**3. Angiogenesis and Prognosis**

Increased MVD, lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic factors have been demonstrated in non-neoplastic (multinodular goiter, toxic multinodular goiter, Graves' hyperplasia) and neoplastic conditions (follicular adenoma, papillary thyroid carcinoma, incidental papillary microcarcinoma, follicular carcinoma, and medullary carcinoma) [48,96]. No clear relationship between MVD measurement and thyroid pathology has been demonstrated. In fact, de la Torre et al. showed that MVD is decreased in all thyroid diseases, and LVD is increased in papillary thyroid carcinoma and incidental papillary microcarcinoma [48]. A second study found an increased MVD in PTC compared to normal controls [96]. Other studies demonstrated a high MVD in differentiated thyroid cancers (DTCs) compared to poorly differentiated thyroid cancers and other thyroid tissue samples [97–99]. More consistent results have been obtained with the evaluation of VEGF. Increased distribution and intensity of VEGF-A and VEGF-C have been demonstrated in thyroid cancers compared to normal samples and autoimmune and inflammatory diseases [48,96,98,99]. However, this increased expression was not indicative of multifocal disease, distant metastases at diagnosis, or increased tumor size [48].

Examining angiogenic processes connecting the thyroid cancer cell to its microenvironment could improve many thyroid cancer management steps. The first could be identifying cytologically indeterminate nodules prone to surgical treatment, thus reducing diagnostic thyroidectomies. The second could be the efficient differentiation between aggressive and indolent DTC so that the treatment extension and approach and the follow-up modalities could be correctly adapted. An improvement to correctly identify thyroid cancer in nodules with indeterminate cytology, ruling in or out malignancy, and selecting patients for surveillance, conservative or radical surgery, was the introduction of gene classifiers to be performed on the fine needle aspiration specimen (or even micro-biopsies or surgical specimens) [100–105]. BRAF mutations, and RET-PTC, RAS and PAX8-PPARG, are the most studied factors in this field. The complete genomics of thyroid cancer subtypes will be unveiled, but caution should be exercised in the interpretation and application of the many variants and mutations that are being discovered, because many of them can also be found in benign lesions; there are also thyroid cancers that show none of the known genetic alterations [106]. BRAF mutational status, along with membranous and nuclear galectin-3, HBME-1, CK19, and estrogen receptor beta, had been associated with DTC with aggressive behavior [107]. BRAFV600E mutated microcarcinomas are associated with adverse prognostic factors, whereas BRAF wild-type ones are associated with indolent behavior and a low probability of recurrence [108]. BRAFV600E is associated with tall cell variant PTC, along with mutations of COL5A1, COL1A1, COL10A1, COL11A1, CCL20, and CXCL5 [109]. BRAF is the most prevalent genetic alteration in radioiodine refractory metastatic thyroid cancer patients. BRAF mutation seems to positively influence the median progression-free survival (PFS) in radioiodine refractory patients treated with tyrosine kinase inhibitor, while having a negative prognostic impact in radioiodine-sensitive PTC patients [110]. BRAFV600E mutation is associated with central neck nodal metastases, but concerns were raised for its utility as a stand-alone marker in this field. Indeed, its use along with the analysis of miR-146b-3p, miR-146b-5p, and miR-222 was found to be prognostic of central neck nodal metastases preoperatively [111]. miRNA classifiers are indeed another option to identify and stratify thyroid cancer [53,112,113]. BRAF V600E appears to identify a subgroup of solitary intraglandular PTC larger than 2 cm and smaller than 4 cm, with a high risk of recurrence, for which a more aggressive treatment should be recommended [114]. There is evidence that BRAFV600E reduces TSP-1 expression in anaplastic thyroid cancer, and this appears to be linked to enhanced proliferation, adherence to collagen, migration, and invasion of the neoplastic thyroid cell. Arguably, this is due to the activation of pericytes in the microenvironment of thyroid cancer, which contributes to stabilizing new vessels through the secretion of PDGRFbeta, VEGF, and other factors [115–117].

VEGF-C and angiopoietin-2, together with their tyrosine kinase receptors KDR, Flt-4, and TEK, were found to be increasingly expressed in thyroid cancers, especially in the tran-

sition from a prevascular to vascular phase, and this was also correlated to the tumor size, nodal invasion, and, along with a reduced expression of TSP-1, to distant metastases [50]. It is known that VEGF overexpression correlates with increased microvascular density and, similarly, a reduced expression of TSP-1 is associated with the increased microvascular count [40]. Serum VEGF levels were significantly higher in patients with metastatic thyroid cancer than in healthy subjects and patients in remission [118]. Conversely, VEGF-D serum levels were decreased in patients with metastatic thyroid cancer, and this should be linked to other factors produced by the cancer cell that inhibits the usual production from other tissues of VEGF-D [119]. Moreover, anaplastic tumors show augmented expression of VEGF [45]. The evidence of high VEGF mRNA expression and of high FAL1 expression and cyclin D1 protein levels also shows how angiogenic processes are driven along with enhanced cell proliferation in papillary thyroid cancer [120,121]. In the context of prognostic examinations for patients affected by radioiodine refractory metastatic thyroid cancer, integrin αvβ3 is essential for tumor angiogenesis, and its expression is high on the surface of activated endothelial cells in newly formed blood vessels. It has been used to trace, using 99mTc-3PRGD2 imaging, metastases that are highly neovascularized. This new angiogenesis imaging modality can provide a new tool to monitor the efficacy of antiangiogenetic therapy [122]. TSH stimulation exerts a regulatory effect on VEGF production from thyroid cancer, probably through the interaction of tissues other than the thyroid itself, and this might have prognostic and therapeutic applications, exploiting the effects of recombinant human TSH administration [60,118,123,124]. The great interest in angiogenesis for prognostic purposes in cancer appears to be tightly connected to the need for circulating markers; this is even true for thyroid cancers, thus avoiding biopsies and other procedures to stratify patients. A possible application for this aim is the assay of miRNA in exosomes, especially miR-21-5p [52,125], which is a decisive proangiogenic factor produced by thyroid cancer cells. VEGF-A and PDGF-BB have been recently indicated as potential circulating biomarkers of PTC treatment [94].

MTC lesions show overexpression of VEGF-A, VEGFR-1, and VEGFR-2 [126], but their prognostic significance is uncertain, although VEGFR-2 and EGFR seem to be related to metastasis [127]. MTC originates from embryologically different tissue, and its management is different from that of DTC. HIF-1α has been associated with an adverse prognosis for MTC [128]. HIF1-1α expression is induced by hypoxia or aberrant signaling and stimulates the expression of VEGF and angiogenesis.

Moreover, MTC shows PSMA expression in the neovasculature, and microvessels showing positivity for PSMA are prognostically favorable. PSMA may become a target for imaging and peptide radioligand therapy [129]. It has been shown that collagen deposition and cross-linking and fibroblast presence in the thyroid cancer microenvironment play a prognostic role, indicating an advanced or aggressive disease, and are driven by PTEN loss BRAFV600E [20,130–133]. The overexpression of MMPs leads to increased VEGF and FGF secretion, related to tumor growth and invasiveness [134,135]. The ratio of MMP-2 to TIMP-2 expression is a prognostic factor for surgically treated MTC, and both of these metalloproteinases play a role in PTC [136–138]. MMP-9 expression is upregulated in PTC and might be a prognostic indicator for more advanced-stage cancer [139–142]. The prognostic role of other components of the extracellular matrix of thyroid cancer tangentially involved in angiogenetic processes, such as macrophages and T lymphocytes, is yet to be clarified [15].
