Thyroid International

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1 Thyroid International Edited by Peter PA Smyth, UCD, Dublin Published by Merck KGaA, Darmstadt, Germany Gothic Church Santa Maria della Spina Targeted Therapies For Thyroid Cancer David Viola and Rossella Elisei

2 Disclaimer This scientific publication is published by and with financial support from Merck KGaA, Darmstadt, Germany subject to the requirement that article(s) published are of a scientific nature. The views expressed by the author(s) do not necessarily reflect the views, ideas or policy of Merck KGaA. This publication is provided for general reference only and is intended solely for healthcare professionals. As a result of ongoing medical advances and developments, the information in this material may not always be completely up to date and, for this reason, such information is provided on an "as is" and "as available" basis. Merck KGaA makes no warranties, representations or gives any undertakings either express or implied about any content of this publication. It may refer to pharmaceutical products, therapeutics or indications not registered or approved in a given country. Please always refer to the full prescribing information applicable in your country for any pharmaceutical product. Registration conditions, warnings and precautions for pharmaceutical products differ from country to country. Copyright in this publication is expressly owned by Merck KGaA and/or its Affiliates (except for any third party content which has been identified as such). All rights reserved. Production Date: December 2013

3 Thyroid International Targeted Therapies For Thyroid Cancer David Viola and Rossella Elisei Address for Correspondence: David Viola, Endocrine Unit, Department of Clinical and Experimental Medicine WHO Collaborating Center for the Study and Treatment of Thyroid Diseases and other Endocrine and Metabolic Disorders, University of Pisa Via Paradisa, 2 Pisa 56124, Italy violadavid@hotmail.it

4 2 Thyroid International David Viola was born in 1976 in Pisa, Italy. In 2005 he graduated in Medicine at the University of Pisa and in 2010 got his specialty degree in Endocrinology and Metabolism at the Department of Endocrinology and Metabolism of Pisa. He was trained under the mentorship of Prof. Aldo Pinchera and Prof. Rossella Elisei at the same University. He is the author of over 15 articles published in impacted and peer-reviewed journals and over eighty conference papers. His work was recognized with oral presentations and awards at national and international meetings. In 2012 he was awarded the European Society of Endocrinology International Endocrine Scholar Program that spent as a basic researcher at the Institute of Biomedical Research Alberto Sols, a center from the Spanish National Research Council and the Autonomous University of Madrid. His research activity is dedicated to oncogenes, thyroid carcinogenesis and their prognostic and therapeutic implications. Since 2007 he has been an investigator in several phase II/III clinical trials on drugs for advanced thyroid cancer treatment. H t Thyr idology ETA s journal on hot and controversial topics Free access: Thyroid International Editor-in-Chief: Peter PA Smyth, UCD, Dublin This is the title of a publication series published by and with financial support from Merck KGaA, Darmstadt, Germany. The Editor-in-Chief is publishing papers from renowned international thyroid experts in order to pass on the extensive experience which the authors possess in their field to a wide range of physicians dealing with the diagnosis and ther apy of thy roid dis eases. Please refer to the full disclaimer on the inside front cover. Respon sible at Merck KGaA, Darmstadt, Germany: Gernot Beroset Thyroid International Merck KGaA, Darmstadt, Germany, D Darm stadt ISSN Rossella Elisei, MD Education: Degree: April 3, 1985 M.D. degree (magna cum laude), University of Pisa, Italy Speciality degree in Endocrinology : July 9, 1988 (magna cum laude), University of Pisa,Italy. Present Professional Position: Associate Professor of Endocrinology, Department of Endocrinology, University of Pisa, Italy. Research Experience In Foreign Institutions: January 1989-December 1990: Postdoctoral Research Fellow at the I.R.I.B.H.N, Endocrinology Branch, Université Libre de Bruxelles, Belgium. March 1995-June 1995: Postdoctoral Assistant, Division of Endocrino logy, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA, USA. July 1995-June 1997: Postdoctoral Assistant, Division of Endocrinology and Metabolism, University of Cincinnati, College of Medicine, Cincinnati, OH, USA. January 1998: participant of the CEC Course on Current Techniques in Radiation Mutagenesis, at the MGC Dept of Radiation Genetics and Chemical Mutagenesis of Leiden University, The Netherlands. Professional International Awards: 2006 International award for excellence in published clinical research in the Journal of Clinical Endocrinology and Metabolism. Title of the awarded publication: Radioiodine ablation of thyroid remnants after preparation with recombinant human thyrotropin in differentiated thyroid carcinoma: results of an international, randomized, controlled study. Vol 91: The ETA (European Thyroid Association) Max Pierre Konig Poster Award in recognition of the best poster in clinical thyroidology titled: Patients with DTC who underwent radioiodine remnant ablation with low activity of 131-I either with rhtsh or after LT4 withdrawal show the same outcome after 10 years follow up Light of Life Foundation international award dedicated to the best thyroid cancer researcher of the year, Sloan Kettering Memorial Cancer Center di New York, February 7th, International award for excellence in published clinical research in the Journal of Clinical Endocrinology and Metabolism. Title of the awarded publication: The timing of total thyroidectomy in RET gene mutation carriers could be personalized and safely planned on the basis of serum calcitonin: 18 years experience at one single center. Vol 97: Publications: Author of 352 publications including 132 papers in Peer-Reviewed Journals, 50 manuscripts in Proceedings of Meetings and Book Chapters and 172 Abstracts for National and International Meetings. Clinical Trials: From 1997 up to 2007: subinvestigator in 4 clinical trials on drugs for thyroid cancer treatment From 2005 to the present: Principal Investigator in 10 phase II/III clinical trials on drugs for thyroid cancer treatment, both medullary and dedifferentiated thyroid cancer.

