Advances in Brief Alterations of Rb Pathway (Rb-p16-Cyclin D1) in Preinvasive Bronchial Lesions

Lung cancer results from a stepwise accumulation of genetic and molecular abnormalities with unknown temporal relationships to precursor bronchial lesions. In a search for biomarkers of malignant progression, we analyzed the expression of the tumor suppressor gene Rb and of the proteins regulating its phosphorylation and function in G1 arrest, p16 and cyclin D1, in preinvasive bronchial lesions accompanying cancer in 75 patients, in comparison with similar lesions in 22 patients with no cancer history. Rb was constantly expressed in preinvasive lesions, including carcinoma in situ (CIS). In contrast, p16 expression was lost in moderate dysplasia (12%) and in CIS (30%) in patients with lung cancer. p16 loss occurred exclusively in patients who displayed loss of p16 expression in their related invasive carcinoma. Loss of p16 expression was not seen in nine patients with dysplasia but no cancer progression. Cyclin D1 overexpression was seen in hyperplasia and metaplasia (6%), mild dysplasia (17%), moderate dysplasia (46%), and CIS (38%) in patients with cancer but was lost in 5% of the patients during the process of invasion; it was also observed in patients with no cancer progression (14%). Our results indicate that Rb protein function can be invalidated before invasion through alteration of the Rb phosphorylation pathway, by p16 inhibition, and/or by cyclin D1 overexpression and suggest a role for p16 and cyclin D1 deregulation in progression of preinvasive bronchial lesions to invasive carcinoma. Introduction Lung cancer is the leading cause of cancer-related death in industrial countries, and cigarette smoking is its main risk factor. Most patients cannot be cured because they present with advanced stages of the disease, and prognosis remains poor despite therapeutic improvements (1–3). Much evidence has been provided that invasive lung cancer is the end result of the stepwise accumulation of genetic alterations. The accumulation of 10–20 successive mutations should allow progression to invasive carcinoma (4). Morphological changes accompanying this transformation process have been described in detail in smokers (5). They progress from hyperplasia to metaplasia, which are rather common reactive lesions, to dysplasia and CIS, which are considered to be at risk for cancer development (5, 6). However, despite increasing risk of malignant transformation with histopathological grade, all these lesions are able to regress, including CIS (7, 8). In contrast, minimal lesions, such as hyperplasia and dysplasia, have been shown to display genetic and molecular changes (9, 10), and two recent studies demonstrated loss of one allele of chromosomes 3p, 9p, and 17p in normal bronchial mucosa of current and former smokers (11, 12). Thus, the morphological classification has a predictive value but cannot predict exactly for each individual case. It is believed that multiple intraepithelial lesions develop at various times in patients exposed to carcinogens, which supports the idea that the entire bronchial mucosa is damaged by carcinogens. This phenomenon is referred to as the “field cancerization” process. At present, neither the temporal sequence of the genetic abnormalities nor their relationship to specific morphological states has been precisely established. Because effective chemoprevention may be the most promising clinical approach, elucidating intermediate biomarkers to stratify patients for individual risk of progression and measure the success of these therapies is of importance. The malignant transformation of bronchial epithelial cells is driven by activation of oncogenes and growth factors and even more evidently by tumor suppressor gene inactivation. In this regard, genes of the p53-Rb pathway of G1 arrest are the most commonly affected genes in lung cancer. Rb gene inactivation, reflected by absence of Rb protein expression, has been reported in a minority of NSCLC (13, 14) but in the majority of small cell lung carcinoma. Although Rb expression is maintained in at least 80% of NSCLC, Rb functions on G1 arrest can be invalidated by mechanisms that alter the Rb phosphorylation pathway. Only the underphosphorylated form of Rb protein is able to mediate G1 arrest. Rb phosphorylation at G1-S transition is driven by Cdks Cdk4 and Cdk6, in protein complexes with cyclin D1. These complexes are controlled by potent inhibitors, Received 8/3/98; revised 11/2/98; accepted 11/3/98. 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 grants from Conseil Régional Rhône-Alpes, Groupement des Entreprises Françaises dans la Lutte contre le Cancer, Association pour la Recherche contre le Cancer, and Projet Hospitalier de Recherche Clinique. 2 To whom requests for reprints should be addressed, at Laboratoire de Pathologie Cellulaire, BP 217, 38043 Grenoble Cedex 9, France. Phone: (33) 4 76 76 54 86; Fax: (33) 4 76 76 59 49; E-mail: Elisabeth. [email protected]. 3 The abbreviations used are: CIS, carcinoma in situ; NSCLC, non-small cell lung carcinoma; Cdk, cyclin-dependent kinase. 243 Vol. 5, 243–250, February 1999 Clinical Cancer Research Research. on April 12, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from the Cdk inhibitors p16, p15, and p18, the inactivation of which may deregulate Rb phosphorylation. Cumulative results implicate p16 as a tumor suppressor gene, because p16 is frequently inactivated in lung cancer through loss of one allele (80–100%) and inactivation of the remaining allele by three alternative mechanisms: homozygous deletion, hypermethylation of the 59 end of the gene, and mutation, in decreasing order of frequency (15–19). In a previous study, we showed that loss of protein expression was highly concordant (95%) with one of these mechanisms of inactivation (20). An inverse correlation between alterations in the expression of Rb and p16 are observed in many tumor types, including lung cancer (21–26), which reflects a functional redundancy of Rb and p16 on a common p16/Rb