Recent expression profile analyses revealed that lung adenocarcinomas canbe divided into several subgroups with diverse pathological features. Because cellular heterogeneity of tumors can confound these analyses, we used laser capture microdissection and microarray expression analysis to characterize the molecular profiles of lung adenocarcinomas. We found 45 genes delineating smokers and nonsmokers that were located at chromosomal loci frequently altered in non-small cell lung cancers, and 27 genes, which were differentially expressed between survivors and nonsurvivors 5 years after surgery. These results are consistent with the hypothesis that the abnormal expression of genes involved in maintaining the mitotic spindle checkpoint and genomic stability, e.g., hBUB3, hZW10, and APC2, contribute to the molecular pathogenesis and tumor progression of tobacco smoke-induced adenocarcinoma of the lung.

Lung cancer is the leading cause of male and female cancer deaths in the United States. Lung carcinoma is classified usually as small cell lung carcinoma or NSCLC3 (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma). In particular, adenocarcinoma is the most common type of lung cancer in women and nonsmokers, and it is increasingly associated with tobacco smoking as well.4 Since the 1950s, steady rises in the incidence of adenocarcinoma of the lung have been observed in many developed countries with ethnically diverse populations (1). Recent reports (2, 3) used the gene expression profiling to divide lung adenocarcinomas into several subgroups and to discriminate primary cancers from metastases of extrapulmonary origin. Lung adenocarcinomas, when compared with squamous cell lung carcinomas or small cell lung carcinomas, show striking differences in the expression patterns (2, 3). In the analysis of clinical tumor specimen in these and other studies, a significant confounder is the cellular heterogeneity of normal and diseased tissue. To overcome this problem, LCM was developed to analyze clinical samples (4, 5, 6, 7). We combined the LCM with microarray gene expression analysis to identify the genes differentially expressed in lung adenocarcinoma associated with the following clinical phenotypic subgroups: prognosis, smoking, and gender, and to generate the hypotheses concerning the molecular pathogenesis and tumor progression of lung carcinoma.

Patients and Surgically Resected Frozen Tissues.

The study was approved by the Institutional Review Boards of the University of Maryland, Baltimore, and the National Cancer Institute. Three pathologists diagnosed the tumor tissues with adenocarcinomas (8). We chose lung adenocarcinoma cases with tumor stages I or II by Tumor-Node-Metastasis classification to minimize any secondary or tertiary effects related to tumor stages III or IV. In Table 1, we summarized the characteristics of patients. Among the 19 patients with adenocarcinoma, 6 survived 5 years or more after surgery, 12 were nonsurvivors that died of a recurrence of lung cancer, and we could not find the exact cause of death for 1 patient. After the classical criteria, patients were defined as nonsmokers if they smoked <100 cigarettes in their life time. Five were nonsmokers, and 14 were smokers, and the pack-years among 14 smokers were 20–125 (mean ± SD; 48 ± 27). Ten were males and 9 were females.

RNA Extraction, LCM, and T7 Amplification.

From each flash-frozen tumor tissue, we prepared 8-μm thick frozen sections. Total RNA was extracted from one section, and the quality was evaluated (Fig. 1,A). Serial frozen sections (∼18/case) were used for the subsequent analysis. We used the PixCell II LCM System from Acturus Engineering (Mountain View, CA) for laser capture and followed the manufacturer’s protocol with several modifications. The sections were immersed in the relevant fixatives or staining solutions (70%, 95%, or 100% ethanol, hematoxylin, eosin, or RNase-free water) for ∼10 s each, followed by dehydration with xylene for 1 min. We recorded the pathological images for each case (Fig. 1, C–M). We repeated reverse transcription and T7 amplification twice to get two-round aRNA as probes for microarray analysis as reported previously (5).

RNA Reference Pool.

As an experimental control, we used the BEAS-2B cell line that was isolated from normal human bronchial epithelium obtained from an autopsy of a noncancerous individual, which was immortalized with an adenovirus 12-SV40 virus hybrid (9).

Microarray and Acquisition of Data.

We used cDNA microarray slides generated by Amersham Pharmacia Biotech (Piscataway, NJ) and fabricated in Laboratory of Molecular Medicine, University of Tokyo. Four sets of array slides contained 18,432 cDNAs selected from the UniGene database with 15,737 unique genes. The cDNAs were amplified by reverse transcription-PCR without any repetitive or poly(A) sequences. The PCR products were spotted in duplicate using an Array Spotter Generation III (Amersham). Each slide commonly contained 48 housekeeping genes. We labeled 2.5 μg of two-round aRNA from LCM-captured tissues or BEAS-2B by reverse transcription with Cy3-dCTP or Cy5-dCTP, respectively. Probes were hybridized to the microarrays in the Automated Slide Processor (Amersham), and signal intensities were quantified using ArrayVision software (Imaging Research, Inc., St. Catharines, Ontario, Canada). Four sets of signal intensities (Cy3 or Cy5, left panel or right panel) from duplicate spots were transformed to log2 scale, and the duplicate log-ratios were averaged. We excluded clones for which the Cy3:Cy5 ratios for duplicate spots differed by more than 2-fold. The log-ratios of nonexcluded clones were normalized by a median centering the 48 housekeeping genes on each array. We selected 6,216 clones with consistent duplicate measurements on at least 15 of the 19 arrays for additional analysis. The analyses were performed using BRB-ArrayTools.5

Hierarchical Clustering.

We selected 2270 clones with consistent log-ratios present in at least 15 of the 19 samples, and which showed more variant expression patterns among the 19 samples. Then, we performed average linkage hierarchical clustering for 19 samples and 2270 clones with centered correlation as the distance metric (10).

