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(Chest. 2003;123:181-186.)
© 2003 American College of Chest Physicians

Nuclear Accumulation of p53 Is a Potential Marker for the Development of Squamous Cell Lung Cancer in Smokers*

Chandrika J. Piyathilake, BDS, MPH, PhD; Andra R. Frost, MD; Upender Manne, PhD; Heidi Weiss, PhD; Douglas C. Heimburger, MD and William E. Grizzle, MD, PhD

* From the Departments of Nutrition Sciences (Drs. Piyathilake and Heimburger) and Pathology (Drs. Frost, Manne, and Grizzle), and the Comprehensive Cancer Center (Dr. Weiss), the University of Alabama, Birmingham, AL.

Correspondence to: Chandrika J. Piyathilake, BDS, MPH, PhD, Department of Nutrition Sciences, Division of Nutritional Biochemistry and Molecular Biology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294; e-mail: piyathic{at}uab.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: To determine whether the nuclear accumulation of p53 in patients with early bronchial neoplasia represents an altered susceptibility for the development of lung cancer.

Patients and measurements: We evaluated the percentage of cells accumulating nuclear p53 immunohistochemically in squamous cell carcinoma (SCC) of the lung, the associated uninvolved bronchial mucosa, and epithelial hyperplasia in 60 archival lung specimens of smokers and in the normal bronchial epithelium and hyperplastic lesions of 60 smokers who had not developed lung cancer.

Results: The percentage of cells accumulating p53 was significantly higher in SCC-associated uninvolved bronchial epithelia of (mean [± SD], 4 ± 0.9%) and in specimens from patients with epithelial hyperplasia (mean, 9 ± 2%) compared to the percentage of cells from the bronchial epithelia of (mean, 0.5 ± 0.2%) and in specimens from patients with epithelial hyperplasia (mean, 1.5 ± 0.5%) who were smokers who had not developed lung cancer (p = 0.0002 and p = 0.0004, respectively). We also observed a statistically significant stepwise increase in the percentage of cells accumulating p53 from SCC-associated uninvolved bronchial epithelium to those from a patient with epithelial hyperplasia to those from a patient with SCC (mean, 35 ± 4%), suggesting the involvement of p53 accumulation in the development of SCC (p <= 0.05 for all comparisons). The accumulation of p53 in SCC cells was not significantly associated with the size of the tumor, nodal involvement, the stage of the disease, the presence or absence of metastasis, the grade of differentiation, or survival of the disease, indicating its lack of association with the clinical progression of the disease.

Conclusions: These results suggested that p53 accumulation is an early event in lung carcinogenesis and potentially could be useful in the identification of smokers who are at risk of developing SCC, but not in the estimation of survival of the disease.

Key Words: bronchial lesions • lung cancer • p53


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A loss of tumor suppressor function of p53 is the most common event leading to the development of human cancers. Genetic alterations in p53 have been identified in > 50% of different types of human cancer including cancer of the lung. Mutations of the p53 gene are one of the most common genetic abnormalities found in all types of tumors of the lung, suggesting a crucial role in bronchial carcinogenesis. The extent of p53 mutations, however, varies among different types of cancer of the lung (70% in cases of small cell undifferentiated carcinoma compared to 47% in cases of non-small cell lung cancer, including 65% of cases of squamous cell carcinoma [SCC] of the lung and 60% of cases of large cell carcinoma, but only 33% of cases of adenocarcinomas).1

