Putative association of the single nucleotide polymorphisms in RASSF1A promoter with Korean lung cancer

Lung Cancer (2008) 61, 301—308

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Putative association of the single nucleotide polymorphisms in RASSF1A promoter with Korean lung cancer Jae Sook Sung a,b, Sle Gi Lo Han b, Young Mi Whang b, Eun Soon Shin c, Jae Won Lee d, Hyo Jung Lee d, Jeong-Seon Ryu e, In Keun Choi f, Kyong Hwa Park f, Jun Suk Kim a,f, Sang Won Shin f, Elizabeth K. Chu g, Yeul Hong Kim a,b,f,∗ a

Department of Internal Medicine and Division of Brain Korea 21 Project for Biomedical Science, Seoul, Republic of Korea Genomic Research Center for Lung and Breast/Ovarian Cancers, Korea University Anam Hospital, Seoul, Republic of Korea c DNA Link Inc., Seoul, Republic of Korea d Department of Statistics, Korea University, Seoul, Republic of Korea e Department of Internal Medicine, Inha University College of Medicine, Incheon, Republic of Korea f Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea g Emory University, Atlanta, GA, USA b

Received 29 September 2007; received in revised form 10 January 2008; accepted 14 January 2008

KEYWORDS Lung cancer; RASSF1A; Single nucleotide polymorphism (SNP); Haplotype; Transcriptional activity

Summary The RASSF1 gene, a putative tumor suppressor gene located on human chromosome 3p21, has attracted a great deal of attention because of frequent allelic loss and gene silencing via promoter hypermethylation in a variety of human malignancies. To evaluate the role of RASSF1A gene in lung cancer risk, genotypes of the RASSF1A promoter region (−710 C > T and −392 T > C) were determined in 410 lung cancer patients and 410 normal subjects. Furthermore, to examine potential effects of the common haplotypes (C—C, T—T and C—T haplotypes) on RASSF1A transcription, luciferase reporter assays were performed in H2009 and H358 non-small cell lung cancer (NSCLC) cell lines. We found that ht2 C—T haplotype was associated with susceptibility to the risk of lung cancer in dominant (odds ratio (OR): 0.69; 95% CI: 0.46—0.99) model. In particular, we found that C—T haplotype showed a decreased risk of lung cancer in males (codominant OR: 0.59; 95% CI: 0.38—0.93 and dominant OR: 0.58; 95% CI: 0.35—0.96) and in smokers (codominant OR: 0.58; 95% CI: 0.36—0.93 and dominant OR: 0.56; 95% CI: 0.33—0.96). Interestingly, C—T haplotype induced transcriptional activity by 50—60% compared with other

∗ Corresponding author at: Division of Oncology/Hematology, Department of Internal Medicine, Korea University Anam Hospital, 126-1 Anam-dong 5 ka, Seongbuk-ku, Seoul 137-705, Republic Korea. Tel.: +82 2 920 5569; fax: +82 2 926 4534. E-mail address: [email protected] (Y.H. Kim).

0169-5002/$ — see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2008.01.012

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J.S. Sung et al. haplotypes in NSCLC cell lines. These results suggest that RASSF1A promoter polymorphisms affect RASSF1A expression, further contributing to the genetic susceptibility to lung cancer. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer has the highest mortality rate among cancers, and has the second highest incidence among cancers in Korea [1,2]. The RASSF1 locus is located within the 120 kb minimal homozygous deletion region on 3p21.3 chromosome, and the RASSF1 gene encodes two major transcripts, RASSF1A and RASSF1C, which are produced by alternative promoter selection and alternative mRNA splicing. The RASSF1A gene is frequently inactivated in primary lung cancers by the de novo methylation of CpG islands in the promoter region [3—9]. Indeed, many studies showed that the RASSF1A is silenced by DNA methylation in over 50% of all solid tumors and in cell lines of different histological types as well as two subsets of hematological tumors [6,7,10]. The RASSF1A has also been noted to be expressed in normal bronchial epithelial cells, but not in 90% of small cell lung cancers (SCLCs) and in 40% of non-small cell lung cancers (NSCLCs) [4,8]. Moreover, mutations in the RASSF1A gene are uncommon, whereas lack of its expression is common in lung cancer [4,6]. Recently, it was reported that the RASSF1A exerts its tumor suppressing effect by blocking oncogene-mediated JNK activation in lung cancer cells [11]. However, the function of RASSF1A and the molecular mechanism have not yet been fully clarified. Single nucleotide polymorphisms (SNPs) represent an important class of genetic variation and affect an individual’s susceptibility to disease in certain circumstances [12—15]. Moreover, SNPs in the coding regions or the regulatory regions of genes are more likely to cause functional differences than SNPs located elsewhere. This study was conducted to examine the SNPs in RASSF1A, and its relations with the risk of lung cancer occurrence in Korean lung cancer patients. The clinical effects of SNPs on lung cancer patients were assessed by analyzing the relationships between gender, smoking status and genotype. In addition, the effects of these polymorphisms on RASSF1A promoter transcription were evaluated in several NSCLC cell lines. This study is the first study to determine an association of the RASSF1A promoter polymorphisms in Korean lung cancers.

