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Clinical Cancer Research Vol. 11, 8235-8242, December 1, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Genome-Wide Screening of Genomic Alterations and Their Clinicopathologic Implications in Non–Small Cell Lung Cancers

Tae-Min Kim1, Seon-Hee Yim2, Jung-Sook Lee1, Mi-Seon Kwon3, Jae-Wook Ryu4, Hyun-Mi Kang1, Heike Fiegler5, Nigel P. Carter5 and Yeun-Jun Chung1

Authors' Affiliations: 1 Department of Microbiology, College of Medicine, Catholic University of Korea, Socho-gu, Seoul; 2 Korea National Cancer Center, Research Institute, Division of Cancer Control and Epidemiology, Gyeonggi-do; Departments of 3 Pathology and 4 Thoracic and Cardiovascular Surgery, College of Medicine, Dankook University, Cheonan, Chungnam, Republic of Korea; and 5 The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom

Requests for reprints: Yeun-Jun Chung, Department of Microbiology, College of Medicine, Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Republic of Korea. Phone: 82-2590-1214; Fax: 82-2596-8969; E-mail: yejun{at}catholic.ac.kr.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Although many genomic alterations have been observed in lung cancer, their clinicopathologic significance has not been thoroughly investigated. This study screened the genomic aberrations across the whole genome of non–small cell lung cancer cells with high-resolution and investigated their clinicopathologic implications.

Experimental Design: One-megabase resolution array comparative genomic hybridization was applied to 29 squamous cell carcinomas and 21 adenocarcinomas of the lung. Tumor and normal tissues were microdissected and the extracted DNA was used directly for hybridization without genomic amplification. The recurrent genomic alterations were analyzed for their association with the clinicopathologic features of lung cancer.

Results: Overall, 36 amplicons, 3 homozygous deletions, and 17 minimally altered regions common to many lung cancers were identified. Among them, genomic changes on 13q21, 1p32, Xq, and Yp were found to be significantly associated with clinical features such as age, stage, and disease recurrence. Kaplan-Meier survival analysis revealed that genomic changes on 10p, 16q, 9p, 13q, 6p21, and 19q13 were associated with poor survival. Multivariate analysis showed that alterations on 6p21, 7p, 9q, and 9p remained as independent predictors of poor outcome. In addition, significant correlations were observed for three pairs of minimally altered regions (19q13 and 6p21, 19p13 and 19q13, and 8p12 and 8q11), which indicated their possible collaborative roles.

Conclusions: These results show that our approach is robust for high-resolution mapping of genomic alterations. The novel genomic alterations identified in this study, along with their clinicopathologic implications, would be useful to elucidate the molecular mechanisms of lung cancer and to identify reliable biomarkers for clinical application.

Lung cancer is the most common incident form of malignancy and is also the leading cause of cancer death worldwide (1, 2). A primary lung cancer is classified into four major histologic subtypes; squamous cell carcinomas, adenocarcinomas, large cell and small cell lung cancers. The former three classes, which are grouped as non–small cell lung cancers (NSCLC), make up almost 80% of all total lung cancer cases. Among the NSCLC, squamous cell carcinomas and adenocarcinomas are the two major subtypes. Histologically different subtypes have different clinical courses, and might require individual therapeutic approaches.

Some genomic aberrations in tumors have been suggested to be prognostic markers or can be used to identify the target genes for treatment or prevention (3, 4). Likewise, in other solid tumors, chromosomal aberrations are thought to be critical molecular events in the pathogenesis of lung cancer (5, 6). However, clinically applicable screening tools or prognostic markers are still underdeveloped. Because the lack of efficient screening methods and therapy accounts for the poor outcome of lung cancer, genome-wide assessment of aberrations could help in developing more accurate diagnostic and therapeutic strategies.

