Molecular Alterations in Thyroid Cancer: From Bench to Clinical Practice

Thyroid cancer comprises different clinical and histological entities. Whereas differentiated (DTCs) malignancies are sensitive to radioiodine therapy, anaplastic (ATCs) and medullary (MTCs) tumors do not uptake radioactive iodine and display aggressive features associated with a poor prognosis. Moreover, in a majority of DTCs, disease evolution leads to the progressive loss of iodine sensitivity. Hence, iodine-refractory DTCs, along with ATCs and MTCs, require alternative treatments reflective of their different tumor biology. In the last decade, the molecular mechanisms promoting thyroid cancer development and progression have been extensively studied. This has led to a better understanding of the genomic landscape, displayed by thyroid malignancies, and to the identification of novel therapeutic targets. Indeed, several pharmacological compounds have been developed for iodine-refractory tumors, with four multi-target tyrosine kinase inhibitors already available for DTCs (sorafenib and lenvatinib) and MTCs (cabozantib and vandetanib), and a plethora of drugs currently being evaluated in clinical trials. In this review, we will describe the genomic alterations and biological processes intertwined with thyroid cancer development, also providing a thorough overview of targeted drugs already tested or under investigation for these tumors. Furthermore, given the existing preclinical evidence, we will briefly discuss the potential role of immunotherapy as an additional therapeutic strategy for the treatment of thyroid cancer.


Introduction
Thyroid cancer represents 2.5% of all cancers and about 90% of endocrine tumors [1]. The incidence of this neoplasia, three-fold higher in women and in individuals aged 25 to 65 years [2], has increased by 4.5% per year over the last decade [3], possibly because of diagnostic techniques improvement, environmental (e.g., radiation, pollution) and lifestyle changes [4,5]. Histologically, thyroid tissue consists of two different epithelial populations, the follicular and para-follicular [also known as C (clear)] cells, with different embryological origins and function. Follicular cells, organized in functional In order to better classify the molecular alterations detected in thyroid cancer, we will initially discuss RTK-related upstream signaling pathways involved in tumorigenesis and subsequently focus on the effectors of these pathways. Finally, we will describe alterations contributing to thyroid carcinogenesis that involve pivotal cellular functions.   [25,31,52]. These alterations are associated with increased phosphorylation of AKT, leading to activation of the PI3K pathway [31] (Figure 3). Interestingly, no mutations have been reported in these genes, with the exception of a single case of PTC displaying the G735S EGFR point mutation, which causes a conformational change of the kinase domain leading to its constitutive activation [53]. Fibroblast growth factor receptor 2 (FGFR2) and FMS-like tyrosine kinase 3 (FLT3) missense mutations have been identified in 11% and 17% of PDTCs, respectively [54]. Lastly, FGFR2 fusions may occur in PTCs with very low frequency (<1%) [30,35], while FGFR4 may be overexpressed in PTCs, FTCs and MTCs [52].  [25,31,52]. These alterations are associated with increased phosphorylation of AKT, leading to activation of the PI3K pathway [31] ( Figure 3). Interestingly, no mutations have been reported in these genes, with the exception of a single case of PTC displaying the G735S EGFR point mutation, which causes a conformational change of the kinase domain leading to its constitutive activation [53]. Fibroblast growth factor receptor 2 (FGFR2) and FMS-like tyrosine kinase 3 (FLT3) missense mutations have been identified in 11% and 17% of PDTCs, respectively [54]. Lastly, FGFR2 fusions may occur in PTCs with very low frequency (<1%) [30,35], while FGFR4 may be overexpressed in PTCs, FTCs and MTCs [52].

Alterations in the PI3K Pathway
Enhanced PI3K signaling is a common feature of thyroid cancer, in particular in the FTC subtype [25] (Figure 3). Alterations in this pathway involve the GTPase RAS, the alpha catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), the serine-threonine protein kinase AKT and the phosphatase and tensin homolog phosphatase (PTEN). While RAS mutations are