2. Subjects and methods 2.1. The study subjects Between August 2001 and June 2006, blood samples were collected from 820 subjects, including 410 lung cancer patients and 410 normal controls without cancer. Lung cancer patients were recruited from the patient pool at the Genomic Research Center for Lung and Breast/Ovarian Cancer and Inha University Medical Center, and 410 control subjects were randomly selected from a pool of healthy volunteers who had visited the Cardiovascular Genome Center, Genomic Research Center for Allergy and Respiratory

Diseases and Keimyung University Dongsan Medical Center. Detailed information on diet, smoking status, drinking status, lifestyle, and medical history were collected by trained interviewer using a structured questionnaire. Out of 410 cases, 399 smoking status, 308 drinking status, 326 stage and 389 cell types were available for the characteristic information. Moreover, out of 410 controls, 308 smoking status and 220 drinking status were available for the characteristic information. All study subjects provided written consents and were ethnic Koreans, and all participating Institutional Review Board approval for the study protocol.

2.2. Preparation of genomic DNA and direct sequencing Genomic DNA was prepared from peripheral blood samples using a Puregene blood DNA kit (Gentra, Minneapolis, MN), following the manufacture’s protocol. For identification of frequent polymorphism sites in RASSF1A gene promoter in Korean population, human genomic DNA was isolated from the whole blood of 48 samples for direct sequencing. Of the entire RASSF1 gene at 3p21.3, we amplified 2 kb of its promoter. PCR amplifications were performed in a PTC-225 Peltier Thermal cycler (MJ Research Inc., Waltham, MA) using AmpliTagGold (Roche, Branchburg, NJ). All amplifications were performed using 35 cycles of 30 s at 95 ◦ C, 1 min at 64 ◦ C and 1 min at 72 ◦ C, followed by a single 10 min extension at 72 ◦ C. PCR products were purified using a Montage PCR96 Cleanup kit (Millipore, Bedfore, MA) and eluted in 20 ␮l of nuclease free H2 O. DNA cycle sequencing was carried out using a BigDye Terminator V 3.1 Cycle Sequencing kit (PerkinElmer, Foster City, CA). For dye terminator removal, Multiscreen SEQ 384 well filter plates were used and sequences were analyzed on an Applied Biosystems 3700 DNA analyzer. All SNPs and sequence alignments were analyzed using Polyphred.

2.3. Genotyping The genotypes of the sample were assayed using single base primer extension assay using a SNaPShot assay kit (ABI, Foster City, CA). Briefly, the genomic regions containing interested SNPs in RASSF1A were amplified by PCR using 5 -CATGTTCAGCCCCTCAG-3 (forward) and 5 AGTCTCGAGCCTTCACTTGG-3 (reverse) primer pairs for −710 C > T, and 5 -GAATGACCCCTGAACCTC-3 (forward) and 5 -CGTTGGCACGCTCCAG-3 (reverse) for −392 T > C. PCRs were performed using an initial denaturation at 95 ◦ C for 10 min, 35 amplification cycles (30 s at 95 ◦ C, 1 min annealing at 63.9 ◦ C and 1 min at 72 ◦ C), followed by a single 7 min extension cycle at 72 ◦ C. The SNP genotyping primers were 5 -TGCCATCCTCAATACCCA-3 (forward) for −710 C > T and 5 GTTGCTTCAGCAAACCGG-3 (forward) for −392 T > C. PCR