Genes That Distinguish Phenotype Classes.

Among 19 cancer cases, to identify genes that discriminate between two phenotype subclasses (e.g., smoker versus nonsmoker), the F test with 0.0025 as a nominal significance level (P) was computed on log2-expression ratios of each clone. Next, phenotype labels were randomly permuted among the 19 samples, the F statistic for each gene in the permuted data set was recomputed, and the number of clones significant at the nominal 0.0025 level was counted. This process was repeated 2000 times, and the proportion of the 2000 replications with at least as many genes significant at the 0.0025 level, provided a global test of the null hypothesis that the expression profile is unrelated to phenotype class.

Following the strategy described in Fig. 1,A, we recorded the tumor pathology (Fig. 1,C), and the number of sections and spots used for LCM, and calculated the yield of aRNA after two rounds of amplification (Fig. 1,B). Within the context of hierarchical clustering among 19 samples and 2270 clones (Fig. 2), 4 of the adenocarcinomas (cases 3, 8, 9, and 15) that clustered separately were diffusely invasive types and the population of cancer cells in LCM-captured tissues was relatively low.

When we performed the F test (P = 0.0025) for smokers and nonsmokers, 45 candidate genes were identified. The probability of obtaining 45 or more such genes in random partitions of the 19 expression profiles into two groups of 14 and 5 samples was only 0.045 (Table 2; Fig. 3,A). Among the significant genes, 30 were known genes and 15 were ESTs or genes of unknown function. To compare the expression levels of genes on the chromosomal band 3p21.3 region between 5 nonsmokers and 14 smokers, we selected 21 known genes that were reported to be in this region and passed the same filtering as our other analyses (AbsLogRatioDifference <1, missing value <4). For those genes, we calculated the fold-difference in geometric means of 14 smokers divided by 5 nonsmokers (Table 3).

Because the exact cause of death for case 15 was unknown, we eliminated this case from the molecular profiling of genes related to prognosis. Using the 18 tumor cases, we performed the univariate F test between survivors and nonsurvivors 5 years after surgery. We found 27 genes differentially expressed between survivors and nonsurvivors with P < 0.0025 (Table 4; Fig. 3 B). Under the null hypothesis of no relationship between the expression profile and survival, the probability of obtaining at least 27 genes significant at the 0.0025 level individually is 0.07. Among the significant genes, 18 were known genes and 9 were unknown genes.

Additionally, to show the reliability of the F test and to estimate the number of false positives with our experimental set, we divided our 19 cases into 10 pairs of random subgroups and repeated the F test. With these random sets, we did not get any significant results, and the number of false positives ranged from 6 to 10 (P = 0.0025). We also used the F test to identify genes that classify gender or specific morphological features, but we have not been able to find a definitive result with our current experimental set.

Analysis of clinical samples is difficult because of the heterogeneity of cellular components in the tissues and their diverse pathological features. In analyzing genetic alterations or expression profiles in cancer cells, the contamination of normal epithelia or stromal cells may confound the analysis, especially when using tissues such as lung, brain, kidney, ovary, mammary gland, or prostate (5, 7, 11, 12), which are unlike the more homogenous fluorescence-activated cell sorted blood cells (13). The use of LCM should improve the sample preparation for microarray expression analysis (4). One disadvantage of microdissection is that RNA amplification is essential for the preparation of an adequate amount of RNA for probe generation. The reproducibility and usefulness of T7 amplification in gene expression profiling has been reported previously (14, 15). In our hands, the gene expression profile from two independent two-rounds of amplification of BEAS-2B RNA was highly correlated (r2 = 0.91; data not shown).

Several of the genes with lower expression among smokers are located in the chromosomal regions where genomic imbalance in NSCLC has been observed previously with high frequency. In Table 2, we summarized the reported data of genomic imbalance including homozygous deletion, which was analyzed with comparative genomic hybridization, LOH analysis, and other methods. For example, chromosome 3p21.3 is a well-known region for a frequent homozygous deletion in lung cancers. Lerman and Minna (16) analyzed 25 genes localized in the 630-kb homozygous deletion region on 3p21.3. Using the F test for smoking, we found two genes, 101F6 and CACT, located on 3p21 region. In addition to 3p21.3, 11q23–24, where NCAM1 is also located, is known for a high frequency of LOH, and putative tumor suppressor genes may be found in this region (17). In addition, a high frequency of LOH in lung cancers has been reported in chromosomes 19p12–13, 19q13.3, and 4q, where C19ORF3, EDG4, SIGLEC5, and RRH are located (18, 19). Mori et al.(20) showed recently that microsatellite instability was observed frequently in the coding region of PA2G4 on chromosome 12q13. The inactivation of these genes may be related to tobacco carcinogenesis. The high expression of RAB4, DJ1, MCT, and ribosomal protein L22 (RPL22) also may be related to the tobacco smoke-induced adenocarcinoma. RAB4 gene shares biochemical properties with the Ras gene superfamily and encodes GTP-binding proteins. DJ1 cooperates with H-RAS in transforming NIH3T3 cells (21). The MCT protein is a novel candidate oncogene sharing a homology with cyclin H, which increases G1 cyclin/cyclin-dependent kinase activity (22). DNA amplification of chromosome 3q26.1-q26.3 is detected frequently in lung squamous cell carcinomas, where RPL22 is also located (23). Decreased expression of glioma tumor suppressor candidate region gene 2 (GLTSCR2) and high expression of candidate oncogene EDG4 among nonsmokers may contribute to the carcinogenesis of nonsmokers. The mitochondrial enzyme TK2 is also higher expressed in adenocarcinomas in nonsmokers than in smokers. TK2 is active throughout the cell cycle and is most likely correlated with mitochondrial content in the tumors. TK2 contributes to the metabolic activation of 2′,2′-difluorodeoxycytidine (gemcitabine), which has significant activity against non-small cell cancers (24). These data suggest the hypothesis that gemcitabine may be more active in the TK2 higher-expressing adenocarcinoma in nonsmokers when compared with smokers.

