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Carcinogenesis Advance Access originally published online on February 24, 2008
Carcinogenesis 2008 29(5):957-963; doi:10.1093/carcin/bgn048
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Association of KRAS polymorphisms with risk for lung adenocarcinoma accompanied by atypical adenomatous hyperplasias

Takashi Kohno1, Hideo Kunitoh2, Kenji Suzuki3, Seiichiro Yamamoto4, Aya Kuchiba4,5, Yoshihiro Matsuno6,8, Noriko Yanagitani1,7 and Jun Yokota1,*

1 Biology Division, National Cancer Center Research Institute, Tokyo 1040045, Japan
2 Thoracic Oncology Division
3 Thoracic Surgery Division, National Cancer Center Hospital, Tokyo 1040045, Japan
4 Cancer Information Services and Surveillance Division, Center for Cancer Control and Information Services, National Cancer Center, Tokyo 1040045, Japan
5 Department of Biostatistics/Epidemiology and Preventive Health Sciences, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 1130033, Japan
6 Diagnostic Pathology Division, National Cancer Center Hospital, Tokyo 1040045, Japan
7 First Department of Internal Medicine, Gunma University School of Medicine, Showa-machi, Gunma 3718511, Japan
8 Present address: Department of Surgical Pathology, Hokkaido University Hospital, Sapporo 0608648, Japan

* To whom correspondence should be addressed. Tel: +81 3 3542 2511; Fax: +81 3 3542 0807;Email: jyokota{at}gan2.ncc.go.jp.


   Abstract
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 Abstract
Introduction
 Materials and methods
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 Discussion
 Supplementary material
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The pulmonary adenoma susceptibility 1 (Pas1) gene affects susceptibility to the development of lung adenomas in mice with a subset of the adenomas progressing to adenocarcinoma (ADC). In this study, genotype distributions for 10 polymorphisms in the human counterparts for three mouse candidate Pas1 genes, KRAS, CASC1/LAS1 and LRMP, were examined in a hospital-based case–control study consisting of 364 lung ADC cases and 253 controls. All the ADC cases were subjected to lobectomy and subsequent pathological investigation of atypical adenomatous hyperplasia (AAH), a putative precursor for peripheral lung ADC, including bronchioloalveolar carcinoma, in the resected lobes. Eighty-one (22%) of the ADC cases carried at least one AAH lesion in addition to the primary ADC and 34 (9%) of them carried multiple AAH lesions. None of the 10 polymorphisms examined showed significant associations with overall lung ADC risk (P > 0.05). However, minor allele carriers for two polymorphisms in the KRAS gene, KRAS-1 and -6, showed significantly increased odds ratios (ORs) for ADC accompanied by multiple AAHs [OR = 3.0; 95% confidence interval (CI) = 1.4–6.2, P = 0.004 and OR = 2.4; 95% CI = 1.1–4.7, P = 0.02, respectively]. Minor haplotypes including the minor allele for the KRAS-6 polymorphism showed increased ORs for ADC accompanied by multiple AAHs, and KRAS transcripts from the minor allele for this polymorphism were more abundantly detected in lung tissues than those from the major allele. Thus, KRAS polymorphisms were indicated to be involved in risk for the development of AAHs that progress to ADC by causing differential KRAS oncogene expression in the lungs.

Abbreviations: AAH, atypical adenomatous hyperplasia; ADC, adenocarcinoma; BAC, bronchioloalveolar carcinoma; Casc, cancer susceptibility candidate; cDNA, complementary DNA; CI, confidence interval; Kras, Kirsten rat sarcoma oncogene; Las, lung adenoma susceptibility; LD, linkage disequilibrium; LRMP, lymphoid-restricted membrane protein; OR, odds ratio; Pas1, pulmonary adenoma susceptibility 1; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism


   Introduction
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 Abstract
 Introduction
Materials and methods
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Adenocarcinoma (ADC) is now the most common type of non-small cell lung carcinoma, followed by squamous cell carcinoma and small cell carcinoma (1,2). Development of lung ADC is less associated with smoking compared with squamous cell carcinoma and small cell carcinoma. Thus, effective ways of preventing ADC are being searched for (1,2). Identification of genes responsible for susceptibility to lung ADC is considered to be indispensable to establish novel and efficient ways of preventing the disease. However, only a few metabolic and DNA repair genes, such as CYP1A1 and OGG1, have been shown to be associated with risk for lung ADC (1,3–6), and therefore, genes responsible for lung ADC susceptibility are largely unknown.

