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Clinical Cancer Research Vol. 10, 1984-1991, March 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Skp2 Gene Copy Number Aberrations Are Common in Non-Small Cell Lung Carcinoma, and Its Overexpression in Tumors with ras Mutation Is a Poor Prognostic Marker

Chang Qi Zhu1, Fiona H. Blackhall2,6, Melania Pintilie2, Pratibha Iyengar3, Ni Liu1, James Ho1, Taylor Chomiak1, Davina Lau1, Timothy Winton2,5, Frances A. Shepherd2,6 and Ming-Sound Tsao1,2,3,4

1 University Health Network, Ontario Cancer Institute and 2 Princess Margaret Hospital, and 3 Departments of Laboratory Medicine and Pathobiology, 4 Medical Biophysics, 5 Surgery, and 6 Medicine, University of Toronto, Toronto, Ontario, Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Skp2 plays a critical role in cell cycle progression, especially at the G1-S transition, putatively through its control of several cell cycle regulator proteins. The Skp2 gene is located on a region of chromosome 5p that is commonly overrepresented in lung cancer. The present study aimed to evaluate Skp2 abnormalities and their prognostic value in non-small cell lung cancer (NSCLC).

Experimental Design: In total 16 NSCLC cell lines and 163 primary tumors were included in studies to measure Skp2 relative gene copy number, mRNA abundance, and protein level. The tumors were also evaluated for p27 protein expression level and ras mutation. These values were correlated with the clinical and pathological features of the patients.

Results: Skp2 relative gene copy number aberrations were found in 88 and 65% of NSCLC cell lines and primary tumors, respectively. Overrepresentation was especially common among squamous cell carcinoma (74%). Both gene copy overrepresentation (13%) and loss (35%) were found in adenocarcinoma. Skp2 relative gene copy number was significantly correlated with mRNA and protein levels, but none of these were correlated with p27 protein levels. Neither high Skp2 protein expression nor ras mutation was prognostically significant. In NSCLCs with ras mutation, however, high Skp2 protein expression was a significant independent poor prognostic marker.

Conclusion: There appears to be a synergistic interaction between high Skp2 protein expression and ras mutation with negative impact on the survival of NSCLC patients.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The S-phase kinase-associated protein (Skp2) is a member of the F-box protein family that forms the substrate recognition unit of the Skp1/Cul1/F-box protein, a multicomponent RING-type ubiquitin ligase E (E3) complex. The Skp1/Cul1/F-box protein complex consists of four subunits: the adaptor protein Skp1, the scaffold protein Cul1/Cullin, the RING-domain protein RbX1/Roc1, and the F-box protein that binds the substrate. More than 70 F-box proteins have been identified in human and mouse genomes, and Skp2 represents the best-studied F-box protein that is intimately involved in regulating the cell cycle (1) . The most important substrates for Skp2 include p27kip1 cyclin-dependent kinase inhibitor and cyclin E, both of which interact with cyclin-dependent kinase 2 to regulate G1-S transition (2) . Recently, Skp2 has also been implicated in regulating the proteosome-mediated degradation of c-myc (3 , 4) , p21cip1 (5) , p57kip2 (6) , and p130-Rb2 (7 , 8) . Elevated Skp2 expression has been demonstrated in various tumor tissues (9 , 10) and cell lines (11 , 12) . Skp2 expression has been correlated with proliferation index (10 , 13) and tumor stage (13 , 14) and with extracutaneous involvement in Kaposi’s sarcoma (15) . In addition, Skp2 overexpression was correlated with poor prognosis in soft tissue sarcoma (9) and oral squamous cell carcinoma (16) . An inverse correlation between Skp2 and p27kip1 protein level has been reported in various tumors (13 , 14 , 17, 18, 19) , and a low p27 protein level has been reported as a poor prognostic marker in several human tumors (20, 21, 22, 23, 24) .

