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
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