5 Targeted Therapies For Thyroid Cancer 3 Abstract Thyroid cancer is the most common endocrine malignancy. In the majority of cases the disease is curable by means of surgery and, when appropriate, by radioiodine treatment. However, some thyroid tumors became radioiodine-refractory (RAI-R) or they are RAI-R from the beginning because of the lack of differentiation or because they originate from parafollicular C cells (medullary thyroid cancer). Until 2011 no effective therapeutic options were available for these tumors. Moreover anaplastic thyroid cancer that is by definition RAI-R is, despite the multidisciplinary approach which includes surgery, external beam radiotherapy and chemotherapy, almost invariably lethal. Currently, novel therapeutic agents targeting the critical oncogenes (e.g. BRAFV600E and RET) and pathways (e.g. MAPK and PI3K/AKT-mTOR) involved in thyroid cancer pathogenesis and tumor progression have been developed. These drugs known as tyrosine kinase inhibitors (TKI) bind to membrane receptors and determine a growth arrest by blocking tumoral cell proliferation. This review offers an overview of the state of the art on the use of targeted therapies in any type of advanced, progressive and RAI-R thyroid tumors. Introduction Thyroid cancer is the most common endocrine malignancy and accounts for 1% of all human malignancies. The incidence of differentiated thyroid carcinomas (DTC) has increased in the last decades and it is estimated to reach 60,220 new cases in 2013 in United States becoming the fifth most common tumor in females and the third as rate of increase. 1-3 The majority of these tumors (85%) are well differentiated whereas a small proportion (about 5-7%), that loose the features of the cell of origin, are classified as poorly differentiated (PDTC) or undifferentiated/anaplastic (ATC) thyroid carcinomas depending on the grade of differentiation. DTC are then distinguished into papillary (PTC, 85%) and follicular (FTC,15%) thyroid carcinoma according to the morphological features. Both of them arise from follicular cells and maintain their physiological features as they continue to produce thyroglobulin (Tg), take up and organify iodine, and to be sensitive to thyroid stimulating hormone (TSH). About 5-8% of all thyroid carcinoma are represented by medullary thyroid cancer (MTC) derived from parafollicular or calcitonin-producing C cells. Because of its origin, MTC are completely different from DTC: they do not produce Tg but calcitonin (Ct), they are not able to take up iodine and are not responsive to TSH. DTC are the most curable cancers by means of surgery and radioactive iodine (131I) therapy. Only 5-10% of them have distant metastases at diagnosis which is, together with an advanced age, the most unfavorable prognostic factor. 4 Moreover, about 10-15% of PTC and FTC are or became radioiodine-refractory (RAI-R) thus representing the real therapeutic challenge at the present together with PDTC and ATC. Fortunately, with the exception of ATC, patients with an early diagnosis and adequate treatment can have a quite long survival despite the persistence of the disease. The median survival is much dependant on the degree of differentiation being of only 6 months in ATC, 5 years in PDTC and 10 years in dedifferentiated DTC. In this last group a very important poor prognostic factor is represented by the ability of metastatic lesions to take up 18-FDG at PET rather than 131-I at whole body scan. 5 Until recently no therapeutic options were available for patients with advanced RAI-R thyroid cancer. Conventional therapies such as external beam radiotherapy (EBRT) and/or chemotherapy may be attempted but there are numerous evidences of low efficacy and significant toxicity so that nowadays they are used only in selected cases. 6

6 4 Thyroid International The novel insights in the pathogenesis, differentiantion and cell proliferation of thyroid cancer and the recent successful development of the first tyrosine kinase inhibitor (TKI) imatinib created an interest in selectively targeting cancer cells while sparing normal ones. Targeted cancer therapies are drugs that block the growth of cancer by interfering with specific molecules involved in tumor growth. Targeting molecular and cellular changes that are specific to cancer may be more effective than other types of treatment, including classical chemotherapy and EBRT, and may be less harmful to normal cells. Many targeted cancer therapies have been already approved by the U.S. Food and Drug Administration (FDA) for the treatment of specific types of human cancer, many others are still under study in clinical trials, and many more are in preclinical testing (for clinical trials please visit the site Targeted cancer therapies are being studied in monotherapy and less extensively, in combination with other targeted therapies, or in combination with other anticancer treatments such as chemotherapy. Table 1 Drugs Main Molecular Targets Axitinib VEGFR 1-3; PDGFRβ and c-kit Cabozantinib VEGFR-2; MET; RET and c-kit Imatinib Bcr-Abl; c-kit and PDGFR Lenvatinib VEGFR 1-3; FGFR 1-3; RET, c-kit, PDGFRβ and RET gene fusions Motesanib VEGFR 1-3; PDGFR; c-kit and RET Pazopanib VEGFR 1-3; PDGFR and c-kit Selumetinib MEK Sorafenib VEGFR-2,-3; PDGFR; Raf and c-kit Sunitinib VEGFR 1-3; PDGFR; c-kit and RET Vandetanib VEGFR-2; EGFR; c-kit and RET Vemurafenib BRAFV600E kinase, CRAF Bcr-Abl: Abelson and breakpoint cluster region fusion gene; CSF-1R: colony stimulating factor 1 receptor; EGFR: epidermal growth factor receptor; FGFR: fibroblast growth factor receptor; KIT: v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene; Raf: v-raf murine sarcoma viral oncogene homolog; BRAFV600E: valine to glutamic acid substitution of BRAF gene; CRAF: v-raf murine sarcoma viral oncogene homolog 1; MEK: mitogen activated protein kinase kinase; MET: hepatocyte growth factor [HGF] receptor; PDGFR: platelet-derived growth factor receptor; RET: Rearranged during Transfection receptor; RET gene fusions, KIF5B-RET, CCDC6-RET and NcoA4-RET; VEGFR-1,-2,-3: vascular endothelial growth factor receptor rs. The two main categories of targeted therapies are small molecules such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mabs). TKIs usually cannot penetrate the cell s plasma membrane and are generally directed against receptors with tyrosine kinase activity that are on cell surface and participate in signal transduction. These drugs could bind specifically to one or multiple receptors with tyrosine kinase activity, with different affinities for each receptor. The other target of these drugs are proteins (i.e. Bcr-Abl) with tyrosine kinase activity (i.e. imatinib). As shown in Table 1 the majority of these drugs are multikinase inhibitors and it is likely that their activity is achieved by the inhibition of more than one target receptor: in particular they can exert their antineoplastic activity both as antiangiogenic and antioncogenic drugs. Only a few drugs are monotarget like selumetinib which specifically inhibit mitogen activated protein kinase (MEK). A different mechanism of action is operated by mabs since they can interfere with growth factors such as bevacizumab that binds and blocks VEGF, or can facilitate the triggering of the immune system to destroy cancer cells and/or induce apoptosis such as rituximab that recognizes CD20 or can deliver radiolabeled molecules specifically to cancer cells such as tositumomab and ibritumomab against CD20. At the present, the most studied drugs in thyroid cancer field are TKIs and the rationale for their use rely in the fact that several targets of these drugs are the products of the oncogenes involved in thyroid cancer pathogenesis and disease progression such BRAFV600E, RAS point mutations, RET-PTC rearrangement, Met overexpression etc. It is well known that these alterations promote thyroid follicular cell growth, tumoral transformation and proliferation through the activation of the MAP kinase pathway and thus their inhibition, as determined by several TKIs, can result in a growth arrest by blocking tumoral cell proliferation. 8 Another important target in cancer are kinases involved in angiogenesis such as vascular endothelial growth factor receptors (VEGFRs). Many TKIs are also able to inhibit the activity of VEGFRs thus acting as antiangiogenic drugs. It is very likely that the combination

