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J Thorac Cardiovasc Surg 1999;118:529-535
© 1999 Mosby, Inc.

GENERAL THORACIC SURGERY

INDEPENDENT PROGNOSTIC ROLE OF p16 EXPRESSION IN LUNG CANCER

Angela M. Groegera,b, Mario Caputic, Vincenzo Espositoa,c, Antonio De Lucaa,d, Luigi Bagellaa, Carmen Pacilioa, Walter Klepetkob, Giovan G. Giordanoe, Feliciano Baldie, Hans E. Kaiserf, Ernst Wolnerb, Antonio Giordanoa

This work was supported by grants from Sbarro Institute for Cancer Research and Molecular Medicine and grants from National Institute of Health to A.G. A.D.L. is the recipient of a grant FIRC. V.E. is supported by a fellowship from the II Universitá di Napoli (Dottorato di Ricerca in Broncopneumologia).

Address for reprints: Antonio Giordano, MD, PhD, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Sbarro Institute for Cancer Research and Molecular Medicine, 1020 Locust St, Room 226, Philadelphia, PA 19107.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 References
 
Objectives: The cyclin-dependent kinase p16 (also known as Ink4A, Mts1, Cdkn2, and Cdkn4i) has been proposed as a tumor suppressor gene mapped on chromosome segment 9p21. This study evaluated p16 protein expression in 135 lung cancer specimens and investigated potential genetic alterations occurring in this gene.
Results: We found altered p16 immunohistochemical expression to be a frequent event in lung cancer and to be independent of either the histologic type or any other clinical-pathologic feature. Western blot analyses performed on about one third of the specimens correlated highly with these results. In addition, we found p16 immunohistochemical expression to be a favorable prognostic factor in lung cancer in that its reduction or loss correlated with a worse outcome for the patients. Polymerase chain reaction amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not necessarily linked to mutational events of these genes.
Conclusions: We conclude that p16-altered expression is both an independent and frequent event in lung cancer and may have an important role in tumorigenesis and in malignant progression of a significant proportion of these cancers. However, the actual incidence and relevance of p16 mutations in this neoplasm continues to be debated, and its analysis seems inconclusive. Our results suggest a prognostic role for the immunodetection of this protein on formalin-fixed and paraffin-embedded specimens. They further suggest its routine use in the evaluation of the frequently unpredictable behavior of lung cancer.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 References
 
The pathogenesis of lung cancer is characterized by its multiplicity of origins. Different agents (biological, chemical, and physical), by acting as initiating or promoting factors, have been associated with increasing lung cancer risk. A common molecular basis, however, underlies all of the different pathogenetic theories. The external environment manipulates cellular proliferation and differentiation by stimulating or inhibiting certain signal transduction pathways. Each component of the cell cycle machinery, being the final executors in cell division, potentially could be able to elicit or to contribute to a neoplastic phenotype. These phenomena are even more relevant in lung cancer, a neoplasm that affects the respiratory system, which is interacting continuously with the external environment.

The recent discovery of a new family of cell cycle regulators called cyclin-dependent kinase inhibitors (CKIs) has opened up new avenues for study of different cell cycle functions. CKIs have the ability to stop cell cycle progression by interacting directly with several cell cycle regulators and also are considered to be potential tumor suppressor genes. At present, 2 different groups of CKIs can be separated on the basis of sequence homology criteria. The first group includes p21 (WAF1, Cip1, Cap20, Sdi1, mda6), the first CKI to be discovered, p27 (Ick, Kip1 and Pic2), and p57 (also called Kip2). The second group includes p16 (Ink4A, Mts1, Cdkn2, and Cdkn4i), p15 (Ink4B, Mts2), p18 (Ink4C and Ink6A), and p19/p20 (also called Ink4D and Ink6B).

The p16 gene encodes a 16-kd protein first identified in transformed cell lines.Go 1 The detection of significant levels of p16 in late G1 and S phase only confirms its role in the inhibition of the cell cycle machine.Go 2 In addition, p16 is believed to block the activation of cyclin-dependent kinases 4 and 6 by competing for D cyclin binding.Go 1 Because p16 is considered to be a specific regulator of D-type cyclin-dependent kinases and because the retinoblastoma gene is the best characterized substrate of the G1 cyclin-dependent kinases, a feedback between these 2 cell cycle regulators has been hypothesized.Go Go 3,4 This theory is supported by the finding that increased levels of p16 are always detected in cells Rb –/–, blocking the interactions between cyclin-dependent kinases 4 and 6 and the D cyclins.Go 4 However, the transcription factor that should regulate this mechanism is yet to be identified.