Although mutations in p53 or detection of the accumulation of p53 in the nuclei of cells by immunohistochemistry has been reported mostly in malignant tumors, changes in p53 can be observed in premalignant lesions as well, including Barrett lesions of the esophagus,2 colorectal adenomas,3 dysplastic squamous epithelium of oral mucosa,4 and preneoplastic lesions of the lung.5 6 7 Most studies of lung cancer, however, have concentrated on invasive carcinomas and advanced premalignant lesions (ie, severe dysplasia and carcinoma in situ) and have not identified sufficient other early bronchial lesions to estimate the frequency and timing of the nuclear accumulation of p53. The carcinoma-associated, uninvolved, histologically normal bronchial epithelium, which is also at high risk for the development of cancer, rarely has been investigated. Therefore, little is known about the timing of the appearance of p53 mutations, their maintenance through cancer progression, or their relevance to patient survival. The present study was undertaken to accomplish the following: (1) to examine the pattern of nuclear accumulation of abnormal p53 protein in SCCs of the lung and associated uninvolved bronchial epithelia and cases of epithelial hyperplasia, metaplasia, and dysplasia compared to normal bronchial epithelia and epithelial hyperplastic lesions of noncancer specimens of the human lung; and (2) to correlate the nuclear accumulation of p53 in SCCs of the lung with established prognostic indicators (ie, tumor differentiation, tumor volume, nodal status, and clinical stage) and with the survival of subjects.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and Tissue Specimens
Formalin-fixed, paraffin-embedded sections of human lung were obtained from the archival collections of the Veterans Affairs Medical Center and University of Alabama Hospital in Birmingham, AL. From a list of patients with SCC of the lung who had undergone surgery between 1989 and 1996, 60 cases were selected at random. The mean (± SD) age of subjects in whom SCC was diagnosed was 65 ± 9 years. Among SCC patients, 83% were men, 17% were women, 88% were white, and 12% were black. Sixty noncancer specimens of the lung were selected at random from a list of lung surgeries performed between 1988 and 1998 for emphysema and/or fibrosis of the lung. The mean (± SD) age of noncancer subjects was 52 ± 11 years. Among these SCC subjects, 53% were men, 47% were women, 90% were white, and 10% black. Both cancer and noncancer subjects were either previous or current smokers. A detailed smoking history was not available for patients in either group. One or more tissue blocks from each patient were selected to provide sections that contained samples of uninvolved bronchial mucosa, or from the lungs of patients with epithelial hyperplasia, metaplasia, and dysplasia (when present) or invasive carcinoma.

Histologic Criteria and Classification of Lesions
Bronchial epithelium was classified histologically as normal or uninvolved (ie, histopathologically normal), hyperplastic, metaplastic, or dysplastic by the pathologists involved in the study. All SCCs of the lung exhibited squamous differentiation with varying degrees of keratinization, although all were either well-differentiated or moderately differentiated. Tumor staging was evaluated by using the TNM system.8

Immunohistochemical Analysis
Five-micrometer sections were deparaffinized and treated with 3.0% H2O2 for 4 min to quench endogenous peroxidase activity. Sections were incubated with preimmune rabbit serum (1%) for 20 min at room temperature to suppress nonspecific staining. Sections were incubated with a monoclonal antibody to p53 (clone Bp53.12, 1:100 dilution) [Ab-8; Oncogene Research Products; San Diego, CA] for 1 h at room temperature. Companion matching slides stained with no primary antibody served as controls (deletes). The remainder of the staining procedure was performed using a biotin-streptavidin detection system (BioGenex; San Ramon, CA). The substrate diaminobenzidine tetrahydrochloride was used for visualization of the antigen-antibody complex (a brown reaction product), and sections were counterstained lightly with hematoxylin.

Assessment of p53 Nuclear Accumulation
The numbers of specimens that contained adequate tissues for the evaluation of SCC, SCC-associated uninvolved bronchial epithelium, and epithelial hyperplasia were 56, 57, and 30, respectively. The number of SCC-associated dysplastic lesions (five) or metaplastic lesions (three) that were detected was too small to be included in the final data analysis. Among the 60 noncancer specimens, 57 specimens contained adequate normal bronchial epithelium and 8 specimens also contained evidence of epithelial hyperplasia. In all tissue types, the p53 accumulation was mainly nuclear. Occasional cytoplasmic staining was noted, but only nuclear staining was classified as positive. p53 nuclear accumulation was scored by three observers (CJP, ARF, and UM). Each observer estimated the percentage of cells stained in each of three epithelial components (uninvolved bronchial mucosa, epithelial hyperplasia, and SCC). The final score was reported as the average of the three observers.

