene [20], and inflammation-related genes [21] play a role in RILI development. However, to our knowledge, the influences of NOS2 variants on RILI have not been reported so far. In this study, we hypothesized that SNPs of the NOS2 gene are biomarkers for predicting susceptibility to RILI among patients with lung cancer who receive thoracic radiation. To test this hypothesis, 21 SNPs of NOS2 were performed in this study. The aim is to figure out the associations between RILI risk and common variants of NOS2 in lung cancer patients, and to predict RILI susceptibility before the initiation of radiation therapy.
2006), as follows: Grade 0, no change; Grade 1, asymptomatic radiographic findings only; Grade 2, symptomatic, not interfering with activities of daily living; Grade 3, symptomatic, interfering with activities of daily living, oxygen required; Grade 4, life-threatening ventilatory support required; and Grade 5 if the patient died from RILI. The time to the endpoint development was based on the duration from the beginning of radiation treatment to occurrence of toxicity, and patients who did not experience the endpoint were censored at the last follow-up.
SNP selection and genotyping Materials and methods Patient population and clinical data collection In this study, a cohort of 301 newly diagnosed lung cancer patients were treated with definitive radiation between December 2009 and January 2011 at Shandong Cancer Hospital (Jinan). The eligible patients were those with histologically or cytologically confirmed lung cancer, Karnofsky performance status (KPS) P60, and an expected survival >6 months. Each patient signed an informed consent before starting therapy. Detailed clinical information and dosimetric factors (MLD, V20) were collected. This study was approved by the Shandong Cancer Hospital institutional review board. We complied with Health Insurance Portability and Accountability Act regulations. Follow-up and evaluation of RILI All patients included in this study were examined and evaluated by their radiation oncologists weekly during radiotherapy and 4–6 weeks after completion of treatment. Patients were then followed up every 3 months for the first 3 years unless they had symptoms that required immediate examination or intervention. Radiographic examination by chest X-ray or computerized tomography was performed at each follow-up visit after completion of treatment. RILI was assessed by three radiation oncologists according to the symptoms and imaging information of each patient, and graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 3.0 (August 9,
Table 1 Characteristics of 21 SNPs in the NOS2 gene. SNP ID
Chromosome
Position
Allele
Function class
rs2297515 rs2297518
17 17
23117460 23120724
A/C A/G
rs2779248 rs3794763 rs4795067 rs8072199 rs2314809 rs7208775 rs16949 rs3730013 rs9906835 rs11080344 rs944722 rs944725 rs1137933
17 17 17 17 17 17 17 17 17 17 17 17 17
23151959 23135353 23130802 23140975 23119505 23109235 23148826 23150045 23113501 23128638 23116164 23133698 23130059
C/T A/G A/G C/T C/T C/G C/T C/T A/G C/T C/T C/T C/T
rs2072324 rs2248814 rs2255929 rs2314810 rs10459953 rs17722851
17 17 17 17 17 17
23141023 23124448 23112094 23128237 23151645 23134963
A/C A/G A/T C/G C/G A/T
Intron CDSnonsynonymous Promoter Intron Intron Intron Intron Intron Intron Intron Intron Intron Intron Intron CDSsynonymous Intron Intron Intron Intron 50 UTR Intron
0
0
Abbreviations: 5 UTR, 5 untranslational region; CDS, Coding Sequence.
tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP tagSNP
We chose tag SNP through the tagger software included in the Haploview software 4.2 and the common SNPs based on previous literature in NOS2. The tag SNP was selected based on ability to tag surrounding variants (NOS2, chr17) in the Han Chinese panel (Beijing, China) of the International HapMap project, NCBI build B36 assembly HapMap phase II + III (http://www.hapmap.org). The tag SNP was obtained with a minor allele frequency (MAF) of
Table 2 Baseline clinical characteristics of patients (n = 301). Characteristic Sex Female Male Age, years Median Range KPS 90–100 80 <80 Histopathology Squamous cell Adenocarcinoma NSCLC, NOS SCLC Tumor location Peripheral Central Clinical stage I–II III IV Smoking status Nonsmoker CSI P 600 CSI < 600 COPD Yes No Treatment RT C + RT CRT C + CRT Radiation technique 2-Dimensional RT 3D-CRT IMRT Radiation dose, Gy Median Range MLD, Gy (n = 259) Median Range V20 of both lungs, % Median Range
No.