Inbred strains of mice exhibit a difference in their susceptibilities to both spontaneous and carcinogen-induced lung adenoma development (7). To date, dozens of genetic loci have been shown to be linked to mouse lung adenoma susceptibility (Las) through linkage analyses, including pulmonary adenoma susceptibility (Pas), pulmonary adenoma resistance and susceptibility to lung cancer (8–12). Recently, we reported that a non-synonymous (associated with amino acid change) single-nucleotide polymorphism (SNP) in POLI, the human counterpart for a candidate pulmonary adenoma resistance 2 gene, was associated with lung ADC risk (13). Thus, it was suggested that human counterparts for mouse Las genes also play a role in human lung ADC risk. Pas1 is a major locus accounting for ~50% of variances in Las in mice, and three genes of Kirsten rat sarcoma oncogene (Kras) 2, cancer susceptibility candidate (Casc) 1/Las1 and lymphoid-restricted membrane protein (Lrmp) have been identified from this locus as strong candidates determining the susceptibility (11,14–18). A case–control study of lung ADC in an Italian population showed that a SNP in the KRAS gene (KRAS-1 in Table I) was associated with lung ADC risk (19); however, the association was not reproduced in the following studies (20,21). SNPs in the LRMP and CASC1 genes did not show associations with lung ADC risk in a recent study (22). Instead, the above SNP in KRAS and a SNP in LRMP (LRMP-6 in Table I) showed associations with prognosis of lung ADC patients (19,20,22). Thus, it is still controversial how the human counterparts for the mouse Pas1 genes are involved in the development and progression of lung ADC in humans.


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  		Table I. Ten polymorphisms in the KRAS, CASC1 and LRMP genes, and primers and conditions for pyrosequencing