The Skp2 gene is located on chromosome 5p13, a region that is commonly overrepresented in lung cancer (25, 26, 27, 28) . Using a fluorescence in situ hybridization technique, Yokoi et al. (11) reported Skp2 gene amplification in 44% and mRNA overexpression in 83% of small cell lung carcinoma (SCLC). They also reported that down-regulation of Skp2 suppressed the growth of a SCLC cell line, suggesting that Skp2 might play an important role in regulating the growth of SCLC. We report here the frequency of Skp2 genomic and expression changes in non-small cell lung carcinoma (NSCLC) and their prognostic significance.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Materials and Patients.
This study used tissue materials from a snap-frozen and paraffin-embedded lung tumor bank that was established at the Princess Margaret Hospital and Toronto General Hospital in 1996, after approval by the Research Ethics Board of the University Health Network. The materials included 163 tumor tissues obtained from patients who had undergone primary lung cancer resection without presurgical radiation or chemotherapy and 29 corresponding nonneoplastic lung parenchymal tissues from a subset of these patients. Patients’ demographic and clinical follow-up information was also obtained after Research Ethics Board approval for chart reviews.

Cell Lines.
Sixteen NSCLC cell lines were used in this study, including those previously established in our laboratory (MGH-4, MGH-7, MGH-8, MGH-13, MGH-24, MGH-30, and RVH-6849) and those obtained from the American Type Culture Collection (Rockville, MD). The latter included NCI-H157, NCI-H226, NCI-H358, NCI-H520, A549, NCI-H1264, NCI-H125, NCI-H661, and NCI-H460. Cell lines are routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA).

Genomic DNA Isolation from Tissue and Laser-Captured Microdissection (LCM).
DNA was extracted from formalin-fixed and paraffin-embedded sections of 79 samples, including 19 nonneoplastic lung and 60 NSCLCs. For tumors, DNA was isolated from the tumor cells by microdissection using the Arcturus Pixcell II (Mountain View, CA) LCM equipment. The captured tumor cells were incubated in the DNA extraction buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.1 mg/ml of gelatin, 0.45% NP40, 0.45% Tween 20, and 0.4 mg/ml of proteinase K. DNA was isolated by phenol-chloroform extraction technique, as described previously (29) . Genomic DNA was also extracted from 37 snap-frozen tumor tissues that had been verified by their representative H&E slides to show tumor cells occupying >50% of tissue area and from a subset of 9 corresponding nonneoplastic lung tissue specimens.

Relative Gene Copy Number Determined by Quantitative-PCR (Q-PCR).
Q-PCR was performed using the ABI Prism 7700 sequence Detection System (Applied Biosystem, Foster City, CA) and the SYBR Green technique. All primers were designed and tested for their specificity using the Primer Express v1.5 (Applied Biosystems; Ref. 30 ). The target amplicons were 61–125 bp in intron 1 of each gene. The sequences of the genomic primers used for subsequent Q-PCR assays are listed in Table 1Citation . Quantitation was performed using the comparative CT method (30) . To normalize the Q-PCR data against aneuploidy that commonly occurs in NSCLCs, the CT values of the Skp2 gene were normalized against those of the phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) gene, which is located on 5q12–13 and was reported previously as showing no amplification in NSCLCs (31) . The Skp2 and PIK3R1 concentrations of a sample DNA (tumor, cell line, or individual normal lung) were also normalized to those obtained from DNA of a calibrator sample to eliminate potential differences in primer efficiencies. The calibrator sample was a pooled DNA from seven normal lung samples. The Skp2 relative gene copy numbers higher or lower than two times the SD from the mean of normal lung tissues were defined as Skp2 gene copy number overrepresentation or loss, respectively.


