Antineoplastic activity of the multitarget tyrosine kinase inhibitors CLM3 and CLM94 in medullary thyroid cancer in vitro Silvia Martina Ferrari, MSc, a Poupak Fallahi, MD, a Concettina La Motta, PhD, b Guido Bocci, MD, PhD, a Alda Corrado, MSc, a Gabriele Materazzi, MD, c David Galleri, MD, c Simona Piaggi, PhD, d Romano Danesi, MD, a Federico Da Settimo, PhD, b Paolo Miccoli, MD, c and Alessandro Antonelli, MD, a Pisa, Italy Background. We report the antineoplastic and anti-angiogenic activity of the pyrazolo[3,4-d]pyrimidine derivative CLM3 and the cyclic amide CLM94, both multiple tyrosine kinase inhibitors (TKIs), in human primary medullary thyroid cancer (P-MTC) cells, and in vitro in the medullary thyroid cancer (MTC) cell lines TT (harboring a RET C634W activating mutation) and MZ-CRC-1 (carrying the MEN2B RET mutation Met891Thr). Methods. The antiproliferative and proapoptotic effects of CLM3 and CLM94 (1, 5, 10, 30, and 50 mmol/L) were tested in P-MTC cells obtained at operation, and in TT cells. In addition, the antiproliferative effects of CLM3 and CLM94 (0.005, 0.05, 0.5, and 5 mmol/L) were tested in TT and MZ-CRC-1 cells after 7 days of treatment to compare the results with those previously reported in the literature. Results. CLM3 and CLM94 (30 or 50 mmol/L) inhibited (P < .01) the proliferation of the P-MTC cells, TT cells, and MZ-CRC-1 cells and increased the level of apoptosis in a dose-dependent manner at 10, 30, and 50 mmol/L (P < .001), while having no effect on migration or invasion. The inhibition of proliferation by CLM3 and CLM94 was similar among P-MTC cells with/without RET mutations, and similar effects were observed regarding the increased level of apoptosis. Furthermore, CLM3 and CLM94 significantly decreased vascular endothelial growth factor-A expression in TT cells. Conclusion. The antitumor activities of the multiple TKIs CLM3 and CLM94 were demonstrated in both primary MTC cultures as well as 2 established MTC cell lines in vitro, opening an avenue for future clinical evaluations. (Surgery 2014;156:1167-76.) From the Departments of Clinical and Experimental Medicine, a Pharmacy, b Surgical, Medical, Molecular Pathology and Critical Area, c and Translational Research and of New Technologies in Medicine and Surgery, d University of Pisa, Pisa, Italy MEDULLARY THYROID CANCER (MTC), a neuroendo- crine neoplasm arising from thyroid parafollicular C cells, is the third most common thyroid cancer. 1 The prognosis of MTC is poorer compared with papillary and follicular thyroid cancers, but better than anaplastic thyroid carcinoma. 2 The available treatments, such as cytotoxic chemotherapy and external-beam radiation, are essentially ineffective for advanced MTC, which can persist or recur with local or distant, often fatal, metastases. Gain-of-function mutations in the RET (REar- ranged during Transfection) kinase, such as M918T, C634W/R, or V804M/L, are specific and key oncogenic events during the development of MTC. 3 In particular, somatic RET mutations have been found in 40–50% of MTCs, and a clear rela- tionship between mutations and poor prognosis has been reported. 4 A number of other molecular alterations, such as an increased secretion of vascular endothelial growth factor (VEGF), or an augmented expression of epidermal growth factor receptor (EGFR) also play crucial roles in the tumorigenesis process of MTC. 5 Several molecules with tyrosine kinase inhibi- tory activity have been developed, some of which Accepted for publication May 12, 2014. Reprint requests: Alessandro Antonelli, Professor, Department of Clinical and Experimental Medicine, University of Pisa, Via Savi, 10, I-56126, Pisa, Italy. E-mail: alessandro.antonelli@med. unipi.it. 0039-6060/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2014.05.005 SURGERY 1167