 
The mouse Pas1 gene affects susceptibility to lung adenoma developments in mice, and a subset of the adenomas progress to ADC that is analogous to bronchioloalveolar carcinoma (BAC) in humans (7). Thus, it is possible that variations in human counterparts for Pas1 are also involved in susceptibility to the development of lung adenoma and/or BAC (18). Atypical adenomatous hyperplasia (AAH) is a lesion with a monoclonal nature (23) and has been considered as a precursor for peripheral lung ADC, including BAC, the adenoma in an adenoma–carcinoma sequence in the peripheral lung (24). AAH is a frequent incidental histologic finding in lungs bearing primary lung ADC (24,25). Such an accompaniment of AAH was detected in 16–35% of ADC cases. AAH was also detected in the lungs of autopsy cases without cancer by histological examination. In two studies, AAH was examined in hospital autopsy cases and was detected in 2.0 and 3.4% of the cases without cancer, respectively (26,27). In another study, administration autopsy cases were examined to estimate the prevalence of AAH in the general population, and AAH was detected in 2.8% of the cases (28). Therefore, AAH is indicated to be present in the lungs of individuals without cancer; however, the frequency of having AAH in those individuals is considerably lower than that in ADC patients (24). The results indicate that the susceptibility to the development of AAH is also associated with that of lung ADC in humans, and therefore, the lungs of humans with primary ADCs accompanied by AAH, particularly those accompanied by multiple AAHs, are analogous to those of mice with the susceptible Pas1 allele. Thus, in the present study, polymorphisms of the KRAS, CASC1 and LRMP genes were examined for association with risk for the development of lung ADC after subclassification of the subjects according to AAH accompaniment and BAC component involvement.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Case and control subjects
All cases and controls were Japanese. The cases consisted of 364 ADC patients treated at the National Cancer Center Hospital, Tokyo. All ADC cases, who received lobectomies from 1999 to 2004 and from whom informed consents as well as blood samples were obtained, were consecutively included in this study without any particular exclusion criteria. All the cases were diagnosed as ADC by histological examinations according to the World Health Organization classification (24,29). Information on the AAH in the ADC patients was obtained by routine surgical pathology examination as follows. Resected lungs were inflated with 10% formalin through bronchial cut ends and after fixation for a few days were serially sliced at intervals of 5 mm, and each cut surface was macroscopically examined. Sliced lungs containing a lesions suspected for AAH were further examined microscopically. Even in cases without macroscopic lesions, at least one tissue block was prepared from all sliced lungs and subjected to microscopic examination. The criteria for AAH were as follows as described previously (27,28): (i) a localized lesion with well-defined boundaries; (ii) an alveolar wall slightly thickened with mild infiltration of inflammatory cells but without scar formation; (iii) proliferating atypical epithelial cells abutting each other but not as compact as in ADC; (iv) atypical epithelial cells that were cuboidal to low columnar or peg shaped in appearance, resembling either type II pneumocytes or non-ciliated bronchiolar epithelial cells (Clara cells) and (v) the presence of some atypical cells with two or more nuclei, most of which had relatively smaller and smoother contours than those of ADC. These criteria are compatible with those described in the reference of World Health Organization classification of lung tumors as a proposal (24,30). The controls consisted of cancer-free patients of National Cancer Center Hospital. All the control subjects were selected with a criterion of no history of cancer. Smoking history of cases and controls was obtained via interview using a questionnaire. Smoking habit was expressed by pack-years, which was defined as the number of cigarette packs smoked daily multiplied by years of smoking, both in current smokers and former smokers. Smokers were defined as those who had smoked regularly for 12 months or longer at any time in their life, whereas non-smokers were defined as those who had not. From each individual, a 20 ml whole-blood sample was obtained. The study was approved by the Institutional Review Boards of the National Cancer Center.

DNA extraction, polymorphism search and genotyping
Genomic DNAs were isolated from whole-blood samples using a QIAamp DNA Blood Maxi kit (Qiagen, Tokyo, Japan). DNAs from 24 lung ADC cases and 24 controls, respectively, were subjected to a search for polymorphisms in exons of the KRAS, CASC1 and LRMP genes by resequencing according to the procedure described previously (13). SNPs in introns of these three genes with minor allele frequencies >0.1 in the Japanese population were selected from SNPs deposited in the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/). Genotyping was performed with 10 ng of genomic DNA by the pyrosequencing method according to the procedure described previously (13).

Statistical analysis
Hardy–Weinberg equilibrium tests were performed using the SNPAlyze version 3 software (DYNACOM Co., Ltd, Chiba, Japna). SNPs with a P value for deviation >0.01 were considered to be in Hardy–Weinberg equilibrium. Calculation of the D' values and haplotype estimation were undertaken using the expectation-maximization (EM) algorithm using the same software. The strength of association of genotypes with ADC risks was measured as crude odds ratios (ORs) and ORs adjusted for gender, age (<49, 50–59, 60–69 and >70) and smoking dosage (pack-years: 0, 1–49 and >50) with 95% confidence intervals (CIs) by unconditional logistic regression analysis (31). Statistical analyses were performed using the JMP version 6.0 software (SAS Institute, Cary, NC). ORs for carrying a copy of a haplotype were also calculated by the bootstrap method with 5000 resampling. The statistical analyses were performed using the SAS version 9 software (SAS Institute). Statistically, a level of P < 0.05 for an OR was considered significant, whereas a level of 0.05 ≤ P < 0.10 for an OR was considered marginally significant.

