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Cancer Epidemiology Biomarkers & Prevention Vol. 15, 373-380, February 2006
© 2006 American Association for Cancer Research

Molecular Profiling of Computed Tomography Screen-Detected Lung Nodules Shows Multiple Malignant Features

María J. Pajares1,6, Isabel Zudaire1,7, María D. Lozano2, Jackeline Agorreta1,6, Gorka Bastarrika3, Wenceslao Torre4, Ana Remírez1, Ruben Pio1,8, Javier J. Zulueta5 and Luis M. Montuenga1,6

1 Oncology Division, Center for Applied Medical Research; Departments of 2 Pathology, 3 Radiology, 4 Thoracic Surgery, and 5 Pulmonary Medicine, Clínica Universitaria de Navarra; and Departments of 6 Histology and Pathology, 7 Genetics and 8 Biochemistry, Schools of Medicine and Sciences, University of Navarra, Pamplona, Spain

Requests for reprints: Luis M. Montuenga, Área de Oncología. Centro para la Investigación Médica Aplicada, University of Navarra, Avenida Pío XII, 55, 31008 Pamplona, Spain. Phone: 34-948194700; Fax: 34-948194714. E-mail: lmontuenga{at}unav.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rationale and Purpose: Low-dose spiral computerized axial tomography (spiral CT) is effective for the detection of small early lung cancers. Although published data seem promising, there has been a significant degree of discussion concerning the potential of overdiagnosis in the context of spiral CT–based screening. The objective of the current study was to analyze the phenotypic and genetic alterations in the small pulmonary malignancies resected after detection in the University of Navarra/International Early Lung Cancer Action Project spiral CT screening trial and to determine whether their malignant molecular features are similar to those of resected lung tumors diagnosed conventionally.

Experimental Design: We analyzed 17 biomarkers of lung epithelial malignancy in a series of 11 tumors resected at our institution during the last 4 years (1,004 high-risk individuals screened), using immunohistochemistry and fluorescence in situ hybridization (FISH). A parallel series of 11 gender-, stage-, and histology-matched lung cancers diagnosed by other means except screening was used as control.

Results: The molecular alterations and the frequency of phenotypic or genetic aberrations were very similar when screen-detected and nonscreen-detected lung cancers were compared. Furthermore, most of the alterations found in the screen-detected cancers from this study were concordant with what has been described previously for stage I-II lung cancer.

Conclusions: Small early-stage lung cancers resected after detection in a spiral CT-based screening trial reveal malignant molecular features similar to those found in conventionally diagnosed lung cancers, suggesting that the screen-detected cancers are not overdiagnosed. (Cancer Epidemiol Biomarkers Prev 2006;15(2):373–80)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is responsible for close to 20% of all cancer deaths. In the United States, >160,000 new lung cancers were identified in 2004 (1). Despite improvements in the treatment of advanced disease, the 5-year survival of lung cancer patients is <15%, causing more deaths than breast, prostate, and colon cancers combined (1). Among the main reasons behind the dismal statistics of lung cancer is the lack of effective techniques for early detection. Less than 20% of patients are diagnosed in early stages when surgical intervention is possible. Since the seminal study by Henschke et al. (2) on spiral computed tomography (spiral CT)–based early detection, and as a result of rapid technical refinements, localized small lung cancers are being found more frequently and excitement about the possibility of screening high-risk individuals has grown (3).