Alterations in the MAPK Pathway
The MAPK pathway is frequently altered in thyroid cancer, particularly in PTCs [63]. In most cases, mutations involve RAS (previously described) and the B-Raf proto-oncogene (BRAF), in addition to the upstream receptors described above, which activate different signaling cascades ( Figure 3). BRAF: Alterations in BRAF are an early tumorigenic event in PTCs (40-80%) although they have also been reported in PDTCs (5-35%) and ATCs (10-50%) [64]. Point mutations in this serine/threonine kinase activate the MAPK pathway resulting in loss of differentiation, tumor progression and inhibition of apoptosis [65,66]. The most common BRAF mutation is the V600E substitution, found in 45% of PTCs, which causes constitutive activation of the proto-oncogene [67]. Rare mutations may occur around codon 600, the most frequent the K601E substitution, and display an inferior oncogenic potential [30,67]. BRAF fusions have also been found in radiation-associated PTCs and, at a lower frequency, in PDTCs and ATCs [30,35]. The first to be identified was the AKAP9-BRAF rearrangement, found in 10% of radiation-induced PTCs, resulting in a fusion protein lacking the auto-inhibitory N-terminal portion of BRAF that exhibits elevated kinase activity [68].

Alterations in the TP53 Pathway
The tumor suppressor TP53 is a transcription factor involved in the control of the cell cycle and apoptosis ( Figure 3). More than 75% of TP53 mutations are small nucleotide changes that inactivate the protein's function. These changes are mostly located in the DNA-binding domain (residues 92-292) [72]. Considering the high prevalence of TP53 mutations in PDTCs (10-35%) and ATCs (40-80%), TP53 inactivation is considered a final step in tumor progression. Indeed, p53 deficiency, in association with activating mutations of oncogenes, such as RAS and BRAF, accounts for the high proliferation rate and increased aggressiveness of the more aggressive forms of thyroid cancer [73].

Other Molecular Alterations in Thyroid Cancer
EIF1AX: Mutations in the eukaryotic translation initiation factor 1A (EIF1AX) cause defects in the formation of the 43S pre-initiation complex for protein translation (Figure 3). Alterations of this gene are clustered in exons 2, 5 and 6, and the most common is the A113 splice mutation at the intron 5/exon 6 splice site, followed by a cluster of mutations in exon 2 [74]. EIF1AX mutations were detected in 1-2% of PTCs, largely occurring in a mutually exclusive manner with BRAF and RAS mutations [30], and more frequently in PDTCs (11%) and ATCs (9%) in which, on the contrary, they are strongly associated with RAS mutations [39]. IDH1: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are highly prevalent in thyroid carcinoma (16%) in particular in FTCs (5%) and ATCs (11%) [75,76] (Figure 3). Even if the most recurrent IDH1 mutation involves arginine at codon 132, no R132 amino acid change has been reported in thyroid tumors. All IDH1 substitutions found in thyroid samples concern five hotspot mutations in exon 4: G70D, G123R, I130M, H133Q and A134D. For some of these mutants, a reduced enzymatic activity has been demonstrated, suggesting a potential tumorigenic role of the IDH1 system in thyroid cancer [76].
PPARγ: Peroxisome proliferator activated receptor gamma (PPAR-γ) is a nuclear transcription factor that enhances apoptosis by activating caspases, up-regulating Bax and down-regulating bcl-2, survivin and c-myc [77,78]. Besides RAS mutations, rearrangements of the PPARγ gene are the most frequent alterations found in FTCs (20-50%) and may represent an initiating event in the transformation of follicular-derived cells [79]. PPARγ fusions may involve PAX8 and CREB3L2, but the PAX8-PPARγ rearrangement is the most frequent [35,80]. The PAX8-PPAR fusion protein (PPFP) acts as a dominant negative inhibitor of wild type PPARγ, thereby constitutively activating the transcription of a subset of PPARγ and PAX8 responsive genes [80] (Figure 3).
TERT: Activating mutations in the promoter of the telomerase reverse transcriptase (TERT) are mostly a late event in thyroid tumorigenesis [39,81]. They are more common in PDTCs (40%) and ATCs (70%), even if they can also be found in PTCs (10%) and FTCs (20%) [81], where they are associated with a poor prognosis [82]. Two mutually exclusive TERT promoter mutations are recurrent in thyroid cancer, one at position -124 (c228t) and one at position -146 (c250t) upstream of the TERT translation start site. Both mutations generate a consensus-binding site in the TERT promoter for E-twenty-six (ETS) transcription factors, which increase TERT transcriptional activities [81] (Figure 3). Finally, TERT mutations may co-occur with BRAF and RAS mutations in PDTCs and ATCs [82,83].