Polymorphisms of RASSFIA promoter and lung cancer products were subsequently purified by incubating them with 10 units of ExoI (USB, Cleveland, OH) and 1 unit of shrimp alkaline phosphatase (Roche, Indianapolis, IN) at 37 ◦ C for 1 h and at 72 ◦ C for 15 min. Extension reactions with 1 ␮l of purified PCR product, 0.15 pmol of genotyping primer, and a SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems, Foster City, CA) were carried out by repeating the following cycle 25 times: 96 ◦ C for 10 s, 50 ◦ C for 5 s, and 60 ◦ C for 30 s. Extension products were incubated with 1 unit of shrimp alkaline phosphatase (Roche, Indianapolis, IN) at 37 ◦ C for 1 h and then at 72 ◦ C for 15 min. Nine microliters of deionized formamide was mixed with 1 ␮l of the purified extension product, and electrophoresed on an ABI Prism 3700 genetic analyzer (Applied Biosystems, Foster City, CA). Results were analyzed using GeneScan analysis, version 3.7 (Applied Biosystems, Foster City, CA).

2.4. Plasmid construct for RASSF1A promoter assay To examine the potential effects of −710 C > T and −392 T > C polymorphisms on the RASSF1A transcription activity, the promoter activity of the three haplotypes (C—C, T—T and C—T) was compared. The fragments of the RASSF1A promoter region (from −1051 to +91, translation start site of exon 1 counted as +1) were synthesized by PCR using gDNA with the following primers (Supplement Fig. I): 5 -CATGTGTTCAGCCCCTCAG-3 (forward) and 5 CGTTGGCACGCTCCAGC-3 (reverse). The PCR products were inserted upstream of the luciferase gene in the pGL3-basic plasmid (Promega, Madison, WI). The structure of each construct was verified by sequencing.

Table 1

303

2.5. Transient transfection and luciferase reporter assay The NCI-H2009 and NCI-H358 NSCLC cell lines used in this study were purchased from the American Type Culture Collection (ATCC). Cells were plated at a density of 1 × 105 cells/35 mm in six-well plates the day before transfection. When cells reached 75% of confluence, they were transfected with 1 ␮g DNA using 3 ␮l of the LipofectamineTM 2000 reagent (Gibco BRL, Rockville, MD). The pGL3-basic vector, which lacks both promoter and enhancer, was used as a negative control in each of the transfection experiments. The pGL3-control vector (Promega, Madison, WI), containing SV40 promoter and enhancer, was used as a positive control. One hundred nanograms of ␤-galactosidase luciferase plasmid (Promega, Madison, WI) were included in each transfection to check transfection efficiency. Cells were collected 48 h after transfection and lysed with 5× lysis buffer (Promega, Madison, WI). Luciferase activities were determined using the Luciferase Assay System (Promega, Madison, WI), and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA), and normalized against the activity of the ␤galactosidase luciferase gene. ␤-Galactosidase assays were performed using a Luminescent ␤-galactosidase Detection kit II (Clontech, Hampshire, UK) [14,16—18]. A minimum of four independent experiments were performed for each plasmid.

2.6. Statistical analysis Allele frequencies, genotype frequencies, and departures of genotype distributions from Hardy—Weinberg equilibrium for each SNP were analyzed using the chi-square test or

Baseline characteristics of study subjects

Variable Age (years)

Cases (n = 410) 61.13 ± 9.91

Controls (n = 410) 60.58 ± 10.85

Gender Male Female

303 (73.9%) 107 (26.1%)

301 (73.4%) 109 (26.6%)

Smoking statusa Non-smoker Smoker

111 (27.8%) 288 (72.2%)

165 (53.6%) 143 (46.4%)

Drinking status Non-drinker Drinker

121 (39.3%) 187 (60.7%)

77 (35%) 143 (65%)

Stage 1-3a 3b-4

93 (28.5%) 233 (71.5%)

Cell types Adenocarcinomas Squamus cell carcinomas Other carcinomasb

157 (40.4%) 123 (31.6%) 109 (28%)

a b

p < 0.001. Including the small cell, large cell, and mixed cell carcinomas or undifferentiated carcinomas.

