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Loss of pRb2/p130 Expression Is Associated with Unfavorable Clinical Outcome in Lung Cancer¹

Department of Medical-Surgical, Cardiological, Respiratory, and Thoracic Sciences [M. C., V. E., A. M.] and Department of Biochemistry and Biophysics "F. Cedrangolo," Section of Anatomic Pathology [F. B., E. W.], Second University of Naples, Naples, Italy; Department of Cardio-Thoracic Surgery, University of Vienna, Vienna, Austria [A. M. G.]; Laboratory for Cell Metabolism and Pharmacokinetics, Center for Experimental Research, Institute Regina Elena, Rome, Italy [A. D. L.]; Department of Pathology, Anatomy, and Cell Biology, Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, Pennsylvania [V. M., A. G.]; and Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122 [A. G.]

	ABSTRACT

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Altered expression of cell cycle regulators represents a frequent event in both small cell and non-small cell lung cancer (NSCLC). Despite several studies that reported involvement of tumor suppressor genes, such as p53 and pRb, in the development and progression of lung cancer, contrasting opinions exist about the prognostic role of this protein in this neoplasm. We developed an immunohistochemical assay suitable for the detection of pRb2/p130, the last discovered member of the retinoblastoma gene family, on formalin-fixed and paraffin-embedded sections. We evaluated the immunohistochemical expression of pRb2/p130 in 135 lung cancer specimens, and performed Western blot analysis in a subset of 30 corresponding tumor lysates. A high correlation between immunohistochemical data and Western blot results (P = 0.0004) was found. We statistically analyzed the relationship between overall survival (OS) time and pRb2/p130 expression according to the different histological types in 105 patients. We did not find any correlation between pRb2/p130 expression and OS in small cell lung cancers, whereas in NSCLCs a direct relationship between pRb2 and OS was found in both adenocarcinoma (P = 0.0002) and squamous cell carcinoma (P = 0.0002) histotypes. According to univariate analysis, pRb2/p130 was a prognostic factor of which the lost or reduced expression correlated with a shorter OS (P < 0.0000). At multivariate analysis, pRb2/p130 expression was an independent predictor of OS (P = 0.0001) when considered together with histotype.

This study demonstrates for the first time the potential independent prognostic value of pRb2/p130 expression on formalin-fixed, paraffin-embedded sections from lung cancer patients. pRb2/p130 immunoreactivity can be used to predict OS in patients with NSCLC and, therefore, may represent a new prognostic marker.

	INTRODUCTION

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The retinoblastoma gene family consists of a group of genes sharing a high percentage of sequence homology. At present, the family comprises three members, the most investigated one being the RB³ gene, the prototype for tumor suppressor genes, which codifies for a protein called pRb. The other genes in the retinoblastoma gene family are p107 and Rb2/p130.

The product of the retinoblastoma gene, pRb/p105, is a nuclear phosphoprotein with a molecular weight of M_r 105,000, which is ubiquitously expressed in vertebrates (1) . Use of RB1 cDNA has confirmed, on one hand, the alteration of this gene in all retinoblastomas, and on the other hand, has shown that mutations in both alleles of this gene also are present in several other tumors, including osteosarcomas, bladder carcinomas, prostate carcinomas, breast carcinomas, lung carcinomas, cervical carcinomas, and leukemias (2) . Because the presence of an intact RB gene prevents the formation of retinoblastoma, and because introduction of a functional RB gene into cell lines derived from tumors lacking functional pRb results in a reversal of the tumor phenotype (3 , 4) , the RB gene was termed a "tumor suppressor gene" and became the prototype of this category.

The ability of pRb/p105 to exert its growth-suppressive activity is dependent on its interaction with transcription factors, such as the family of E2F/DP molecules, of which the function is to promote the transcription of genes required for cell cycle progression (5) . pRb binds to E2F/DP heterodimers during the G₁ phase of the cell cycle and blocks their transcriptional activities (6 , 7) . Cell cycle-dependent phosphorylation of pRb, as well as complex formation with a number of DNA tumor viral oncoproteins (such as adenovirus E1A, SV40 T antigen, and papillomavirus E7) impairs its ability to bind to E2F/DP complexes. This eventually results in the entry of the cells into the S phase of the cell cycle (8, 9, 10, 11) . The other two members of the retinoblastoma gene family, p107 and pRb2/p130, have been cloned recently (12 , 13) . They share function and a high percentage of sequence homology with the Rb gene. The most strongly conserved regions of pRb, p107, and pRb2/p130 correspond to the "pocket region," which is needed by these proteins for binding to the viral oncoprotein. The "spacer" region, located between the two domains of the "pocket," is conserved between p107 and pRb2/p130, and shares 40% homology, whereas the protein sequence of the "spacer" of pRb is shorter and shares little homology with the other two members of the family. The regions of these three proteins required for interaction with viral transforming proteins are the same required for interaction with several cellular proteins, such as the family of E2F transcription factors and various cyclins (5 , 14) .