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  		Table 1 Primer sequences and conditions for quantitative PCR

 
Real-Time PCR Quantitation of mRNA Expression.
To measure the mRNA expression level, we performed quantitative reverse transcription-PCR on total cellular RNA isolated from 131 primary NSCLCs and 18 corresponding nonneoplastic lung tissues of these cases, using the ABI PRISM 7700 Sequence Detection System, as described previously (29) . To avoid amplification of contaminating genomic DNA sequences, primers were designed to span two adjacent exons. The primers for Skp2 and 18S transcript assay are also listed in Table 1Citation . All assays were done using duplicate samples of each reverse transcriptase product, and the assay was repeated three times. The sample:sample variation of RNA:cDNA quantity was normalized using the {Delta}CT = [CT(Skp2) - CT(18S)] method and 18S ribosomal RNA as the housekeeping gene (29) . Using the mean {Delta}CT of 18 nonneoplastic lung tissues, we also calculated the relative mRNA transcript levels in tumor tissues as fold changes compared with the mean of non-tumor lung tissue, as described previously (29) .

ras Genotyping.
All tumor samples had their ras genotype determined by the allelic-specific oligonucleotide hybridization technique, as described previously (32) .

Tissue Microarray Construction.
The H&E slides of resected lung tumors were reviewed for the presence of carcinoma. The slides that best represented the tumor were selected, and the respective paraffin blocks were retrieved. Using the manual tissue arrayer (Beecher Instruments, Silver Spring, MD), we constructed tissue microarray blocks containing cores of these tumors. Tissue cores of 1.0 mm were successfully extracted from 95 cases. They included 55 adenocarcinomas (ADCs), 34 squamous cell carcinomas (SQCCs), and 5 large cell carcinomas (LCCs). On average, three to four cores were obtained from each tumor, and these were arrayed into four recipient tissue microarray blocks. Each block also included non-pulmonary tissue cores to serve as controls and for orientation purposes.

Immunohistochemistry.
Serial 4-µm-thick sections from the tissue microarray blocks were cut and dried in a 60°C oven overnight. Sections were dewaxed in xylene and rehydrated through graded alcohol to water. Endogenous peroxidase was blocked in 3% hydrogen peroxide. After performing microwave antigen retrieval in 10 mM citrate buffer (pH 6.0) in a pressure cooker, slides were blocked for endogenous biotin with Vector’s biotin blocking kit. After blocking with normal serum, sections were covered in primary antibody for 16 h at room temperature in a moist chamber. The Skp2 antibody (ZYMED, South San Francisco, CA) was used at 1:100 dilution, and the p27 antibody (BD bioscience, Mississauga, Ontario, Canada) was used at 1:1000 dilution. After washing in PBS, secondary antibody incubation was carried out using the multi-species link (Ultra-Streptavidin detection system; Signet Pathology System, Dedham, MA), which was followed by incubation with streptavidin-horseradish peroxidase. Immunoreactivities were revealed by incubation in Nova Red substrate (Vector Laboratories, Burlingame, CA) for 5 min. Slides were counterstained in Mayer’s hematoxylin and mounted in Permount.

To score the immunoreactivity of Skp2 and p27 proteins, only nuclear staining was evaluated for both the staining intensity and extent of staining. The staining intensity was scored into three grades: 0 for complete absent staining, 1 for weak staining, and 2 for strong staining. The extent of positively stained nuclei was scored into four grades: 0 for <10% nuclei staining, 1 for 10% to <25%, 2 for 25% to <50%, and 3 for tumors with 50% or greater tumor cell nuclei staining positive. The final score for each tumor sample represents the sum of staining intensity and extent. Two pathologists independently evaluated the immunostaining.

Statistical Analysis.
Statistical analyses were performed with SAS v8.2 and S-plus. Skp2 copy number was categorized into low, normal, and high groups, whereas mRNA abundance and protein levels were treated as continuous variables. The association between the molecular factors was evaluated using the Spearman correlation coefficient and Wilcoxon test. Kappa statistics and the Spearman correlation coefficient were calculated to assess the degree of agreement between the scoring for the immunohistochemistry analysis done by the two pathologists. Two outcome measures were considered: overall survival and disease-free survival. Overall survival was evaluated from the date of surgery to the date of last follow-up or until death occurred. Disease-free survival was calculated from the date of surgery to the date of relapse. No patient died before experiencing a relapse. The survival curves were based on the Kaplan-Meier estimates. The association between the molecular factors and outcome was investigated using the Cox proportional hazards model. The Martingale residuals were used to examine the functional form of the molecular parameters with the outcome. The molecular factors were tested in both univariate and multivariate models where the statistically significant clinical factors were considered. We also tested the interaction between ras and Skp2 with respect to their association on clinical outcome. Because this interaction was found to be significant, Skp2 was tested in the wild-type and mutant ras subgroups.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor samples from 163 NSCLC patients with pathological stages ranging from stages I–III were used in this study, but smaller subsets of overlapping yet randomly chosen samples were used in various molecular analyses. The sample cohorts analyzed by different techniques showed comparable clinical pathological characteristics (Table 2)Citation .