Thus far, spiral CT-based lung cancer screening studies have reported high rates of detection of small cancers in early stages, 70% to 90% of the tumors being stage I or II, with an average size of 1.5 cm (2, 4-10). Although these data are very promising, there has been intense discussion concerning the possibility of overdiagnosis. Some authors suggest that CT screening protocols may find a high proportion of indolent cancers that may not cause a significant reduction in the life expectancy of screenees (11, 12). According to this view, these tumors could remain latent and localized for the entire life of an individual. A way to test the existence of overdiagnosis is by analyzing whether the biological traits and the natural history of the resected tumors are identical to cancers diagnosed conventionally. If in the setting of a screening trial, a high proportion of resected lung tumors were not fatally malignant, then the benefits of the screening program would be minimal due to overdiagnosis. To address this question, molecular profiling of the lesions resected within a screening protocol may be very helpful. This analysis will provide a clearer picture of the phenotypic and genetic alterations present in these lesions and an indication of their potential lethality. The aim of this study was to perform biological profiling of the tumors resected within our institutional International Early Lung Cancer Action Project spiral CT screening trial. A number of molecular changes leading to lung cancer have already been described (13). To assess the biological aberrance present in the screen-detected tumors, a panel of biomarkers mostly related to the "hallmarks of cancer" (14) were selected. Specifically, markers involved in cell cycle (Ki-67, p16, cyclin D1, cyclin E, p53, and p21), apoptosis (Bcl-2 and Bax), invasion and angiogenesis [matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF)], and mRNA processing [heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP K, and {alpha}CP-4] were analyzed. Additionally, chromosomal imbalances using a set of lung cancer-related probes (15) were investigated. Finally, comparisons between the molecular traits of these tumors with those of a control group of gender-, stage-, and histology-matched lung cancers diagnosed conventionally (not in the setting of a screening program) were carried out.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Population
One thousand and four current and former smokers of at least 40 years of age with no symptoms of lung cancer participated in the screening program. A background questionnaire and written informed consent were obtained. The study protocol was approved by the ethics committee of our center. Eighteen screen-detected lung tumors were diagnosed and resected, 11 of which were available for biomarker studies. Histologic diagnosis and classification of the specimens were defined based on WHO criteria (16). Pathologic staging of the tumors was done according to the international system for staging lung cancer (17). A second series of gender-, stage-, and histology-matched nonscreening diagnosed lung tumors was also studied. These additional patients were diagnosed with lung cancer after presenting with symptoms or because of a casual finding in a diagnostic work-up for other conditions that included either a chest X-ray or a CT.

Immunohistochemistry
For immunohistochemical analysis, 3 µm formalin-fixed, paraffin-embedded sections were dewaxed in xylene and rehydrated through a graded alcohol series. Endogenous peroxidase activity was blocked by placing sections in 3% hydrogen peroxide in water for 10 minutes. Sections were rinsed in deionized water, and, then, when necessary, antigen retrieval was carried out. Tissues were washed in TBS [0.01 mol/L Tris and 0.1 mol/L NaCl (pH 7.36)] and incubated in 5% normal serum for 30 minutes at room temperature to block nonspecific staining. Later, the diluted primary antibody was applied and left overnight at 4°C. Technical conditions of immunohistochemical procedure (type of retrieving buffer and conditions, source of primary antibodies, and dilutions) are indicated in Table 1. Sections were washed in TBS and incubated with monoclonal or polyclonal Envision complex (DAKO, Glostrup, Denmark) for 30 minutes at room temperature. After washing the slides, development of peroxidase with diaminobenzidine was performed. Counterstaining was carried out with hematoxylin or methyl green. Sections were dehydrated through graded alcohol steps and mounted in DPX mounting medium.


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  		Table 1. Dilutions, retrieval conditions, and sources of primary antibodies used for the immunohistochemical assays

 
Positive and negative controls were included in each experiment. Negative controls were done, leaving out the primary antibody. As positive control, we used tissue previously shown to express the antigen of interest. When possible, internal positive controls were used to interpret the intensity of the staining on the tumor cells.

Immunostaining Evaluation
Two observers (M.J. Pajares and M.D. Lozano) evaluated the extension and intensity of the staining for each antibody in all the samples. Both observers evaluated the samples independently and blinded to the method used for diagnosis. The extension was scored as percentage of positive tumor cells (0-100%) and the intensity of staining was assessed compared with a known internal or external positive control (1+, mild labeling; 2+, moderate; and 3+, intense labeling). Discordant independent reading was resolved by simultaneous review by the two observers.

We chose the cutoff point for each investigated biomarker based on previously published reports. Ki-67 labeling index was defined as high if >20% of the tumor nuclei were positive and low if ≤20% were positive (18, 19). For p16 immunostaining, tumors were considered positive when >10% of the cells showed nuclear staining (20). Cyclin D1, cyclin E, p53, and p21 were considered overexpressed when >5% of the cells showed nuclear immunoreactivity (21-24). A tumor was considered positive for Bcl-2, Bax, and MMP-9 if staining intensity was 2+ or more in at least 20% of the tumor cells (25, 26). Immunostaining scores for VEGF were calculated by the product of the percentage of labeled tumor cells multiplied by the intensity of the staining. The tumors were considered positive when the score was ≥10. The mean staining score was used as the cutoff value to define two groups, one presenting high levels and the other low levels of VEGF expression (27). Tumors were considered positive for hnRNP A1 and K if >50% of the cells showed nuclear immunostaining (28). For {alpha}CP-4 immunostaining, tumors were considered positive when >10% of the cells showed cytoplasmic staining (29).