Both pRb2/p130 and p107, like pRb, display a cell cycle-regulated phosphorylation pattern (15) , and form complexes with different members of the E2F family of transcription factors with a varying temporal order of complex formation (16, 17, 18) . Interplay between the Rb family and the E2F family is hypothesized to regulate transcription and progression of the cell cycle. Several data sources suggest that pRb, p107, and pRb2/p130 associate with distinct E2F species (19) . These structural identities are reflected in similar functional properties. Both pRb2/p130 and p107, like pRb, display growth-suppressive properties, although the growth arrest mediated by the three pocket proteins is not identical (20, 21, 22) . This suggests that, although the different members of the RB gene family may complement each other, they are not fully functionally redundant (21) .

The human Rb2/p130 gene is positioned on chromosome 16q12.2, an area frequently altered in some human cancers, such as breast, ovarian, hepatic, and prostatic carcinomas (23) . This suggests a role for the involvement of Rb2/p130 as a tumor suppressor in human cancers. Its encoded protein pRb2/p130, like the other pocket proteins, is localized mainly in the nuclear compartment of the cell (2) . However, a recent study (24) suggests that mutations may disrupt the nuclear localization of pRb2/p130 in human tumor cell lines and primary tumors.

With regard to lung cancer, in an immunohistochemical study on 77 lung cancer specimens (25) , we showed that pRb2/p130 protein is undetectable in a higher percentage of patients compared with the other two pocket proteins. In an additional study (26) , we found a negative correlation between histological grading and pRb2/p130 staining in 158 specimens of human lung cancer. These and more recent data (27) suggest an important role for the altered expression of this gene in lung cancer.

On the basis of these findings, we evaluated the expression of Rb2/p130 by immunohistochemistry in a large series of lung cancer specimens to confirm our previous results and to evaluate a possible prognostic role of this protein in lung tumors.

	MATERIALS AND METHODS

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Population Study.
Archival formalin-fixed, paraffin-embedded lung cancer specimens (105) were obtained from patients who underwent surgical resection for lung cancer (lobectomy or pneumonectomy) in the Department of Cardio-Thoracic Surgery of the University Hospital of Vienna (Vienna, Austria) between 1989 and 1993. In addition, 30 fresh-frozen lung cancer specimens and the corresponding paraffin-embedded tissues were collected at the Department of Thoracic Surgery of the V. Monaldi Hospital (Naples, Italy) from patients who underwent surgical resection during the same period. Patients did not receive any chemo- or radiotherapy before surgical resection. Outcome data were collected from the "Central Institute of Statistics of Austria," hospital charts and periodic interviews with patients and their families. The histological diagnosis and tumor classification were based on the WHO criteria (28) . The postoperative pathologic TNM stage was determined according to the guidelines of the American Joint Committee on Cancer (29) .

Immunohistochemistry.
We assessed the immunohistochemical expression of pRb2/p130 in 135 lung cancer specimens, as described previously (26) . Polyclonal rabbit immune serum ADL-1 was used for the detection of pRb2/p130 protein. Characterization of this antibody and its suitability for immunohistochemical studies have been described elsewhere (25 , 26) . Diaminobenzidine was used as the final chromogen, and hematoxylin was used as the nuclear counterstain. For the negative control, preimmune serum was substituted for the primary antibody ADL-1 in each tissue section. Immunostaining of SaOs-2 cells was used as an external positive control (data not shown). All of the samples were processed under the same conditions. Three pathologists (A. D. L., F. B., and A. G.) independently evaluated the staining pattern of the protein, and each scored the specimens for the percentage of positive nuclei. As described previously (28) , tissue samples were scored as negative for pRb2/p130 expression when nuclear staining was undetectable in cancer cells, regardless of cytoplasmic staining, whereas internal non-neoplastic elements showed nuclear immunoreactivity. If the latter were negative as well, the staining was considered uninterpretable. A cutoff of 1% of positive cells was also adopted. Thus, specimens with <1% of positive cells were also included in the first group (score 0, undetectable expression). All of the specimens considered positive were scored using the sequential arbitrary cut-offs: 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, >60% of positive cells (high expression level). At least 20 high-power fields were chosen randomly, and 2000 cells were counted.