Targeted Therapies in Thyroid Cancer
Due to an improved understanding of thyroid cancer biology, in recent years, a plethora of targeted molecules have been tested while other compounds are currently under investigation. Herein, Genes 2019, 10, 709 9 of 33 we thoroughly review the current landscape of targeted therapies for thyroid cancer. Ongoing trials awaiting preliminary results are also reported.

Multi-Target Agents
Several molecules inhibiting tyrosine kinases involved in cell proliferation, survival and angiogenesis have shown clinical efficacy in both advanced RAIR DTCs and MTCs, while promising results are also beginning to emerge in ATCs (Figures 4 and 5). To date, four drugs have received FDA approval, two for advanced RAIR DTCs (sorafenib and lenvatinib) and two for metastatic MTCs (vandetanib and cabozantinib). However, numerous trials have investigated additional tyrosine kinase inhibitors (TKIs), whereas many others are currently ongoing (Table 1). 9 chromatin remodeling complex, mutated in 6% of PDTCs and 36% of ATCs [39]. Finally, specific histone deacetylase (HDAC) subtypes are associated with different thyroid cancer characteristics and behaviors [84] (Figure 3).

Targeted Therapies in Thyroid Cancer
Due to an improved understanding of thyroid cancer biology, in recent years, a plethora of targeted molecules have been tested while other compounds are currently under investigation. Herein, we thoroughly review the current landscape of targeted therapies for thyroid cancer. Ongoing trials awaiting preliminary results are also reported.