Statistical Analysis
Descriptive statistics such as mean, median, and SEM were calculated for the nuclear accumulation of p53 for each type of tissue. Differences in the nuclear accumulation of p53 were calculated between types of tissues, and either the Wilcoxon sign rank test or the Wilcoxon rank sum test was employed to determine whether there was differential nuclear accumulation between different types of tissues. The nuclear accumulation of p53 in SCCs was compared according to different histopathologic characteristics (ie, nodal status, tumor size, stage, and grade of differentiation). The Wilcoxon rank sum test was employed to evaluate significant differences in the nuclear accumulation of p53 for each histopathologic parameter. The overall survival after resection of SCCs in two groups defined by the nuclear accumulation of p53 in invasive cancers (greater than the median and less than or equal to the median) was determined by applying the log rank test.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The percentage of cells accumulating p53 was significantly higher in SCC-associated uninvolved bronchial epithelia (uninvolved cells, 4 ± 0.9%) and specimens of patients with epithelial hyperplasia (hyperplastic cells, 9 ± 2%) compared to normal bronchial epithelia (normal cells, 0.5 ± 0.2%) and specimens of patients with epithelial hyperplasia (noncancerous hyperplastic cells, 1.5 ± 0.5) from smokers who had not developed lung cancer (p = 0.0002 and p = 0.0004, respectively). Figure 1 shows the immunohistochemical staining for p53 in the different tissue types that were evaluated. We observed a statistically significant stepwise increase in the percentage of cells accumulating p53 from SCC-associated uninvolved bronchial epithelia to specimens from patients with epithelial hyperplasia, to specimens from patients with SCC (SCC, 35 ± 4%), suggesting the involvement of p53 in the development of SCC (p <= 0.05 for all comparisons) [Fig 2 ]. The association between the percentage of SCC-associated, uninvolved bronchial epithelial cells and hyperplastic cells that were positive for p53 was positive and statistically significant (r = 0.33; p = 0.01). Similarly, the associations of p53 positivity between SCC-associated hyperplasia and SCC (r = 0.38; p = 0.04), and SCC-associated, uninvolved bronchial epithelia and SCC (r = 0.47; p = 0.003) were also significant. The accumulation of p53 in SCCs was not significantly associated with the size of the tumor, nodal involvement, the stage of the disease, the grade of tumor differentiation (Table 1 ), or survival from the disease (Fig 3 ).



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  		Figure 1.. Immunohistochemical staining (a brown reaction product) for p53 in normal bronchial cells (top left, A) and in epithelial hyperplasia (top middle, B) of noncancer subjects and in SCC-associated, histologically normal, uninvolved bronchial epithelium (top right, C), epithelial hyperplasia (bottom left, D), and SCC (bottom right, E).

 


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  		Figure 2.. Significant stepwise increase in the percentage of cells testing positive for p53.

 

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  		Table 1.. The Percentage of Cells Accumulating Nuclear p53 in Relation to Histopathologic Characteristics of SCC of the Lung

 


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  		Figure 3.. Overall survival after the resection of SCCs in two groups defined by the nuclear accumulation of p53 in patients with invasive cancer (ie, <= median and > median).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SCC is thought to develop through multiple stages that are recognized histologically. The earliest histopathologic changes include bronchial hyperplasia, squamous metaplasia, followed by dysplasia, carcinoma in situ, and microinvasive and invasive cancer. Although there is some evidence that these lesions represent, to different extents, precursor steps in the development of carcinomas of the lung,9 it is still unclear at what stage in this progression the lesion should be regarded as premalignant.