For this reason, previous cytogenetic studies using conventional comparative genomic hybridization (CGH) or fluorescence in situ hybridization have focused on identifying the chromosomal aberrations associated with NSCLC. Recurrent genomic alterations have been observed in NSCLC, including the gains of partial or whole chromosomal arms on 1q, 3q, 5p, and 8q along with the losses on 3p, 6q, 8p, 9p, 13q, and 17p (7–11). However, the ~10 Mb resolution of conventional CGH is insufficient for the precise identification of submicroscopic changes (12). As accumulating evidence suggests that changes in the genomic dosage contribute to tumorigenesis by altering the expression levels of the cancer-related genes (13, 14), more detailed analyses with sufficient resolution are required.

For enhancing the resolution, array CGH using mapped bacterial or P1 artificial chromosomes (BAC/PAC) rather than metaphase chromosomes, has been recently developed (15–17). This technique provides a high resolution that is directly related to the genomic density and insert size of the arrayed clones. Array CGH has emerged as a useful tool for detecting and mapping the genomic aberrations, which may contain putative oncogenes or tumor suppressor genes and for performing a molecular classification of tumors (18).

To see genomic alterations and their clinicopathologic implications in NSCLC, we applied genome-wide array CGH to the genomic DNA extracted from the microdissected tissues of 29 squamous cell carcinoma and 21 adenocarcinoma cases, on which the association study was done. Using this strategy, the genomic copy number changes specific to NSCLC including novel minimally altered regions (MAR) were identified. Those genomic alterations are likely to be related to tumorigenesis or the clinical outcomes of lung cancer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study materials. Frozen tissues were obtained from 50 NSCLC patients, who underwent surgical resection at Dankook University Hospital, Cheonan, Korea. Tissue collection and the full procedure of genetic analyses were done under the approval of Institutional Review Board of Kangnam St. Mary's Hospital, The Catholic University of Korea. The 50 NSCLC cases were histologically classified into squamous cell carcinomas (29 cases) and adenocarcinomas (21 cases). Tumor staging was done according to the standard tumor-node-metastasis classification in the American Joint Committee on Cancer guidelines. Of 50 patients whose mean age was 60 years, 88% (44 cases) were male. Other clinical information on the 50 patients is also available in Supplementary Table S1.

Tissue preparation. After surgical resection, tumor and adjacent normal tissues from the same patient were collected separately and snap-frozen in a deep freezer. Frozen sections were prepared of 10 µm thickness on a gelatin-coated slide using 2800 Frigocut (Reighert-Jung, Germany). After H&E staining, tumor cell–rich area (>60% of tumor cells) and histologically normal cell area were selected under the microscope and dissected manually. Microdissected tissues were transferred into the cell lysis buffer (1% proteinase-K in TE buffer) and DNA was extracted. DNA from normal tissue was used as reference DNA for array CGH. Extracted DNA was purified using a DNA purification Kit (Solgent, Daejeon, Korea) and used for dye labeling reactions.

Array comparative genomic hybridization and image analysis. We used human large insert clone arrays with 1 Mb resolution across the whole genome printed by the Sanger Institute Microarray Facility (19). DNA labeling, prehybridization, hybridization, and posthybridization processes were done as described previously (19, 20). Arrays were scanned using GenePix 4100A scanner (Axon Instruments, Union City, CA) and the image was processed using GenePix Pro 6.0.

Data processing, normalization, and mapping of BAC clones. Normalization and re-aligning raw array CGH data were done using the web-based array CGH analysis interface, ArrayCyGHt (http://genomics.catholic.ac.kr/arrayCGH/; ref. 21). Mapping of large insert clones was done according to the genomic location in the UCSC genome browser (May 2004 freeze). In total, 2,987 successfully mapped BAC clones out of initial 3,014 clones were processed subsequently. All the genomic coordinates such as cytogenetic bands or gene positions described in this study are based on the same version of the human genome available on the UCSC genome browser.

Data analysis for chromosomal alterations. To set the cutoff value for chromosomal alterations of individual large insert clones, we did a series of four independent normal hybridizations (three sex-matched and one male versus female hybridizations) as a control. The average SD value of the control batch was 0.081. Adopting the criteria of a previous study (22), the cutoff value for the copy number aberrations was set to be ±0.2 in log2 ratio in this study, >2-fold of control SD. The entire chromosome arm gain or loss was determined as previously described (23). Regional copy number change was defined as DNA copy number alteration limited to part of a chromosome. High-level amplification of clones was defined when their intensity ratios were >1.0 in log2 scale, and vice versa for homozygous deletion. The boundary of copy number change was assigned to be halfway between the two neighboring clones.