Among the 21 genes on chromosome 3p21.3 listed in Table 3, 16 genes including PL6, 101F6, HYAL2, FUS2, and RBM6, which are located in 630 kb with a high frequency of homozygous deletions (16), showed lower expression in smokers. This result supported our hypothesis that genes with lower expression among smokers are located in the chromosomal regions with a high frequency of LOH, including 3p21.3.

Among the 27 genes that were differentially expressed between 5-year survivors or more and nonsurvivors (Table 4), several of the genes are involved in the metaphase-anaphase transition and the mitotic spindle checkpoint. Whereas APC2 was expressed at a higher level in cases with a poor prognosis, hBUB3, hZW10, and proteasome 26S subunit, ATPase 1 and 5 (PSMC1 and PSMC5) were expressed higher in cases with a better prognosis. The APC/C is activated by Cdc20 and Cdh1, and promotes metaphase-anaphase transition by ubiquitinating cyclins and the anaphase inhibitor, which are subsequently degraded by the 26S proteasome, in which PSMC1 and PSMC5 gene products are subunits (25). APC2 is an evolutionarily conserved component of the APC/C (26). In Saccharomyces cerevisiae, temperature-sensitive APC2 mutants arrest in metaphase (27). hBUB3 is a component of the mitotic checkpoint complex that inhibits APC/C (28), a multisubunit E3 ubiquitin ligase that targets proteins of which the proteolytic cleavage of cyclins and anaphase inhibitors is necessary for sister chromatid separation and the exit from mitosis (29, 30). BUB3 is evolutionarily conserved in eukaryotes (31, 32, 33). Mutation of BUB3 inactivates the mitotic spindle cell checkpoint in both the budding yeast S. cerevisiae(34) and the mouse (35). Murine BUB3 gene knockout embryos accumulate mitotic errors including lagging chromosomes and micronuclei by 4.5 days and do not survive past 7.5 days (35). ZW10 recruits dynactin and dynein to the centromeric kinetochore (36), a multiprotein-DNA complex responsible for attachment to spindle microtubules, prometaphase chromosome congression, anaphase initiation, and poleward movement. Mutation in the DrosophilaZW10 gene results in both lagging chromosomes and aneuploidy (36). The positive correlation between poor prognosis and the reduced expression of hBUB3 and hZW10 is consistent with the hypothesis that a defective mitotic spindle checkpoint, and the resultant abnormal segregation and increasing aneuploidy would enhance tumor progression. Bhattacharjee et al.(3) also have found that increased expression of hBUB3 is associated with a better prognosis in lung adenocarcinoma cases.

Our study and others (2, 3) generate hypotheses for both clinical and laboratory investigations. The combined use of LCM with microarray expression analysis should decrease the confounder of tissue heterogeneity. Laboratory studies of differentially expressed genes associated with prognosis may identify genes and their function involved in metastasis.

Fig. 1.

Experimental course of LCM and T7 amplification. A, flow chart of tissue preparation, LCM, and two-round T7 amplification. ss cDNA, single-strand cDNA; ds cDNA, double-strand cDNA. B, the summary of LCM and two-round aRNA yields. C–M, pathological images of LCM. Left panels, before LCM; middle panels, after LCM; and right panels, the tissues captured on LCM caps. C, case 1; D, case 2; E, case 3; F, case 5; G, case 7; H, case 8; I, case 11; J, case12; K, case 15; L, case 18; and M, case 19.

Fig. 1.

Experimental course of LCM and T7 amplification. A, flow chart of tissue preparation, LCM, and two-round T7 amplification. ss cDNA, single-strand cDNA; ds cDNA, double-strand cDNA. B, the summary of LCM and two-round aRNA yields. C–M, pathological images of LCM. Left panels, before LCM; middle panels, after LCM; and right panels, the tissues captured on LCM caps. C, case 1; D, case 2; E, case 3; F, case 5; G, case 7; H, case 8; I, case 11; J, case12; K, case 15; L, case 18; and M, case 19.

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Fig. 2.

A hierarchical clustering and gene expression cluster view of 19 samples and 2270 cDNA clones. Green, transcript levels below the median; black, equal to the median; red, greater than median; and gray, missing data for filtering.

Fig. 2.

A hierarchical clustering and gene expression cluster view of 19 samples and 2270 cDNA clones. Green, transcript levels below the median; black, equal to the median; red, greater than median; and gray, missing data for filtering.

Close modal
Fig. 3.

A, an image plot view of 45 genes listed in Table 2. ID numbers of genes are identical to Table 2. Green, transcript levels below the median; black, equal to the median; red, greater than median; and gray, missing data for filtering. B, an image plot view of 27 genes listed in Table 4. ID numbers of genes are identical to Table 4. Green, transcript levels below the median; black, equal to the median; red, greater than median; and gray, missing data for filtering.

Fig. 3.

A, an image plot view of 45 genes listed in Table 2. ID numbers of genes are identical to Table 2. Green, transcript levels below the median; black, equal to the median; red, greater than median; and gray, missing data for filtering. B, an image plot view of 27 genes listed in Table 4. ID numbers of genes are identical to Table 4. Green, transcript levels below the median; black, equal to the median; red, greater than median; and gray, missing data for filtering.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by Japan Society for the Promotion of Science Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH, 69811 (to K. M.).