7 Targeted Therapies For Thyroid Cancer 5 of both actions (i.e. anti-vegfr and anti-specific tyrosine kinase receptors) makes these drugs particularly useful for the treatment of advanced thyroid cancer. 9 Although the majority of currently available TKIs are directed against receptors involved in the MAP kinase pathway, new TKIs able to interfere with molecules belonging to the other important pathway for the development of thyroid tumors that is the PI3K/AKT/mTOR pathway are now available. 10 This pathway is also activated in some thyroid cancer and is closely connected to the MAP kinase pathway (Fig. 1). In particular, two small molecules, everolimus and temsirolimus, that inihibit the serine/threonine kinase called mtor (mammalian target of rapamycin), have recently been approved by the Food and Drug Administration (FDA) for the treatment of subependymal giant cell astrocytoma (everolimus), in patients who have tuberous sclerosis and cannot be treated with surgery, and of advanced renal cell carcinoma, in patients who have already been treated with sunitinib or sorafenib with no benefit (everolimus and temsirolimus). In this review, we report on the results of the most important clinical trials performed to date in different histological types of thyroid tumors employing the new targeted drugs. We offer an overview of the state of the art on the use of targeted therapies in any type of advanced, progressive and radioiodine refractory thyroid tumors. Figure 1: MAP kinase and PI3K/AKT/mTOR pathways are both involved in thyroid proliferation: TKI molecular targets and specific drugs able to inhibit them are shown. HGF VEGF GDNF Cell membrane MET VEGFR RET RAS Cabozantinib PI3K RAF Cabozantinib Lenvatinib Vandetanib PDK1 AKT Axitinib Lenvatinib Motesanib Pazopanib Sorafenib Suntinib Vandetanib Sorafenib MEK ERK Vemurafenib mtor Selumetinib Nuclear membrane Everolimus Temsirolimus Cell growth, proliferation and survival Inhibition of apoptosis Invasion and metastases

8 6 Thyroid International Differentiated Thyroid Carcinoma After the initial surgical treatment, if needed, DTC are treated with radioactive iodine (131I). Unfortunately, about 10-15% of DTC cannot be cured with 131I because the tumoral cells lose completely or in part the capacity to take up iodine (131I). The high frequency of BRAFV600E mutation found in 29-69% of PTC, in 13-35% of PDTC 11 as well as in 44% of ATC and in 95% of metastatic RAI-R thyroid cancer 12 have made this mutation an attractive target for TKI therapy in advanced thyroid cancer. The presence of this mutation in fact was clearly associated to persistent/ recurrent disease and mortality In addition, the presence of other genetic abnormalities (RAS mutation, RET/PTC and NTRK1 rearrangements) and overexpression of some tyrosine kinase receptors (VEGFR, EGFR, MET and FGFR) led to investigate the therapeutic role of TKIs in these tumors. 9 The phase II trial of motesanib used in both RAI-R DTC and MTC was the first one to be conducted and published. 16 Motesanib is a multikinase inhibitor of VEGFR, PDGFR, c-kit and RET. Among the 93 DTC patients enrolled, 14% showed a partial response (PR), while the stabilization of the disease (SD) was achieved in 67% of the patients and it was maintained for 24 weeks or longer in 35% while only 8% had progressive disease (PD) as the best response. The estimate of median progression-free survival (PFS) was 40 weeks. 16 It is worth to note that this was the first drug showing good results in terms of PR (14%) and clinical benefit (49%) which was never reached before with conventional chemotherapies. However, even if the study was completed in 2006 the drug is still not commercially available and no other phase III studies have been started. Axitinib is a multikinase inhibitor with activity predominantly against VEGFR. In a phase II study for all histological subtypes (PTC, FTC, MTC and ATC), 18/60 (30%) enrolled patients experienced PR and 23/60 (38%) SD for more than 4 months. In the PTC subpopulation PR was achieved in 8 cases (8/22, 36%), SD in 12 (12/22, 55%) and PD in 2 (2/22, 9%). In the FTC subpopulation PR was achieved in 6 cases (6/14, 43%), SD in 7 (7/14, 50%) and PD in 1 (1/14, 7%). 17 Despite the good percentage of clinical benefit (i.e. PR+SD) observed in this study, neither motesanib or axitinib are not currently commercially available. The activity of sunitinib, a TKI that targets VEGFR, PDGFR and c-kit, was evaluated in a phase II study on 35 subjects (7 MTC, 28 DTC) treated with 37.5 mg daily dose administered on a continuous basis. The median duration of treatment for all patients was 8.5 months. Table 2: Response Evaluation Criteria in Solid Tumors (RECIST) Target Lesions Response Category Complete Remission (CR) Partial Remission (PR) Stable Disease (SD) Progressive Disease (PD) Definition disappearance of all target lesions 30% decrease in the sum of the longest diameter neither PR nor PD criteria met 20% increase in the sum of the longest diameter Non-Target Lesions Response Category Complete Remission (CR) Stable Disease (SD) Progressive Disease (PD) Definition disappearance of all Non-target lesions persistence of one or more non-target lesions one or more new lesions and/or unequivocal progression of existing non-target lesions