%
69 232
22.9 77.1 60 25–87
152 111 38
50.5 36.9 12.6
105 70 69 57
34.9 23.3 22.9 18.9
169 132
56.1 43.9
28 151 122
9.3 50.2 40.5
77 132 92
25.6 43.9 30.6
42 259
14.0 86.0
24 126 24 127
8.0 41.9 8.0 42.2
42 163 96
14.0 54.0 32.0 59.6 40–80.6 14.6 3.2–35.6 26 2–75
Abbreviations: NOS, not otherwise specified; CSI, cigarette-smoke index; CRT, concurrent chemoradiation; 3D-CRT, three-dimensional conformal radiation therapy; IMRT, intensity modulated radiotherapy.
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
3
J. Zhang et al. / Radiotherapy and Oncology xxx (2014) xxx–xxx
0.05, a pairwise correlation coefficient r2 of 0.80. The characteristics of 21 selected SNPs were shown in Table 1. A 5 mL wholeblood sample was obtained from each patient before starting therapy, and genomic DNA was extracted from whole-blood cells using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen, USA). Genotyping analyses were conducted by using the Sequenom MassArray system (San Diego, USA). Statistical analysis The end point for this analysis was the development of RILI grade P2. Statistical analysis was performed using IBM SPSS 19.0. Student’s T-test or chi-square test was used to examine the differences in clinical characteristics. Hazard ratios (HRs) with 95% confidence intervals (CIs) of genotype for RILI were computed by the Cox proportional hazards model. In addition, further multivariate Cox regression analysis was performed to adjust other covariates. Kaplan–Meier analysis was performed to calculate the estimated cumulative RILI probability. Comparisons were made with the log-rank test. The Bonferroni method was used to adjust for multiple comparisons. A P value of 0.05 or less was considered statistically significant in two-tails of the T-test. Results Patient characteristics and association with RILI Table 2 lists the baseline clinical characteristics of the 301 patients, of whom 92.0% (N = 277) received combined platinumbased chemoradiotherapy. Of 277 patients, 57 (20.6%) patients received an etopside/cisplatin regimen and 220 (79.4%) patients received a gemcitabine/cisplatin or taxane/cisplatin or vinorelbine/ cisplatin regimen. The median radiation dose was 59.6 Gy (range, 40–80.6 Gy); 82.4% (N = 248) of patients received 50–70 Gy. By the time of the final analysis (January 2012), the median follow-up time had been 16 months (range, 3–25). After radiotherapy, 81 patients (26.9%) had PGrade 2 RILI (Grade 2, 3, and 5 were observed in 48, 28 and 5 patients, respectively). The median time in the development of RILI was 2 months. Table 3 shows the associations between patient-, tumor-, therapy-related characteristics and RILI grade P2 by univariate and multivariate analyses. Our results demonstrate that sex and radiation dosimetric factors (radiation dose, MLD, V20) are associated with RILI in univariate and multivariate analyses. Univariate and multivariate analyses Among the 21 SNPs studied, a total of four SNPs in NOS2 were found to be significantly associated with risk of RILI (grade P2)
Table 3 Associations Between patient-, tumor-, and therapy-related characteristics and RILI Grade P2. Parameter
Univariate analysis HR
Sex Female 1.000 Male 1.935 Age, years <65 1.000 P65 1.048 KPS 90–100 1.000 80 0.698 <80 0.839 Stage IV 1.000 I, II, III 1.037 COPD No 1.000 Yes 1.141 Tumor location Central 1.000 Peripheral 1.459 Histology Squamous cell 1.000 Adenocarcinoma 1.142 SCLC 1.082 NSCLC, NOS 1.190 Tobacco use Never 1.000 CSI < 600 0.571 CSI P 600 1.114 CRT No 1.000 Yes 0.867 Radiation dose, Gy <60 1.000 P60 1.774 MLD, Gy* (n = 259) <17.7 1.000 P17.7 2.051 V20 (n = 259) < 30% <26% 1.000 26–37% 1.147 P37% 2.568
Multivariate analysis
95% CI
P
1.023–3.659
HR
95% CI
P
1.000 0.042 2.099
1.101–4.003
0.024
0.663–1.656
1.000 0.840 1.140
0.687–1.889
0.612
0.425–1.146 0.423–1.665