3

The abbreviations used are: NSCLC, non-small cell lung carcinoma; hBUB3, human BUB3; LCM, laser capture microdissection; T7 amplification, T7-based RNA amplification; aRNA, amplified antisense RNA; LOH, loss of heterozygosity; TK2, thymidine kinase-2; hZW10, human ZW10; APC/C, anaphase-promoting complex or the cyclosome.

4

Internet address: http://www.cdc.gov/tobacco/sgr_forwomen.htm.

5

Simon, R., and Peng, A. BRB-ArrayTools Users Guide. National Cancer Institute. Internet address: http://linus.nci.nih.gov/BRB-ArrayTools.html.

Table 1

Characteristics of patients

IDAgeGenderPrognosisaSmokingPack-years
75 Db 
54 40 
48 50 
66 47 
74 60 
59 125 
72 60 
53 20 
55 80 
10 66 40 
11 49 25 
12 73 
13 78 60 
14 61 46 
15 70 Unknown 60 
16 57 
17 38 
18 63 
19 63 30 
IDAgeGenderPrognosisaSmokingPack-years
75 Db 
54 40 
48 50 
66 47 
74 60 
59 125 
72 60 
53 20 
55 80 
10 66 40 
11 49 25 
12 73 
13 78 60 
14 61 46 
15 70 Unknown 60 
16 57 
17 38 
18 63 
19 63 30 
a

The status 5 years after surgery.

b

D, dead; A, alive; N, no; Y, yes.

Table 2

Forty-five genes that classify smokers and nonsmokers and the information of genomic imbalance