growth suppressor pathway. Cyclin D1 gene product is part of the family of Cdk-cyclin complexes that allow Rb phosphorylation at G1-S transition. Somatic deregulation of cyclin D1 either by amplification or by transcriptional up-regulation has been demonstrated in many tumor types (27–31) and may play a role in the progression of lung cancer (32, 33). This is, after p16 inactivation, the secondmost reported mechanism responsible for Rb epigenetic inactivation in lung cancer through inappropriate phosphorylation. Although increased cyclin D1 protein expression has been recently shown in proliferative and preinvasive breast lesions (34), neither Rb nor p16 or cyclin D1 overexpression has been reported in mucosa and specifically bronchial preneoplasia. The aim of this study was to investigate preinvasive lesions for Rb, p16, and cyclin D1 overexpression to assess their role in lung cancer initiation and early progression. Because early lesions such as hyperplasia and metaplasia have been shown to carry genetic abnormalities and, specifically, loss of allele (loss of heterozygosity) at 3p and 9p chromosomes (9, 10), we included them in the spectrum of preinvasive lesions studied. To investigate the specificity of these phenotypic abnormalities for cancer progression in preinvasive lesions, we compared the frequency of p16 and cyclin D1 alterations in patients with previous, synchronous, or metachronous lung cancer with those observed in identical intraepithelial lesion of patients without previous cancer history or cancer development over a 3-year follow-up period. Materials and Methods Patients and Samples. Bronchial specimens with preinvasive lesions were obtained from lung resection performed for lung cancer in 46 patients. Preinvasive lesions were classified according to classical criteria (35, 36). Because both severe dysplasia and CIS are sometimes difficult to differentiate and cannot be distinguished only by thickness, we grouped them in a common group of severe preinvasive lesions. Four of the 46 patients with surgical resection were nonsmokers. Tobacco consumption ranged from 7 to 125 pack-years for the 38 others. Tissues were fixed in formalin and embedded in paraffin. Frozen sections were systematically performed on cancer and bronchial resection margins, but preinvasive lesions could be observed by chance on this frozen material in only 20 cases. In addition, 57 bronchial biopsies presenting at least one area of metaplasia obtained at fiberoptic bronchoscopy in 51 patients were also included in the study. In 29 of these patients, a lung cancer was diagnosed over a period of time extending from 4 years before to 1 year after biopsy. Two of these 29 patients never smoked. In the 22 other patients, neither previous or synchronous cancer nor metachronous cancer had been diagnosed within a 3-year follow-up. This was considered the control group with no cancer history. In this control group, 12 patients were nonsmokers, and bronchial biopsies were performed because of respiratory symptoms and abnormal chest X-ray in the clinical setting of benign disease, including infectious or inflammatory process, sarcoidosis, amyloidosis, chronic obstructive bronchopulmonary diseases, and benign tumors. Bronchial biopsies were fixed in Bouin fixative and embedded in paraffin, for conventional histopathological diagnosis. There was obviously more than one lesion per patient because one to five distinct anatomical area of preinvasive lesions present on the same or different blocks from bronchial margins were examined for each patient and selected for the study. The distance of each area of intraepithelial lesion from invasive carcinoma was determined by the thickness separating serial blocks in paraffin section and precise localization of the frozen section. Lesions present on the same section as invasive carcinoma were always considered adjacent to invasive carcinoma. Preinvasive lesions located more than 1 cm distant from the margin of invasive carcinoma and separated from it by normal strands of bronchial epithelium were considered distant from invasive carcinoma. Invasive carcinoma were classified according to the WHO classification (35). Basaloid carcinoma refers to a recently described histological class accounting for 5% of NSCLC (37). Large cell neuroendocrine carcinoma is a high grade neuroendocrine lung tumor recently individualized by Travis et al. (38). Immunohistochemistry. The sources of primary antibodies and dilutions used in the study as well as retrieval methods are indicated in Table 1. Immunohistochemistry was performed on frozen and formalin-fixed sections or Bouin-fixed bronchial biopsies. On paraffin sections, endogenous peroxidase activity was quenched with 3% hydrogen peroxide at room temperature for 10 min. After overnight incubation at 4°C with the primary antibody, slides were washed in PBS and then exposed to the secondary antibody, biotinylated donkey F(ab9)2 antirabbit (1:1000; The Jackson Laboratory, West Grove, PA) or antimouse (1:400; The Jackson Laboratory), for 1 h at room temperature. They were then washed in PBS and incubated with the streptavidin-biotin-peroxidase complex (1:400; DAKO, Copenhagen, Denmark) for 1 h at room temperature. The chromogenic substrate of peroxidase was a solution of 0.05% 3,39diaminobenzidine tetrahydrochloride, 0.03% H2O2, and 10 mmol/liter imidazole in 0.05 mol/liter Tris buffer (pH 7.6). Normal rabbit or mouse IgG at the same concentration as the primary antibodies served as negative controls. For cyclin D1, immunostaining was enhanced using a tyramine kit (DAKO) according to manufacturer’s instructions, with minor modifications: 0.5 dilution of the amplification reagent and the streptavidin-peroxidase solution. Statistical Analysis. Differences between independent groups were determined by means of the Kruskal-Wallis test. Differences between proportions were evaluated using Fisher’s exact test. 244 Rb Pathway in Bronchial Preinvasive Lesions Research. on April 12, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from