Definition of minimally altered regions. To define MARs of chromosomal gain or loss, we used CGH-Miner (http://www-stat.stanford.edu/~wp57/CGH-Miner/) to smooth the raw intensity ratio and to identify the breakpoints of chromosomal alterations (24). A series of four normal hybridizations were combined as a control and the analysis was done with recommended program variables. The significant gains or losses reported by the program were directly used for subsequent aligning procedures. Minimal regions of chromosomal gains and losses were determined by altered segments recurring for at least seven samples.

Statistical analysis. The significance of the differences in chromosomal arm changes between squamous cell carcinomas and adenocarcinomas was tested by two-sided Fisher's exact test. The correlations between recurrent genetic changes on minimally altered regions were assessed using univariate pairwise Pearson's correlation. For multiple comparisons, the step-down Sidak method was used to adjust the overall level of significance. In this case, the pairs of genetic changes on the same chromosomal arm were excluded for the concordance analysis. The correlations between genetic alterations and clinical variables were analyzed by two-sided Fisher's exact test. All the MARs as well as chromosomal arm changes were included in the analysis. For comparison, four kinds of clinical variables were treated as categorical variables such as age (<60 versus ≥60 years), stage (stages I and II as early versus stages III and IV as advanced), lymph node status (negative versus positive), and the disease recurrence (presence versus absence). Kaplan-Meier method was used for survival analysis and the difference between survival curves was compared using the log-rank test in univariate model. To identify independent prognostic factors after adjusting clinical variables such as age, sex, stage, treatment, metastasis, and recurrence, Cox regression was done. In all statistical analyses, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Comprehensive profiling of genomic alterations in non–small cell lung cancers. The overall genomic alterations observed in the 50 NSCLC cases (29 squamous cell carcinomas and 21 adenocarcinomas) are illustrated in Fig. 1A. The frequency plots of the chromosomal changes in the 50 NSCLC cases show that they are not randomly distributed but clustered in several hot regions across all the chromosomes (Fig. 1B). Eight chromosomal arms were frequently gained: 19q (40%, 20 of 50 cases), 20q (26%), 22q (24%), 3q (22%), 19p (22%), 1q (20%), 5p (20%), and 17q (20%). Also, six chromosomal arms were frequently lost: Yp (52%), Yq (46%), 9p (42%), 3p (26%), 17p (24%), and 4q (20%). Six chromosomal changes differentially distributed between squamous cell carcinomas and adenocarcinomas. Gains of 3q and 12p as well as losses of 3p, Yp, and Yq were found to be specific to squamous cell carcinomas, whereas a gain of 6p was found to be specific to adenocarcinomas (see Supplementary Table S2). The array CGH signal intensity ratio (in log2 scale) data of the 50 NSCLC can be downloaded from our web site (http://lib.cuk.ac.kr/micro/CGH/lung.htm).



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  		Fig. 1. Genome-wide copy number alterations in 50 cases of NSCLC. A, genomic profiles of 29 squamous cell carcinomas (top) and 21 adenocarcinomas (bottom). Fifty NSCLC cases are represented in individual lanes with corresponding sample numbers in two subtypes. Intensity ratios are schematically plotted in different color scales reflecting the extent of genomic gains (red) and losses (green) as indicated in the reference color bar. A total of 2,987 BAC clones were ordered (x-axis) according to the map positions and the chromosomal order from 1pter to Yqter. B, the genome-wide frequencies of all significant gains (>0.2 of intensity ratio, top plot) and losses (<–0.2 of intensity ratio, bottom plot) for each clone are shown for 29 cases of squamous cell carcinomas (black, above the x-axis) and 21 cases of adenocarcinomas (gray, below the x-axis), respectively. The boundaries of individual chromosome and the location of centromere are indicated by vertical bars and dotted lines below the plots, respectively.