For Ki-67, cyclin D1, cyclin E, p53, and p21, appropriate external positive controls were used. Fibroblast and endothelial cells as well as normal adjacent lung epithelium showing nuclear reactivity were used as positive internal controls for p16. Other internal positive controls were tumor-infiltrating lymphocytes for Bcl-2, normal bronchial epithelium for Bax, and stromal fibroblast and infiltrating macrophages for MMP-9. Smooth muscle cells and hyperplastic type II pneumocytes were used as internal positive controls for VEGF. Normal bronchiolar cells were used as an internal positive control for hnRNP A1 and {alpha}CP-4.

Fluorescence In situ Hybridization
Interphase FISH analysis was done on 3 µm paraffin-embedded tissue sections. Briefly, slides were deparaffinized and pretreated with 10 mmol/L citrate acid buffer (pH 6) and 0.5 mg/mL pepsin solution. The sections were dehydrated through a series of ethanol washes and codenatured with LAVysion set probe at 70°C for 10 minutes. Posthybridization washes were done for 2 minutes at 72°C in 2x SSC. Later, tissues were counterstained with 4',6-diamidino-2-phenylindole. The samples were evaluated on a Zeiss Axioplan2 fluorescent microscope (Zeiss, Welwyn Garden City, United Kingdom) and multifocus images were acquired for all probes using different focal planes with the ISIS software (Metasystems, Altlussheim, Germany).

FISH Data Evaluation
Evaluation of FISH signals was done by counting 60 intact and nonoverlapping nuclei per slide belonging to several different areas. To establish the cutoffs for abnormality, four normal-appearing bronchiolar epithelium sections were also analyzed. The average number of signals per cell and the percentage of abnormal cells were scored from the three groups (normal lung tissue, screen-detected tumors, and nonscreen-detected tumors). Statistical significant cutoffs were calculated in the normal-appearing lung tissues as the average number of signals per cell plus thrice the SD for each probe scored.

Statistical Analysis
To determine whether the molecular profile of the screen-detected tumors shared similarity with nonscreen-detected tumors, a Fisher's exact test was done to compare the results of each immunohistochemical abnormality. The Kruskall-Wallis test was used to compare the FISH profile within the three groups. Independent groups were compared using the Mann-Whitney U test. Data analysis was done using SPSS software version 11.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pathologic Findings
All resected screen-detected nodules were diagnosed as malignant lung tumors. No resected nodule was diagnosed as benign. From a histologic point of view, five squamous cell carcinomas and six adenocarcinomas were diagnosed among the screen-detected tumors. All the adenocarcinomas were moderately differentiated tumors, showing epithelial malignant cells with some degree of glandular differentiation. The squamous cell tumors were composed of sheets and cords of epithelial atypical cells without glandular differentiation. One of the screen-detected squamous carcinomas presented a significant lymphoid infiltrate and was classified as a lymphoepithelioma like carcinoma (patient SDT3). According to the international system for staging of lung cancer, 10 tumors were staged as IA and one tumor was staged as IB. The control series of nonscreen-diagnosed lung tumors was matched by histology and stage with the series of screen-detected tumors. Clinical and pathologic features of both series of patients are described in Table 2. There were no statistically significant differences in age, tumor size, and pack-years between the two groups.


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  		Table 2. Clinicopathologic features of the 22 non–small cell lung cancer patients

 
Immunohistochemical Assays
Table 3 summarizes the results on the 13 immunohistochemical markers in the two series of lung tumors investigated.


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  		Table 3. Results of the immunohistochemical analysis

 
Cell Cycle–Related Proteins. Comparing screen-detected (n = 11) with nonscreen-detected (n = 11) lung cancers (Fig. 1), the following abnormalities were found: eight screen-detected and seven conventionally detected tumors presented high levels of Ki-67; expression of p16 protein was lost in six tumors in each group; cyclin D1 was altered in five and six tumors, respectively; and cyclin E was overexpressed in seven and six tumors, respectively. Most of the cancers (10 in screen-detected and 11 in nonscreen-detected tumors) had alterations in at least one protein of the Rb pathway. Four tumors out of 11 in each group of cancers were positive for p53 accumulation, whereas five cancers in each group had lost expression of p21. At least one of the p53-p21 pathway proteins analyzed was altered in eight cases in both the screen-detected and nonscreen-detected cancers. Normal lung epithelium, both alveolar and bronchiolar, was negative for all the studied cell cycle–related proteins.