IDDescriptionPGenBank accession no.CytobandGenomic imbalance at cytoband in NSCLCaReferences
High in nonsmokers and low in smokers       
 1 thymidine kinase 2, mitochondrial 0.00004 AA429631 16q22 gain(16q22, 45%)  19  
 2 putative tumor suppressor, 101F6 0.00010 AC002481 3p21.3 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss (3cen-p26, >98%)  16, 37, 38 
 3 UDP-Gal:βGlcNAc β 1,3-galactosyltransferase, polypeptide 4 0.00034 AA987754 6p21.3 loss(6p21.3, 42–69%), gain(6p11-p22, 27%), loss(6cen-p25, 100%) 18, 19, 37 
 4 integrin, α 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41B 0.00081 M34480 17q21.32 gain(17q11-q24, 82%), gain(17q21, 37%), loss(17q21, 53%), loss(42%) 19, 394041 
 5 ESTs 0.00113 H24302    
 6 neural cell adhesion molecule 1 0.00122 S71824 11q23.1 loss(11q23-q24, 71%), loss(11q23.2, 54%), loss(11q23) 424344 
 7 retinal pigment epithelium-derived rhodopsin homolog, RRH 0.00122 AF012270 4cen-4q12 loss(4q33-q35, 47%), loss(43%), loss(4cen-q35, 100%) 18, 38, 45 
 8 SELENOPHOSPHATE SYNTHETASE; Human selenium donor protein 0.00129 U34044 Xq13 loss(Xp-q21, 67%)  45  
 9 sialic acid binding Ig-like lectin 5, SIGLEC5 0.00191 U71383 19q13.3 loss(19q13.3, 39%), loss(19cen-q13.4, 73%), gain(19cen-q13.4, 39%) 18, 19, 46 
 10 EST 0.00200 AA663323    
 11 cytochrome P450, subfamily IID, polypeptide 7a 0.00224 X16866 22q13.2-q13.31 loss(22q13-qter, 57%), loss(22q11.2-q13.1, 75%) 18, 47 
 12 proliferation-associated 2G4 38kD, PA2G4 0.00227 U59435 12q13 loss(12q24.1, 63%)  47  
 13 carnitine/acylcarnitine translocase, CACT 0.00228 AA521247 3p21.31 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss(3cen-p26, >98%) 16, 37, 38 
 14 hypothetical protein FLJ23443 0.00230 W94201 3p22-p21.33 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss(3cen-p26, >98%) 16, 37, 38 
 15 ESTs 0.00234 AA532514    
 16 chromosome 19 open reading frame 3, C19ORF3 0.00242 AF028824 19p13.1 loss(19p13.2, 75–83%), gain(82%), loss(19p12-p13.2, 77%), loss(19p13.3, 58%) 18, 19, 45, 48 
 17 EDG4 0.00243 AF011466 19p12 loss(19p13.2, 75–83%), gain(82%), loss(19p12-p13.2, 77%), loss(19p13.3, 58%) 18, 19, 45, 48 
High in smokers and low in nonsmokers       
 18 maternal G10 transcript 0.00025 S77329 7q22.1   
 19 MCT protein 0.00033 AI336165 Xq22-q24 gain(Xq22-q25, 45%), homozygous deletion(Xq22, 67%) 19, 45 
 20 3-hydroxyisobutyrate dehydrogenase 0.00041 W95267 7p11.2-p21   
 21 RAB4, member RAS oncogene family 0.00048 F05353 1q42-q43 gain(1q21.1-q44, 100%), gain(43%) 38, 46 
 22 ribosomal protein L22, RPL22 0.00055 AA962580 3q26 gain(3q24-qter, 91%), gain(3cen-q29, 35%), gain(3cen-q29, 35%) 19, 37, 46 
 23 destrin (actin depolymerizing factor) 0.00059 S65738 20p11.23   
 24 ESTs 0.00069 W23535    
 25 TATA-binding protein-binding protein 0.00072 N64689 6p21.33 loss(6p21.3, 42–69%), gain(6p11-p22, 27%), loss(6cen-p25, 100%) 18, 19, 37 
 26 TERF1 (TRF1)-interacting nuclear factor 2 0.00078 AA041364 14q12-q21.3 loss(14q12, 31%), loss(14q21, 18%), loss(14q11-q12, 38%) 18, 19, 40 
 27 ESTs 0.00096 AA478650    
 28 tubulin, α, ubiquitous 0.00106 AA492218 12q12-q14.3 loss(12q24.1, 63%)  47  
 29 ESTs 0.00116 H95968    
 30 Homo sapiens cDNA: FLJ22712 fis, clone HSI13435 0.00128 AI192127    
 31 Homo sapiens cDNA: FLJ21880 fis, clone HEP02743 0.00149 T71056 9q34.13 loss(90%), loss(9q34, 50%), loss(40%) 37, 38, 45 
 32 NADH dehydrogenase (ubiquinone) Fe-S protein 4 (18kD) 0.00178 AI131435 5q11.1 loss(5q21, 35%), loss(5cen-q35, 100%) 38, 45 
 33 ESTs 0.00180 AI206756    
 34 ESTs 0.00180 N54851    
 35 glioma tumor suppressor candidate region gene 2, GLTSCR2 0.00182 AA459728 19q13.3 loss(19cen-q13.4, 73%), gain(19cen-q13.4, 39%), loss(19q13.3, 39%) 18, 19, 46 
 36 hypothetical protein FLJ20585 0.00189 AI336134 2p14-q23.3   
 37 general transcription factor IIA, 2 (12kD subunit) 0.00192 U21242 15q11.25-q22.33   
 38 RNA-binding protein regulatory subunit, DJ1 0.00196 AA576779 1p36.33-p36.12 loss(1p36, 40%)  38  
 39 ASB protein 0.00196 H40863 2q37 loss(2q33, 31%), loss(2q36-q37, 27%), loss(2q32, 63%) 18, 19, 47 
 40 vesicle-associated soluble NSF attachment protein receptor 0.00211 AA777761 14q23.3   
 41 DKFZP547E1010 protein 0.00212 AI039693 1p36.1 loss(10q23-q25, 43%), loss(1p36-pter, 64%) 18, 45 
 42 hypothetical protein FLJ20027 0.00228 AA679314 10pter-q21.2 loss(10p11-q11, 36%), loss(10cen-q25, 100%) 38, 45 
 43 signal peptidase complex (18kD) 0.00228 AA742432 15q11.2 loss(15q11, 50%), gain(15cen-q26, 52%) 45, 46 
 44 ESTs 0.00232 AI276082    
 45 CGI0 protein 0.00246 AA889621 14q22.1-q22.3   