304 Table 2

J.S. Sung et al. Association analysis of RASSF1A promoter polymorphisms and haplotypes in lung cancer

Group

Loci

Genotype

Cases

Controls

Over all

−710 C > T

CC CT TT

121 225 63

−392 T > C

TT CT CC

ht1 C—C

Male

Female

Non-smoker

Codominant OR (95% CI)a

Dominant OR (95% CI)

Recessive OR (95% CI)

143 194 68

1.04 (0.82—1.31)

1.26 (0.90—1.76)

0.78 (0.51—1.19)

96 225 89

109 206 94

1.11 (0.88—1.40)

1.38 (0.96—1.98)

0.93 (0.64—1.36)

−/− +/− +/+

97 223 89

108 202 94

1.10 (0.87—1.39)

1.38 (0.96—1.98)

0.92 (0.63—1.34)

ht2 C—T

−/− +/− +/+

346 60 3

319 81 4

0.70 (0.48—1.03)

0.69 (0.46—0.99)b

0.48 (0.10—2.24)

ht3 T—T

−/− +/− +/+

121 226 62

143 193 68

1.03 (0.82—1.30)

1.26 (0.90—1.77)

0.77 (0.50—1.17)

−710 C > T

CC CT TT

86 172 45

111 140 46

1.13 (0.84—1.51)

1.47 (0.98—2.22)

0.79 (0.47—1.33)

−392 T > C

TT TC CC

69 168 66

81 144 75

1.09 (0.83—1.45)

1.51 (0.97—2.34)

0.82 (0.52—1.29)

ht1 C—C

−/− +/− +/+

70 167 66

81 140 75

1.09 (0.83—1.45)

1.54 (0.99—2.39)

0.80 (0.51—1.27)

ht2 C—T

−/− +/− +/+

260 41 2

229 63 4

0.59 (0.38—0.93)c

0.58 (0.35—0.96)c

0.29 (0.05—1.64)

ht3 T—T

−/− +/− +/+

86 173 44

111 139 46

1.12 (0.84—1.50)

1.48 (0.98—2.24)

0.77 (0.45—1.30)

−710 C > T

CC CT TT

35 53 18

32 54 22

0.85 (0.57—1.25)

0.84 (0.46—1.54)

0.72 (0.35—1.48)

−392 T > C

TT TC CC

27 57 23

28 62 19

1.19 (0.78—1.81)

1.14 (0.60—2.18)

1.39 (0.69—2.81)

ht1 C—C

−/− +/− +/+

27 56 23

27 62 19

1.16 (0.76—1.77)

1.08 (0.57—2.07)

1.39 (0.68—2.81)

ht2 C—T

−/− +/− +/+

86 19 1

90 18 0

1.15 (0.56—2.34)

1.07 (0.51—2.25)

—

ht3 T—T

−/− +/− +/+

35 53 18

32 54 22

0.84 (0.46—1.54)

0.84 (0.46—1.54)

0.72 (0.35—1.48)

−710 C > T

CC

40

56

0.81 (0.56—1.17)

0.81 (0.48—1.37)

0.69 (0.35—1.36)

Polymorphisms of RASSFIA promoter and lung cancer

305

Table 2 (Continued ) Group

Smoker

Loci

Genotype

Cases

Controls

Codominant OR (95% CI)a

Dominant OR (95% CI)

Recessive OR (95% CI)

CT TT

54 16

79 29

−392 T > C

TT TC CC

23 61 27

40 88 37

1.24 (0.86—1.80)

1.34 (0.74—2.43)

1.31 (0.12—2.37)

ht1 C—C

−/− +/− +/+

23 60 27

39 88 37

1.22 (0.84—1.77)

1.28 (0.70—2.33)

1.31 (0.72—2.37)

ht2 C—T

−/− +/− +/+

91 18 1

136 27 1

1.06 (0.57—1.96)

1.02 (0.53—1.97)

2.37 (0.13—43.08)

ht3 T—T

−/− +/− +/+

40 54 16

56 79 29

0.81 (0.56—1.17)

0.81 (0.48—1.37)

0.69 (0.35—1.36)

−710 C > T

CC CT TT

77 166 45

51 63 25

1.22 (0.89—1.67)