A candidate molecule for the investigation of this poorly understood molecular process is the p53 protein, which regulates cellular proliferation and apoptosis. As stated earlier, the nuclear accumulations of both p53 and p53 mutations are shown to occur in dysplastic lesions of the lung. However, the nuclear accumulation of p53 rarely has been examined in other bronchial premalignant lesions associated with SCC. Fontanini et al10 reported no p53 immunoreactivity in normal, hyperplastic, or metaplastic lesions of the lung. A few studies,11 12 however, have reported the nuclear accumulation of p53 in metaplastic lesions and in the normal bronchial mucosa. Our study investigated the nuclear accumulation of p53 in a series of cases involving patients with uninvolved bronchial mucosa and/or epithelial hyperplasias, which is a larger group than hitherto investigated, and also compared the degree of nuclear accumulation of p53 in these lesions with similar lesions from noncancer subjects.

The noncancer subjects identified in our study are likely to have had exposures to carcinogens from cigarette smoke similar to those among SCC subjects. This is supported by their reported long-term smoking habits and by the frequent presence of emphysematous changes of the lung, which are associated with cigarette smoking through the activation of elastase leading to elastic tissue damage.13 Histologically, the hyperplastic lesions detected in smokers who developed cancer and in those who did not were similar. This observation suggests that the detection of the presence of hyperplastic lesions in a clinical setting may only indicate a similar degree of exposure to cigarette smoke rather than a difference in susceptibility to the development of SCC in smokers. In contrast, the evaluation of nuclear accumulation of p53 in those same lesions may provide valuable information on the likelihood of developing cancer. A prospective follow-up study of smokers, however, is needed to confirm these results. If these results are confirmed, the nuclear accumulation of p53 in early lesions such as epithelial hyperplasia could be used to identify the smokers who are at greater risk for the development of SCC.

Although the relevance of epithelial hyperplasia in the pathogenesis of lung cancer is not widely appreciated, at present, it could constitute a fertile soil in which cancerous proliferation may eventually arise. We previously have reported14 15 16 an elevated and frequent expression of several other biomarkers in epithelial hyperplastic lesions that are associated with SCCs, suggesting that epithelial hyperplasia of the lung may be a premalignant condition. We now report that the epithelial hyperplastic lesions of smokers who developed lung cancer express significantly higher percentages of p53-positive cells compared to similar lesions from smokers who did not develop lung cancer. These observations are important since the loss of heterozygosity in chromosomal regions that frequently are deleted in lung cancer patients have been reported in hyperplastic lesions associated with non-small cell lung cancer,17 18 19 suggesting a relationship between hyperplasia and lung carcinogenesis.

The mean percentage of cells with nuclear accumulation of p53 increased among tissues of uninvolved bronchial mucosa, epithelial hyperplasia, and SCC, suggesting an involvement of p53 in the development of SCCs. The positive correlations of the percentage of p53-positive cells among tissues of uninvolved bronchial epithelium, epithelial hyperplasia, and SCC also are suggestive of an involvement of p53 accumulation in the development of SCCs. These observations also may suggest that the nuclear accumulation of p53 in epithelial hyperplastic lesions may lead to the transformation of these lesions to invasive cancer. The lack of associations among p53 accumulation, the histopathologic features of SCCs, and patient survival suggested that the alteration of p53 by itself may not be significant in the progression of SCCs in smokers. Most of the published data on SCCs of the head and neck have shown no positive relationship between p53 expression and the histologic grading of tumors, the staging of tumors, or tumor size.20

In summary, our observations suggested that the nuclear accumulation of p53, an early event in lung carcinogenesis, potentially could be useful in the identification of smokers who are at greater risk of developing SCC of the lung, but not in the estimation of survival from the disease. A lack of detailed information on smoking history (eg, the number of pack-years of smoking) and a limited number of noncancer specimens with preneoplastic lesions are limitations of the study. The replication of this study in other populations is necessary to increase the scientific credibility of the observed results.


