**4. Preclinical and Clinical Evidence for Antiangiogenic Therapy**

Therapeutic options for thyroid cancers are variable following clinical-pathological staging. Localized and low-risk DTC benefit from surgical treatment (total thyroidectomy) followed by thyroid hormone suppression and adjuvant thyroid ablative therapy with radioactive iodine (RAI) treatment. This management could be applied to 85% of papillary and follicular subtypes [143,144].

In metastatic disease, patients are usually managed with a combination of surgery and RAI treatment. The prognosis depends on metastasis location (if it is suitable for a complete surgical resection) and post-operative radioiodine uptake in the tumor tissue. In the same setting of total thyroidectomy, indeed, patients could be submitted to therapeutic central neck dissection or lateral neck dissection in case of preoperative or intraoperative inspection of lymph nodes that are deemed to be pathologic [145]. However, some tumors are refractory to T4-mediated TSH suppression as well as RAI therapy. Approximately two-thirds of these patients may develop distant metastases resulting in much poorer overall survival rate and a poor prognosis [143,144].

In recent years, the scientific community's effort has been focused on the study of several molecular pathways involved in cancer development. Proliferation pathways, cell cycle control pathways, and angiogenesis processes have been largely evaluated, allowing the identification of mediators that may be useful targets for new anticancer treatments [146]. Multiple tyrosine kinase inhibitors (TKIs) have been identified, and their efficacy on different molecular pathways has been the primary end point of several studies in search of further treatment options for recurrent/metastatic thyroid cancer.

Vandetanib was the first TKI approved by the Food and Drug Administration (FDA) for the treatment of patients with symptomatic, unresectable, locally advanced, or metastatic MTC in the USA (2011) and Europe (2013). This drug acts on EGF, RET, VEGF, and VEGF receptors, leading to a downregulation of proliferative, angiogenic pathways and mediation on apoptosis pathways. The effects have been demonstrated in two phase 2 clinical trials and one phase 3 clinical trial (ZETA trial) in which patients with advanced unresectable MTC were blindly randomized to receive vandetanib at 300 mg daily or placebo [147].

This study demonstrated a significantly longer median PFS duration compared to the placebo group, with a partial response in 44% of cases. A significant difference in the objective response rates and disease control rates as well as in the biochemical response was also discussed. Moreover, vandetanib has also been tested in RAI-refractory DTC patients. A randomized, double-blinded, placebo-controlled phase 2 clinical trial evaluated the effect of this TKI, showing a statistically significant increase in the PFS of patients treated with vandetanib compared to the placebo group [143,148,149].

The second TKI, approved by the FDA in 2012 for the management of advanced and symptomatic MTC, was cabozantinib. This is a TKI with function on the hepatocyte growth factor receptor, RET, and VEGF2 receptors, leading to downregulation or inhibition of angiogenic, proliferative, and apoptotic pathways.

A phase 3 study (EXAM study) evaluated the effect of cabozantinib, showing a statistically significant longer median of PFS in patients treated with the drug (140 mg per day) with respect to those treated with placebo (11.2 versus 4.0 months). In this study, cabozantinib was associated with significant but manageable toxicity [143,149,150].

Sherman et al. published the results of an exploratory analysis of phase 3 trial data evaluating the influence of rearranged during transfection (RET) and RAS (HRAS, KRAS, and NRAS) mutations on cabozantinib clinical activity. They concluded that cabozantinib provides the most significant clinical benefit to patients with MTC who have RET M918T or RAS mutations [151].

Comparing vandetanib with cabozantinib, it seems that vandetanib is more tolerable than cabozantinib, making it the drug of choice for fragile and older patients. This is probably due to differences in patient selection for the two studies in phase 3. In the ZETA study, non-progressive but symptomatic patients were also studied, while the EXAM study enrolled only progressive cases, with adverse events due to cabozantinib higher than the adverse events observed in the ZETA trial. Cabozantinib has stronger antiangiogenic effects than vandetanib; therefore it could be strongly considered in cases of rapid MTC progression.

Cabozantinib and vandetanib have been tested on DTC, without approval [143,149].

In November 2013, sorafenib was the first multiple TKI to be approved by the FDA for the treatment of progressive metastatic DTC refractory to RAI treatment.

It has a documented efficacy in inhibiting all RAF kinases with a specific function on VEGF receptors 1–3, PDGFRB, and RET, which confers to sorafenib proapoptotic propriety and angiogenic effect fitting a molecular rationale for the treatment of all histological subtypes of thyroid cancer. Indeed, some studies have analyzed the use of Sorafenib in patients with metastatic thyroid cancer not suitable for curative surgery, RAI, or radiotherapy with a dose of 400 mg twice a day; these studies report partial response in 32% of cases, stable disease beyond six months, and a toxicity profile similar to that observed in previous studies and managed with dose delay or reduction [152]. Brose et al. investigated sorafenib in a multicenter, randomized, double-blind, placebo-controlled, phase 3 trial (DECISION). Sorafenib was used with the same administration in patients with RAI-refractory, locally advanced, or metastatic DTC that had progressed within the previous 14 months. The results showed how significantly sorafenib improved PFS compared to placebo [153].