 
High-level amplification and homozygous deletion. In total, 98 large insert clones showed high-level amplifications at least in one case and they clustered in 36 different genomic segments. The genomic size of the amplicons ranged from 0.31 to 14.78 Mb. All the identified amplicons along with the putative cancer-related genes located in these amplicons are summarized in Table 1. Figure 2A shows an example of a high copy number gain observed recurrently around 3q26-q28. The first (3q26), which is as large as 14.78 Mb, harbors several putative oncogenes such as EVI1, SKIL, ECT2, and PIK3CA. The other (3q28), which is 10.31 Mb in size, contains the putative oncogenes, BCL6 and HES. In all 50 NSCLC cases, only three homozygous deletions were identified (Table 1). Among them, a homozygous deletion of RP11-765C10 (10q23.31) harbors the tumor suppressor gene PTEN (Fig. 2B).


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  		Table 1. Genomic segments representing high copy number changes in NSCLC

 


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  		Fig. 2. Individual profiles of high copy number changes. A, high-level amplifications on 3q21-q29 for SqCs9 and SqCs22. B, a homozygous deletion on 10q23.31 for SqCs2. In the intensity ratio profiles, the x-axis represents the map position of corresponding clone according to the UCSC human genome (May 2004 freeze) and the intensity ratios were assigned to the y-axis. The schematic presentation of cytogenetic bands as well as a map position is shown below the plot.

 
Minimal regions of recurrent genomic changes. High copy number changes were relatively rare among the 50 cases. However, single copy number changes were more common and widespread. In total, 13 MAR gains (MAR-G) and 4 MAR losses (MAR-L) were identified. Table 2 lists the map position, size, and cancer-related genes located in the 17 MARs. Examples of MAR-G and MAR-L are illustrated in Fig. 3. The MAR-G on 1p36-p34, which was observed in 12 cases, contains several putative cancer-related genes such as PAX7, FGR, LCK, and MYCL1. In addition, another MAR-G on 1p32.3, which was found in nine cases, contains the putative cancer-related gene, TTC4 (Fig. 3A). The MAR-L on 5q23.2-q31.1 in seven cases includes several putative tumor suppressor genes such as IRF1, CDKL3, and RAD50 (Fig. 3B).


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  		Table 2. Minimal regions of recurrent copy number changes

 


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  		Fig. 3. Examples of minimal regions of genomic gain or loss. MAR was defined as a commonly altered segment recurring for at least seven cases. Each sample is represented as an individual lane. MARs are schematically shown as a colored box below the cytogenetic bands: red, copy number gain; green, copy number loss; black, no change. A, two minimally gained regions on chromosome 1p with different genomic sizes in 16 cases. B, minimal region of losses on chromosome 5q common to seven NSCLC samples.

 
Correlation between minimally altered regions. Pairwise correlation analysis between the MARs was done to determine if such genomic changes appeared concordantly in a set of NSCLC cases. For comparison, all possible combinations between the 17 MARs were considered except for pairs on the same chromosomal arms. A significantly positive correlation was observed for three pairs of MARs (see Supplementary Table S3). The MAR-G on 19q13.1 correlated with the MAR-Gs on 6p21.3-p21.1 (r = 0.549; P = 0.0482) and 19p13.2-p13.1 (r = 0.672; P = 0.0016). Another significant association was found between two MAR-Gs on 8p12.2-p12.1 and 8q11.2-12.1 (r = 0.610; P = 0.0370).

Association between genomic aberrations and clinical characteristics. Four types of clinical variables (age, stage, lymph node, and recurrence) were analyzed for their association with the genomic alterations identified (see Supplementary Table S4). Significant associations were observed for the MAR-L on 13q21 with cancers from those aged <60 and an advanced stage (stages III and IV). The chromosomal gain of Xq was also found to be associated with an advanced stage and disease recurrence. The MAR-G on 1p32 and a chromosomal gain of Yp were associated with being lymph node–negative.