Western Blot Analysis.
One gram of each fresh-frozen lung cancer tissue was sectioned and quickly homogenized at 4°C in 250 mM NaCl, 50 mM Tris (pH 7.4), 5 mM EDTA, 0.1% (v/v), Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 0.5 mM Na3VO4, 10 mg/ml leupeptin, and 50 mg/ml aprotinin. The homogenates were cleared by centrifugation for 15 min at 13,000 x g at 4°C, and the amount of total protein in the extracts was determined. pRb2 expression was evaluated by Western blot analysis in all 30 of the lung cancer specimens. Western blots were performed as described previously (21) using ADL-1 antibody at a dilution 1:1,000 for 14 h at 4°C.

Statistical Analysis.
We performed statistical analysis to investigate the relationship between pRb2/p130 expression, clinico-pathologic parameters (age, sex, histotype, TNM status, tumor stage, histological grading, and postoperative radio- and/or chemotherapy), and patient survival time. Linear-by-linear and Kruskal-Wallis association tests were used to examine any possible correlation between clinico-pathologic parameters, pRb2/p130 expression levels as assessed by Western blot, and immunohistochemical data. Statistical significance was established at P < 0.05.

Life tables were computed using the product-limit estimate by Kaplan and Meier. The log-rank test was used to examine the dependence of survival on either histotype or pRb2 status, taken one at a time. Proportional hazard regression analysis was used to incorporate both histotype and Rb2 status in the same model. The stepwise procedure and likelihood ratio tests were used to select those variables with the greatest prognostic value. The analysis was carried out using Stata 5.0. (Stata Corp., College Station, TX).

	RESULTS

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Table 1 summarizes the clinico-pathologic information regarding all of the evaluated patients. Gender was distributed unevenly with females accounting for 21.5% (29 patients). The mean age was 62 years. Only 16% of the tumors were classified as SCLCs, and nodal involvement was reported in 50% of the population.

View larger version (101K): [in this window] [in a new window]   Fig. 1. Representative immunostainings for pRb2/p130 in lung cancer. a, pRb2/p130 nuclear staining in a well-differentiated lung squamous cell carcinoma (x150). b, pRb2/p130 nuclear staining in a well-differentiated lung squamous cell carcinoma (x300). c, pRb2/p130 nuclear staining in a well-differentiated adenocarcinoma of the lung (x500).

View larger version (41K): [in this window] [in a new window]   Fig. 2. Western blot analysis of a representative panel of lung cancer specimens showing different expression levels of pRb2/p130.

In Table 2 , we show that percentage of pRb2/p130-positive cells, but no other investigated factors, correlated with patient outcome (P < 0.0000) according to the log-rank test.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Kaplan-Meyer survival percentage curves in lung cancer patients plotted according to different pRb2/p130 percentages of positive cells as stratified by histotype: (a) adenocarcinomas, (b) squamous cell carcinomas, and (c) SCLC.

	DISCUSSION

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We demonstrated that the expression of the retinoblastoma family member pRb2/p130 is a strong predictor of OS in a large series of lung cancer patients. This finding is consistent with several other studies demonstrating the prognostic role of pRb2/p130 in several tumor types. In particular, loss of pRb2/p130 expression significantly correlated with a negative prognosis in endometrial (30) , oral squamous cell carcinomas (31) , choroidal melanoma (32) , and malignant lymphomas (33) .

Whereas pRb2/p130 expression appears to be an important predictor of clinical behavior, evidence to date suggests that loss of pRb2/p130 protein in cancer is attributable to genomic mutations occurring in cell lines, such as lymphoid (24) and nasopharyngeal, and in human specimens of primary nasopharyngeal carcinomas (34) , lung tumors (35) , and Burkitt’s lymphomas.

