Advances in Brief Alterations of Rb Pathway (Rb-p16 INK4 -Cyclin D1) in Preinvasive Bronchial Lesions 1 Elisabeth Brambilla, 2 Sylvie Gazzeri, Denis Moro, Sylvie Lantuejoul, Sylvie Veyrenc, and Christian Brambilla Lung Cancer Research Group, Institut Albert Bonniot, CHU-C.J.F. Institut National de la Sante ´ et de la Recherche Me ´dicale, 97-01 [E. B., S. G., D. M., S. V., C. B.]; Laboratoire de Pathologie Cellulaire, Ho ˆpital Albert Michallon, BP217 [E. B., S. L.]; and Service de Pneumologie, Ho ˆpital Albert Michallon, BP217 [D. M., C. B.], 38043 Grenoble Cedex 9, France Abstract Lung cancer results from a stepwise accumulation of genetic and molecular abnormalities with unknown tempo- ral 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 G 1 arrest, p16 INK4A 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 path- way, 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 car- cinoma. Introduction Lung cancer is the leading cause of cancer-related death in industrial countries, and cigarette smoking is its main risk fac- tor. 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, 3 which are considered to be at risk for cancer development (5, 6). However, despite increasing risk of malignant transfor- mation 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 ge- netic 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 carcino- gens. This phenomenon is referred to as the “field canceriza- tion” process. At present, neither the temporal sequence of the genetic abnormalities nor their relationship to specific morpho- logical states has been precisely established. Because effective chemoprevention may be the most promising clinical approach, elucidating intermediate biomarkers to stratify patients for indi- vidual 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 G 1 arrest are the most commonly affected genes in lung cancer. Rb gene inacti- vation, 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 main- tained in at least 80% of NSCLC, Rb functions on G 1 arrest can be invalidated by mechanisms that alter the Rb phosphorylation pathway. Only the underphosphorylated form of Rb protein is able to mediate G 1 arrest. Rb phosphorylation at G 1 -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 Re ´gional Rho ˆne-Alpes, Groupement des Entreprises Franc ¸aises 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. Brambilla@ujf-grenoble.fr. 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 November 26, 2021. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from