Survival analysis was done to assess the prognostic values of the genetic aberrations identified. Using Kaplan-Meier methods, we identified that six genetic aberrations associated with a relatively poor survival (Fig. 4); gain of 10p (P = 0.0091) and 16q (P = 0.0262), loss of 9p (P = 0.0082) and 13q (P = 0.0019), and the MAR-Gs on 6p21 and 19q13 (P = 0.0265 and 0.0295, respectively). Multivariate analyses using all the genetic alterations identified, as well as the clinical variables such as age, gender, stage, treatment, metastasis, and recurrence showed that four genetic alterations and three clinical variables remained independent factors to be significantly associated with a poor survival outcome (Table 3). One of the four genetic alterations was a MAR-G on 6p21 and the other three were a loss of 9p, and gains of 7p and 9p.



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  		Fig. 4. Kaplan-Meier survival curves. The survival curves for the cases with (thick line) or without (thin line) specific genomic changes are plotted using the Kaplan-Meier method. The chromosomal changes associated with relatively poor survival are presented with the significance level; gain of 10p (A) and 16q (B), loss of 9p (C) and 13q (D), and MAR-Gs on 6p21 (E) and 19q13 (F).

 

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  		Table 3. Independent predictors of poor survival in 50 NSCLCs

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Using whole-genome array CGH strategy, we successfully identified novel chromosomal aberrations as well as previously identified ones in NSCLC. This study focused on the potentially meaningful genomic changes such as recurrent single copy changes as well as high-level amplifications or deletions. For this, microdissection was used to remove the nontumor tissues, and the microdissected DNA was hybridized to arrays without performing whole genome amplification, which reduced any possible bias due to a random amplification.

The frequent chromosomal changes in this study are largely consistent with previous cytogenetic analysis (7–11), including a loss of the Y chromosome in male patients (5, 6). It is notable that the copy number alterations on the small chromosomes such as 19, 20, and 22 were much more frequent in our study. This might be due to the differences in the analytic methods. However, it is more likely to reflect the potential of array CGH to improve the low resolution of conventional CGH as described elsewhere (25). The genomic size of the high copy number changes ranged from 0.31 to 14.78 Mb, and most of them were <5 Mb. Genomic changes <5 Mb are likely to be novel because they cannot be detected using conventional CGH (12).

To date, two array-based CGH analyses of lung cancer have been published (26, 27). Our results are in general agreement with the previous array CGH results. For example, the 30-Mb sized recurrently gained region around 3q26 reported by Massion et al. (26) overlapped with the MAR-G on 3q26-q28 in this study. Other metaphase CGH studies have also shown frequent amplifications around the same region in squamous cell carcinomas (9–11). However, some of the MARs identified were not consistent with them. Gain of 3q26 was the only recurrent alteration in Massion et al.'s study, whereas we identified not only 3q26 but also 16 more MARs across various chromosomes. The difference is thought to be due to the resolution. This study placed 2,987 large insert clones in 1 Mb intervals (average of 125 clones per chromosome), whereas they used 348 BAC clones (average of 15 clones per chromosome, at most 78 clones on chromosome 3). That might explain why more MARs could be defined. In Jiang et al.'s study (27), the recurrent genomic alterations were only partly compatible with this result, and there was no clear minimal recurrent gain on 3q. They used a cDNA microarray rather than a BAC/PAC array for CGH analysis. Despite its advantages, the sensitivity and reliability for detecting copy number changes, particularly single copy changes, is known to be limited (18).

Several interesting cancer-related genes are located in the genomic alteration regions identified in this study. For example, PIK3CA on 3q26-q28, which is one of the genes in the most common MAR-G in this study, is believed to contribute to the tumorigenesis of squamous cell carcinomas by involving in the phosphoinositide-3-OH kinase signaling pathway (26). The ECT2 oncogene, which is located in the same locus, is known to activate the Rho signaling pathway leading to a malignant transformation (28). In our unpublished data,6 ECT2 is frequently overexpressed in primary lung cancers. Although, there has been no report of ECT2 overexpression in lung cancer, this suggests the involvement of ECT2 in malignant transformation or the progression of lung cancer. Most high-level amplifications were usually observed in one or two cases with the exceptions of the amplifications on 3q. They might reflect the individual nature of the genomic evolution for the respective NSCLC cases. Among the putative cancer-related genes in the high-level amplification region (Table 1), the expression of the AF1Q, TPM3, REL, SKIL, ECT2, BCL6, MLLT6, YES1, and HKR genes have not been reported in lung cancer.