9 Targeted Therapies For Thyroid Cancer 7 Among the 26 DTC patients who could be evaluated for disease response, the objective response rate (ORR) per Response Evaluation Criteria In Solid Tumors (RECIST) (Table 2) was present in 8 out of 26 patients (31%). In particular, 1 CR (4%), 7 PR (27%), 14 SD (54%) and 4 PD (15%) were observed. 18 Once again, the percentage of the overall clinical benefit was very interesting but at present sunitinib has not yet been approved for thyroid cancer treatment and can be used both in the USA and EU only for the treatment of renal cell carcinoma and imatinib-resitant gastrointestinal stromal tumor. Pazopanib is a TKI targeting VEGFR, PDGFR and c-kit. The efficacy and safety of this drug was evaluated in a phase II study in 39 patients with metastatic, rapidly progressive (within 6 months from enrolment) RAIrefractory DTC (papillary, Huerthle cells and follicular). Of the thirty-seven patients that were evaluable for response 18 had PR (49%), 8/11 (73%) were FTC, 5/11 (45%) were affected by Huerthle cell tumors and 5/15 (33%) were PTC. The median duration of PFS was 11.7 months and the median overall survival at one year was 81%. 19 An international phase II study of pazopanib in patients with advanced thyroid cancer is currently recruiting participants. Lenvatinib is an oral TKI that targets VEGFR receptors, fibroblast growth factor receptors (FGFR) 1-3, RET, c-kit, and PDGFRβ. Recently it was demonstrated to inhibit the autophosphorylation of three RET gene fusions, KIF5B-RET, CCDC6-RET and NcoA4-RET that represents the most frequent rearrangement involved in PTC pathogenesis. 20 In a phase II study, RAI-R advanced DTC patients progressing within 12 months were treated with 24 mg once daily until disease progression or unacceptable toxicity. Among the 58 patients enrolled and assessable for response 29 (50%) had PR. Response rate was higher for patients that were naive (54%) compared to patients previously treated with VEGFR-targeted drugs (41%). The median PFS was 12.6 months. 21 The good antitumor activity and acceptable toxicity profile observed in this phase II study led to a phase III multicenter, randomized, double-blind placebo-controlled trial in RAI-R progressive DTC that is still ongoing although the recruitment of patients was completed several months ago. An interim analysis of the data is in process and should be published rather soon. The most studied TKI in RAI-R thyroid cancer is sorafenib, an inhibitor of VEGFR, PDGFR, BRAF, RET, and c-kit whose efficacy was explored in four phase II studies The good results in terms of safety and efficacy in monotherapy in these phase II studies promoted the development of a phase III international, randomized, controlled clinical trial versus placebo in advanced/metastatic RAI-R DTC (DECISION study). 26 The results of this trial were presented at the 2013 American Society of Clinical Oncology ASCO meeting. A total of 417 patients with locally advanced or metastatic RAI-refractory DTC that had progression within the previous 14 months as defined by RECIST criteria and that did not undergo previous treatment with targeted therapy or chemotherapy were enrolled and randomly assigned 1:1 to take 400 mg of sorafenib or placebo, orally twice daily. Disease progression was assessed every 8 weeks, and in the case of disease progression on placebo, patients were allowed to cross over to the sorafenib arm. The treatment and study arms were well balanced regarding all the clinical-pathological features and previous radioiodine activity administered. The study met its primary endpoint in terms of PFS, with a statistically significant difference in the hazard ratio in the sorafenib arm compared to the placebo arm (10.8 vs 5.8 months; HR= [95% CI, ]). The median overall survival (OS) that was a secondary endpoint of the study has not been reached yet, however it will be affected by the large proportion of patients in the placebo arm (71%) who have crossed over to treatment during the study. Nevertheless additional analyses of OS have been planned. At the end of the study PR was observed in 12.2% of patients in the sorafenib arm compared to 0.5% in the placebo arm, the median duration of PR being 10.2 months. No patient experienced CR. Interestingly sorafenib treatment was able not only to reduce target lesion size in 73% of patients compared with 27% in the placebo arm but this shrinkage was often sufficient to alleviate disease related symptoms in many patients. 27 The results of sorafenib treated patients in monotherapy make it the best option for advanced, metastatic RAI-R DTC patients at the present.