IDDescriptionPGenBank accession no.CytobandGenomic imbalance at cytoband in NSCLCaReferences
High in nonsmokers and low in smokers       
 1 thymidine kinase 2, mitochondrial 0.00004 AA429631 16q22 gain(16q22, 45%)  19  
 2 putative tumor suppressor, 101F6 0.00010 AC002481 3p21.3 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss (3cen-p26, >98%)  16, 37, 38 
 3 UDP-Gal:βGlcNAc β 1,3-galactosyltransferase, polypeptide 4 0.00034 AA987754 6p21.3 loss(6p21.3, 42–69%), gain(6p11-p22, 27%), loss(6cen-p25, 100%) 18, 19, 37 
 4 integrin, α 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41B 0.00081 M34480 17q21.32 gain(17q11-q24, 82%), gain(17q21, 37%), loss(17q21, 53%), loss(42%) 19, 394041 
 5 ESTs 0.00113 H24302    
 6 neural cell adhesion molecule 1 0.00122 S71824 11q23.1 loss(11q23-q24, 71%), loss(11q23.2, 54%), loss(11q23) 424344 
 7 retinal pigment epithelium-derived rhodopsin homolog, RRH 0.00122 AF012270 4cen-4q12 loss(4q33-q35, 47%), loss(43%), loss(4cen-q35, 100%) 18, 38, 45 
 8 SELENOPHOSPHATE SYNTHETASE; Human selenium donor protein 0.00129 U34044 Xq13 loss(Xp-q21, 67%)  45  
 9 sialic acid binding Ig-like lectin 5, SIGLEC5 0.00191 U71383 19q13.3 loss(19q13.3, 39%), loss(19cen-q13.4, 73%), gain(19cen-q13.4, 39%) 18, 19, 46 
 10 EST 0.00200 AA663323    
 11 cytochrome P450, subfamily IID, polypeptide 7a 0.00224 X16866 22q13.2-q13.31 loss(22q13-qter, 57%), loss(22q11.2-q13.1, 75%) 18, 47 
 12 proliferation-associated 2G4 38kD, PA2G4 0.00227 U59435 12q13 loss(12q24.1, 63%)  47  
 13 carnitine/acylcarnitine translocase, CACT 0.00228 AA521247 3p21.31 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss(3cen-p26, >98%) 16, 37, 38 
 14 hypothetical protein FLJ23443 0.00230 W94201 3p22-p21.33 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss(3cen-p26, >98%) 16, 37, 38 
 15 ESTs 0.00234 AA532514    
 16 chromosome 19 open reading frame 3, C19ORF3 0.00242 AF028824 19p13.1 loss(19p13.2, 75–83%), gain(82%), loss(19p12-p13.2, 77%), loss(19p13.3, 58%) 18, 19, 45, 48 
 17 EDG4 0.00243 AF011466 19p12 loss(19p13.2, 75–83%), gain(82%), loss(19p12-p13.2, 77%), loss(19p13.3, 58%) 18, 19, 45, 48 
High in smokers and low in nonsmokers       
 18 maternal G10 transcript 0.00025 S77329 7q22.1   
 19 MCT protein 0.00033 AI336165 Xq22-q24 gain(Xq22-q25, 45%), homozygous deletion(Xq22, 67%) 19, 45 
 20 3-hydroxyisobutyrate dehydrogenase 0.00041 W95267 7p11.2-p21   
 21 RAB4, member RAS oncogene family 0.00048 F05353 1q42-q43 gain(1q21.1-q44, 100%), gain(43%) 38, 46 
 22 ribosomal protein L22, RPL22 0.00055 AA962580 3q26 gain(3q24-qter, 91%), gain(3cen-q29, 35%), gain(3cen-q29, 35%) 19, 37, 46 
 23 destrin (actin depolymerizing factor) 0.00059 S65738 20p11.23   
 24 ESTs 0.00069 W23535    
 25 TATA-binding protein-binding protein 0.00072 N64689 6p21.33 loss(6p21.3, 42–69%), gain(6p11-p22, 27%), loss(6cen-p25, 100%) 18, 19, 37 
 26 TERF1 (TRF1)-interacting nuclear factor 2 0.00078 AA041364 14q12-q21.3 loss(14q12, 31%), loss(14q21, 18%), loss(14q11-q12, 38%) 18, 19, 40 
 27 ESTs 0.00096 AA478650    
 28 tubulin, α, ubiquitous 0.00106 AA492218 12q12-q14.3 loss(12q24.1, 63%)  47  
 29 ESTs 0.00116 H95968    
 30 Homo sapiens cDNA: FLJ22712 fis, clone HSI13435 0.00128 AI192127    
 31 Homo sapiens cDNA: FLJ21880 fis, clone HEP02743 0.00149 T71056 9q34.13 loss(90%), loss(9q34, 50%), loss(40%) 37, 38, 45 
 32 NADH dehydrogenase (ubiquinone) Fe-S protein 4 (18kD) 0.00178 AI131435 5q11.1 loss(5q21, 35%), loss(5cen-q35, 100%) 38, 45 
 33 ESTs 0.00180 AI206756    
 34 ESTs 0.00180 N54851    
 35 glioma tumor suppressor candidate region gene 2, GLTSCR2 0.00182 AA459728 19q13.3 loss(19cen-q13.4, 73%), gain(19cen-q13.4, 39%), loss(19q13.3, 39%) 18, 19, 46 
 36 hypothetical protein FLJ20585 0.00189 AI336134 2p14-q23.3   
 37 general transcription factor IIA, 2 (12kD subunit) 0.00192 U21242 15q11.25-q22.33   
 38 RNA-binding protein regulatory subunit, DJ1 0.00196 AA576779 1p36.33-p36.12 loss(1p36, 40%)  38  
 39 ASB protein 0.00196 H40863 2q37 loss(2q33, 31%), loss(2q36-q37, 27%), loss(2q32, 63%) 18, 19, 47 
 40 vesicle-associated soluble NSF attachment protein receptor 0.00211 AA777761 14q23.3   
 41 DKFZP547E1010 protein 0.00212 AI039693 1p36.1 loss(10q23-q25, 43%), loss(1p36-pter, 64%) 18, 45 
 42 hypothetical protein FLJ20027 0.00228 AA679314 10pter-q21.2 loss(10p11-q11, 36%), loss(10cen-q25, 100%) 38, 45 
 43 signal peptidase complex (18kD) 0.00228 AA742432 15q11.2 loss(15q11, 50%), gain(15cen-q26, 52%) 45, 46 
 44 ESTs 0.00232 AI276082    
 45 CGI0 protein 0.00246 AA889621 14q22.1-q22.3   
a