Analysis of KRAS transcripts
Genomic DNA and total RNA were extracted from eight non-cancerous lung tissues of eight lung ADC patients heterozygous for an insertion–deletion polymorphism, KRAS-6 (see Table I). Complementary DNA (cDNA) was synthesized by reverse transcription of 1 mg of total RNA using the Superscript First-Strand Synthesis System. Ten nanograms of genomic DNA and cDNA corresponding to 50 ng of total RNA were subjected to polymerase chain reaction (PCR) in duplicate. The PCR was performed using a set of primers, 5'-CAGGAACTGCAGTGCTTATG-3' and 5'-TTAAGGCTGTAATAATTAGGTAAC-3' (fluorescein isothiocyanate labeled), for 30 cycles consisting of denaturation at 95°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min. PCR products were electrophoresed using an ABI PRISM 3700 Genetic Analyzer and analyzed by Gene Scan software (Applied Biosystems, Foster City, CA). Ratios of the minor allele (i.e. T allele) to the major allele (i.e. – allele) products in each sample were calculated from the height of peaks corresponding to the minor and major alleles, respectively. The ratio for each sample was expressed by the mean ratios of the two independent PCRs. The difference in the mean ratios between genomic DNA and cDNA in eight cases was tested by the paired t-test.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
We searched for polymorphisms located in exons of the KRAS, CASC1 and LRMP genes by the resequencing of their exons in 48 Japanese individuals, and identified a 1 bp insertion–deletion polymorphism in the KRAS gene and three non-synonymous SNPs in the CASC1 and LRMP genes (Table I). The insertion–deletion polymorphism was novel, while the three SNPs had been deposited in the dbSNP database. Six other SNPs in introns of these three genes whose minor allele frequencies were >0.1 in the Japanese population were selected from SNPs deposited in the dbSNP database (Table I). In total, 10 polymorphisms dispersed in the KRAS, CASC1 and LRMP genes were selected for the present study (Figure 1).


Figure 1
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  		Fig. 1. Polymorphisms in the human Pas1 locus and their LD. Ten polymorphisms are shown on top, and D' values between the SNPs are shown below. D' values >0.9 are marked in red.

 
We prepared 364 ADC cases and 253 hospital-based controls (Table II). The ADC cases received lobectomies at National Cancer Center Hospital and were subjected to the pathological search for AAH in the resected lobes. In the lobes, one or more (i.e. multiple) AAH lesions were detected in 81 cases (22%), while no AAH lesion was detected in the remaining 283 cases (78%). The frequency of AAH accompaniment in these ADC cases was consistent with those in previous studies (24). Representative microphotographs of multiple AAH lesions detected in an ADC patient are shown in supplementary Figure 1 (available at Carcinogenesis Online). In 34 of the 81 cases (9%), 2 or more (i.e. multiple) AAH lesions were detected. The 364 ADC cases included 173 cases of small-sized ADC (i.e. <2 cm in maximum diameter), and the information on the presence of BAC components in the tumor was available (Table II). One hundred and fifty-two cases (88%) contained BAC components in the tumor, whereas the remaining 21 cases (12%) did not. It was difficult to histologically define AAHs in primary ADC lesions, even in ADCs with BAC components. Thus, it was not possible to pick up cases of ADCs in AAH lesions, so-called ‘carcinoma in adenoma’. Accordingly, cases with AAHs were defined as having AAHs that existed independently from primary ADCs in the present study.


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  		Table II. Lung ADC cases and controls used in this study

 
All the cases and controls were subjected to genotyping for the 10 polymorphisms, and all the polymorphisms were in Hardy–Weinberg equilibrium both in the cases and controls. Genotypic differentiation for the 10 polymorphisms was examined between cases and controls. The differentiation was examined for all ADC cases as a whole and cases categorized by AAH accompaniment. The differentiation was also examined for small-sized ADC cases with BAC components. The number of small-sized ADC cases without BAC components was small; therefore, they were excluded from the analysis. The differentiation was also examined after dividing ADC subjects into smokers and non-smokers. To increase statistical power, genotypic differentiation was examined by assessing OR of minor allele carriers against homozygotes for the major allele (i.e. non-carriers).