1.66 (1.06—2.59)b

0.86 (0.50—1.50)

−392 T > C

TT TC CC

68 161 59

44 64 34

1.03 (0.76—1.39)

1.37 (0.86—2.17)

0.75 (0.46—1.24)

ht1 C—C

−/− +/− +/+

69 160 59

44 60 34

1.03 (0.76—1.39)

1.40 (0.88—2.22)

0.73 (0.45—1.21)

ht2 C—T

−/− +/− +/+

248 38 2

105 30 3

0.58 (0.36—0.93)b

0.56 (0.33—0.96)b

0.32 (0.05—1.95)

ht3 T—T

−/− +/− +/+

77 167 44

51 62 25

1.22 (0.89—1.67)

1.67 (1.07—2.61)b

0.84 (0.48—1.46)

Logistic regression models were used to calculate the ORs, 95% CIs and the corresponding p values of codominant (minor allele homozygotes vs. heterozygotes vs. major allele homozygotes), dominant (minor allele homozygotes + heterozygotes vs. major allele homozygotes), and recessive (minor allele homozygotes vs. heterozygotes + major allele homozygotes) models whilst controlling for age, gender and smoking status as covariates. a ORs and 95% CI were calculated by logistic regression and adjusted for age, gender and smoking status. b p < 0.05. c p < 0.03 and p < 0.05. c

Fisher’s exact test. A p-value of <0.05 was considered statistically significant. Linkage disequilibrium (LD) was tested on pairwise combinations of SNPs using the absolute value of the standardized measure of linkage disequilibrium, D , calculated by the Haploview program version 3.2 [19]. The haplotypes and their frequencies were estimated by the Haploview program version 3.2. Genotype-specific risks were estimated as odds ratios (ORs) with associated 95% confidence intervals by unconditional logistic regression (SAS Institute, Cary, NC) and adjusted for age and gender. When comparisons were made, the pc values were also calculated for multiple testing using Bonferroni’s inequality method. Differences in haplotype-specific luciferase activity were tested using the Student’s t-test.

3. Results Table 1 shows the clinicopathological features of the cases and controls. By direct sequencing of RASSF1A promoter region (∼2 kb), we discovered two polymorphisms of the RASSF1A promoter region among 48 lung cancer patient samples (−710 C > T and −392 T > C loci). The minor allele frequencies were 0.370 and 0.440, respectively. Further analyses were then performed on 362 lung cancer patients and 410 controls. The genotype distributions of polymorphisms among the controls were in Hardy—Weinberg equilibrium. Association of the risk of lung cancer with RASSF1A promoter polymorphisms and haplotypes was analyzed

306

J.S. Sung et al.

Fig. 1 Comparative analysis of transcription activity using polymorphism constructs of RASSF1A promoter. In this schematic, the translation initiation site for the RASSF1A protein is indicated as +1. The LD block organization of RASSF1A promoter region is in Korean population (A). Promoter activities of the constructs were measured by transient transfection in H2009 (B) or H358 (C) cells. The p values were calculated using Student’s t-test. *p < 0.005, **p < 0.025.

(Table 2). The genotypes of RASSF1A promoter polymorphisms were not associated with the risks of lung cancer in three alternative models. The two polymorphisms were in linkage disequilibrium (|D | value; 0.99). The subsequent analysis revealed that ht2 C—T haplotype was associated with susceptibility to the risk of lung cancer in dominant (OR: 0.69; 95% CI: 0.46—0.99) model. The association of the polymorphisms with the risk of lung cancer was further examined after stratifying the subjects according to gender and smoking status. The protective effect of the C—T haplotype on the risk of lung cancer was displayed in males (codominant OR: 0.59; 95% CI: 0.38—0.93 and dominant OR: 0.58; 95% CI: 0.35—0.96) and in smokers (codominant OR: 0.58; 95% CI: 0.36—0.93 and dominant OR: 0.56; 95% CI: 0.33—0.96). In order to elucidate possible mechanisms for the observed association between RASSF1A promoter polymor-

phisms and lung cancer, we measured the promoter activity using the luciferase assay system and compared the activities of the RASFF1A haplotypes in H2009 and H358 cell lines using transient transfection assay: the promoter activities of the three haplotypes were compared because these two polymorphisms were in LD (Fig. 1A). In H2009 cells, the C—T haplotype showed significantly increased promoter activity compared to the C—C and T—T haplotypes (Fig. 1B). Similarly, transcriptional activities of the C—T haplotype in H358 cells also revealed significantly increased promoter activity compared to the C—C and T—T haplotypes (Fig. 1C).