Abnormalities in the above-described p16/Rb pathway have been reported in different tumors.Go Go 5-9 It is widely known that lung cancers of morphologic similarities may behave differently in any assigned stage group, which may affect markedly the clinical management of each patient. WeGo Go 10,11 have reported the involvement of p27 and p21 in lung cancer pathogenesis and progression. Drawing on our previous findings, we decided to assess the p16 status in a group of 135 patients who had surgical resection for lung cancer in an attempt to identify a prognostic marker for this frequent and aggressive neoplasm whose behavior often seems to be unpredictable.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 References
 
Tumor specimens.
A total of 135 formalin-fixed and paraffin-embedded lung cancer specimens were included in the study. All specimens were obtained from patients who underwent surgical resection (lobectomy or pneumonectomy) and complete mediastinal lymphadenectomy in the Department of Cardio-Thoracic Surgery at the University Hospital of Vienna (Austria) or in the Department of Thoracic Surgery of V. Monaldi Hospital, Naples (Italy). One surgeon (A.M.G. or W.K.) from our research group was always the principal surgeon of the surgical team.

Neoplastic freshly frozen tissues also were available from 54 patients who had not undergone any adjuvant therapy. Sex was unevenly distributed, with women accounting for 21.5% of the population, and the mean patient age was 62 years.

Follow-up data were collected from the Central Institute of Statistics of Austria, from hospital charts, and from periodic interviews with patients and their families. The histologic diagnoses and classifications of the tumors were based on the World Health Organization criteria.Go 12 The postoperative pathologic TNM stage was determined according to the guidelines of the American Joint Committee on Cancer.Go 13

Polymerase chain reaction (PCR) and direct sequencing.
Genomic DNA of normal tumor tissues was extracted from 54 micro-dissected frozen specimens, and exons 1 and 2 of the p16 gene were amplified as previously described.Go 14 PCR products then were purified (QIAquick PCR purification kit, Germany) and sequenced by Dyedeoxy terminator reaction chemistry for sequence analysis on the Applied Biosystem model 377 DNA sequencing system (Foster City, Calif).

Western blot analysis.
One gram of each frozen lung cancer tissue sample was sectioned and quickly homogenized at 4°C in 250 mmol/L NaCl, 50 mmol/L tromethamine (Tris, pH 7.4), 5 mmol/L EDTA, 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L NaF, 0.5 mmol/L Na3VO4, 10 mg/mL leupeptin, and 50 mg/mL aprotinin. The homogenates were cleared by centrifugation for 15 minutes at 13,000g at 4°C, and the total protein in the extracts was determined. Next, 50 mg of protein was denatured by boiling in 2X Laemmli sample buffer and separated by electrophoresis in a 15% sodium dodecyl sulfate–polyacrylamide gel, followed by electrophoretic transfer of the proteins to a PVDF membrane (Millipore, Bedford, Mass) in CAPS buffer (10 mmol/L CAPS, 20% methanol, pH 11). The membrane then was blocked with 5% milk in TBS-T buffer (2 mmol/L tromethamine [Tris], 13.7 mmol/L NaCl, 0.1% Tween-20, pH 7.6) and washed in TBS-T. Monoclonal mouse anti-p16 (F-12) dilution 1 µg/mL (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) was incubated with the membrane in 3% milk and then washed in TBS-T. Secondary antibody, anti-mouse coupled to horseradish peroxidase, was incubated with the membrane and then washed in TBS-T. The presence of secondary antibody bound to the membrane was detected by using the ECL system (DuPont NEN Company, Boston, Mass).

Immunohistochemistry.
Sections from each specimen were cut at 3 to 5 µm, mounted on glass, and dried overnight at 37°C. All sections were deparaffinized in xylene, rehydrated through a graded alcohol series, and washed in phosphate-buffered saline solution. This buffer was used for all subsequent washes and for dilution of the antibodies. Sections were quenched sequentially in 0.5% hydrogen peroxide and blocked with diluted 10% normal horse anti-mouse serum (Vector Laboratories, Burlingame, Calif). A monoclonal antibody raised against p16 (F-12) (Santa Cruz Biotechnology) was used (dilution 1:100). The incubation time was 60 minutes at room temperature. After being washed in phosphate-buffered saline solution, the slides were incubated with diluted horse anti-mouse biotinylated antibody (Vector Laboratories) for 30 minutes at room temperature.

