[CANCER RESEARCH 63, 8108 – 8112, December 1, 2003] Advances in Brief FGFR3 and TP53 Gene Mutations Define Two Distinct Pathways in Urothelial Cell Carcinoma of the Bladder Ashraf A. Bakkar, 1 Herve Wallerand, 1 Franc ¸ois Radvanyi, 3 Jean-Baptiste Lahaye, 1 Serge Pissard, 2 Laure Lecerf, 1 Jean Claude Kouyoumdjian, 2 Claude C. Abbou, 1 Jean-Claude Pairon, 1 Marie-Claude Jaurand, 1 Jean-Paul Thiery, 3 Dominique K. Chopin, 1 and Sixtina Gil Diez de Medina 1 1 Equipe mixte Inserm Institut National de la Sante ´ et de la Recherche Me ´dicale, and Service d’Urologie; 2 De ´partement de Biochimie et Ge ´ne ´tique, Universite ´ Paris XII, Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital Henri Mondor; and 3 Unite ´ mixte de Recherche, Centre National de la Recherche Scientifique, Institut Curie, Paris, France Abstract FGFR3 and TP53 mutations are frequent in superficial papillary and invasive disease, respectively. We used denaturing high-performance liq- uid chromatography and sequencing to screen for FGFR3 and TP53 mutations in 81 newly diagnosed urothelial cell carcinomas. Tumors were classified as follows: 31 pTa, 1 carcinoma in situ, 30 pT1, and 19 pT2-T4. Tumor grades were as follows: 10 G1, 29 G2, and 42 G3. FGFR3 muta- tions were associated with low-stage (P < 0.0001), low-grade (P < 0.008) tumors, whereas TP53 mutations were associated with high-stage (P < 0.003), high-grade (P < 0.02) tumors. Mutations in these two genes were almost mutually exclusive. Our results suggest that FGFR3 and TP53 mutations define separate pathways at initial diagnosis of urothelial cell carcinoma. Introduction UCC 4 of the bladder is a spectrum disease characterized by unpre- dictability in its biological behavior and response to treatment. UCCs are classified into two broad types: superficial and invasive tumors. Most UCCs present as superficial bladder cancer, confined in 75% of cases to the epithelium (CIS, pTa) or lamina propria (pT1). The remaining (25%) tumors present as muscle invasive disease (pT2), with no history of superficial disease (1). However, superficial blad- der cancer covers many lesions with different biological potentials, and morphologically similar tumors of the same stage may display completely different patterns of behavior (1). It has been suggested that different genetic changes account for these different forms of UCC and for the observed differences in biological behavior (2). Losses of chromosome 9 have been described as the most common finding in superficial papillary tumors, but such losses are in fact observed at all stages and grades. Alterations of chromosome 9q occur very early in the disease and seem to be involved in the development of bladder cancer. Regions of interest (at 9p21, 9q12–31, 9q32–33, and 9q34) harboring candidate tumor suppressor genes have been identified on both arms of chromosome 9 (reviewed in Ref. 2). However, the prognostic significance of these findings remains un- clear. TP53 mutations are frequent in bladder tumors of high stage and grade, and have been associated with invasiveness (3). It has also been shown that loss of heterozygosity on chromosome 17p, to which TP53 maps, is more frequent in high-grade than in low-grade tumors. The p53 protein functions as a transcription factor, regulating the expres- sion of several downstream genes. Two major functions of p53 are cell cycle arrest and apoptosis (reviewed in Ref. 4). Spruck et al. (5) suggested that UCCs progress via two molecular pathways, with TP53 mutations and loss of heterozygosity on chro- mosome 17 more frequent in CIS and invasive tumors. In contrast, selective deletions of chromosome 9 are more common in superficial papillary tumors. It was suggested that CIS is the most likely precur- sor of invasive tumors (5). However, the role and the position of FGFR3 mutations in current models of bladder carcinogenesis still need to be clarified. FGFR3 receptors belong to a family of highly conserved, structurally related genes, which is classified into four subtypes (FGFR1, 2, 3, and 4) that bind fibroblast growth factors with different affinity (6). These tyrosine kinase receptors regulate several cellular processes including cell growth, differentiation, migration, wound healing, and angiogenesis, and this depends on the target cell type and the developmental stage (7, 8). FGFR3 is located at 4P16.3 and comprises 19 exons spanning 16.5 Kb (9). FGFR3 activating mutations identified in thanatophoric dysphasia were also found in cervix and bladder cancer (10). Currently, it is believed that these mutations result in constitutive activation of the receptor (11). An oncogenic role has been attributed to these mutations in bladder neoplasms, whereas they have an inhibitory role in skeletal diseases (10, 12). The coding sequence of FGFR3 spanning exons 2–19 was investigated previously, and somatic mutations in bladder tumors were localized in exons 7, 10, and 15 (10, 13). FGFR3 somatic mutations were shown to be the most frequent gene mutation in low-stage bladder tumors, which underlie their importance in bladder cancer (10, 13) The high frequency of FGFR3 and TP53 mutations in superficial papillary and invasive bladder cancers, respectively, led us to compare these two genetic alteration in 81 bladder cancer tumors of all stages and grades at initial diagnosis, to determine whether they correspond to different disease pathways and could be used for molecular classification of these tumors. In this study, we screened for FGFR3 (exons 7, 10, and 15) and TP53 (exons 2–11) mutations by DHPLC analysis and sequencing in consecutive primary tumors. We found that mutations in FGFR3 and TP53 were almost mutually exclusive, and defined several groups of tumors consistent with the TNM classification in terms of tumor aggressiveness: (a) superficial papillary UCCs with FGFR3 mutations and no TP53 mutations; (b) superficial papillary UCCs with no mutation in either gene; (c) su- perficial papillary UCCs with TP53 mutations but no FGFR3 muta- tions; and (d) invasive UCCs with and without TP53 mutations. This observation provides a framework for the molecular classification of bladder cancer. Received 7/18/03; revised 9/18/03; accepted 10/16/03. Grant support: This research was funded by: INSERM, Universite ´ Paris 12-BQR, Association de la recherche contre le Cancer (ARC), De ´le ´gation a ` la recherche Clinique, Assistance Publique Ho ˆpitaux de Paris (AP-HP; PHRC AOA 94015), L’association Claude Bernard, le ministe `re du Travail, UMR 144-Laboratoire Associe ´, Ligue Nationale Contre Le Cancer, Comite ´ de Paris. 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. Requests for reprints: Dominique K. Chopin, EMI Institut National de la Sante ´ et de la Recherche Me ´dicale, 03–37, Faculte ´ de Me ´decine, Centre de Recherches Chirurgicales, 8 rue du Ge ´ne ´ral Sarrail, 94000, Cre ´teil, Cedex, France. Phone: 33-1-498-13551; Fax: 33-1-498-13552; E-mail: chopin@univ-paris12.fr. 4 The abbreviations used are: UCC, urothelial cell carcinoma; CIS, carcinoma in situ; FGFR3, fibroblast growth factor receptor 3; DHPLC, denaturing high-performance liquid chromatography; TNM, Tumor-Node-Metastasis; mut, mutated; wt, wild type. 8108 Research. on August 23, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from