1.000 0.155 0.661 0.616 0.759
0.386–1.132 0.361–1.594
0.131 0.466
0.662–1.625
1.000 0.875 0.981
0.597–1.612
0.939
0.617–2.110
1.000 0.674 1.370
0.660–2.841
0.398
0.920–2.314
1.000 0.108 1.484
0.866–2.543
0.151
0.629–2.074 0.570–2.052 0.661–2.144
1.000 0.662 1.478 0.810 1.514 0.561 1.063
0.743–2.941 0.748–3.065 0.575–1.966
0.266 0.249 0.846
0.203–1.606 0.497–2.499
1.000 0.288 0.642 0.793 1.110
0.319–1.293 0.629–1.959
0.215 0.719
0.557–1.349
1.000 0.527 1.064
0.658–1.720
0.802
1.126 to 2.795
1.000 0.013 1.717
1.033–2.852
0.037
1.275–3.298
1.000 0.003 1.829
1.072–3.119
0.027
0.709–1.854 1.318–5.004
1.000 0.577 1.1519 0.690–1.918 0.006 2.475 1.193–5.136
0.590 0.015
NOTE: Multivariate analyses were adjusted for all factors listed in this table. Abbreviations: NOS, not otherwise specified; CSI, cigarette-smoke index; CRT, concurrent chemoradiation. * Either total radiation dose, MLD or V20 was used in multivariate analyses, but not together.
in the univariate Cox regression analyses (Table 4). In multivariate analysis, this effect is virtually unchanged after adjustment for other covariates (P value = 0.001, 0.000092, 0.024, 0.001, respectively). After further Bonferroni corrections (P value = 0.019,
Table 4 Associations between NOS2 genotypes and Grade P2 RILI. Polymorphisms and genotypes
rs2297518 AA or AG GG rs1137933 CC CT or TT rs3794763 AA AG or GG rs16949 CC or CT TT
No. of patients
300 68 232 300 231 69 301 33 268 300 73 227
Events
Univariate analysis (Crude)
Multivariate analysis (Adjusted)
HR
95% CI
P
HR
95% CI
P
10 70
1 2.551
1.274–5.107
.008
1 3.585
1.711–7.510
72 8
1 0.321
0.154–0.666
.002
1 0.209
0.095–0.458
14 66
1 0.516
0.300–0.919
.025
1 0.488
0.262–0.909
.024
10 69
1 2.480
1.277–4.814
.007
1 3.481
1.707–7.099
.001
.001
9.2E05
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
4
Variants in NOS2 gene are associated with RILI
In addition, Table 4 lists the Kaplan–Meier incidences of grade P2 in patients with different genotypes of 4 SNPs at 12 months from starting radiation. In general, RILI developed more often in patients exhibiting GG in rs2297518, CC in rs1137933, AA in rs3794763, or TT in rs16949 genotypes, with RILI rates of 30.2%, 31.2%, 42.4%, and 30.3%, respectively, than in those having AG/AA in rs2297518, CT/TT in rs1137933, AG/GG in rs3794763, or CC/CT in rs16949 genotypes, for which the incidences were 14.7%, 11.6%, 24.6%, and 13.7%, respectively. Cumulative probability of RILI grade P2 as a function of time from the beginning of radiotherapy by genotypes in rs2297518, rs1137933, and rs16949 was shown in Fig. 1. Subgroup analysis for dosimetric factors Since dosimetric factors were also independent risk factors for RILI, we then investigated the subgroup analysis results by radiation dose and MLD. The cumulative RILI grade P2 probability as a function of time was analyzed according to genotypes and radiation dose (Supplementary files-Fig. 1A–C), MLD (Supplementary files-Fig. 1D–F). The results suggest that the effect of the genetic variants on RILI grade P2 was independent on dosimetric parameters. Haplotype analysis Since rs2297518, rs1137933, and rs16949 are in strong linkage disequilibrium (r2 = 0.57–0.73), we further constructed three-marker haplotypes (Supplemental file-Table 1). Compared to the most common GCT haplotype, only the ATC haplotype is associated with reducing risk of developing RILI grade P2, with the adjusted OR being 0.157 (95% CI, 0.047–0.522; P < 0.001). Discussion
Fig. 1. Cumulative probability of RILI grade P2 as a function of time from the beginning of radiotherapy by genotypes. (A) rs2297518; (B) rs1137933; (C) rs16949. The rs2297518:GG, rs1137933:CC and rs16949:TT genotype was associated with a statistically significant higher incidence of RILI compared with other genotypes, respectively.