If the minimal region and the percentage of genomic imbalance involvement were known, they were written in the parentheses. The data of references 37 and 38 was analyzed with adenocarcinomas only. The data of references 18, 40414243, 45, 47, and 48 was analyzed with LOH analysis with polymorphic markers. The data of references 19, 37, 38, and 46 was analyzed with comparative genomic hybridization.

Table 3

Genes on chromosomal band 3p21.3 and the fold-difference of their expression levels

FoldaDescription of genes on 3p21.3GenBank accession no.Symbol
High in nonsmokers and low in smokers    
 0.32 protein tyrosine phosphatase, non-receptor type 23 C75369 PTPN23 
 0.46 carnitine/acylcarnitine translocase AA521247 CACT 
 0.48 g20 protein AI300189 LOC51161 
 0.48 hypothetical protein FLJ23443 W94201 FLJ23443 
 0.51 putative tumor suppressor AC002481 101F6 
 0.56 ubiquitin specific protease 4 (proto-oncogene) U20657 USP4 
 0.60 endonuclease G-like 1 AA743829 ENDOGL1 
 0.68 mitogen-activated protein kinase-activated protein kinase 3 U09578 MAPKAPK3 
 0.68 PL6 protein U09584 PL6 
 0.69 hyaluronoglucosaminidase 2 U09577 HYAL2 
 0.76 glutathione peroxidase 1 M21304 GPX1 
 0.77 putative tumor suppressor AF040704 101F6 
 0.82 putative tumor suppressor AF040705 FUS2 
 0.86 ubiquitin specific protease 4 (proto-oncogene) AA255621 USP4 
 0.87 ras homolog gene family, member A L25080 ARHA 
 0.88 interferon-related developmental regulator 2 U09585 IFRD2 
 0.91 RNA binding motif protein 6 AF069517 RBM6 
High in smokers and low in nonsmokers    
 1.16 HYA22 protein D88153 HYA22 
 1.31 SH3 protein AA702730 AF3P21 
 1.56 laminin receptor 1 (67kD, ribosomal protein SA) J03799 LAMR1 
 2.04 villin-like D88154 VILL 
FoldaDescription of genes on 3p21.3GenBank accession no.Symbol
High in nonsmokers and low in smokers    
 0.32 protein tyrosine phosphatase, non-receptor type 23 C75369 PTPN23 
 0.46 carnitine/acylcarnitine translocase AA521247 CACT 
 0.48 g20 protein AI300189 LOC51161 
 0.48 hypothetical protein FLJ23443 W94201 FLJ23443 
 0.51 putative tumor suppressor AC002481 101F6 
 0.56 ubiquitin specific protease 4 (proto-oncogene) U20657 USP4 
 0.60 endonuclease G-like 1 AA743829 ENDOGL1 
 0.68 mitogen-activated protein kinase-activated protein kinase 3 U09578 MAPKAPK3 
 0.68 PL6 protein U09584 PL6 
 0.69 hyaluronoglucosaminidase 2 U09577 HYAL2 
 0.76 glutathione peroxidase 1 M21304 GPX1 
 0.77 putative tumor suppressor AF040704 101F6 
 0.82 putative tumor suppressor AF040705 FUS2 
 0.86 ubiquitin specific protease 4 (proto-oncogene) AA255621 USP4 
 0.87 ras homolog gene family, member A L25080 ARHA 
 0.88 interferon-related developmental regulator 2 U09585 IFRD2 
 0.91 RNA binding motif protein 6 AF069517 RBM6 
High in smokers and low in nonsmokers    
 1.16 HYA22 protein D88153 HYA22 
 1.31 SH3 protein AA702730 AF3P21 
 1.56 laminin receptor 1 (67kD, ribosomal protein SA) J03799 LAMR1 
 2.04 villin-like D88154 VILL 
a

Fold-difference in geometric means of 14 smokers divided by 5 nonsmokers.

Table 4

Twenty-seven genes that classify survivors and nonsurvivors, and the information of genomic imbalance