Figure 1
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  		Figure 1. Examples of alteration patterns of proteins in screen-detected lung tumors (A, C, E, and G) compared with nonscreen-detected ones (B, D, F, and H). A and B, Ki-67 expression. C and D, p16 loss of expression. E and F, p53 protein accumulation. G and H, VEGF expression.

 
Markers of Apoptosis. Bcl-2 was positive in 5 of 11 tumors in both screen-detected and nonscreen-detected tumors. Immunohistochemical labeling for Bax protein was found in seven screen-detected tumors and in six conventionally detected tumors. Normal bronchial epithelium was positive for Bcl-2 and Bax in all the cases studied.

Markers of Invasion and Angiogenesis. Eight of 11 tumors were positive for MMP-9 in both screen-detected and nonscreen-detected tumors. VEGF was highly expressed in four tumors in each group. Normal bronchial epithelial cells expressed high levels of VEGF and MMP-9.

RNA-Binding Proteins. All the lung cancers from both study groups overexpressed at least one of the RNA-binding proteins investigated. All the tumors from both groups showed hnRNP A1 expression in most of the cells. Intensity of immunostaining was higher in the tumor cells than in the normal bronchiolar epithelial cells. In seven cancers from each group, hnRNP K expression was found in neoplastic cells but not in normal cells. Expression of {alpha}CP-4 was observed in the cytoplasm of normal bronchiolar or bronchial cells and was lost in six and eight cancers from the screen-detected and the nonscreen-detected group, respectively.

In summary, all the tumors had abnormalities in at least three of the four investigated pathways (cell cycle, apoptosis, invasion and metastasis, and RNA processing) and in at least 7 of the 13 lung cancer-related proteins analyzed. Statistical analysis comparing the frequencies of aberrant expression showed that there were no significant differences for any of the biomarkers studied by immunohistochemistry between screen-diagnosed and nonscreen-diagnosed tumors (P > 0.05, Fisher's exact test).

FISH Analysis
Successful hybridization was observed in 11 (100%) screen-detected tumors and 10 (90.9%) nonscreen-diagnosed tumors. Control tumor number 19 was excluded from the FISH analysis because of the suboptimal FISH signal intensity. Three screen-detected tumors and one control matched case also showed suboptimal results for epidermal growth factor receptor (EGFR) probe due to weakness of the FISH signals. The average number of signals per cell and the percentage of abnormal cells are summarized in Table 4. The results of the FISH analysis of the normal-appearing lung tissues established the cutoff values of 2.2 for the 5p15.2 probe, 1.9 for EGFR, 2.1 for MYC, and 2.3 for the centromeric probe of chromosome 6. With the exception of two screen-detected tumors, which had a normal number of copies of chromosome 6, and one conventionally diagnosed tumor with normal EGFR and CEP6 signals, all the other tumors analyzed showed an abnormal pattern of signals for all four probes (Fig. 2).


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  		Table 4. Number of FISH signals for each probe and frequencies of abnormal cells in the tumors

 

Figure 2
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  		Figure 2. Examples of FISH analysis. Sequence from chromosome 5p15 is labeled with SpectrumGreen, centromere 6 probe is labeled with SpectrumAqua (blue dots), EGFR probe is labeled with SpectrumRed, and CMYC probe is labeled with SpectrumGold (yellow dots). A, Screen-detected lung tumor (case SDT 5). B, Nonscreen-detected tumor (case NSDT 20).

 
Considerable intercellular heterogeneity was observed among the tumors, with copy number per cell in screen-detected tumors ranging from 1 to 20 for 5p15.2, 1 to 12 for centromere 6, 1 to 10 for EGFR, and 1 to 18 for MYC. Conventionally detected tumors showed a similar range of heterogeneity in the signals.