Multi-Target Agents
Several molecules inhibiting tyrosine kinases involved in cell proliferation, survival and angiogenesis have shown clinical efficacy in both advanced RAIR DTCs and MTCs, while promising results are also beginning to emerge in ATCs (Figures 4 and 5). To date, four drugs have received FDA approval, two for advanced RAIR DTCs (sorafenib and lenvatinib) and two for metastatic MTCs (vandetanib and cabozantinib). However, numerous trials have investigated additional tyrosine kinase inhibitors (TKIs), whereas many others are currently ongoing (Table 1).      Anlotinib: A multi-target TKI, displaying an affinity for VEGFR 2-3, FGFR 1-4, PDGFR α/β, c-KIT and RET [85]. After a phase I trial defined an MTD of 12 mg daily in a 2/1 schedule, a phase IIA study tested the drug on 58 chinese patients with advanced MTC. Partial responses (PR) occurred in 57% of patients, whereas median PFS was not reached at the time of data cut-off [85,86]. Results from a phase IIB randomized trial on 91 metastatic MTC patients were recently reported: mPFS was 20.7 months in the experimental arm and 11 months in the placebo arm (HR 0.53; p < 0.03), with a considerable benefit in terms of ORR (48% with anlotinib vs. 3.5% with placebo, p < 0.0001). Overall survival data are still immature. Adverse events were consistent throughout the trials, with hand-foot syndrome, hypertension and hyper-triglyceridemia representing the more frequent toxicities [87].
Axitinib: A VEGFR1-2-3 inhibitor [88] that also targets PDGFR-β and c-KIT. Its activity in thyroid tumors of any histology has been explored in two phase-II trials, comprehensively, including 112 patients. Results were consistent in the two studies, with an ORR of 30% in the first one and of 35% in the second, and median PFS of 18.1 and 16 months, respectively [89,90].
Cabozantinib: A selective inhibitor of MET, VEGFR-2 and RET [91]. In a phase I trial cabozantinib showed encouraging results in a cohort of 37 heavily pre-treated MTC patients [92]. These promising findings were further confirmed in the phase III EXAM study that randomized 330 patients with hereditary or sporadic advanced MTC to receive cabozantinib 140 mg daily or placebo, demonstrating a significant PFS benefit for subjects in the experimental arm (11.2 months vs. 4 months, HR 0.28, CI 0.19-0.40, p < 0.001). However, OS did not differ significantly between the two arms. The drug seems more effective in patients carrying a RET mutation [93,94]. In 2012 FDA approved cabozantinib for the treatment of advanced MTC [95]. More recently, the potential role of cabozantinib as salvage treatment in RAIR DTCs progressing on a VEGFR inhibitor has been explored in a phase II trial. Twenty-five enrolled patients presented a 40% ORR and a 92% disease control rate (DCR), with 12.7 months of median PFS and 34.47 months of median OS [96].
Imatinib: A multiple kinases inhibitor that targets ABL, c-KIT and PDGFR. This drug was tested both in advanced or relapsed ATCs and MTCs. A pilot trial with 11 ATC patients with proven PDGFR overexpression by IHC showed that 800 mg Imatinib/daily determined 2 PR and four assessments of stable disease [97]. However, in two additional studies, a total of 24 advanced MTCs received imatinib 600 mg daily with dismal results (i.e., no OR and sporadic disease stabilization) [98,99].
Lenvatinib: The drug inhibits several targets, including VEGFR 1-2-3, FGFR 1-2-3-4, PDGFR-α, RET and c-KIT [100]. Its activity in advanced RAIR DTCs has been proven in a phase II trial enrolling 58 patients, treatment naïve or pre-treated, and then confirmed in phase III SELECT trial, which randomized 261 subjects to receive 24 mg lenvatinib daily or placebo. Median PFS was 18.3 months with lenvatinib versus 3.6 months with placebo (HR 0.21; 95% CI 0.14 to 0.31; p < 0.0001), with a 64% OR rate, including four complete responses (CR). Median OS was not reached at the time of data cutoff. Most frequent adverse events were hypertension, diarrhea, fatigue, appetite and weight loss and nausea, with a 14% discontinuation rate [101,102]. Because of its favorable efficacy and safety profile, in 2015 lenvatinib was granted approval for the treatment of advanced RAIR DTCs and is currently the preferred therapeutic choice for this disease in the first line setting [103]. The drug also showed activity in advanced MTCs, since a phase II trial on 59 patients reported 36% OT rates (CI 24-49%) with 80% DCR (CI 67-89%) and nine months of median PFS (CI seven months-NE) [104].
Sorafenib: A multi-kinase inhibitor used against VEGFR 1-2-3, RET, RAF, PDGFR-β, c-KIT and FLT3 [112]. In a single arm phase II trial, Schneider and colleagues reported 18 months of median PFS [mPFS (CI 7-29 months)] and 34.5 months median OS (CI 19-50 months) in 31 patients with advanced RAIR DTC [113]. The subsequent phase III, multicenter, randomized, double blinded, placebo-controlled DECISION trial enrolled 417 advanced RAIR DTC patients in the first line setting. It showed significantly longer PFS in the experimental arm (10.8 months) compared with the placebo arm (5.8 months) [HR 0.59; 95% CI 0.45 to 0.76; p < 0.0001] and a 12% ORR, but failed to demonstrate a survival benefit in the group of patients treated with sorafenib 400 mg bis in die, which reported considerable toxicities leading to dose reduction or treatment discontinuation in more than 60% of cases [114]. Nevertheless, sorafenib is currently approved both by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of advanced RAIR DTC. Additionally, in a cohort of 20 pre-treated ATC patients, sorafenib induced durable PR in two patients and stable disease in five subjects, whereas it led to 1 PR and 14 disease stabilizations in 16 sporadic advanced MTCs [115,116]. However, the use of this drug in anaplastic and medullary thyroid carcinomas remains off label.
Sunitinib: A compound that displays a broad spectrum of activity against VEGFR 1-2, c-KIT, RET, PDGFR-β and FLT3 [117]. Preliminary results from a phase II trial on 33 evaluable patients with advanced RAIR DTC (n = 26) or MTC (n = 7) receiving sunitinib 37.5 mg on a continuous schedule indicated that 11 patients (31%) experienced a PR and 16 patients (46%) had SD, with a median time to progression of 12 months [118]. Another phase II study of continuous sunitinib in 23 RAIR DTC patients confirmed a good activity profile in this subset of patients. In fact, ORR was 26% and clinical benefit rate (CBR) 83% [119]. Additionally, the phase II THYSU trial investigated sunitinib activity at 50 mg/daily four weeks on followed by two weeks off, in 71 previously untreated advanced thyroid carcinomas of any histology. Nine out of 39 evaluable DTC patients had a disease response (1 CR and 8 PR; ORR 22%), whereas 10 of the 24 MTC patients reported a PR (ORR 38.5%). Of the four enrolled patients with ATC, only two were evaluable and experienced disease stabilization. PFS was 13.1 months and 16.5 months in DTCs and MTCs, respectively [120].
Vandetanib: A RET, VEGFR 2-3, c-KIT and EGFR inhibitor primarily tested in MTC [121,122]. Preliminary efficacy data came from a phase II trial on 30 locally advanced or metastatic hereditary MTC patients, 22 of which yielded PR or disease stabilization >24 weeks (73% DCR) with vandetanib 300 mg/daily [123]. The following phase III randomized, placebo-controlled study (ZETA trial) included 331 patients with hereditary or sporadic advanced MTC and confirmed the drug's efficacy. Median PFS was 30.5 months in the vandetanib arm compared with 19.3 months in the placebo arm [HR 0.46, CI 0.31-0-69, p = 0.001], while OS results were still immature. Overall, a trend of enhanced efficacy in RET mutated disease emerged. Diarrhea, skin rash, nausea, hypertension and headache were the most frequent adverse reactions. Additionally, QT prolongation, potentially evolving in torsade de pointes and sudden death, represented an infrequent, but critical adverse event [124]. In 2011 vandetanib was the first FDA-approved drug for the treatment of symptomatic or progressive MTC in patients with unresectable, locally advanced, or metastatic disease. A special "black box" warning about QTc prolongation risk exists, hence the drug can only be prescribed by certified physicians [125]. Even though vandetanib is not indicated in advanced RAIR DTCs, it has been tested in a phase II randomized trial in this population, demonstrating a good activity profile (median PFS 11.1 months vs 5.9 months in patients receiving placebo, HR 0.63, 95% CI 0.54-0.74, p = 0.008) [126]. A phase III trial (VERIFY, NCT01876784) randomizing 238 subjects with RAIR DTC to receive vandetanib or placebo completed accrual, but no results are yet available.