0.0017, 0.019, respectively), three SNPs (rs2297518, rs1137933 and rs16949) remained significant for its association with RILI (grade P2).
The human NOS2 gene comprises 27 exons, exon 1–13 code for the oxygenase domain and exon 14–27 encode for the reductase domain of the protein. Previous studies have indicated that even single amino acid change in iNOS may have dramatic effects on enzymatic activity [22,23]. The rs2297518 (Ser608Leu, missense), located in exon 16 of the NOS2 gene, has been associated with risk of silicosis [24] and asthma [25], and the C > T substitution may lead to a more activated iNOS in the target cells, finally elevating NO to a higher level [26]. In spite of being a silent mutation, rs1137933 (exon 10, D385D) has also been implicated with Crohns disease [27], asthma [25] and multiple sclerosis [28]. In our study, we used symptomatic RILI as the end point and found that rs2297518 (Ser608Leu), rs1137933 (D385D), and rs16949 (intron) were significantly associated with risk of developing RILI. And their associations were independent of radiation dose and MLD. Our observations are consistent with data reported by Salam et al. [25], stating that genetic variations, mainly located in coding and downstream region of the NOS2 gene, but not in NOS1 and NOS3 genes, were the determinants of exhaled nitric oxide(FeNO) levels in children. Compared to eNOS encoded by NOS3, iNOS can produce much higher levels of NO and thus the resulting cytotoxicity [29]. In addition, Leu/Leu homozygotes of rs2297518 have been reported to confer higher enzymatic activity and NOS2 expression [22,30], resulting in increased NO production and inflammation. Our results that patients with rs2297518:CC has a higher risk of developing RILI, suggested that this functional SNP may influence RILI through inducing the expression of NOS2. However, although rs2297518 is of functional significance and most likely to be the susceptibility site, the observed association with RILI might be by linkage disequilibrium with other SNPs rather than in a single effect manner. Further haplotype analysis of three
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001
J. Zhang et al. / Radiotherapy and Oncology xxx (2014) xxx–xxx
SNPs evidenced an association of haplotype ATC with decreased RILI susceptibility, may support the hypothesis that rs1137933 and rs16949 may have a role in RILI, perhaps not as a single etiologic polymorphism but because of strong LD with rs2297518. Although there are many possible mechanistic explanations for these findings, it is clear that these results should be validated in future study. Despite these positive findings and relatively large sample size, some limitations of the study need to be mentioned. Firstly, there is no further validation or replication in the current study. Secondly, we used a candidate polymorphism approach, combinations of tag SNPs and common SNPs reported in the literatures, but did not cover all SNPs in the entire gene. Some important SNPs may have been missed or the observed association may result from genetic linkages with other unidentified SNPs. Thirdly, because some genetic markers are ethnic specific, our results also should be validated among different ethnic populations in the future. Fourthly, since CTCAE scoring was utilized, data presented do not distinguish between acute radiation pneumonitis and early lung fibrosis. Such a ‘mixed phenotype’ may overestimate the incidence of radiation pneumonitis. Fifthly, recurrent disease may mimic RILI, so we assume that patients are censored in case of a recurrence. However, this may underestimate the incidence of radiation-induced lung injury. In conclusion, to our knowledge, this report is the first study to demonstrate that polymorphisms of the NOS2 gene have a significant effect on the risk of developing RILI in lung cancer patients who received definitive radiotherapy. Our results suggest that in addition to measures of clinical and radiation dosimetric factors, the naturally occurring genetic variants in the NOS2 gene might be useful to predict the risk of RILI. However, our conclusion should be interpreted with caution in clinical practice because of some of the problems previously encountered in the field [31–33]. Furthermore, we need to validate and replicate our results, and we are planning to do so more recently at our institution. Conflict of interest The authors declare that they have no competing interests. Acknowledgements This work was supported, in part, by the National Nature Science Foundation (Grant No. 30970861) and by Science and Technology Project of the Shandong Province (2009GG10002011 and 2011GGC03054).