The Kruskal-Wallis test detected significant differences in the FISH quantitative results between normal tissue and the tumor groups for all four probes (P = 0.010 for 5p15, P = 0.006 for EGFR, P = 0.008 for MYC, and P = 0.041 for CEP6). Using the Mann-Whitney U test, significant differences between normal lung tissue and screen-detected tumors, and between normal lung tissue and nonscreen-detected tumors, were found. No statistically significant difference in any of the probes was found between screen- and nonscreen-detected tumors (Table 5).


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  		Table 5. Comparison of FISH results between normal lung tissues, screen–detected and nonscreen–detected tumors

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Spiral CT can detect lung cancer as small as several millimeters in diameter and at very early stages (2, 10, 30). However, it has been suggested that a proportion of these early diagnosed lung cancer may be overdiagnosed cases (benign nodules that are treated as malignant or slow-growing tumors that would not cause the demise of the patient). The present work summarizes data on the phenotypic and genetic alterations present in the tumors resected in the setting of a spiral CT screening trial, and suggests that these lesions can be considered fully neoplastic according to their biomarker profile. Although the size of the series investigated is limited, it is obtained from a screened cohort of >1,000 individuals, thus providing relevant information on the malignant features from a molecular point of view of tumors found in a screening setting. These data support the view that screen-detected tumors by spiral CT share the same molecular profile as conventionally detected lung cancers of the same clinicopathologic stage.

A wealth of information has accumulated over the last decade on the alterations in the expression of several genes in lung cancer cells compared with normal lung epithelium. The current view of lung carcinogenesis is a multistep process starting as a normal epithelium, which progressively accumulates molecular abnormalities from the preneoplastic to the neoplastic stages (13). To evaluate the molecular abnormalities in screen-detected lung cancers resected at our institution, 17 biomarkers frequently involved in lung cancer were analyzed: 13 phenotypic molecular alterations and 4 genetic aberrations. Among the former, phenotypic alterations analyzed included the expression of proteins related to cell cycle, apoptosis, invasion, angiogenesis, and mRNA metabolism. Changes in cell cycle–related proteins, especially Ki-67 overexpression, dysregulation of the Rb-cyclin D1-p16 pathway and p53-p21WAF1 alterations, are very common biomarkers for lung cancer (31). In fact, several reports have shown that 90% of lung tumors have at least one of the Rb pathway proteins altered (32-34). In our series of early lung tumors, we have evaluated three Rb pathway proteins and, in agreement with other reports, we have observed modifications of at least one protein of this pathway in 80% to 90% of the tumors both in the screening and the nonscreening groups. Similarly, most of these tumors showed alterations in the p53-p21 pathway. In relation to apoptosis, our data showed alterations in the expression of at least one of the two markers studied, Bcl-2 and Bax, in around 75% of the cases in both groups. Other key mechanisms in lung cancer development and progression include the dysregulation of angiogenesis and invasion. VEGF, an angiogenic factor (27, 35), and MMP-9, an invasion marker especially active in extracellular matrix degradation (25), are overexpressed in most of the tumors analyzed. The relevance of modifications in alternative mRNA splicing has been recently stressed (36), and in this regard, the hnRNP family is involved in the regulation of transcription, mRNA metabolism, and translation. Overexpression of hnRNP A2/B1, a member of the hnRNP family, has been reported as a potential biomarker for early detection of lung cancer (37), whereas other members of the same family have been shown to be abnormally expressed in lung cancer biopsies and cell lines (28, 29). In the present work, immunohistochemical analysis showed an alteration of the RNA processing mechanisms in both types of tumors studied. Finally, the analysis of genetic alterations by FISH using the LAVysion set also shows a close similarity between the two groups of tumors studied. This panel of probes has been shown to be highly sensitive and specific for lung cancer (15, 38). In this series, all but three tumors showed an aberrant genetic profile for all four probes. Although multicolor FISH analysis is a laborious technique in particular on paraffin-embedded tissues, these results suggest that it could become a very useful tool for the diagnosis of early solid lung tumors. In summary, all the molecular markers studied are similarly altered in screen-detected tumors and in stage I cancers diagnosed outside a screening program.