Single-Target Agents
Although multi-kinase inhibitors represent the most studied agents for the treatment of thyroid cancer, several drugs selectively blocking a single altered protein may also be effective in oncogene-addicted disease ( Figure 6). Different trials have already investigated the role of single target agents, whereas many others are still ongoing (Table 2). However, in the absence of a reliable predictive biomarker, the use of single-target molecules generally leads to unsatisfactory results.

Single-Target Agents
Although multi-kinase inhibitors represent the most studied agents for the treatment of thyroid cancer, several drugs selectively blocking a single altered protein may also be effective in oncogene-addicted disease ( Figure 6). Different trials have already investigated the role of single target agents, whereas many others are still ongoing (Table 2). However, in the absence of a reliable predictive biomarker, the use of single-target molecules generally leads to unsatisfactory results. Apatinib: Two small chinese trials evaluated apatinib, a selective VEGFR-2 inhibitor, in 30 patients with advanced RAIR DTC. The drug showed promising activity, both in terms of thyroglobulin levels reduction and tumor shrinkage [127,128]. Hence, further studies (phase II and III) assessing the role of apatinib for RAIR DTC treatment are currently ongoing (Table 2).
Dabrafenib and Trametinib: Combining a BRAF inhibitor (dabrafenib) with a MEK inhibitor (trametinib) may be an effective strategy for BRAF V600E mutated ATC, as reported in a phase II trial recruiting 16 pre-treated ATC patients. The reported 69% ORR, including a CR, along with the estimated 90% rate of ongoing responses at 12 months, represent unprecedented results for this aggressive disease [129,130]. Clinical trials employing dabrafenib in combination with trametinib or lapatinib are ongoing (Table 2). Apatinib: Two small chinese trials evaluated apatinib, a selective VEGFR-2 inhibitor, in 30 patients with advanced RAIR DTC. The drug showed promising activity, both in terms of thyroglobulin levels reduction and tumor shrinkage [127,128]. Hence, further studies (phase II and III) assessing the role of apatinib for RAIR DTC treatment are currently ongoing (Table 2).
Dabrafenib and Trametinib: Combining a BRAF inhibitor (dabrafenib) with a MEK inhibitor (trametinib) may be an effective strategy for BRAF V600E mutated ATC, as reported in a phase II trial recruiting 16 pre-treated ATC patients. The reported 69% ORR, including a CR, along with the estimated 90% rate of ongoing responses at 12 months, represent unprecedented results for this aggressive disease [129,130]. Clinical trials employing dabrafenib in combination with trametinib or lapatinib are ongoing (Table 2).