[5]
[6]
[7] [8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
[21]
[22] [23]
[24]
[25]
Appendix A. Supplementary data
[26]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.radonc. 2014.03.001.
[27]
[28]
References [29] [1] Mehta V. Radiation pneumonitis and pulmonary fibrosis in non-small-cell lung cancer: pulmonary function, prediction, and prevention. Int J Radiat Oncol Biol Phys 2005;63:5–24. [2] Kim M, Lee J, Ha B, Lee R, Lee KJ, Suh HS. Factors predicting radiation pneumonitis in locally advanced non-small cell lung cancer. Radiat Oncol J 2011;29:181–90. [3] Claude L, Perol D, Ginestet C, et al. A prospective study on radiation pneumonitis following conformal radiation therapy in non-small-cell lung cancer: clinical and dosimetric factors analysis. Radiother Oncol 2004;71:175–81. [4] Robnett TJ, Machtay M, Vines EF, McKenna MG, Algazy KM, McKenna WG. Factors predicting severe radiation pneumonitis in patients receiving
[30]
[31] [32]
[33]
5
definitive chemoradiation for lung cancer. Int J Radiat Oncol Biol Phys 2000;48:89–94. Rancati T, Ceresoli GL, Gagliardi G, Schipani S, Cattaneo GM. Factors predicting radiation pneumonitis in lung cancer patients: a retrospective study. Radiother Oncol 2003;67:275–83. Yamada M, Kudoh S, Hirata K, Nakajima T, Yoshikawa J. Risk factors of pneumonitis following chemoradiotherapy for lung cancer. Eur J Cancer 1998;34:71–5. Tucker SL, Liu HH, Liao Z, et al. Analysis of radiation pneumonitis risk using a generalized Lyman model. Int J Radiat Oncol Biol Phys 2008;72:568–74. Nozaki Y, Hasegawa Y, Takeuchi A, et al. Nitric oxide as an inflammatory mediator of radiation pneumonitis in rats. Am J Physiol 1997;272:L651–8. Giaid A, Lehnert SM, Chehayeb B, Chehayeb D, Kaplan I, Shenouda G. Inducible nitric oxide synthase and nitrotyrosine in mice with radiation-induced lung damage. Am J Clin Oncol 2003;26:e67–72. Zhang H, Han G, Liu H, et al. The development of classically and alternatively activated macrophages has different effects on the varied stages of radiationinduced pulmonary injury in mice. J Radiat Res 2011;52:717–26. McCurdy MR, Wazni MW, Martinez J, McAleer MF, Guerrero T. Exhaled nitric oxide predicts radiation pneumonitis in esophageal and lung cancer patients receiving thoracic radiation. Radiother Oncol 2011;101:443–8. Morgan DL, Shines CJ. Alveolar macrophage cytotoxicity for normal lung fibroblasts is mediated by nitric oxide release. Toxicol In Vitro 2004;18:139–46. Kang JL, Park W, Pack IS, et al. Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation. J Appl Physiol 2002;92:795–801. Giri SN, Biring I, Nguyen T, Wang Q, Hyde DM. Abrogation of bleomycininduced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice. Nitric Oxide 2002;7:109–18. Yung GL, Kriett JM, Jamieson SW, et al. Outpatient inhaled nitric oxide in a patient with idiopathic pulmonary fibrosis: a bridge to lung transplantation. J Heart Lung Transpl 2001;20:1224–7. Geller DA, Nussler AK, Di Silvio M, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A 1993;90:522–6. Yuan X, Liao Z, Liu Z, et al. Single nucleotide polymorphism at rs1982073:T869C of the TGFbeta 1 gene is associated