CLINICAL STUDY BRAF V600E mutation and expression of proangiogenic molecular markers in papillary thyroid carcinomas Cosimo Durante*, Giovanni Tallini 1, *, Efisio Puxeddu 2 , Marialuisa Sponziello, Sonia Moretti 2 , Claudia Ligorio 1 , Antonio Cavaliere 3 , Kerry J Rhoden 4 , Antonella Verrienti, Marianna Maranghi, Laura Giacomelli 5 , Diego Russo 6 and Sebastiano Filetti Dipartimento di Medicina Interna e Specialita ` Mediche, Universita ` di Roma ‘Sapienza’, V.le del Policlinico, 155, 00161 Roma, Italy, 1 Dipartimento di Ematologia e Scienze Oncologiche ‘L. e A. Seragnoli’, Universita ` di Bologna, 40139 Bologna, Italy, 2 Dipartimento di Medicina Interna and 3 Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita ` di Perugia, 06126 Perugia, Italy, 4 U.O. Genetica Medica, Dipartimento di Scienze Ginecologiche Ostetriche e Pediatriche, Universita ` di Bologna, 40139 Bologna, Italy, 5 Dipartimento di Scienze Chirurgiche, Universita ` di Roma ‘Sapienza’, 00161 Roma, Italy and 6 Dipartimento di Scienze Farmacobiologiche, Universita ` di Catanzaro ‘Magna Graecia’, 88100 Catanzaro, Italy (Correspondence should be addressed to S Filetti; Email: sebastiano.filetti@uniroma1.it) *(C Durante and G Tallini contributed equally to this work) Abstract Objective: Tyrosine kinase inhibitors (TKIs) are evaluated for treatment of radioiodine refractory thyroid cancer. Their effects in this setting are based on blockade of proangiogenic signaling mediated by receptors for vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGF). Most TKIs also block other cancer-relevant kinases, such as B-type Raf kinase (BRAF), which are constitutively activated in approximately half of papillary thyroid carcinomas (PTCs), but the impact of these effects is not clear. Design: The aim of our study was to investigate the impact of BRAF V600E on proangiogenic gene expression and microvascular features of PTCs. Methods: mRNA levels for VEGFA, VEGF receptors, and coreceptors (VEGFRs 1, 2, and 3, neuropilin- 1), and PDGF receptor b (PDGFRb or PDGFRB) were measured with real-time PCR in BRAF V600E (nZ55) and wild-type BRAF (BRAF-wt; nZ35) PTCs. VEGF and VEGFR protein expression and microvessel densities (MVD) and lymphatic vessel densities (LVDs) were assessed by immuno- histochemistry in 22 of the 90 PTCs (including 11 BRAF V600E cases). Angiogenic gene expression was also studied in vitro after induction/silencing of the BRAF V600E mutation in thyrocyte lines. Results: Transcript levels of proangiogenic factors were significantly lower in BRAF V600E PTCs versus BRAF-wt PTCs (P!0.0001), but MVD and LVDs were not significantly different. VEGFA mRNA levels in thyroid cell lines decreased when BRAF V600E mutation was induced (PZ0.01) and increased when it was silenced (PZ0.01). Conclusions: Compared with BRAF-wt PTCs, those harboring BRAF V600E exhibit downregulated VEGFA, VEGFR, and PDGFRb expression, suggesting that the presence of BRAF mutation does not imply a stronger prediction of response to drugs targeting VEGF and PDGFB signaling pathways. European Journal of Endocrinology 165 455–463 Introduction Approximately 90% of differentiated thyroid cancers (DTCs) can be successfully managed with surgical resection and radioactive iodine ( 131 I) ablation (1). However, when distant metastases occur and the cancer becomes refractory to radioiodine therapy mainly for the loss of the sodium/iodide symporter expression (2), survival rates plummet, because effective alternatives to radioiodine are lacking (3, 4). Hopes have been raised by the development of novel, molecularly targeted cancer therapies, in particular tyrosine kinase inhibitors (TKIs) or agents targeting epigenetic alterations, which can block critical oncogenic signal transduction pathways (5). Two first-generation TKIs, sorafenib and sunitinib, have been approved for treatment of several solid tumors and are now being tested in patients with thyroid cancer. Promising results have already emerged from phase II studies of sorafenib for the treatment of metastatic, radioiodine-resistant thyroid cancer (6, 7), and the 2009 American Thyroid Association (ATA) guidelines for the management of DTCs strongly recommend TKIs for progressive or symptomatic metastatic disease (1). However, responses to these drugs vary and they can have serious adverse effects, so they need to be used where they are most likely to produce benefits. The TKIs undergoing clinical testing for DTCs all have antiangiogenic effects that are mediated in large part by European Journal of Endocrinology (2011) 165 455–463 ISSN 0804-4643 q 2011 European Society of Endocrinology DOI: 10.1530/EJE-11-0283 Online version via www.eje-online.org