IDDescriptionPGenBank accession no.CytobandGenomic imbalance at cytoband in NSCLCaReferences
High in survivors and low in nonsurvivors       
 1 BUB3 (budding uninhibited by benzimidazoles 3, yeast) homolog 0.00011 AF047472 10q26 loss(10q26.13-qter, 36%)  45  
 2 ZW10 (Drosophila) homolog, centromere/kinetochore protein 0.00054 U54996 11q23.2 loss(11q23-q24, 71%), loss(11q23.2, 54%), loss(11q23) 424344 
 3 dCMP deaminase 0.00109 L39874 4q35.1 loss(4q31-qter, 64%), loss(4q34-q35, 50%), loss(4q32-qter, 100%) 19, 38, 45 
 4 keratin 9 (epidermolytic palmoplantar keratoderma) 0.00113 Z29074 17q21.1-q21.2 gain(17q11-q24, 82%), gain(17q21, 37%), loss(17q21, 53%), loss(42%) 19, 394041 
 5 KIAA1008 protein 0.00145 AA156488 13q21.23-q22.2 loss(13q11-q14, 62%), loss(13cen-qter, 90%), loss(13cen-qter, 21%) 38, 45, 46 
 6 proteasome 26S subunit, ATPase, 5 0.00155 D44467 17q23-q25 gain(17q11-q24, 82%), gain(17q21, 37%), loss(17q21, 53%), loss(42%) 19, 394041 
 7 ESTs 0.00166 AA394152    
 8 eyes absent (Drosophila) homolog 2 0.00184 AF055015 20q13.1   
 9 nuclear RNA export factor 1 0.00191 U80073 11q12-q13 gain(11q13, 27%), gain(11q13, 100%) 19, 38 
 10 hypothetical protein KIAA1165 0.00219 AA037467 13q22.2 loss(13cen-qter, 90%), loss(13cen-qter, 21%) 38, 46 
 11 ancient ubiquitous protein 1 0.00226 AF100746 2p13 gain(2p11-p15, 45%)  19  
 12 NADH dehydrogenase (ubiquinone) Fe-S protein 3 (30kD) 0.00228 AF067139 11p11.11   
 13 proteasome 26S subunit, ATPase, 1 0.00232 L02426 19p13.3 loss(19p13.3, 77%), loss(19p13.3, 58%) 45, 48 
High in nonsurvivors and low in survivors       
 14 anaphase-promoting complex 2 0.00021 AB037827    
 15 acetylserotonin O-methyltransferase-like 0.00041 Y15521 Xp22.3, Yp11.3 loss(Yp, 44%), loss(Xp-q21, 67%) 19, 45 
 16 ribosomal protein L24 0.00051 M94314 3q12 gain(50%), gain(35%), gain(3cen-qter, 35%) 37, 41, 46 
 17 ESTs 0.00067 BC007849    
 18 ESTs, Weakly similar to KIAA0748 protein [H. sapiens] 0.00091 BAA34468    
 19 α-B glycoprotein 0.00094 W25099 19cen-q13.2   
 20 hypothetical protein FLJ20548 0.00096 AK000555 9q34.13 loss(90%), loss(9q34, 50%), loss(40%) 37, 38, 45 
 21 seven transmembrane domain orphan receptor 0.00117 AB037108 3q21.3 gain(50%), gain(35%), gain(3cen-qter, 35%) 37, 39, 46 
 22 KIAA0747 protein 0.00125 BC004998 12q13.13 loss(12q24.1, 63%)  47  
 23 nucleolar protein 1 (120kD) 0.00145 M32110 12p13 gain(12p12, 27%)  19  
 24 ribosomal protein L15 0.00161 M77875 3p24.1 loss(3cen-p26, 95%), loss(3cen-p26, >98%) 37, 38 
 25 SH3 protein 0.00192 AF178432 3p21.31 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss(3cen-p26, >98%) 16, 37, 38 
 26 ESTs 0.00209 W02794    
 27 hypothetical protein AL 133206 0.00247 AL133206 1p35.3-p34.1 gain(1p33-p35, 45%)  19  
IDDescriptionPGenBank accession no.CytobandGenomic imbalance at cytoband in NSCLCaReferences
High in survivors and low in nonsurvivors       
 1 BUB3 (budding uninhibited by benzimidazoles 3, yeast) homolog 0.00011 AF047472 10q26 loss(10q26.13-qter, 36%)  45  
 2 ZW10 (Drosophila) homolog, centromere/kinetochore protein 0.00054 U54996 11q23.2 loss(11q23-q24, 71%), loss(11q23.2, 54%), loss(11q23) 424344 
 3 dCMP deaminase 0.00109 L39874 4q35.1 loss(4q31-qter, 64%), loss(4q34-q35, 50%), loss(4q32-qter, 100%) 19, 38, 45 
 4 keratin 9 (epidermolytic palmoplantar keratoderma) 0.00113 Z29074 17q21.1-q21.2 gain(17q11-q24, 82%), gain(17q21, 37%), loss(17q21, 53%), loss(42%) 19, 394041 
 5 KIAA1008 protein 0.00145 AA156488 13q21.23-q22.2 loss(13q11-q14, 62%), loss(13cen-qter, 90%), loss(13cen-qter, 21%) 38, 45, 46 
 6 proteasome 26S subunit, ATPase, 5 0.00155 D44467 17q23-q25 gain(17q11-q24, 82%), gain(17q21, 37%), loss(17q21, 53%), loss(42%) 19, 394041 
 7 ESTs 0.00166 AA394152    
 8 eyes absent (Drosophila) homolog 2 0.00184 AF055015 20q13.1   
 9 nuclear RNA export factor 1 0.00191 U80073 11q12-q13 gain(11q13, 27%), gain(11q13, 100%) 19, 38 
 10 hypothetical protein KIAA1165 0.00219 AA037467 13q22.2 loss(13cen-qter, 90%), loss(13cen-qter, 21%) 38, 46 
 11 ancient ubiquitous protein 1 0.00226 AF100746 2p13 gain(2p11-p15, 45%)  19  
 12 NADH dehydrogenase (ubiquinone) Fe-S protein 3 (30kD) 0.00228 AF067139 11p11.11   
 13 proteasome 26S subunit, ATPase, 1 0.00232 L02426 19p13.3 loss(19p13.3, 77%), loss(19p13.3, 58%) 45, 48 
High in nonsurvivors and low in survivors       
 14 anaphase-promoting complex 2 0.00021 AB037827    
 15 acetylserotonin O-methyltransferase-like 0.00041 Y15521 Xp22.3, Yp11.3 loss(Yp, 44%), loss(Xp-q21, 67%) 19, 45 
 16 ribosomal protein L24 0.00051 M94314 3q12 gain(50%), gain(35%), gain(3cen-qter, 35%) 37, 41, 46 
 17 ESTs 0.00067 BC007849    
 18 ESTs, Weakly similar to KIAA0748 protein [H. sapiens] 0.00091 BAA34468    
 19 α-B glycoprotein 0.00094 W25099 19cen-q13.2   
 20 hypothetical protein FLJ20548 0.00096 AK000555 9q34.13 loss(90%), loss(9q34, 50%), loss(40%) 37, 38, 45 
 21 seven transmembrane domain orphan receptor 0.00117 AB037108 3q21.3 gain(50%), gain(35%), gain(3cen-qter, 35%) 37, 39, 46 
 22 KIAA0747 protein 0.00125 BC004998 12q13.13 loss(12q24.1, 63%)  47  
 23 nucleolar protein 1 (120kD) 0.00145 M32110 12p13 gain(12p12, 27%)  19  
 24 ribosomal protein L15 0.00161 M77875 3p24.1 loss(3cen-p26, 95%), loss(3cen-p26, >98%) 37, 38 
 25 SH3 protein 0.00192 AF178432 3p21.31 homozygous deletion(3p21.3), loss(3cen-p26, 95%), loss(3cen-p26, >98%) 16, 37, 38 
 26 ESTs 0.00209 W02794    
 27 hypothetical protein AL 133206 0.00247 AL133206 1p35.3-p34.1 gain(1p33-p35, 45%)  19  
a

If the minimal region and the percentage of genomic imbalance involvement were known, they were written in the parentheses. The data of references 36 and 45 was analyzed with adenocarcinomas only. The data of references 38394041, 43, 46, and 47 was analyzed with LOH analysis with polymorphic markers. The data of references 19, 36, 38, and 45 was analyzed with comparative genomic hybridization.

We thank Drs. Hiroshi Murakami, Ryuichi Wada, Atul Kumar, Ivan Rosas, Masayuki Shiseki, Remy Pedeux, Shu Okamura, Stefan Ambs, Hasan Seker, Xin Wang, and Makoto Nagashima for technical advice and suggestions, Dr. John Gillespie for helping with the pathological diagnosis, Dr. Joanna Shih for helpful discussion on the statistical analysis, the microarray group of Dr. Nakamura’s laboratory for the fabrication of microarrays, Mary G. McMenamin for cell culture, Audrey Salabes for collecting the clinical information, and Dorothea Dudek for editorial assistance.

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