Minor allele carriers for a SNP, KRAS-12, showed a marginally significant increase in the OR for the risk for overall ADC (P = 0.06), while none of the other nine polymorphisms showed significant increases or decreases in the OR (Table III). When the ADC cases were categorized by AAH accompaniment, ORs of minor allele carriers for six polymorphisms, KRAS-1, -6, CASC-1, -4, -5 and LRMP-7, against non-carriers were higher for ADC with AAH than for that without. The ORs were even higher for ADC with ≥2 AAHs. Increases in adjusted ORs for two of the six polymorphisms, KRAS-1 and -6, were statistically significant (OR = 3.0; 95% CI = 1.4–6.5 and 2.4; 95% CI = 1.1–5.1, respectively), and those for three other polymorphisms, CASC-1, -4 and LRMP-7, were marginal (Table III).


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  		Table III. KRAS, CASC1 and LRMP genotypes and risks for ADC

 
We next calculated ORs for the risk for small-sized ADC with BAC components. Minor allele carriers for the KRAS-1 SNP, which showed the most significantly increased OR of 1.3 for ADC with ≥2 AAHs, showed a slightly increased OR against non-carriers for the risk for ADC with BAC components, but the increase was not statistically significant (P = 0.3) (Table III). ORs for the other nine polymorphisms were not significant, either. When the ADC cases were divided into smokers and non-smokers, ORs were not apparently different between them. Increases or decreases in ORs were not significant, either.

Linkage disequilibrium (LD) among the 10 polymorphisms was then estimated. Five polymorphisms, KRAS-1, -6, CASC1-1, -4 and -5, were in LD with one another (D' > 0.9) (Figure 1), and the size of the region with LD was 94 kb. Thus, the distribution of haplotypes consisting of these five polymorphisms was evaluated in cases and controls (Table IV). Three haplotypes were deduced to comprise ~97% of chromosomes among controls and cases. The major haplotype consisting of major alleles for all the five polymorphisms (i.e. haplotype 1 in Table IV) was less prevalent in ADC cases with ≥2 AAHs than in controls, whereas a minor haplotype consisting of minor alleles for the five polymorphisms (i.e. haplotype 2) was more prevalent. Haplotype 3 was almost evenly distributed in cases and controls. Adjusted ORs for the risk for ADC accompanied by ≥2 AAHs by carrying one copy of haplotypes 2 and 3 was calculated based on the estimated copy number of haplotypes for each subject by the bootstrap method. The ORs for haplotypes 2 and 3 were 2.6 (95% CI = 1.1–5.9, P = 0.02) and 1.8 (95% CI = 0.6–5.6, P = 0.3), respectively. Therefore, both haplotypes showed increased ORs, while only increase in the OR for haplotype 2 was statistically significant.


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  		Table IV. Frequency of the Pas1 haplotypes in cases and controls

 
In mice, it was shown that KRAS transcripts from the susceptible Pas1 allele in lung tissue are more abundant than those from the resistant allele, and such a difference was proposed to underlie the difference in the susceptibility to adenoma development (18). As described above, hapolytypes 2 and 3, which showed significantly and insignificantly increased ORs for ADC accompanied by multiple AAHs, respectively, contained a minor allele for KRAS-6, a 1 bp insertion–deletion polymorphism in the 3'-untranslated region (3'-UTR) of the KRAS gene. This result prompted us to compare the amounts of KRAS transcripts between susceptible and resistant alleles by using the KRAS-6 polymorphism. Genomic DNA and cDNA prepared from non-cancerous lung tissues of eight heterozygotes for this polymorphism were subjected to PCR using a set of primers that amplifies the same DNA/cDNA fragments encompassing the KRAS-6 site. Relative amounts of the susceptible (i.e. T) allele products to the resistant (i.e. –) allele products were greater in cDNA than in genomic DNA in all the eight cases (Figure 2). The mean for the ratio of the minor allele products to the major allele products in genomic DNA and cDNA was 1.02 and 1.13, respectively, and the difference was statistically significant (P = 0.005 by the paired t-test). Thus, it was indicated that transcripts from the KRAS-6 minor allele were more abundant than those from the major allele in lung tissues.