10 8 Thyroid International However, similar to other TKI, after a variable period of PFS many patients experience disease progression (i.e. escape phenomenon). For this reason the role of sorafenib in combination with other agents should be explored since the combination to other agents may help to overcome this mechanism of escape and hopefully could help to re-sensitize these cells to radiation. Moreover its capacity to induce apoptosis in different tumors could be used to potentiate the cytotoxic effects of classical chemotherapies that for many years were considered not adequate and can help in reducing the dose of the drugs and alleviate their side effects. Unfortunately up to now the degree to which this synergism can help in reducing toxicities and increasing the efficacy remains to be defined. To our knowledge the only data available on the combination of sorafenib with other targeted drugs is a phase I study in association with a farnesyltransferase inhibitor, tipifarnib, in both DTC and MTC. Each 28 day cycle consisted of the administration of 28 days of sorafenib and 21 days of tipifarnib. The majority of DTC patients (17 out of 22) had progression determined by RECIST before study treatment. The patients were quite heterogeneous regarding histology: 16 PTC, 5 FTC, and 1 PDTC. In the DTC subgroup, 1 out of 15 (7%) of subjects was in PR while 8 out of 15 (53%) were in SD at 6 months. The median time to treatment failure was 9 months with a median PFS of 20 months. Likewise all other study treatments the median OS has not been reached. 28 Despite the good efficacy of this combination these results were not better than the results previously reported in the phase II trials with sorafenib in monotherapy. affected by BRAFV600E positive progressive DTC. In fact vemurafenib is a potent inhibitor of BRAFV600E kinase and at higher concentrations also of CRAF. In the first phase I study in solid tumors, among the three patients affected by PTC one experienced PR with a duration of response of 7.6 months and a time to progression of 11.7 months, the other two patients experienced SD with a time to progression of 13.2 and 11.4 months. 29 For this reason an open-label, multicenter exploratory phase II study is ongoing to evaluate the clinical activity of this potent selective RAF inhibitor in patients with metastatic or unresectable PTC positive for the BRAFV600E mutation. The most recent and one of the most promising drugs under evaluation for DTC is selumetinib. This is a TKI that inhibits the activity of the mitogen activated protein kinase kinase (MAPKK), also known as MEK, that is one of the kinases in the mitogen activated protein kinase (MAPK) pathway. This inhibition was recently demonstrated to increase RAI uptake in patients affected by RAI-R thyroid cancer. 30 The impressive results obtained in that pilot study determined the activation of an international multicentric phase III study to explore the ability of selumetinib to potentiate the 131-I ablation activity. The study has started few weeks ago and it is expected to give results in months. Although a specific activity of the drug on the tumoral growth cannot be completely excluded the possibility to revert and/ or potentiate the ability to take up iodine may suggest a greater use of this drug compared to the others. More recently, the good results in some phase I and phase III studies of vemurafenib in patients affected by BRAFV600E melanoma sparked the interest in patients Anaplastic Thyroid Carcinoma Prognosis of ATC is almost invariably fatal due to incomplete surgical resection or inoperability. This is due to its rapid growth and infiltration of surroundings tissue such as vessels, trachea, esophagus and larynx. Moreover when the tumor is extrathyroidal the surgical approach is still controversial and definitively not curative. 31 The proof of ATC aggressiveness is emphasized by the fact that the median overall survival is about 6 months and has not improved over the last 50 years and the possibility to cure ATC patients is still a dream at the present. 32 The only therapeutic agent approved for metastatic radioiodine refractory thyroid cancer since 1974

11 Targeted Therapies For Thyroid Cancer 9 is doxorubicin that, due to the poor results obtained in patients with unresectable tumors, has no more or little use in this setting. For this reason the ATC patients who are in good general condition should be considered for surgery (if possible) and, soon after, for clinical trials with new and experimental drugs. Several targets such as tyrosine kinase receptors, PPARgamma rearrangements and Aurora kinases were hypothesized to be involved in ATC pathogenesis and disease progression. However despite the demonstration of the inhibitory activity of some drugs that target the above mentioned molecular alterations in ATC cell lines and mice xenografts in terms of inhibition of proliferation and induction of apoptosis only a few of them were tested in clinical studies involving a few ATC patients with unsatisfactory results. Because of the overexpression and overactivation of c-kit and PDGFR in ATC, in 2009 some Authors investigated the clinical efficacy of imatinib in a phase II study in which 11 patients affected by advanced ATC were enrolled. Among those evaluable for response, 2 patients (25%) experienced PR and 4 (50%) SD with a 6-month PFS of 36% and a 6-month OS rate of 45%. 33 The presence of BRAFV600E mutation was demonstrated in about 30% of ATCs. In a preclinical study sorafenib was demonstrated to inhibit the growth of ATC xenografts and improve survival. These data led to design a phase II study with sorafenib in 20 patients affected by ATC. Among them 2 patients (10%) experienced PR and 5 (25%) SD with an overall median PFS of 1.9 months. 34 The evidence that fosbretabulin (combretastatin-a4; CA4P), a novel vascular disrupting agent, was able to elicit microtubule depolymerization, endothelial cell mitotic arrest and apoptosis, promoted its use in ATC preclinical models. This agent, caused significant cytotoxicity in eight human ATC cell lines and in xenograft tumors from four ATC cell lines when injected in athymic nude mice. 35 These data were confirmed in an openlabel Phase II study in patients with advanced/relapsed ATC, in which approximately a quarter of patients treated with single-agent CA4P experienced a progression free survival of more than 3 months. The promising results of these studies stimulated the design of a multicenter, open-label, randomized, phase II/III trial to evaluate the safety and efficacy of CA4P in combination with paclitaxel and carboplatin (CP) in comparison with CP alone in ATC patients. Twenty-six patients received fosbretabulin 45 mg/m2 as an intravenous infusion on days 1, 8, and 15 of a 28-day cycle and the treatment continued until disease progression. Despite the acceptable safety profile of this drug there were no PR and no CR. However SD was obtained in 7 patients (26%) with a median duration of SD of 12.3 months. The median survival was 4.7 months and with 23% of the patients that survived at 1 year. 36 Similar results were obtained in a very large study in which 80 patients were enrolled: median OS of 5.2 months for those treated with chemotherapy in combination with fosbretabulin (CP+fosbretabulin) and 4.0 months for those treated with chemotherapy alone (CP) (p=0.22). One-year survival for CP+fosbretabulin was 26% compared to 9% for CP. However no significant difference in PFS was observed in the two arms 37 likely due to the relatively low number of cases enrolled. More recently another phase II trial investigated the efficacy of sorafenib in 20 patients with ATC who had failed on previous therapies (classical cytotoxic chemotherapy with or without fosbretabulin). Partial response was obtained in 2 out of 20 patients (10%) and in 5 (25%) had stable diseases. The median duration of response was 4 months in patients with SD and 10 and 27 months in the two patients with PR. Progression free survival was 15% at 6 months and 10% at 12 months with a median of 1.9 months. Overall survival was 30% at 6 and 20% at 12 months with a median of 3.9 months. Interestingly the patient that experienced PR on sorafenib for 10 months had previously progressed on fosbretabulin. 34 The safety, tolerability and progression free survival (PFS) of a similar drug, Crolibulin (EPC2407), that derives from CA4P was investigated in combination with cisplatin in a phase I/II study. This is a microtubulin inhibitor that has been shown to have direct antitu-

