Molecular and Cellular Pathobiology Secondary Tumors Arising in Patients Undergoing BRAF Inhibitor Therapy Exhibit Increased BRAF–CRAF Heterodimerization Lise Boussemart 1,2 , Isabelle Girault 2 ,H el  ene Malka-Mahieu 2,3 , Christine Mateus 1 , Emilie Routier 1 , Margot Rubington 2 , Nyam Kamsu-Kom 2 , Marina Thomas 1 , Gorana Tomasic 4 , Sandrine Agoussi 2 , Marie Breckler 5 ,M elanie Laporte 5 , Ludovic Lacroix 2,5 , Alexander M. Eggermont 1 , Andrea Cavalcanti 6 , Florent Grange 7 , Julien Adam 2,8 , St  ephan Vagner 2 , and Caroline Robert 1,2,3 Abstract BRAF inhibitors (BRAFi) elicit therapeutic responses in met- astatic melanoma, but alarmingly, also induce the formation of secondary benign and malignant skin tumors. Here, we report the emergence and molecular characterization of 73 skin and extracutaneous tumors in 31 patients who underwent BRAFi therapy. The majority of patients presented with classic epider- mal tumors such as verrucous papillomas, keratoacanthomas, and squamous cell carcinomas (SCC). However, 15 patients exhibited new or rapidly progressing tumors distinct from these classic subtypes, such as lymph node metastasis, new melano- mas, and genital and oral mucosal SCCs. Genotyping of the tumors revealed that oncogenic RAS mutations were found in 58% of the evaluable tumor samples (38/66) and 49% of the control tumors from patients not treated with BRAFi (30/62). Notably, proximity ligation assays demonstrated that BRAF– CRAF heterodimerization was increased in fixed tumor samples from BRAFi-treated patients compared with untreated patients. Our findings reveal that BRAF–CRAF complex forma- tion is significantly associated with BRAFi treatment, and may therefore serve as a useful biomarker of BRAFi-induced cuta- neous and extracutaneous tumor formation. Cancer Res; 76(6); 1476–84. Ó2016 AACR. Introduction Activating BRAF mutations occur in about 7% of human malignancies and in approximately 40% of melanomas (1). BRAF inhibitors, vemurafenib and dabrafenib, induce objective tumor responses in 50% to 60% of the patients with BRAF V600 meta- static melanoma, and prolong their overall survival compared with previous standard chemotherapy (2). However, BRAF inhi- bitors have two major drawbacks. First, their action is limited over time with most patients developing secondary resistance in 6 to 8 months (3). Second, they are frequently associated with the emergence of multiple benign and malignant skin tumors: pap- illomas, keratoacanthomas (KA), squamous cell carcinomas (SCC), and more rarely with atypical pigmented nevi and even new melanomas (4). These rather unexpected adverse events had already been reported with the use of a previous less potent and less selective RAF inhibitor, sorafenib (5) that was used for its anti- VEGFR properties. The hypothesis that skin tumors arising with sorafenib were linked to the anti-RAF effect of the drug has already been demonstrated in vitro on keratinocytes that showed para- doxical activation of the MAPK pathway via CRAF activation associated with the presence of BRAF/CRAF heterodimers in the presence of sorafenib (6–8). As might have been expected from these data, more potent and more selective BRAF inhibitors, such as vemurafenib and dabrafenib, which became available some years later, gave rise to a higher incidence of new skin tumors, occurring in up to 15% to 20% of the patients. These secondary tumors harbor a RAS mutation in 21% to 60% of the cases (9, 10). In line with these findings, in vitro data on melanoma cell lines harboring an NRAS mutation but wild type for BRAF, showed paradoxical MAPK pathway activation in the presence of BRAF inhibitors. In these models, the signal was transduced via CRAF, itself transactivated via its dimerization with BRAF (11–13). As RAS mutations can occur in various benign tumors from diverse tissues and organs, the worrisome question of whether extracutaneous cancer could be induced by a BRAF inhibitor 1 D epartement de m edecine oncologique, Gustave Roussy, Villejuif, France. 2 INSERM, U981, Villejuif, France. 3 Facult e de M edecine, Uni- versit e Paris-Sud, Le Kremlin Bicetre, France. 4 D epartement de patho- logie, Gustave Roussy, Villejuif, France. 5 Laboratoire de Recherche Translationnelle et Centre de Ressources Biologiques, AMMICA, INSERM US23/CNRS UMS3655, Gustave Roussy, Villejuif, France. 6 D epartement de chirurgie, Gustave Roussy, Villejuif, France. 7 D epartement de Dermatologie, CHU de Reims, Reims, France. 8 Mod- ule de D eveloppement en Pathologie, SIRICSOCRATE, Gustave Roussy, Villejuif, France. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). L. Boussemart and I. Girault are co-first authors for this article; S. Vagner and C. Robert share senior co-authorship for this article. Current address for L. Boussemart: D epartment de Dermatologie, CHU de Rennes, Université de Rennes, CNRS UMR6290, F-35000, Rennes, France; and current address for S. Vagner, CNRS UMR3348, Institut Curie, Orsay, France. Corresponding Author: Caroline Robert, Gustave Roussy, 114 rue Edouard Vaillant, 94800 Villejuif 94800, France. Phone: 331-4211-4210; Fax: 331-42-11- 5002; E-mail: caroline.robert@gustaveroussy.fr doi: 10.1158/0008-5472.CAN-15-2900-T Ó2016 American Association for Cancer Research. Cancer Research Cancer Res; 76(6) March 15, 2016 1476 on June 5, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 29, 2016; DOI: 10.1158/0008-5472.CAN-15-2900-T