Figure 2
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  		Fig. 2. Differential messenger RNA expression between two polymorphic KRAS alleles. (A) Electrophoregram for genomic DNA (gDNA) and cDNA of a representative case. Relative amounts of PCR products from the minor (T) allele to those from the major (–) allele are shown below as T/– ratios. (B) Relative amounts of PCR products from the minor allele to those from the major allele in genomic DNA and cDNA of non-cancerous lung tissues of eight KRAS-6 hetrozygotes. Mean ± SD for the relative amounts in genomic DNA and cDNA is also indicated with a P value by the paired t-test.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
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In the present study, we examined the association of polymorphisms in the human counterparts for three mouse candidate Pas1 genes with risk for lung ADC. None of the 10 polymorphisms examined showed significant associations with risk for lung ADC as a whole. This result was consistent with recent case–control studies (20,22). However, when ADC subjects were categorized according to AAH accompaniment, two polymorphisms, KRAS-1 and -6, showed associations with risk for ADC accompanied by multiple AAHs. Haplotypes containing the minor allele for the KRAS-6 polymorphism also showed increased ORs for ADC accompanied by multiple AAHs. The results strongly suggested that KRAS is a determinant for susceptibility to ADC accompanied by multiple AAHs in humans. Namely, individuals with minor alleles for these two KRAS polymorphisms could have a higher risk than those without for the development of lung tumors analogous to the tumors in mice with a susceptible Pas1 allele; i.e. multiple adenomatous lesions (multiple AAHs) with a subset of lesions that have progressed to primary ADC. KRAS transcripts from the risk haplotypes were more abundant than those from the resistant haplotype. This result was also analogous to the status of the mouse Pas1 locus (18). KRAS/Kras is an oncogene that is activated by somatic mutations in lung ADC both of humans and mice (32–35). Thus, an abundant KRAS/Kras expression due to polymorphisms might make the lung epithelial cells more susceptible to the development of adenomas, a subset of which progress to ADC, both in humans and mice.

KRAS minor allele carriers showed only a slightly increased and insignificant OR for the risk for development of lung ADC as a whole and even of lung ADC with BAC components, which is considered to develop from AAH. This result is in contrast to the finding in mice that Pas1 has a major role in predisposition to not only lung adenoma but also lung ADC (36). The result may imply that KRAS polymorphisms are responsible for the development of AAH; however, their effects on the development of lung ADC, including BAC, are limited. In this context, however, we should consider the difference in the process of ADC development between mice and humans. In the studies of experimental mouse models, lung ADCs in mice could be exclusively developed through adenomas; therefore, Pas1 might have been judged as having a role in the predisposition to not only lung adenoma but also lung ADC. In humans, in contrast, a subset of ADC, including BAC, may not be developed through AAH. This could be a reason why the KRAS minor allele has not been defined as a risk allele for lung ADC development in several studies, including this one. In fact, fractions of minor allele carriers for KRAS-1 and -6 polymorphisms in ADC cases with multiple AAHs were significantly higher than those in ADC cases without AAH (OR = 3.0; 95% CI = 1.4–6.2, P = 0.004 and OR = 2.2; 95% CI = 1.1-4.7, P = 0.03, respectively). This result further supports that KRAS polymorphisms are responsible for the development of AAH but not of lung ADC. In addition, progression of AAH to ADC in mice can be influenced by several other genetic factors, as indicated by the pulmonary adenoma progression loci, which determine the susceptibility to the progression of adenoma to ADC in the lungs (21,36). Thus, association of polymorphisms in the KRAS gene with risk for lung AAH and ADC should be further examined in a larger number of samples in conjunction with polymorphisms of human counterparts for such modifier loci for Pas1.