Selumetinib:
The drug targets MAP kinases MEK-1 and 2 and reversed iodine refractoriness in 8 of 20 patients with metastatic thyroid cancer, as assessed by 124 I-PET. Selumetinib-treated patients subsequently received RAI achieving PR (n = 5) or SD (n = 3) after the radio-metabolic treatment. Additionally, the NRAS mutation seems to be a predictive biomarker of selumetinib efficacy [131]. Further studies are ongoing (Table 2).
Vemurafenib: A selective BRAF inhibitor, which showed promising results in a phase II trial conducted on 51 patients with advanced RAIR PTC displaying BRAF V600E . The study population comprised two cohorts according to previous treatment with an anti-VEGFR. Ten out of 26 (38.5%) TKI naïve patients achieved a PR, whereas nine patients (35%) maintained an SD for six months or longer, determining a 73% CBR, with a median PFS of 18.2 months. In the second TKI-exposed cohort, six of the 22 evaluable patients (27%) experienced a PR, while other six had SD for at least six months (CBR 54.6%) and a median PFS of 8.9 months [132]. A further study is ongoing (Table 2).

ALK Inhibitors
Due to its oncogenic activity, ALK represents a potential therapeutic target in many solid and hematologic cancers [133,134]. Currently, the therapeutic options for ALK-rearranged tumors include first-(crizotinib) or second-generation (ceritinib, alectinib and brigatinib) inhibitors or immunotherapeutic drugs directed against activated ALK ( Figure 5). Ongoing trials employing ALK inhibitors are described in Table 2.
Ceritinib: A second-generation inhibitor that overcomes secondary resistance, due to acquired ALK mutations, amplification or activation of alternative-ALK-independent-survival pathways (e.g., EGF, IGF, RAS/SRC and AKT/mTOR signaling pathways) [134][135][136]. A study by Guan at al demonstrated limited efficacy of ceritinib in ATC patients with the ALK L1198F mutation in full-length ALK or the EML4-ALK fusion protein [137]. An additional trial is currently evaluating this drug in patients harboring ALK mutations or fusions (NCT02289144) ( Table 2).
Crizotinib: A second generation TKI targeting ALK, MET and ROS1. This drug has been extensively investigated in ALK-fusion-positive tumors [138]. One ALK-translocated ATC patient treated with crizotinib achieved a PR [139].
Jun Ho Ji and colleagues reported a dramatic response with crizotinib in an advanced MTC patient harboring an ALK fusion (NCT01121588) [140]. Additionally, in a phase Ib study (PROFILE 1013; NCT01121588), which enrolled 44 ALK-positive metastatic patients, one individual diagnosed with MTC experienced a PR lasting 16.1 weeks [138].

NTRK Inhibitors
Selective inhibition of TRK signaling may be useful for patients with thyroid tumors that harbor an oncogenic NTRK translocation. Currently, four first and second generations TRK-inhibitors (entrectinib, larotrectinib, LOXO-195 and TPX-0005) have been developed and tested in clinical trials [141,142] (Table 2) ( Figure 5).
Entrectinib: A pan-TRK inhibitor with additional activity against ROS1 and ALK [143]. A phase I/Ib study in children or young adults (aged 2-22 years) with or without NTRK, ROS1 or ALK fusions (STARTRK-NG, NCT02650401) and a phase II "basket trial" in adults with the same rearrangements (STARTRK-2, NCT02568267) enrolled patients with thyroid cancer. Results are still pending.
Larotrectinib: An ATP-competitive pan-TRK inhibitor, which received agnostic approval by the FDA for patients with advanced solid tumors harboring an NTRK gene fusion [144]. Fifty-five patients received larotrectinib in three different trials: A phase I in adults (NCT02122913), a phase I/II in pediatric patients (SCOUT, NCT02637687) and a phase II in adolescents and adults (NAVIGATE, NCT02576431), including five patients (9%) with thyroid cancer [145]. Although data specifically concerning the thyroid population are unavailable, promising results emerged in the intention-to-treat population, whit a 75% ORR (95% CI, 61% to 85%) and 71% of patients free from progression at 12 months. Median DOR and PFS were not reached after a median follow-up of 8.3 months and 9.9 months, respectively.

