Between August 2001 and November 2007, blood samples were collected from 901 subjects, including 443 lung cancer patients and 458 normal controls without cancer. The lung cancer patients were recruited from the patient pool at the Genomic Research Center for Lung and Breast/Ovarian Cancer and the Inha University Medical Center, and the control subjects were randomly selected from a pool of healthy volunteers who had visited the Cardiovascular Genome Center, the Genomic Research Center for Allergy and Respiratory Diseases and the Keimyung University Dongsan Medical Center. Detailed information on diet, the smoking status, the drinking status, lifestyle and the medical history was collected by a trained interviewer with using a structured questionnaire. Out of 443 cases, 432 smoking statuses, 342 drinking statuses, 347 stages and 418 cell types were available for the information on the subjects' characteristics, while 356 smoking statuses and 267 drinking statuses, out of the 458 controls, were available for the information on the controls' characteristics. All the study subjects provided written consent and they were all ethnic Koreans, and all the participating Institutional Review Board approved the study protocol.

Genomic DNA was prepared from the peripheral blood samples using a Puregene blood DNA kit (Gentra, Minneapolis, MN) and following the manufacture's protocol.

For identification of the frequent polymorphism sites in the STK11 gene promoter in this Korean population, human genomic DNA was isolated from the whole blood of 24 samples for direct sequencing. Of the entire STK11 gene at 19p13, we amplified 2 kb of its promoter. PCR amplifications were performed in a PTC-225 Peltier Thermal cycler (MJ Research Inc, Waltham, MA) and with using AmpliTagGold (Roche, Branchburg, NJ). All the amplifications were performed using 35 cycles of 30 sec each cycle at 95℃, 1 min at 64℃ and 1 min at 72℃, and this was followed by a single 10 min extension at 72℃. The PCR products were purified using a Montage PCR96 Cleanup kit (Millipore, Bedfore, MA) and they were eluted in 20 µL of nuclease free H₂O. DNA cycle sequencing was carried out using a BigDye Terminator V 3.1 Cycle Sequencing kit (Perkin Elmer, Foster City, CA). Multiscreen SEQ 384 well filter plates were used for dye terminator removal, and the sequences were analyzed on an Applied Biosystems 3700 DNA analyzer. All the polymorphisms and sequence alignments were analyzed using Polyphred.

After direct sequencing of the STK11 gene, we performed genotyping for the three polymorphisms (-1,795 T>C, -1,494 A>C and -160 G>T). The genotypes of the sample were assayed using a single base primer extension assay and employing a SNaPShot assay kit (ABI, Foster City, CA). Briefly, the genomic regions containing the polymorphisms of interest in the STK11 gene were amplified by PCR. The PCRs were performed using an initial denaturation step at 95℃ for 10 min, 35 amplification cycles (30 sec at 95℃, 1 min annealing at 63.9℃ and 1 min at 72℃), and this was followed by a single 7 min extension cycle at 72℃. The PCR products were subsequently purified by incubating them with 10 units of Exo I (USB, Cleveland, OH) and 1 unit of shrimp alkaline phosphatase (Roche, Indianapolis, IN) at 37℃ for 1 hour and then at 72℃ for 15 min. The extension reactions with 1 µL of purified PCR product, 0.15 pmoles 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℃ for 10 sec, 50℃ for 5 sec and 60℃ for 30 sec. The extension products were incubated with 1 unit of shrimp alkaline phosphatase (Roche, Indianapolis, IN) at 37℃ for 1 hour and then they were incubated at 72℃ for 15 min. 9 µL of deionized formamide was mixed with 1 µL of the purified extension product, and this was electrophoresed on an ABI Prism 3,700 genetic analyzer (Applied Biosystems, Foster City, CA). The results were analyzed using GeneScan analysis, version 3.7 (Applied Biosystems, Foster City, CA).

The allele frequencies, the genotype frequencies and the departures of the genotype distributions from Hardy-Weinberg equilibrium for each SNP were analyzed using the chi-square test or Fisher's exact test. A p-value of <0.05 was considered statistically significant. Linkage disequilibrium (LD) was tested on pairwise combinations of polymorphisms with using the absolute value of the standardized measure of linkage disequilibrium, D', as calculated by the Haploview program version 3.2. The haplotypes and their frequencies were estimated by the Haploview program version 3.2. The genotype-specific risks were estimated as odds ratios (ORs) with associated 95% confidence intervals by unconditional logistic regression analysis (SAS Institute, Cary, NC) and they were adjusted for age, gender and the smoking status.