Two SNPs in the KRAS gene were in LD with three other SNPs in a nearby gene, CASC1. A haplotype consisting of minor alleles of these five polymorphisms were significantly associated with risk for ADC with multiple AAHs. Genotypes with minor alleles for polymorphisms of the CASC1 gene also showed increased ORs for ADC accompanied by multiple AAHs, although they were not statistically significant. Thus, it was also suggested that CASC1 polymorphisms could also be involved in the susceptibility to ADC with multiple AAHs. Similar results were also shown in mice; Casc1 polymorphisms are in LD with KRAS polymorphisms, and Casc1 polymorphisms also showed associations with lung adenoma risk (14,15,37). The Casc1 gene has a polymorphism associated with amino acid substitution. Casc1 protein has a growth-suppressive activity on lung cancer cells, and the activity was indicated to be different between the polymorphic proteins (15). However, differential expression of Casc1 was not observed between polymorphic alleles (18). Thus, it is probably that Casc1 contributes to lung ADC/adenoma susceptibility by expressing polymorphic proteins with differential activity. This result is in contrast to the case of the Kras gene, for which polymorphisms associated with amino acid substitution have not been found, while differential expression between polymorphic alleles was observed (18). Interestingly, the human CASC1 gene also has a polymorphism with amino acid substitution, CASC-4. Therefore, it is possible that the polymorphism also causes a difference in the activity of CASC1 protein as in the case of mouse Casc1 protein. Thus, the functional significance of CASC1 SNPs should be further investigated both on expression level and protein activity to clarify the involvement of the CASC1 gene in ADC/AAH susceptibility, and such a study is in progress in our laboratory.

The present study indicated that KRAS/Kras polymorphisms are involved in the susceptibility to lung tumor development by causing differential expression levels of the KRAS oncogene, not only in mice but also in humans. Thus, a further study should be done to elucidate molecular mechanisms underlying the differential expression between susceptible and resistant KRAS/Kras alleles. In the present study, transcripts from the minor allele for the KRAS-6 polymorphism were shown to be more abundant than those from the major allele. However, it remains unknown whether this polymorphism is responsible for differential expression or not. The KRAS-6 polymorphism is located in a 94 kb LD region covering introns and the 3'-UTR of the KRAS gene (Figure 1), and 10 of other polymorphisms have been identified in this region. Notably, the ratios of the KRAS transcripts between the major and minor alleles for the KRAS-6 polymorphism were different among the eight cases examined; therefore, it is possible that KRAS expression is affected by several polymorphisms. Introns and the 3'-UTR of the KRAS/Kras gene contain several genomic segments with significant homologies between humans and mice (http://genome.ucsc.edu/). Thus, it is possible that such segments have common functions in the expression of KRAS/Kras gene, and therefore, polymorphisms in these regions are responsible for the differential levels of KRAS/Kras gene expression. Notably, genomic fragments of 50 bp in size encompassing the KRAS-1 and -6 polymorphisms, which showed associations with risk for ADC accompanied by multiple AAHs, did not show significant homologies with the mouse genome. Thus, these two polymorphisms are unlikely to be responsible ones. Further functional and genetic studies on KRAS will give us more critical information on the involvement of KRAS in lung tumorigenesis in humans.


   Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Ministry of Health, Labor and Welfare for Research on Human Genome Tailor-made and for Cancer Research (16S-1).


   Acknowledgments
 
We thank Dr Koji Tsuta of the National Cancer Center Hospital for help in pathological examination. We thank Ms Sachiyo Mimaki, Kaoru Toyama and Rumi Ono for technical assistance.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received October 14, 2007; revised January 16, 2008; accepted February 7, 2008.


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