LOXO-195:
A selective TRK inhibitor specifically developed to overcome acquired resistance that may occur in subjects receiving larotrectinib or other TRK inhibitors [145]. LOXO-195 is currently under evaluation in a phase I/II trial (NCT03215511) enrolling patients with NTRK-rearranged tumors-including thyroid cancer-previously treated with a TRK inhibitor.

PI3K/AKT/mTOR Pathway Inhibitors
Activation of the PI3K/AKT/mTOR pathway is a common feature in thyroid cancer [31,146,147]. Preclinical and clinical data suggest that targeting this pathway can be an effective strategy for the treatment of patients with advanced RAIR DTCs and MTCs [148]. To date, several trials have employed mTOR inhibitors in thyroid cancer (Table 3), whereas the phase II MATCH studies are testing PI3K (e.g., taselisib, copanlisib) and AKT-inhibitors (e.g., capivasertib) ( Figure 6).
Buparlisib: A pan-class I PI3K inhibitor that failed to show a significant PFS benefit in 43 advanced, RAIR DTCs. Despite a reduction in tumor growth, the drug did not induce any objective response in the overall population, and 48.8% of patients had progressed after six months. The decrease in tumor growth may suggest an incomplete inhibition of the PI3K oncogenic pathway [149].
Everolimus: A phase II trial tested this mTOR inhibitor in 38 patients with advanced RAIR DTC. The authors reported an 81% DCR and a median PFS of 47 weeks [150]. Two additional phase II studies analyzed everolimus safety and efficacy on seven subjects with MTC, 28 patients with metastatic or locally advanced DTC and seven individuals with ATC. Five patients (71.4%) showed SD, and 4 (57.1%) had an SD lasting >24 weeks [151]. In the second study, 17 patients (65%) showed SD; in 15 of these 17 patients (58%), the response lasted >24 weeks. Median PFS and OS were 9 and 18 months, respectively [152]. A further phase II trial evaluated everolimus efficacy in patients with RAIR thyroid cancer and correlated tumor mutational profiling with response. Median PFS were 12.9, 13.1 and 2.2 for DTC, MTC and ATC cohorts, respectively, and patients with mutations in the PI3K pathway appeared to benefit most from drug treatment [153].
Since activation of the somatostatin receptor (SSTR1-5) also inhibits PI3K/AKT signaling, the somatostatin analog pasireotide has been tested in combination with everolimus [154]. Pasireotide activates SSTRs, in particular, subtype 2, which is the most expressed somatostatin receptor in thyroid cancers [155]. In a phase II trial, 19 patients with advanced MTC began pasireotide, achieving a median PFS of 36 months (95% CI 19.5-52.5). Seven patients with tumor progression received everolimus in combination with pasireotide, experiencing a median PFS of nine months (95% CI 0-21.83) [156]. Another phase II trial combining pasireotide with everolimus completed accrual and results are still awaited (Table 3).
Sirolimus: A retrospective study reported that this drug combined with cyclophosphamide generated PFS rates comparable to the standard of care for RAIR DTC. One-year PFS probability was 0.45 in the sirolimus plus cyclophosphamide cohort and 0.30 in the control population [157].
Temsirolimus: In a phase II trial temsirolimus was combined with sorafenib for the treatment of 36 RAIR thyroid cancer patients. Radiographic response rate was the primary endpoint. A PR was observed in 22% of cases, stable disease in 58% and progressive disease in 3% of patients. Individuals with any prior systemic treatment had a response rate of 10% compared to 38% for subjects with no prior systemic treatment [158].

PPAR-γ Agonist-Efatutazone
Different studies suggest that PPAR-γ agonists may inhibit tumor growth through the induction of terminal cell differentiation, cell cycle arrest, apoptosis, and angiogenesis inhibition [78,159]. Efatutazone, is a PPAR-γ agonist evaluated in combination with paclitaxel in a phase I study accruing patients diagnosed with ATC. Safety, potential effectiveness, and maximally tolerated dose were the end point of this trial. Results demonstrated that the combination was safe, with no dose-limiting toxicities (DLTs) and preliminary evidence of efficacy. Thus, these findings supported the addition of efatutazone to paclitaxel in patients with advanced ATC [160]. A further clinical trial is ongoing, but not yet recruiting (Table 2).