Another TKI, lenvatinib, was approved in February 2015 in many countries to treat advanced DTC refractory to RAI. This is a multitargeted TKI of the VEGFRs 1, 2, and 3, FGFRs 1 through 4, PDGFR α, RET, and KIT signaling networks. In a phase 1 study, lenvatinib was associated with a partial response in thyroid cancer as well as melanoma, endometrial, and renal cancers. Subsequently, Sherman et al. performed a phase 2 study involving patients with DTC refractory to RAI, analyzing clinical activity and efficacy of levantinib [154]. Further, a phase 3, randomized, double-blind, multicenter study involving patients with progressive thyroid cancer refractory to iodine-131 was performed (SELECT trial). Schlumberger et al. randomized 261 patients to receive lenvatinib (at a daily dose of 24 mg per day in 28-day cycles) and 131 patients to receive placebo, showing PFS benefit and a high response rate associated with lenvatinib compared to placebo [155]. In an exploratory analysis, Wirth et al. examined treatment-emergent hypertension and its relationship with lenvatinib efficacy and safety in SELECT. They concluded that TE-HTN was significantly correlated with improved outcomes in patients with radioiodine-refractory DTC, indicating that hypertension may be predictive of lenvatinib efficacy in this population [156].

Based on these studies, sorafenib and lenvatinib are now recommended by the National Comprehensive Cancer Network to treat progressive, RAI-refractory DTC [157].

A multicenter phase II study investigated the efficacy and safety of dovitinib in advanced or RAI refractory thyroid cancer. This is another oral TKI with a documented activity on VEGFR, PDGFR, and RET pathways and a unique feature of inhibiting FGFRs. After a phase I study, in which the antitumor activity of dovitinib was evaluated on metastatic renal cell carcinoma [158], Indeed, Lim et al. tested dovitinib orally 500 mg once daily for five consecutive days followed by a 2-day rest every week in patients with PTC, FTC, and MTC with refractory disease. The study showed that dovitinib has modest activity with manageable toxicity with an overall response rate of 20.5% and a disease control rate of 69.1% compared to a relatively short PFS [144].

Another important multitarget tyrosine kinase inhibitor, anlotinib, has been tested for advanced refractory solid tumors. It has a referred action on tumor angiogenesis and growth with a proved effective target on VEGF and their receptors with an inhibition capacity 500 times stronger than sorafenib. Anlotinib also acts on tumor progression and cell proliferation by inhibiting PDGFR alfa/beta, c-Kit, Ret, Aurora-B, c-FMS, and discoidin

domain receptor 1 and carrying mutations in PDGFR alfa, c-Kit, Met, and EGFR. In a phase I, open-label study on patients with various types of solid tumor including MTC, Sun et al. showed that at a dose of 12 mg once daily on the 2/1 schedule, anlotinib had a toxicity profile in agreement with that reported for sorafenib and a substantial, broad-spectrum antitumor potential [146]. Subsequently, in a phase II clinical trial, the antitumor activity of anlotinib in patients with advanced or metastatic MTC was confirmed, also demonstrating a manageable adverse event profile [159].

A TKI with selective action on VEGFR1-3, axitinib, has been tested in patients with different types of thyroid cancers. Initially approved for the treatment of renal cell carcinoma, it has been tested in two phase 2 clinical trials for thyroid cancers with a recommended dose of 5 mg twice daily. Both studies described a clinical benefit in refractory and progressive thyroid cancer, tolerability, and a safety profile for axitinib as first-line therapy [160,161].

Pazopanib is another antiangiogenic TKI acting on VEGFR1-3, FGF1/2, PDGF, KIT, and RET receptors. After being approved for renal cancers, it has been tested (at a dose of 800 mg daily) in a phase II clinical trial in patients with metastatic, rapidly progressive, radioiodine-refractory DTC. Another phase II clinical trial tested the antitumor activity on MTC and anaplastic thyroid cancer [162–164].

Recently, another phase II clinical trial of pazopanib in RAI refractory DTC patients with progressive disease confirmed the clinical activity and manageable toxicities of the drug, examining in parallel biomarkers that might precede therapeutic response. However, no predictive biomarkers were found to facilitate a robust early identification of patients likely to respond to pazopanib therapy [165].

Sunitinib is an antiangiogenic TKI able to inhibit the VEGF1–3, PDGF, KIT, and RET receptors. As with other TKIs, it was first approved for other cancers such as renal cell carcinoma and pancreatic neuroendocrine carcinoma and then analyzed for RAI refractory DTC and MTC with FDG-PET-avid disease. Three phase 2 clinical trials reported on sunitinib administered at 37.5 mg daily on a continuous basis. All studies concluded that sunitinib exhibits significant anti-tumor activity in patients with advanced DTC and MTC, with some common adverse events such as fatigue, diarrhea, hand-foot syndrome, neutropenia, and hypertension [166–168].

The knowledge of thyroid tumor behavior, with its ability to produce cytokines and chemokines and consequently promote tumorigenesis, has guided the scientific community for many years in identifying the best TKI. This has led to the testing and approval of different therapeutic options for advanced thyroid tumors that previously had no possibility of treatment (Table 1).

Recently, the development and approval of immunotherapeutics for cancer and the identification of immune checkpoint inhibitors have modified the treatment landscape for many malignancies, taking advantage of the capacity of restoring the state of immunosurveillance on some tumors that were able to evade it [169,170].

Starting from the evidence of increased frequency of aggressive regulatory T cells and the correlation between expression of PD-1 ligand and the worse prognosis for recurrent PTC, the immune system components have been largely studied as therapeutic targets useful in the treatment of thyroid cancer. These are the new fields to be explored in the analysis of target therapy [169].


**Table 1.** Drugs studied in treatment of thyroid cancer: Tyrosine kinase inhibitors and their multitarget activity.