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  		Table 2 Clinical-pathological characteristics of the NSCLCa cases used in various molecular studies

 
Skp2 Gene Copy Number Aberrations Were Common in NSCLCs.
Skp2 gene copy number overrepresentation was noted in 88% (14 of 16) of NSCLC cell lines (Fig. 1A)Citation . Initial studies on genomic DNA isolated from snap-frozen primary NSCLC tissue demonstrated low frequency of copy number aberrations, but this was dramatically increased when the measurements were performed on LCM tumor samples. The latter revealed Skp2 gene copy number abnormalities in 65% (39 of 60) of NSCLC samples, with 48% (29 of 60) showing copy number overrepresentation and 17% (10/60) demonstrating gene copy losses. Importantly, overrepresentation was much more frequent in SQCCs (74%; 25 of 34) than in ADCs (13%; 3 of 23). In contrast, gene copy loss was common in ADCs (35%; 8 of 23) but rare among the SQCCs (6%; 2 of 34).



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  		Fig. 1. Skp2 relative gene number (A), mRNA expression (B), and protein (C) levels in a NSCLC cell line and primary tumors. Dotted lines in A, upper/lower limit of normal. Bars in B, medians. LCM, laser-captured microdissection; CL, cell line; N, normal lung; ADC, adenocarcinoma; SQCC, squamous cell carcinoma; LCC, large cell carcinoma.

 
Skp2 mRNA Expression in NSCLCs.
The reverse transcription and real-time PCR analysis of mRNA transcript levels in 131 NSCLC samples and 18 corresponding nonneoplastic lung parenchyma samples demonstrated that the median Skp2 mRNA expression levels relative to the average of non-tumor lung tissue were 1.07 for ADCs, 4.32 for SQCCs, and 1.13 for LCCs, consistent with the Skp2 gene copy number overrepresentation being most commonly found in SQCCs (Fig. 1BCitation ; Table 3Citation ). The median mRNA was 0.90 among those with gene copy loss, 1.40 for those with normal gene copy number, and 3.10 in the group with copy number increase (n = 55; P = 0.027).


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  		Table 3 Association between Skp2 and ras abnormalities with histological typing

 
Skp2 Protein Expression in NSCLCs.
Protein expression levels were assessed by immunohistochemistry. Typical Skp2 immunostaining patterns that showed differing staining intensity are shown in Fig. 2Citation . There were 95 primary NSCLC cases with at least two cores containing tumor cells. The Spearman correlation coefficient between the independent assessments by two pathologists was 0.92, and if the scores are grouped in three categories of 0–1, 2–3, and 4–5, the weighted Kappa statistic is 0.81 with a 95% confidence interval of 0.72–0.9. These statistics suggest a good concordance between the two sets of scores. The sum of the scores from the two pathologists was used for subsequent correlative analyses. Consistent with the Skp2 relative gene copy number and mRNA expression levels, SQCCs had a much higher proportion of high Skp2 protein-expressing tumors than ADCs (median 6 versus 2; P < 0.0001; Table 3Citation ). The median of the Skp2 protein level for the group with loss of gene copy was 2, for the group with normal gene copy number was 4, and for the group with overrepresented copy number was 7 (n = 35; P = 0.033). Among 67 cases for which both immunohistochemistry and Q-PCR analyses were performed, there was a significant correlation between the Skp2 protein and mRNA expression levels (r = 0.49; P < 0.0001).



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  		Fig. 2. Representative Skp2 immunostaining in NSCLC. Strong (+2) nuclear staining was demonstrated in an adenocarcinoma (A) and a squamous cell carcinoma (C). B, an adenocarcinoma with negative nuclear staining. D, a squamous cell carcinoma with diffuse but weak (+1) staining in tumor cells.