12 10 Thyroid International mor effects in vivo and in vitro, destabilizing spindles and inducing apoptosis, resulting in the disruption of neovascular endothelial cells with disruption of blood flow to the tumor. The results of this trial were recently presented at the Annual Meeting of the American Society of Clinical Oncology (ASCO 2013). Twenty-one patients with solid tumors (16 ATCs) were enrolled and were administered cisplatin and crolibulin intravenously up to 100 and 20 mg/m2 respectively. Among the 8 ATCs patients that were treated at dose level 3 (100/20 mg/m2), 1 (13%) experienced CR and 1 (13%) SD while 3 (38%) had PD and 3 were not evaluable. The patient with CR was on study for more than 12 months and remained on crolibulin alone. 38 At present, no new drugs have been approved for the treatment of ATC patients and this thyroid tumor remains a lethal disease. However international guidelines suggest that these patients be treated using a multidisciplinary approach including new drugs which can at least improve the local symptoms and quality of life. 39 Medullary Thyroid Carcinoma The definitive cure of MTC is possible if precocious diagnosis and early surgical treatment is performed. 40 However in the majority of cases MTC metastasizes early in its clinical course without giving any signs or symptoms. Classical chemotherapy for persistent/recurrent MTC has shown limited efficacy. For this reason the use of standard chemotherapeutic agents is not recommended as first-line therapy and patients should be addressed to specialized centers to be enrolled in clinical trials or to use of TKIs. 41 After the identification of RET (REarranged during Transfection) mutation in hereditary MTC and the evidence that this mutation has a key role in the development of MTC some Authors demonstrated that the inhibition of RET has an antitumor activity In addition to RET mutation, other tyrosine kinase receptors (TKRs) such as EGFR, VEGFR, MET and FGFR4 were demonstrated to be overexpressed in MTC and became attractive therapeutic targets in MTC patients, either RETpositive or negative neoplasms. 44, 45 Imatinib was the first TKIs investigated for MTC treatment but did not meet the expected results, in fact, only transient stable disease was achieved and only in some patients. 46 Another published phase II study investigated the effectiveness of motesanib in patients with PD within 6 months or symptomatic disease. Partial remission was observed in only 2% of patients and SD in 81% of cases but only 48% of these patients had a durable response 24 weeks. Moreover considering that MTC has a slow progressive behaviour in the majority of cases and that some patients were enrolled for symptomatic disease and not for disease progression, the high rate of SD could not be correctly interpreted. 47 Sunitinib, a TKI with activity predominantly against RET, VEGFR2, PDGFR, and c-kit, was investigated in an open-label multicenter phase II trial conducted in 7 patients with MTC treated with a continuous daily administration of 37.5 mg. Among the 7 MTC patients who were evaluable for disease response the ORR per RECIST was present in 3 out of 6 patients (50%). Three patients experienced PR (43%), 2 SD (28.5%) and 2 PD (28.5%). 48 The evidence that sorafenib targets not only Raf but also RET, VEGFR and PDGFR has induced some authors to design clinical trials to assess its efficacy in MTC A phase II clinical trial of MTC patients treated with sorafenib in monotherapy was recently published. Among the 41 enrolled patients, 1 out of 16 sporadic MTC had PR (6.3%) while 14 had SD (87.5%) and one was non-evaluable. The median progression-free survival was 17.9 months. In the 25 patients affected by hereditary MTC the treatment was prematurely terminated because of slow accrual. 52