Bianchi et al. (39) have recently reported a cDNA array analysis of screen-detected and nonscreen-detected tumors. Their results show that the two groups of tumors hardly differ in gene expression profiles. In the present work, the focus was put on a panel of markers that have already been related to lung cancer. In agreement with the aforementioned cDNA study, the almost identical percentages of molecular alterations reported herein support the hypothesis that lung cancer detected in a screening program has a similar malignant potential than conventionally, nonscreen-detected cancer. Furthermore, all the data from both series are concordant with the published literature on resectable non–small cell lung cancer in relation to cell cycle–related proteins (18, 21, 23, 24, 33, 40-42), apoptosis regulators (26, 43, 44), invasive and angiogenic markers (26, 27), RNA processing molecules (28, 29), and genetic aberrations (15, 38).

Results found in screen-detected tumors also fit with previously published reports on differences in the expression of biomarkers according to tumor histology. Thus, high levels of Ki-67 (18, 19, 40), loss of p16 (21), accumulation of p53 (45, 46), cyclin E overexpression (40), and Bcl-2 expression (26, 43, 44, 47) were observed more commonly in squamous cell carcinomas than in adenocarcinomas. However, p21 loss was observed more frequently in adenocarcinomas than in squamous cell carcinomas. In genetic analysis by FISH, there were no significant differences between both histologic types, showing that this multi-FISH assay is a useful tool for detection of both types of tumors.

In summary, with respect to protein and genetic profiles, screen-detected lung cancers showed profound molecular alterations that did not differ significantly from conventionally detected tumors. This provides strong support to the hypothesis that spiral CT–detected tumors in the setting of a screening program are bona fide aggressive malignancies. The number of altered protein markers (at least 7 of 13) and genetic markers (at least 3 of 4) found in all of the cancers studied, as well as the fact that 100% of the cases had at least three of four key molecular pathways altered, suggests that none of them was a mere benign hyperproliferative noninvasive disease with minor clinical adverse consequences. Rather, in screen-detected early tumors, most of the biological hallmarks of aggressive cancer are already present.


   Acknowledgments
 
We thank Dr. Natalia Spiteri and Usua Montes for their work in the collection of the samples; Dr. G. Dreyfuss and L. Lecanda for their antibodies: hnRNP1 and hnRNPK respectively; Dr. María José Calasanz and Dr. María Dolores Odero for help and advice in the FISH analysis; Prof. Elisabeth Brambilla for her advice in setting up and validating some of the immunohistochemical biomarkers; María Amada Maset, Virginia Pulido, María Dolores Martínez, and María Antonia Martínez for their technical support; and Borja Sáez for help with statistical analysis.


   Footnotes
 
Grant support: Fundación para la Investigación Médica Aplicada (UTE-CIMA agreement), Fondo de Investigaciones Sanitarias (RITCCC 03/10, PI 04/2128, and PI 02/1116), Departamento de Salud del Gobierno de Navarra (61/2002, 38/2003, and 35/2004), European Union Vth Framework Project (QLRT-2001-01735), and a Prevention Fellowship from the International Association for the Study of Lung Cancer (M.J. Pajares).

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.