A homozygous deletion on 10q23.31 observed in one squamous cell carcinoma case contains the well-known tumor suppressor gene, PTEN. PTEN is known to encode lipid phosphatase, which negatively controls the signaling proteins activated in the phosphoinositide-3-OH kinase pathway (29). This suggests a potential role of the phosphoinositide-3-OH kinase signaling pathway in NSCLC pathogenesis.

In contrast to the high copy number changes that were largely limited to a few samples, single copy changes were found in many more samples, which is indicative of a shared mechanism common to the earlier stage of NSCLC. Minimal recurrent gains and losses were successfully identified using high-resolution array CGH. Seventeen MARs of various sizes were defined. The MAR-Gs on 1p, 2p, 6p, 8p, 19p, and 20p along with MAR-Ls on 5q and 20q are believed to be novel features in lung cancer, which shows the advantage of genome-wide, high-resolution mapping of the genomic alterations. Interestingly, three pairs of MAR-Gs (19q13.1 and 6p21, 19p13 and 19q13.1, and 8p12 and 8q11-12) showed significant correlations among themselves, suggesting a possible collaborative role in the tumorigenesis of NSCLC. Further investigations will be needed to confirm the functional consequences of the associations between the MARs. Some of the MARs showed significant correlations with the clinical features. This suggests that the common single copy changes identified by high-resolution analysis can be useful biomarkers for the clinical characteristics of lung cancer.

Survival analysis revealed that six genetic alterations were associated with a poor survival outcome in the univariate model (Fig. 4). Among those six alterations, a loss of 9p was reported to be associated with a poor survival outcome (30). However, there has been no report about the association between the other five genomic alterations and survival outcomes in lung cancer. These genomic alterations might be a novel genetic indicator of the prognosis of NSCLC after the appropriate validation. In particular, two of these alterations are MARs, which appeared concordantly (P = 0.0482). These two MARs, MAR-Gs on 6p21 and 19q13, contain cancer-related genes such as PIM1, CCND3 (both in 6p21), and HKR1 (19q13). The high expression level of HKR1 after administering platinum drugs has been reported to be associated with the acquisition of resistance to chemotherapy (31). There is no report demonstrating an alteration of CCND3 and PIM1 proto-oncogene in lung cancer. However, both genes are well known to be involved in the tumorigenesis pathways of various tumors. Therefore, further investigations will be needed to evaluate their specific implications in lung cancer.

Subsequent Cox regression analysis identified seven factors, including four genomic alterations such as MAR-G on 6p21, 9p loss, 7q gain, and 9q gain, to be independent indicators of a poor survival outcome. This indicates that in addition to the clinical factors, precisely defined recurrent genetic alterations can be useful biomarkers for the prognosis of NSCLC. However, due to the limited number of samples in this study, further studies with a larger sample size will be needed to confirm the prognostic implication of these genomic alterations and to identify further reliable prognostic markers.

This study showed that a well-designed high-resolution array CGH could define more novel regions possibly associated with the tumorigenesis of lung cancer. Therefore, these results will give a clue for further studies to elucidate lung cancer pathogenesis or to develop biomarkers for predicting the prognosis or treatment response of lung cancer.


   Acknowledgments
 
We thank the Wellcome Trust Sanger Institute Microarray Facility for printing BAC array slides.


   Footnotes
 
Grant support: Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ3-PG6-01GN07-0004).

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.

Note: T-M. Kim and S-H. Yim contributed equally to this paper.

Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

6 M.S. Kwon, H.M. Kang, and Y.J. Chung, manuscript in preparation. Back

Received 5/31/05; revised 8/ 3/05; accepted 9/ 1/05.


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