 
Correlation between Skp2 Abnormalities and p27kip1 Protein Expression Levels.
On the basis of the putative role of Skp2 in regulating p27 degradation, we investigated the relationship between Skp2 and p27 protein levels assessed by immunohistochemistry. There was a complete lack of correlation between the p27 protein levels and Skp2 relative gene copy number, mRNA transcript levels, and protein expression levels. The Spearman correlation coefficients between the p27 protein level with the Skp2 protein level and mRNA transcript level were 0.01 and -0.03, respectively. The median of the p27 protein level for the group with loss of relative gene copy number was 5, for the group with normal copy number was 6, and for the group with overrepresented copy number was 7 (P = 0.32).

Correlation between Skp2 Abnormalities and ras Mutation.
The frequency of ras mutation was 28% (46 of 164), with these mutations occurring predominantly on the Ki-ras gene. Only one H-ras mutation was found. Frequent ras mutations were observed among ADCs (38%; 38 of 100), compared with only 13% (7 of 53) in SQCCs. Although higher levels of Skp2 relative gene copy number, mRNA expression, and immunohistochemistry scores were noted in tumors with wild-type ras gene compared with those with mutant ras, these associations were entirely abolished when adjusted for histology.

Correlation with Clinical Outcome.
In this data set, there were 69 events for survival and 83 for disease-free survival. The median follow-up was 3.2 years (range, 0–6.3 years). The results for overall survival resembled closely the ones for disease-free survival; hence, we will give the results only for the former end point. Among the clinical factors, grade and stage were significant prognostic variables (P = 0.0033 and P < 0.0001, respectively), whereas histology was not (P = 0.22). None of the molecular parameters was significant for survival, whether considered alone in the model (Table 4)Citation or when their effect was adjusted for grade and stage (data not shown). An a priori question was related to the interaction between ras genotype and Skp2 abnormalities. Significant interaction was only found between ras and Skp2 protein for survival (P = 0.018), even after adjusting for stage and grade (P = 0.023). Neither Skp2 mRNA (P = 0.65) nor copy number (P = 0.24) interacted with ras genotype for survival. The significance of this interaction suggested that Skp2 overexpression might function differently in normal ras and mutant ras groups. Indeed, the subgroup analysis found that the Skp2 protein level was not of prognostic significance for the normal ras group, whether the model was unadjusted for grade and stage (P = 0.83) or adjusted (P = 0.15; Fig. 3Citation ). On the other hand, a high Skp2 protein level was significant as a negative prognostic marker in the ras mutant subgroup, both in the unadjusted (P = 0.0021) as well as the adjusted (P = 0.0034; Fig. 3Citation ) analyses.


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  		Table 4 Prognostic significance of various molecular and clinicopathological factors

 


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  		Fig. 3. Kaplan-Meier plots demonstrating interaction between ras mutations and high Skp2 protein expression. High Skp2 protein level was not prognostically significant for NSCLCs with normal/wild-type ras (A) but was significant in tumors with mutant ras (B).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies on NSCLCs have demonstrated frequent amplification occurring on chromosome 5p, where the Skp2 gene is located (25 , 26 , 33) . We have combined LCM and Q-PCR techniques to demonstrate that aberrations in Skp2 gene copy number occur in a high percentage of NSCLC cell lines and primary tumors, and that these changes are reflected at both the mRNA and protein expression levels. More importantly, our data suggest that high Skp2 protein expression is an independent poor prognostic marker for NSCLCs, but only among tumors that harbor a ras mutation. Our findings provide important evidence that the prognostic impact of ras mutation may only be revealed through its interaction with other genetic or gene expression aberrations.