Received 5/ 3/05; revised 9/ 9/05; accepted 12/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA Cancer J Clin 2004;54:8–29.[Abstract/Free Full Text]
  2. Henschke CI, McCauley DI, Yankelevitz DF, et al. Early Lung Cancer Action Project: overall design and findings from baseline screening. Lancet 1999;354:99–105.[CrossRef][Medline]
  3. Mulshine JL, Weinstein JN. Is the gene expression pattern of lung cancer detected by screening with spiral computed tomography different from that of symptom-detected lung cancer? Clin Cancer Res 2004;10:5973–4.[Free Full Text]
  4. Swensen SJ, Jett JR, Sloan JA, et al. Screening for lung cancer with low-dose spiral computed tomography. Am J Respir Crit Care Med 2002;165:508–13.[Abstract/Free Full Text]
  5. Diederich S, Wormanns D, Semik M, et al. Screening for early lung cancer with low-dose spiral CT: prevalence in 817 asymptomatic smokers. Radiology 2002;222:773–81.[Abstract/Free Full Text]
  6. Pastorino U, Bellomi M, Landoni C, et al. Early lung-cancer detection with spiral CT and positron emission tomography in heavy smokers: 2-year results. Lancet 2003;362:593–7.[CrossRef][Medline]
  7. Sone S, Li F, Yang ZG, et al. Results of three-year mass screening programme for lung cancer using mobile low-dose spiral computed tomography scanner. Br J Cancer 2001;84:25–32.[CrossRef][Medline]
  8. Sobue T, Moriyama N, Kaneko M, et al. Screening for lung cancer with low-dose helical computed tomography: anti-lung cancer association project. J Clin Oncol 2002;20:911–20.[Abstract/Free Full Text]
  9. Henschke CI, Naidich DP, Yankelevitz DF, et al. Early lung cancer action project: initial findings on repeat screenings. Cancer 2001;92:153–9.[CrossRef][Medline]
  10. Bastarrika G, Garcia-Velloso MJ, Lozano MD, et al. Early lung cancer detection using spiral computed tomography and positron emission tomography. Am J Respir Crit Care Med 2005;171:1378–83.[Abstract/Free Full Text]
  11. Reich JM. Improved survival and higher mortality: the conundrum of lung cancer screening. Chest 2002;122:329–37.[Free Full Text]
  12. Lenox RJ. To screen or not to screen. Chest 2005;127:1091–2.[Free Full Text]
  13. Sekido Y, Fong KM, Minna JD. Molecular genetics of lung cancer. Annu Rev Med 2003;54:73–87.[CrossRef][Medline]
  14. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.[CrossRef][Medline]
  15. Romeo MS, Sokolova IA, Morrison LE, et al. Chromosomal abnormalities in non-small cell lung carcinomas and in bronchial epithelia of high-risk smokers detected by multi-target interphase fluorescence in situ hybridization. J Mol Diagn 2003;5:103–12.[Abstract/Free Full Text]
  16. Travis WD, Colby TB, Corrin B, Shimosato Y, Brambilla E. Histological typing of tumours of lung and pleura. 3rd ed. New York: Springer-Verlag; 1999.
  17. Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111:1710–7.[Abstract/Free Full Text]
  18. Mehdi SA, Etzell JE, Newman NB, et al. Prognostic significance of Ki-67 immunostaining and symptoms in resected stage I and II non-small cell lung cancer. Lung Cancer 1998;20:99–108.[CrossRef][Medline]
  19. Shiba M, Kohno H, Kakizawa K, et al. Ki-67 immunostaining and other prognostic factors including tobacco smoking in patients with resected nonsmall cell lung carcinoma. Cancer 2000;89:1457–65.[CrossRef][Medline]
  20. Gazzeri S, Gouyer V, Vour'ch C, Brambilla C, Brambilla E. Mechanisms of p16INK4A inactivation in nonsmall-cell lung cancers. Oncogene 1998;16:497–504.[CrossRef][Medline]
  21. Brambilla E, Moro D, Gazzeri S, Brambilla C. Alterations of expression of Rb, p16(INK4A) and cyclin D1 in non-small cell lung carcinoma and their clinical significance. J Pathol 1999;188:351–60.[CrossRef][Medline]
  22. Jeanmart M, Lantuejoul S, Fievet F, et al. Value of immunohistochemical markers in preinvasive bronchial lesions in risk assessment of lung cancer. Clin Cancer Res 2003;9:2195–203.[Abstract/Free Full Text]
  23. Esposito V, Baldi A, Tonini G, et al. Analysis of cell cycle regulator proteins in non-small cell lung cancer. J Clin Pathol 2004;57:58–63.[Abstract/Free Full Text]
  24. Toledo G, Sola JJ, Lozano MD, Soria E, Pardo J. Loss of FHIT protein expression is related to high proliferation, low apoptosis and worse prognosis in non-small-cell lung cancer. Mod Pathol 2004;17:440–8.[CrossRef][Medline]