13 Targeted Therapies For Thyroid Cancer 11 The evidence that vandetanib, an efficient blocker of oncogenic RET kinases, produced a significant dosedependent inhibition of tumor growth in a panel of human tumor xenograft models was the rationale to start clinical trials in MTC. 53, 54 The results of the first two phase II studies demonstrated a good efficacy of this drug and were recently published. 55, 56 The first was a multicenter phase II trial which included patients with unresectable, measurable, locally advanced, or metastatic hereditary MTC harboring a RET germline mutation. Thirty patients were treated with vandetanib (300 mg per day) as monotherapy. The data indicated that 20% (6/30) of patients experienced PR according to RECIST criteria with a median duration of response of 10.2 months and that an additional 53% (16/30) of patients experienced SD for more than 24 weeks. 55 The second phase II trial evaluated the efficacy of vandetanib in 19 patients with hereditary MTC orally treated with 100 mg per day as monotherapy. The patients were allowed to increase the dose to 300 mg daily when experiencing progression. The results indicated that 16% (3/19) of patients experienced PR and 53% (10/19) of patients experienced SD ( 24 weeks). Based on the promising results of these small size phase II trials an international, phase III, randomized, double-blinded, placebo-controlled, multicenter study (ZETA study) was designed to assess the efficacy of vandetanib versus placebo in subjects with unresectable, locally advanced or metastatic MTC. In this study 331 patients were enrolled and randomly assigned to vandetanib (231) or placebo (100). This was the first study enrolling a large population of MTC. Progression free survival was longer in vandetanib treated patients compared with placebo (HR 0.46; 95% CI, 0.31 to 0.69; p= 0.001). A significant efficacy in patients treated with vandetanib compared with those treated with placebo was observed with improvement in ORR (p=0.001) and disease control rate (p=0.001). These data were not accompanied by a statistically significant difference in overall survival rate (HR 0.89; 95% CI, 0.48 to 1.65). However due to the fact that MTC patients are in many cases long survivors, the data at the time of publication were immature for this analysis. 57 This study led to the approval of this orphan drug for symptomatic or progressive MTC locally advanced or metastatic in the USA in April 2011 and in Europe in February At present, vandetanib is the unique TKI approved in the thyroid cancer field and at the present, it use is confined to MTC. An international multicentric phase III study enrolling patients with RAI-R DTC has been recently started. Unfortunately, preclinical studies showed that some mutations of the RET proto-oncogene (i.e.val804) may confer resistance to ZD6474. This observation is not negligible since at least in Italy Val804Met RET mutation is the most common mutation among hereditary cases. 58 In addition, after a variable period of response to the treatment the tumor develop an escape mechanism and the disease starts to progress. For this reason and for the fact that complete remission cannot be achieved with this treatment, the development of multiple lines of treatments is necessary. Cabozantinib (XL184) is a TKI that targets RET, VEGFR2 and MET. A preliminary report of a phase I study on 37 patients affected by MTC showed PR in 29% (10/34) of patients and 68% (25/37) had either PR or prolonged SD for more than 6 months. Responses have been observed in MTC patients with and without RET mutations and in patients naive or previously treated with other TKIs (i.e vandetanib, motesanib and sorafenib). The promising results of this drug stimulated the design of a randomized phase III study in MTC (EXAM study). The results of this have been recently published. 59, 60 Unlike the phase I study in which progressive disease was not an eligibility criterion, in the EXAM trial the subjects needed radiologically documented disease progression within 14 months. A total of 330 patients were enrolled and randomly assigned to cabozantinib or placebo in a 2:1 ratio. In fact PFS for patients receiving cabozantinib as compared to placebo was 11.2 versus 4.0 months (HR=0.28, 95% CI, ; p <0.0001). Similarly to the phase I trial the benefits were independent from previous TKI treatment and RET mutation status. The PFS hazard ratio was 0.24 for RET mutation positive and 0.47 for RET mutation negative cases. At 12 months PFS rate was 47.3% in patients treated with cabozantinib compared to 7.2% treated with placebo and the ORR was 28% and 0% in cabozantinib and placebo

14 12 Thyroid International group, respectively. 59 According to these results, cabozantinib represents a valid alternative to vandetanib for the treatment of advanced and progressive MTC even if patients have been previously treated with another TKI. Side Effects Even if TKIs differ from each other for their pharmacokinetic they share a wide variety of adverse reactions likely due to their very similar spectrum of targeted kinases and mechanism of action (Table 3). Fatigue is probably the most common and unpleasant side effect but only occasionally determines discontinuation of therapy since just a reduction of the daily dose may improve this symptom. Other common side effects include nausea, vomiting, diarrhea, anorexia, dysgeusia and stomatitis that can be the main causes of weight loss. Also for these side effects a reduction of the daily dose can be sufficient to make them acceptable. Nausea and vomiting are generally self-limiting and anti-emetic drugs are rarely needed. For diarrhea in addition to the dietary approach the administration of anti diarrheal agents (e.g. loperamide) is generally enough to control this symptom. In the case of stomatitis simple suggestions such as proper dental care, avoidance of hot beverages and food as well as salty and spicy food could help but some cases could require the use of topical anesthetic or anti-inflammatory paste. The most common dermatologic manifestation is handfoot syndrome followed by palmar-plantar erythrodysestesia and skin rash (Fig. 2, Panel A). This side effect is more frequent in patients treated with sorafenib but the use of skin care creams, the suggestion to avoid heat, fiction, harsh chemicals and to wear loose fitting shoes could help to manage the symptoms and prevent them Table 3: Tyrosine kinase inhibitors (TKIs) and their most common side effects Drugs Axitinib Cabozantinib Imatinib Lenvatinib Motesanib Pazopanib Selumetinib Sorafenib Sunitinib Vandetanib Vemurafenib Most Common Side Effects Diarrhea, Hypertension, Fatigue, Decreased appetite, Nausea and Dysphonia. Anemia, Lymphocytopenia, Creatinine increase, Bicarbonate decreased, Hypocalcemia, ALP increase and Hyperglycemia Diarrhea, Stomatitis, Nausea, Fatigue, Decreased weight, Anorexia, Dysgeusia, Palmar Plantar Erythrdysesthesia, Hair color depigmentation and Hypertension. Increased AST, ALT and ALP, Hypocalcemia, Lymphopenia, Neutropenia and Thrombocytopenia Fluid Retention, Nausea, Muscle Cramps, Musculoskeletal Pain, Diarrhea, Rash, Fatigue, Headache, Joint Pain, Abdominal Pain, Nasopharyngitis and Hemorrhage Anemia, Thrombocytopenia, Neutropenia Increased AST, ALT and ALP Hypertension, Decreased weight, Diarrhea, Fatigue, Voice changes, Anorexia, Weight loss, Headache, Rash and Joint pain Proteinuria, Anemia, Thrombocytopenia and Lymphopenia Diarrhea, Fatigue, Hypertension, Anorexia, Nausea, Abdominal pain, Weight loss, Headache and Hypothyroidism Diarrhea, Fatigue, Dysgeusia, Stomatitis, Weight loss, Anorexia, Rash and Hair color depigmentation Rash, Fatigue, Diarrhea, Peripheral edema, Nausea/vomiting, Stomatitis and Pruritus Increased AST, ALT Diarrhea, Fatigue, Anorexia, Weight loss, Rash/desquamation, Hand-foot skin reaction and Alopecia Diarrhea, Fatigue, Mucositis/stomatitis,Skin discoloration, Abdominal Pain and Anorexia Anemia, Thrombocytopenia and Lymphopenia, Increased AST and ALT Diarrhea, Nausea, Rash, Hypertension, Headache, Fatigue, Dermatitis Acneiform/Acne, Hypoglycemia and Photosensitivity, Hypocalcemia, Increased ALT Musculoskeletal Pain, Joint Pain, Fatigue, Anorexia, Dysgeusia, Rash and Photosensitivity