Histone Deacetylase Inhibitors-Valproic Acid
Several studies have shown that histone deacetylase (HDAC) inhibitors display promising effects for the treatment of several malignancies as they inhibit tumor proliferation, induce apoptosis, cell cycle arrest, and cancer differentiation [161][162][163].
In a phase II study valproic acid (VA) was administered to 13 patients with RAIR thyroid cancer of follicular origin. The primary endpoint of the study was to determine VA antitumor activity by evaluating measurable tumor response and/or decreased thyroglobulin levels. VA did not decrease tumor size and only generated a modest decrease in serum thyroglobulin levels. The secondary endpoint was to determine if VA could increase RAI uptake by the tumor cells, but results were disappointing [164].

Immunotherapy Landscape in Thyroid Cancer
In the last decade, the detailed understanding of the mechanism employed by cancer cells to elude the immune system have fostered a renewed interest for immune based-therapies [165]. Although thyroid carcinomas are not deemed to be "immunogenic" and display a low median tumor mutational burden (about 0.4 mutations/Mb) [166], several observations indicate a possible rationale for the use of immune checkpoint inhibitors in these tumors [167]. Indeed, immune cell infiltration, including natural killer cells, macrophages, mast cells, dendritic cells and T regulatory cells (Tregs) [168][169][170] has been reported in DTCs. Importantly, the relative amount of tumor-infiltrating lymphocytes and T-regs in primary thyroid cancer seems to correlate with prognosis [168,171]. Furthermore, the expression levels of programmed death 1 (PD-1) and programmed death ligand (PD-L1) correlate with higher risk of disease recurrence and reduced DFS [172,173]. To date, PD-L1 expression represents the most useful predictive biomarker to determine immunotherapy efficacy in thyroid tumors [168,174,175], with PD-L1 positivity ranging from 6.1 to 82.5% in PTCs and from 22.2% to 81.2% in ATCs [176].

Immune Checkpoint Inhibitors
Several clinical trials are investigating immune checkpoint inhibitors in thyroid cancer both alone or in combination with other drugs. A phase Ib trial tested the anti PD-1 antibody pembrolizumab on 22 PD-L1-positive patients with RAIR thyroid tumors. Clinical benefit rate was 50% (95% CI 28-72%), even though seven patients experienced early progression (32%; 95% CI 14-55%). Median PFS was seven months (95% CI 2-14 months), whereas median OS was not reached at the time of data cut-off (95% CI 22 months to NR) [177]. Based on these promising results, a phase II basket trial is currently ongoing (NCT02628067). Another study is testing the combination of the anti PD-1 nivolumab and the anti CTLA-4 ipilimumab in RAIR DTCs (NCT03246958).
Immunotherapy is also being evaluated in ATC. However, while the results of a phase II trial employing pembrolizumab are awaited for October 2019 (NCT02688608), a recently presented study failed to demonstrate any efficacy for the combination of the anti-PD-L1 durvalumab with the anti CTLA-4 tremelimumab and stereotactic radiation therapy in 12 patients with ATC [178]. Additional clinical trials are ongoing to establish if immune checkpoint inhibitors may prove of clinical benefit for thyroid cancer (Table 4).

Conclusions
In the last few years, a rapid advance in the knowledge of the molecular mechanisms underlying thyroid tumorigenesis along with the identification of pivotal driver genes contributing to disease progression has led to the introduction of several biological therapies, including TKIs, monoclonal antibodies and antibody-drug conjugates [179][180][181][182][183].
In this wide landscape of potentially targetable genomic alterations, RTKs modulating angiogenesis, proliferation and differentiation have represented the most easily druggable targets. Indeed, small molecules blocking these receptors have provided significant survival benefits for both RAIR DTCs and MTCs. However, these benefits have come at the cost of meaningful clinical and financial toxicities [184,185].
While additional studies are currently investigating other RTK-directed TKIs in DTCs and MTCs, the use of these drugs in ATC has been largely unsatisfactory. On the contrary, the combination of BRAF and MEK inhibitors has generated unprecedented response rates in patients diagnosed with these aggressive thyroid carcinomas, and validation of the published preliminary results is eagerly awaited with the hope that it may provide durable benefits comparable to those reported in BRAF-mutant melanoma.
Finally, despite not being classified as a highly immunogenic, preliminary findings suggest a possible benefit from immune checkpoint inhibitors in thyroid cancer, although these data are still immature. As in other types of solid tumors, it remains to be established which patients will derive meaningful benefits from this therapeutic approach [186].