All slides were processed by the ABC method (Vector Laboratories) for 30 minutes at room temperature. Diaminobenzidine was used as the final chromogen, and hematoxylin was used as the nuclear counterstain. Negative controls for each tissue section were prepared by leaving out the primary antibody. Immunostaining of Saos-2 cells was used as the positive controlGo 15 (data not shown).

All samples were processed under the same conditions. Two pathologists (G.G. and F.B.) independently evaluated the staining pattern of the protein. As previously described,Go Go 15,16 a tumor is considered p16 negative if there is no nuclear staining in any neoplastic cell regardless of cytoplasmatic staining and if admixed non-neoplastic elements do show nuclear immunoreactivity. If the latter are negative as well, the stain is considered uninterpretable. A cutoff of 1% of positive cells also was adopted. Specimens with less than 1% of positive cells also were included in the first group (score 0, undetectable expression). All specimens evaluated as positive were scored according to the sequential arbitrary cutoffs: score 1, from 1% to 30% of positive cells (low expression level); score 2, from 30% to 60% of positive cells (medium expression level); and score 3, more than 60% of positive cells (high expression level). Analysis of the data with these arbitrary cutoffs was highly statistically significant and, therefore, functionally operative. At least 20 high-power fields were chosen randomly and 2000 cells were counted.

Statistical analysis.
We performed a statistical analysis to investigate the relationship between the clinical-pathologic parameters (age, sex, histotype, TNM status, tumor stage, and postoperative radiation or chemotherapy), p16 expression, and the patient survival times. Univariate analysis of survival times was carried out by means of Kaplan-Meier analysis stratified by the individual covariates. In most patients the log rank test was used to test homogeneity of survival over strata. In a few patients, the Wilcoxon test was used because the alternative hypothesis in this last test is that there is a decreasing proportional difference across strata. This test is appropriate when one suspects an effect that may diminish over time. In contrast, the alternative hypothesis in the log rank test is that there is a proportional difference in survival across strata that remains constant. Multivariate Cox proportional hazards models were fit using all variables that were <.15 at the univariate level.

Linear-by-linear and Kruskal-Wallis association tests were used to assess possible associations among clinical-pathologic parameters, Western blot p16 expression levels, and immunohistochemical data. The analysis was performed with the use of Stata 5.0 (Stata Corp 1997, Stata Statistical Software, release 5.0, Stata Corporation, College Station, Tex).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 References
 
Table I summarizes the clinical-pathologic features of the patients included in the study. PCR amplification and direct sequencing performed on the 54 freshly frozen specimens did not show any alteration in p16 exons 1 and 2. The immunohistochemical expression of p16 was detected mostly in the nuclei of both normal and neoplastic cells(Fig 1). Thirty-six specimens were considered uninterpretable according to the criteria described in the "Material and methods" section.


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  		Table I. Frequency of distribution of clinical-pathologic factors
 


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  		Fig. 1. Representative p16 immunostainings of lung cancer specimens with the presence of staining of non-neoplastic elements (positive internal control). A, Lung cancer with high p16 expression level (x200). B, Lung cancer with medium p16 expression level (x200). C, Lung cancer with low p16 expression level (x200). D, Lung cancer negative for p16 expression with a positive internal control (lymphocytes) (x200).

 
The level of agreement between the observers was 91.1% (123 specimens). In the remaining 12 specimens the score was obtained from the opinion of a third observer (A.G.). In 10 specimens (10.1%), p16 expression was undetectable. Score 1 was attributed to 27 specimens (27.3%), score 2 was attributed to 32 specimens (32.3%), and score 3 was attributed to 30 specimens (30.3%). Western blot analysis performed on 54 specimens showed different expression levels of p16 ranging from undetectable levels (6 specimens, 11.1%) to high expression levels (15 specimens, 27.7%)(Fig 2). These results correlated highly with the immunohistochemical findings (P = .0002). No correlation was found between p16 immunohistochemical expression and any clinical-pathologic factor (ie, tumor stage and histotype)(Table II).Tables III andIV show the summary of univariate and multivariate analyses of survival times. At the univariate level, the percentage of p16 positive cells, the tumor stage, and the histologic type correlate with patient overall survival. However, in a multivariate analysis, models controlled for sex, stage, nodal status, and treatment (radiation and chemotherapy) and stratified by histotype show p16 expression to be an independent prognostic factor with its higher expression correlating with longer patient survival time(Fig 3).