15 Targeted Therapies For Thyroid Cancer 13 from worsening. Unpleasant follicular rash can be observed in adults treated with TKI, in particular with sorafenib and vandetanib (Fig. 2, Panel B). Particular attention must be suggested to patients to avoid prolonged sun exposure and to use UV protective creams because some of this drugs, particularly vandetanib, can cause photosensitivity with very severe eritrodermia (Fig. 2, Panel C). As far as the cutaneous side effects are concerned it is worth noting that some anti-braf TKI can induce the development of wart and/or skin tumors such as keratoacantomas and squamous cell carcinomas; for this reason periodic dermatological controls are suggested 61 (Fig. 2, Panel D). However these lesions can be removed surgically without interrupting the treatment. Other common side effects are hypertension, OTc elongation, electrolyte abnormalities, hair hypopigmentation or alopecia and hypothyroidism. Hypertension is generally easily manageable with the introduction of some antihypertensive drugs. Electrolyte abnormalities are generally mild and would not need to be treated per se, however it is feasible that correcting these abnormalities they could reduce the risk of QTc elongation that is a relatively common adverse event (AE) of some of these drugs (particularly vandetanib). Hair loss can be reduced choosing a mild shampoo, a soft hairbrush and cutting short hair before the beginning of the treatment, this will lead to a less dramatic change when hair falls out. As soon as alopecia is a possible AE (particularly sorafenib) some patients could be advised to choose a wig long before the event became manifest. Hair colour change/depigmentation is particularly frequent for some drugs (cabozantinib) but fortunately this could be easily managed. Happily all these AEs are temporary and reversible after stopping the treatment. Figure 2: Common dermatologic side effects of TKI: erythema of the trunk (sorafenib), Panel A; follicular rash (sorafenib and vandetanib), Panel B; skin erythema, xerosis and ulcers due to photosensitivity (vandetanib), Panel C; verruca vulgaris (vemurafenib), Panel D. A B C D

16 14 Thyroid International Some patients experience also the reduction of blood cell count, mainly red blood cells, lymphocytes and neutrophils that is self limited in the majority of cases. Among endocrine side effects, hypothyroidism is the most common. The mechanism responsible of this disorder is not completely elucidated, some Authors hypothesized that the hypothyroidism could be the results of an increase in type 3 deiodination that enhance T4 and T3 metabolism but it is likely that the pathogenesis is multifactorial. 62 This is one of the most frequent but manageable side effect and the referral physician should remember to test thyroid function periodically. As mentioned above, many of these side effects can be safely managed with the introduction of appropriate drugs or with a reduction of the daily dose of the TKI. Sometimes a personalized scheme of administration of the drug can be effective to continue to treat the patient avoiding the major side effects. Conclusions Differentiated thyroid tumors can be treated with surgery and radioiodine in the majority of cases but those cases that are unable to take up radioiodine either because they lost this ability over the time or because they were iodine refractory since the beginning, such as MTC and ATC, represent a real challenge both for doctors and patients. Only recently, after a long period of validation through phase III clinical trials, vandetanib (Caprelsa, from Astra Zeneca) and cabozantinib (Cometriq, from Exelixis) have been finally approved for the treatment of advanced and progressive MTC. For advanced RAI-R DTC no systemic treatment is available at the moment but due to the good results of a phase III study hopefully sorafenib (Nexavar from Bayer) will be approved. For ATC no effective standard treatment exists. However, the clinical management should benefit from a multidisciplinary approach that includes surgical treatment, EBRT, classical chemotherapy possibly associated to new promising targeted drugs. It is worthy of note that in patients with indolent, asymptomatic or slowly progressive disease, TKI administration should be carefully evaluated and, taking into considerations the possible side effects, treatment related risks and quality of life should be considered. Conversely, in the absence of approved drugs or with disease progression, patients with advanced disease should be offered the opportunity to participate in clinical trials with TKI or treated with drugs already approved for the treatment of other human cancers 41 before starting with conventional chemotherapy. Finally, it is of clinical interest to recall that despite the good results in terms of PFS and ORR, until now no significant difference in OS was observed in treated compared to untreated patients. However since the median survival of the majority of these patients is rather long (i.e. up to 5-10 years in both PDTC and dedifferentiated DTC) longer follow up are needed to draw any conclusion.