In lung cancer, our data confirm and extend our preliminary studies by immunohistochemistry on the expression of the Rb family members in a series of 77 (25) and 158 (26) retrospective specimens of lung carcinoma and on fine-needle aspiration biopsies from lung tissues (36) , suggesting a role for the loss of pRb2/p130 in the pathogenesis and progression of this tumor. Moreover, we reported that the expression of pRb2/p130 correlates with a high degree of differentiation and with a lower proliferation rate of lung tumors (26) . After these preliminary results, we assessed pRb2/p130 immunohistochemical expression in a group of 135 followed-up patients to assess the prognostic significance of this protein in lung cancer. pRb2/p130 was found to be an independent prognostic factor of which the loss or reduced expression correlated with a shorter patient survival time. pRb2/p130 expression also correlated with tumor histological grading, as we showed previously (26) . We also performed Western blot analysis on a subset of 30 specimens to additionally confirm data obtained by immunohistochemical assay. We found a high correlation between immunohistochemical data and Western blot analysis results (P = 0.0004). This observation seems to be in contrast with a recent study (37) on 69 lung tumors, where expression of pRB2/p130, as assessed by Western blot, was detected in every sample tested; however, the use of a different antibody may account for the different result from our report. In previous studies, including those on lung cancer (25 , 26) , we (30 , 32) and others (38) have shown the specificity of the rabbit polyclonal immune serum against pRb2/p130 (ADL1), and its suitability for both immunohistochemistry and Western blot analysis. Therefore, it seems logical to use the same antibody in this study.

Our finding of relatively frequent undetectable expression of pRb2/p130 in lung carcinoma (18.4% in this study) could be linked to genomic inactivating mutations, as we showed previously (35) . On the other hand, potential mechanisms of functional inactivation, such as enhanced degradation pathways, must also be considered because they already have been shown to be involved in the reduced expression of p27^kip1, another cell cycle regulator that interacts directly with cyclin-cyclin-dependent kinase complexes in lung cancer (39) .

pRb2/p130 was the last member of the RB gene family to be identified. Like Rb and p107, it has well-characterized cell growth-suppressive properties similar to, yet distinctive from, the other family members (21 , 40) . Using a tetracycline-regulated gene expression system to control the expression of pRb2/p130 in a JC virus-induced hamster brain tumor cell line, we demonstrated that induced expression of pRb2/p130 reduces the tumor mass in nude mice (41) . In another study in nude mice, we showed that ectopic expression of pRb2/p130 suppresses the tumorigenicity of the SKOV3 ovarian cancer cell line overexpressing erbB-2 (42) . Finally, in support of the involvement of pRb2/p130 as a tumor suppressor gene in lung cancer, we showed that in vivo retroviral transduction of pRb2/p130 in established tumors, derived from injection of the lung adenocarcinoma cell line H23 grown in nude mice, reduced the mass 12-fold with respect to the control viruses (35 , 43) .

In summary, in this study, we present evidence for a role of pRb2/p130 in lung cancer patients as an independent prognostic predictor of clinical outcome. Patients with lung cancer that show loss of pRb2/p130 expression are at a higher risk of death from disease and may eventually benefit from more aggressive adjuvant therapy. The reliability of pRb2/p130 as a potential marker in the routine clinical management of patients with lung cancer deserves to be additionally evaluated in long-term follow-up studies. Additional studies on the mechanism responsible for the loss of pRb2/p130 are also required to design novel therapeutic strategies, such as gene therapy, targeting this tumor suppressor gene.

	ACKNOWLEDGMENTS

 
We thank Marie Basso for editing the manuscript and Hao Wang for assistance with statistical analysis.

	FOOTNOTES

 
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.

¹ Supported by NIH Grant RO1 CA60999/01A1, PO1 NS36466/01A1, and PO1 CA56309 (to A. G.). A. D. L. is recipient of an Federazione Italiana Ricerca Cancro grant. V. M. is supported by a fellowship from the Consiglio Nazionale delle Ricerche (CNR) and a training grant from the National Cancer Institute (PHS 5 T32 CA09137).

² To whom requests for reprints should be addressed, at Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, BioLife Sciences Building, Suite 333, 1900 North 12th Street, Philadelphia, PA 19122. Phone: (215) 204-9520; Fax: (215) 204-9519; E-mail: giordano{at}temple.edu

³ The abbreviations used are: RB, retinoblastoma; OS, overall survival; TNM, Tumor-Node-Metastasis; RR, relative risk; CI, confidence interval; SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer.

Received 8/14/00; revised 8/ 5/02; accepted 8/14/02.

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