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  		Fig. 2. Western blot analysis of a representative panel of lung cancer specimens showing different p16 expression levels. Western blot was normalized with monoclonal antibody anti–heat shock protein (HSP 72/73) purchased from Oncogene Science Diagnostics, Inc (Cambridge, Mass).

 

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  		Table II. Relationship between p16 expression and tumor stage
 

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  		Table III. Univariate Kaplan-Meier analysis
 

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  		Table IV. Multivariate Cox proportional hazards
 


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  		Fig. 3. Kaplan-Meier survival percentage curves for patients with lung cancer constructed according to different p16 status.

 

   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
References
 
Lung cancer is a disease characterized by a complex array of biomolecular alterations that drive uncontrolled growth and metastatic spread. Identification of these alterations could provide a variety of molecular markers, and tests could redefine the criteria for cancer diagnosis and provide for new approaches to treatment. Development of new diagnostic assays then could expand the ability of clinicians to accurately stage tumor development and to better predict clinical outcomes by matching patients to different therapeutic treatments.

These changes that lead to loss of cell cycle inhibition by negative regulators, such as p16, which releases lung cancer cells from the constraints of cell division, are regarded now as target events for new biomolecular assays. We found altered p16 immunohistochemical expression to be a rather frequent event in lung cancer and to be independent of the histologic type of cancer or other clinical-pathologic features. Expression of p16 was undetectable in 10.1% of the specimens, expressed in less than 30% of neoplastic cells in 27.3% of the specimens, expressed in 30% to 60% of cells in 32.3% of the specimens, and detectable in more than 60% of cells in only 30.3% of the specimens. Our Western blot analyses highly correlated with these findings (P = .0002). These data are in agreement with the findings of Geradts and Wilson,Go 17 who reported a high frequency of aberrant p16 expression in 104 human breast cancers but did not correlate this finding with histopathologic parameters. Washimi and associates,Go 18 however, described p16 structural alterations that preferentially affected non–small cell lung cancers in vivo, and Kratzke and coworkersGo 16 recently reported a high frequency of p16 aberrant expression in non–small cell lung cancer to be inversely related to the pathologic stage of the disease. In addition, we also conclude that p16 immunohistochemical expression is a favorable independent prognostic factor in lung cancer. Reduction or loss of p16 expression correlates with a worse patient outcome.

These results, obtained on a large number of patients including those with small cell carcinomas, confirm the suggestion of Kratzke and associatesGo 16 that absent or reduced p16 expression is a negative prognostic indicator.

In this study, PCR amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not linked necessarily to mutational events of these gene. Previous studies have reported contrasting results both in lung cancer and in tumors from other anatomic sites. These studies described p16 aberrant expression to be either a frequent consequence of DNA alterationsGo Go Go 5,18,19 or a post-transcriptional event.Go Go Go 8,20-23 DNA hypermethylation has been proposed recently as an alternative mechanism for p16 gene functional inactivation.Go Go 24,25

In conclusion, our study demonstrated that p16-altered expression is a frequent event in lung cancer. These data attribute an important role to this protein during both tumorigenesis and tumor progression in a significant proportion of patients with lung cancer, even though the incidence and the relevance of p16 mutations in this neoplasm remain debatable and its analysis seems inconclusive.

Our results suggest an important role for the immunodetection of this protein on formalin-fixed and paraffin-embedded specimens. If confirmed in larger groups of patients, this simple assay could be of value in the evaluation of the frequently unpredictable behavior of lung cancer.


   Acknowledgments
 
We thank Dr J. J. Gartland, Thomas Jefferson University medical editor, for editing the manuscript.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received for publication July 6, 1998. Revisions requested Oct 22, 1998; revisions received April 19, 1999. Accepted for publication April 22, 1999.


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