Skp2 putatively plays a critical role in regulating cell cycle progression, especially the G1-S checkpoint, by controlling the degradation of p27kip1 (2) , p21cip1 (5) , p57kip2 (6) , p130-Rb2 (7 , 8) , and cyclin E (2) . A high level of Skp2 expression is observed in a variety of cancers, and the prognostic value of Skp2 has been reported in ovarian adenocarcinoma (34) , soft tissue sarcomas (9) , and oral squamous carcinoma (16) but not in hepatocellular carcinoma (35) . In the lung, Inui et al. (20) reported previously that 12 of 15 NSCLC cases they studied had significant Skp2 mRNA overexpression, with expression levels significantly higher in SQCCs compared with ADCs. This is consistent with our observation that Skp2 is amplified and overexpressed in a very high proportion of NSCLCs, especially SQCCs. However, in contrast to observations in other cancers that showed prognostic value of Skp2, Skp2 gene copy number overrepresentation and overexpression by themselves were not prognostically significant in NSCLCs. Skp2 overexpression in hepatocellular carcinoma also failed to be prognostically significant (35) .

Despite the small number of cases with ras mutation, our observation that Skp2 protein overexpression interacts with ras mutations to exert an independent adverse prognostic impact in NSCLC patients is a significant and novel finding. Although the prognostic significance of ras mutations in NSCLCs has been studied extensively, the results remain controversial (36 , 37) . The fact that the ras mutation may interact with other genes to confer an adverse outcome suggests that additional studies focusing on the interactions of ras mutations with other molecular aberrations in a variety of tumors are warranted. A previous study (18) reported that Skp2 overexpression could only transform primary rat embryo fibroblasts when H-ras was also coexpressed. Furthermore, although the expression of Skp2 or N-ras alone failed to induce malignancy, the coexpression of Skp2 and N-ras was capable of inducing lymphoma (19) . Additional studies in a larger set of patients and tumor samples, especially among ADCs, will also be required to confirm the interaction of Skp2 and ras mutation.

Skp2 is required for the ubiquitination and proteolysis of the cell cycle regulatory proteins p27 (38) . Previous studies have reported an inverse relationship between Skp2 mRNA/protein and p27 protein levels in oral squamous cell carcinoma (18) , lymphoma (19) , colorectal carcinoma (17) , and prostate cancer (14) . However, this relationship was not seen in soft tissue sarcomas (9) , Kaposi’s sarcoma (15) , and cervical cancer (39) . Inui et al. (20) also reported an inverse relationship between Skp2 and p27 in 15 primary NSCLCs, but our results with a much larger cohort of samples did not confirm this correlation. The lack of such inverse correlation suggests that the levels of Skp2 protein do not exclusively or predominantly control p27 protein homeostasis, at least in NSCLCs. Alternative ubiquitination or Skp2-independent degradation pathways for p27 have been reported (40 , 41) . Endogenous phosphorylation activities might also play an important role for phosphorylation of p27 at Thr-187, which is necessary for p27 recognition by Skp2 (42) .

Last but not least, we have also demonstrated that Q-PCR can be reliably used to confirm gene copy number aberrations in tumor cells of paraffin-embedded tumor tissue, but this requires a careful choice of reference genes and LCM to increase the purity of tumor cell DNA as a template for PCR amplification. High-throughput multiplex Q-PCR could provide an alternative or complementary strategy to fluorescence in situ hybridization for future validation of genome wide array-comparative genomic hybridization fine mapping studies.


   FOOTNOTES
 
Grant support: This work was supported by National Cancer Institute of Canada Grant 012150 from the Canadian Cancer Society and a Grant-in-Aid from the Ontario Cancer Research Network.

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.

Notes: F. H. Blackhall was supported by an unrestricted educational fellowship from Aventis Pharma Inc. (Laval, Quebec, Canada). F. A. Shepherd is the Scott Taylor Chair in Lung Cancer Research. M.-S. Tsao is the M. Qasim Choksi Chair in Lung Cancer Translational Research. T. Winton is currently at Capital Health Authority, University of Alberta, Edmonton, Alberta, Canada.

Requests for reprints: Ming-Sound Tsao, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, M5G 2M9 Canada. Phone: 416-946-4426; Fax: 416-946-6579; E-mail: Ming.Tsao{at}uhn.on.ca

Received 10/24/03; revised 12/15/03; accepted 12/19/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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