4. Discussion Although smoking plays a critical role in the development of lung cancer [20,21], only a fraction of smokers develop lung

Polymorphisms of RASSFIA promoter and lung cancer cancer during their lifetime. The possible mechanisms instigating lung carcinogenesis in the remainder of the smoking population has not been clearly defined. One such mechanism is the RASSF1 tumor suppressor gene, which is noted for its substantial affiliation with smoking and has commonly been found in an inactivated state in many human cancers, including lung cancer. Recently, several investigations have reported that DNA hypermethylation is associated with exposure to cigarette smoke [22—24], and DNA methylation of the CpG sites in the promoter region of the RASSF1A gene is accountable for 40% of lung cancer in smokers [25]. The loss of heterozygosity and epigenetic inactivation of the RASSF1A promoter have been reported to down-regulate gene expression, subsequently increasing the risk of lung cancer [7—9]. This suggests that genetic constitution of individuals is of importance in determining the susceptibility of individuals to lung cancer [26,27]. There are other plausible mechanisms that can contribute to the onset of lung cancer. In the past, researchers have examined the somatic mutations of the RASSF1A gene in breast and lung cancer patients [4,8,28], however somatic mutations of the RASSF1A in lung cancer tissues are uncommon. It has also been reported that a germ line polymorphism of RASSF1A (A133S) decreased phosphorylation and anti-proliferative activity in NSCLC cells [29]. At this codon 133, recent study showed that a SNP of the RASSF1A gene is associated with the risk of lung adenocarcinoma [30] and breast cancer [31]. However, no SNPs study has been done on the promoter region of RASSF1A. In order to validate this claim, we carried out an experiment on the Korean population, however found no significant correlation between codon 133 polymorphisms and lung cancer (data not shown). In the present study, therefore we determined the existence of a possible association between lung cancer in the Korean population and the polymorphisms in the promoter region of RASSF1A in the blood sample of lung cancer patients. Interestingly, our results demonstrated that the ht2 C—T haplotype of the RASSF1A promoter had a protective effect on the risk of lung cancer, especially in males and smokers. The study on the association with smoking and risk of lung cancer was based on 308 controls, not 410. In NSCLC cell lines, the C—T haplotype showed a significantly increased promoter activity compared to the C—C and T—T haplotypes, which implicates an induction of tumor suppression, consequently diminishing the risk of lung cancer. Polymorphisms often show ethnic variation [32]. In the present study, we observed that the frequencies of the minor alleles of −710 C > T and −392 T > C among healthy Korean population were 0.407 and 0.482, respectively. In comparison, the frequencies in the dbSNP database (www.ncbi.nlm.nih.gov/SNP) in various other ethnicities have been reported to have no frequency data in −710 C > T polymorphism, while that of the −392 C allele has been reported to vary among ethnicities (Europeans: no frequency; Han Chinese: 0.628; Japanese: 0.489 and SubSaharan African: 0.275). In conclusion, this is the first study to disclose an association between the RASSF1A promoter polymorphisms and the risk of lung cancer. The C—T haplotype of the RASSF1A promoter which exhibits a significantly increased transcriptional activity indicates an enhanced suppression of lung

307 carcinogenesis. Moreover, considering the ‘‘two-hit’’ theory of tumor suppression genes, it can be implied that patients with a low level of RASSF1A expression could be more vulnerable to smoking related gene inactivation, via such as promoter methylation. Therefore, we suggest that the C—T haplotype of the RASSF1A promoter may play an important role in genetic susceptibility of the Korean population to lung cancer.

Conflict of interest None.

Acknowledgements We thank Young Lim, Dong-Hoon Shin, Choon-Sik Park and Yangsoo Jang for sample collection and provision. This study was supported by grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A010250), Jae Won Lee and Hyo Jung Lee was supported by Korea Science and Engineering Foundation Grant (R14-2003-00201002-0).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.lungcan. 2008.01.012.

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