  25. Cox G, Jones JL, O'Byrne KJ. Matrix metalloproteinase 9 and the epidermal growth factor signal pathway in operable non-small cell lung cancer. Clin Cancer Res 2000;6:2349–55.[Abstract/Free Full Text]
  26. Cox G, Louise Jones J, Andi A, Abrams KR, O'Byrne KJ. Bcl-2 is an independent prognostic factor and adds to a biological model for predicting outcome in operable non-small cell lung cancer. Lung Cancer 2001;34:417–26.[CrossRef][Medline]
  27. Decaussin M, Sartelet H, Robert C, et al. Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-1 and VEGF-R2-1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival. J Pathol 1999;188:369–77.[CrossRef][Medline]
  28. Pino I, Pio R, Toledo G, et al. Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family in lung cancer. Lung Cancer 2003;41:131–43.[CrossRef][Medline]
  29. Pio R, Zudaire I, Pino I, et al. {alpha}CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant {alpha}CP-4a, is underexpressed in lung cancer. Cancer Res 2004;64:4171–9.[Abstract/Free Full Text]
  30. Kaneko M, Kusumoto M, Kobayashi T, et al. Computed tomography screening for lung carcinoma in Japan. Cancer 2000;89:2485–8.[CrossRef][Medline]
  31. Fong KM, Sekido Y, Minna JD. Molecular pathogenesis of lung cancer. J Thorac Cardiovasc Surg 1999;118:1136–52.[Abstract/Free Full Text]
  32. Tanaka H, Fujii Y, Hirabayashi H, et al. Disruption of the RB pathway and cell-proliferative activity in non-small-cell lung cancers. Int J Cancer 1998;79:111–5.[CrossRef][Medline]
  33. Gugger M, Kappeler A, Vonlanthen S, et al. Alterations of cell cycle regulators are less frequent in advanced non-small cell lung cancer than in resectable tumours. Lung Cancer 2001;33:229–39.[CrossRef][Medline]
  34. Zhou JX, Niehans GA, Shar A, et al. Mechanisms of G1 checkpoint loss in resected early stage non-small cell lung cancer. Lung Cancer 2001;32:27–38.[CrossRef][Medline]
  35. O'Byrne KJ, Koukourakis MI, Giatromanolaki A, et al. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer. Br J Cancer 2000;82:1427–32.[Medline]
  36. Venables JP. Aberrant and alternative splicing in cancer. Cancer Res 2004;64:7647–54.[Abstract/Free Full Text]
  37. Zhou J, Mulshine JL, Unsworth EJ, et al. Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4. J Biol Chem 1996;271:10760–6.[Abstract/Free Full Text]
  38. Varella-Garcia M, Kittelson J, Schulte AP, et al. Multi-target interphase fluorescence in situ hybridization assay increases sensitivity of sputum cytology as a predictor of lung cancer. Cancer Detect Prev 2004;28:244–51.[CrossRef][Medline]
  39. Bianchi F, Hu J, Pelosi G, et al. Lung cancers detected by screening with spiral computed tomography have a malignant phenotype when analyzed by cDNA microarray. Clin Cancer Res 2004;10:6023–8.[Abstract/Free Full Text]
  40. Takahashi S, Kamata Y, Tamo W, et al. Relationship between postoperative recurrence and expression of cyclin E, p27, and Ki-67 in non-small cell lung cancer without lymph node metastases. Int J Clin Oncol 2002;7:349–55.[CrossRef][Medline]
  41. Han H, Landreneau RJ, Santucci TS, et al. Prognostic value of immunohistochemical expressions of p53, HER-2/neu, and bcl-2 in stage I non-small-cell lung cancer. Hum Pathol 2002;33:105–10.[CrossRef][Medline]
  42. Kalomenidis I, Orphanidou D, Papamichalis G, et al. Combined expression of p53, Bcl-2, and p21WAF-1 proteins in lung cancer and premalignant lesions: association with clinical characteristics. Lung 2001;179:265–78.[CrossRef][Medline]
  43. Hanaoka T, Nakayama J, Haniuda M, Sato TA. Immunohistochemical demonstration of apoptosis-regulated proteins, Bcl-2 and Bax, in resected non-small-cell lung cancers. Int J Clin Oncol 2002;7:152–8.[CrossRef][Medline]
  44. Groeger AM, Esposito V, De Luca A, et al. Prognostic value of immunohistochemical expression of p53, bax, Bcl-2 and Bcl-xL in resected non-small-cell lung cancers. Histopathology 2004;44:54–63.[CrossRef][Medline]
  45. Mitsudomi T, Hamajima N, Ogawa M, Takahashi T. Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis. Clin Cancer Res 2000;6:4055–63.[Abstract/Free Full Text]
  46. Grossi F, Loprevite M, Chiaramondia M, et al. Prognostic significance of K-ras, p53, bcl-2, PCNA, CD34 in radically resected non-small cell lung cancers. Eur J Cancer 2003;39:1242–50.
  47. Pezzella F, Turley H, Kuzu I, et al. bcl-2 protein in non-small-cell lung carcinoma. N Engl J Med 1993;329:690–4.[Abstract/Free Full Text]



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