   Footnotes
 
Abbreviation: SCC = squamous cell carcinoma

This study was presented in part at the FASEB Experimental Biology meeting, March 7, 2001.

Received for publication November 30, 2001. Accepted for publication May 21, 2002.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Greenblatt, MS, Bennett, WP, Hollstein, M, et al (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54,4855-4898[Free Full Text]
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  3. Shirasawa, S, Urabe, K, Yanagawa, Y, et al p53 gene mutations in colorectal tumors from patients with familial polyposis coli. Cancer Res 1991;51,2874-2878[Abstract/Free Full Text]
  4. Gusterson, BA, Anbazhagan, R, Warren, W, et al Expression of p53 in premalignant and malignant squamous epithelium. Oncogene 1991;6,1785-1789[ISI][Medline]
  5. Nuorva, K, Soini, Y, Kame, D, et al Concurrent p53 expression in bronchial dysplasias and squamous cell lung carcinomas. Am J Pathol 1993;142,725-732[Abstract]
  6. Bennett, WP, Colby, TV, Travis, WD, et al p53 protein accumulates frequently in early bronchial neoplasia. Cancer Res 1993;53,4817-4822[Abstract/Free Full Text]
  7. Walker, C, Robertson, LJ, Myskow, MW, et al p53 expression in normal and dysplastic bronchial epithelium and in lung carcinomas. Br J Cancer 1994;70,297-303[ISI][Medline]
  8. American Joint Committee on Cancer (AJCC). Fleming, ID Cooper, JS Hensen, DEet al eds. Manual for staging cancer 1997 JB Lippincott Philadelphia, PA.
  9. Woolner, LB Lung. Henson, DE Albores-Saavedra, J eds. Pathology of incipient neoplasia 1986,57-85 WB Saunders Philadelphia, PA.
  10. Fontanini, G, Vignati, S, Bigini, D, et al Human non-small cell lung cancer: p53 protein accumulation is an early event and persists during metastatic progression. J Pathol 1994;174,23-31[CrossRef][ISI][Medline]
  11. Boers, JE, Ten Velde, GP, Thunnissen, FB p53 in squamous metaplasia: a marker for risk of respiratory tract carcinoma. Am J Respir Crit Care Med 1996;153,411-416[Abstract]
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  13. Lipscomb, MF The respiratory system. Kumar, V Cotran, RS Robbins, SL eds. Basic pathology 1992,392-394 WB Saunders Philadelphia, PA.
  14. Piyathilake, CJ, Frost, AR, Manne, U, et al The expression of Ep-CAM (17–1A) in squamous cell carcinomas of the lung. Hum Pathol 2000;31,482-487[CrossRef][ISI][Medline]
  15. Piyathilake, CJ, Frost, AR, Manne, U, et al The expression of fatty acid synthase (FASE) is an early event in the development and progression of squamous cell carcinoma of the lung. Hum Pathol 2000;31,1068-1073[CrossRef][ISI][Medline]
  16. Piyathilake, CJ, Frost, AR, Bell, WC, et al Altered global methylation of DNA: an epigenetic difference in susceptibility for lung cancer is associated with its progression. Hum Pathol 2001;32,856-862[CrossRef][ISI][Medline]
  17. Hung, J, Kishimoto, Y, Sugio, K, et al Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA 1995;273,558-563[Abstract]
  18. Kishimoto, Y, Sugio, K, Hung, JY, et al Allele-specific loss in chromosome 9p loci in preneoplastic lesions accompanying non-small-cell lung cancers. J Natl Cancer Inst 1995;87,1224-1229[Abstract/Free Full Text]
  19. Wistuba,, II, Behrens, C, Milchgrub, S, et al Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma. Oncogene 1999;18,643-650[CrossRef][ISI][Medline]
  20. Raybaud-Diogene, H, Tetu, B, Morency, R, et al p53 overexpression in head and neck squamous cell carcinoma: review of the literature. Eur J Cancer B Oral Oncol 1996;32B,143-149[Medline]



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