with the risk of radiation pneumonitis in patients with non-small-cell lung cancer treated with definitive radiotherapy. J Clin Oncol 2009;27:3370–8. Zhang L, Yang M, Bi N, et al. ATM polymorphisms are associated with risk of radiation-induced pneumonitis. Int J Radiat Oncol Biol Phys 2010;77:1360–8. Yang M, Zhang L, Bi N, et al. Association of P53 and ATM polymorphisms with risk of radiation-induced pneumonitis in lung cancer patients treated with radiotherapy. Int J Radiat Oncol Biol Phys 2011;79:1402–7. Yin M, Liao Z, Liu Z, et al. Genetic variants of the nonhomologous end joining gene LIG4 and severe radiation pneumonitis in nonsmall cell lung cancer patients treated with definitive radiotherapy. Cancer 2012;118:528–35. Hildebrandt MA, Komaki R, Liao Z, et al. Genetic variants in inflammationrelated genes are associated with radiation-induced toxicity following treatment for non-small cell lung cancer. PLoS One 2010;5:e12402. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999;1411:217–30. Johannesen J, Pie A, Pociot F, Kristiansen OP, Karlsen AE, Nerup J. Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes. J Clin Endocrinol Metab 2001;86:2792–6. Qu Y, Tang Y, Cao D, et al. Genetic polymorphisms in alveolar macrophage response-related genes, and risk of silicosis and pulmonary tuberculosis in Chinese iron miners. Int J Hyg Environ Health 2007;210:679–89. Salam MT, Bastain TM, Rappaport EB, et al. Genetic variations in nitric oxide synthase and arginase influence exhaled nitric oxide levels in children. Allergy 2011;66:412–9. Holla LI, Stejskalova A, Znojil V, Vasku A. Analysis of the inducible nitric oxide synthase gene polymorphisms in Czech patients with atopic diseases. Clin Exp Allergy 2006;36:1592–601. Martin MC, Martinez A, Mendoza JL, et al. Influence of the inducible nitric oxide synthase gene (NOS2A) on inflammatory bowel disease susceptibility. Immunogenetics 2007;59:833–7. Barcellos LF, Begovich AB, Reynolds RL, et al. Linkage and association with the NOS2A locus on chromosome 17q11 in multiple sclerosis. Ann Neurol 2004;55:793–800. Marletta MA. Nitric oxide synthase: function and mechanism. Adv Exp Med Biol 1993;338:281–4. Shen J, Wang RT, Wang LW, Xu YC, Wang XR. A novel genetic polymorphism of inducible nitric oxide synthase is associated with an increased risk of gastric cancer. World J Gastroenterol 2004;10:3278–83. Andreassen CN. Searching for genetic determinants of normal tissue radiosensitivity – are we on the right track? Radiother Oncol 2010;97:1–8. Kerns SL, Ruysscher DD, Andreassen CN, et al. STROGAR – STrengthening the Reporting Of Genetic Association studies in Radiogenomics. Radiother Oncol 2013. Andreassen CN, Barnett GC, Langendijk JA, et al. Conducting radiogenomic research – do not forget careful consideration of the clinical data. Radiother Oncol 2012;105:337–40.
Please cite this article in press as: Zhang J et al. Genetic variants in inducible nitric oxide synthase gene are associated with the risk of radiation-induced lung injury in lung cancer patients receiving definitive thoracic radiation. Radiother Oncol (2014), http://dx.doi.org/10.1016/j.radonc.2014.03.001