Abstract 934: BRAF inhibitors induce skin and extra-cutaneous tumors via paradoxical activation of the MAPK pathway: Molecular study of 66 tumors and visualization of BRAF/CRAF protein dimers
Authors: Boussemart Lise , Girault Isabelle , Mateus Christine , Thomas Marina , Routier Emilie , Cazenave Hugo , Tomasic Gorana , Wechlser Janine , Kamsu-Kom Nyam , Roy Séverine , Favre Michel , Lacroix Ludovic , Eggermont Alexander , Vagner Stéphan , Robert Caroline 
Published: May 2015

Abstract
INTRODUCTION :
BRAF inhibitors (BRAFI) are affective anti-melanoma agents in V600BRAF mutant melanoma but have one major adverse event, the paradoxical activation of MAPK pathway in WT BRAF cells, which favors the emergence of benign, borderline and malignant skin cancers: verrucous papillomas, kératoacanthomas (KA), squamous cell carcinomas (SCC) and even new melanomas.
MATERIALS AND METHODS :
Between January 2010 and October 2013 we collected 63 skin tumors, 1 vulvar tumor, 1 gingival tumor, and 1 urothelial tumor in 50 patients treated with BRAFI vemurafenib or dabrafenib. Tumors were characterized histologically, p16 and P-ERK expression were studied by immunohistochemistry. Mutation hotspots for HRAS, KRAS and NRAS were investigated by Sanger analysis; human papilloma virus (HPV) DNA was also investigated and genotyped by PCR.
RESULTS:
The 63 skin tumors consisted of 35 verrucous papillomas, 9 KA, 13 SCC, and 6 melanomas. The other tumors were 1 vulvar and 1 gingival SCC, and 1 urothelial carcinoma. All tumors showed a strong nuclear pERK staining. Using Proximity Ligation Assay (PLA), we visualized, for the first time in human tumors in situ, the dimerization between BRAF and CRAF proteins in BRAFI-induced secondary tumors. BRAF-CRAF dimers were significantly less abundant in control normal skin samples and skin tumors that were not induced by BRAFI. P16 staining, often associated with HPV infection in cervical SCCs, was positive in 37% of papillomas, 78% of KA and 100% of SCC, respectively. However, the prevalence of HPV was not correlated with p16 staining: 9, 11 and 13 %, respectively (vs 25% of a series of 10 KA controls occurring in the absence of any BRAF inhibitor treatment ). HRAS mutation was found in 3% of papillomas and 56% of KA and a KRAS mutation was found in 17% of papillomas. We found NRAS mutations in the secondary melanomas, an HRAS mutation in the vulvar SCC, and a KRAS mutation in the urothelial carcinoma.
DISCUSSION
BRAFI can induce both cutaneous and extra-cutaneous secondary tumors that might have been underestimated in previous studies. Paradoxical activation of the MAPK pathway facilitated by a RAS mutation can be visualized using PLA illustrating in situ the dimerization between BRAF and CRAF. Although most keratinocytic tumors harbor clinical and histological characteristics of viral warts), despite the intensity of the P16 staining, we found a relatively low prevalence of HPV.
CONCLUSION
BRAFI are oncogenic agents that can induce both skin and extra-cutaneous cancers. Differential levels of RAF protein multimers in BRAFI-induced tumors and in control similar “spontaneous” tumors as well as the relatively low incidence of HPV despite indirect virus presence stigmata demonstrate that oncogenesis induced by BRAFI involves mechanisms distinct from previously known cancer initiation pathways.
Citation Format: Lise Boussemart, Isabelle Girault, Christine Mateus, Marina Thomas, Emilie Routier, Hugo Cazenave, Gorana Tomasic, Janine Wechlser, Nyam Kamsu-Kom, Séverine Roy, Michel Favre, Ludovic Lacroix, Alexander Eggermont, Stéphan Vagner, Caroline Robert. BRAF inhibitors induce skin and extra-cutaneous tumors via paradoxical activation of the MAPK pathway: Molecular study of 66 tumors and visualization of BRAF/CRAF protein dimers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 934. doi:10.1158/1538-7445.AM2014-934