The [^99mTc]Tc-MIBI is a lipophilic cation able to cross the cell membrane. It penetrates into the cytoplasm in a reversible way and then irreversibly moves through the membrane of the mitochondria using a different electrical gradient regulated by a high negative inner membrane electric potential. Tumor cells are characterized by a higher negative inner membrane electric potential compared to normal cells. Consequently, it can be helpful to characterize the biological behavior of cytologically indeterminate nodules [16]. A very high to absolute NPV was found in thyroid nodules with a [^99mTc]Tc-MIBI hypoactive pattern, while an increased [^99mTc]Tc-MIBI uptake conferred a significantly higher risk of cancer. However, while a NPV of >96 is achieved, the positive predictive value (PPV) is unsatisfactory as [^99mTc]Tc-MIBI uptake may also be recorded in benign nodules (especially follicular and oxyphilic adenomas) [17,18]. Moreover, a semiquantitative approach to evaluate the tracer washout from the nodule (Washout Index (WOind)) was introduced, providing better results in comparison with qualitative analysis [19].

[¹⁸F]FDG uptake is related to an overexpression of the transmembrane glucose transporter proteins (GLUTs), which transport the tracer into the cell, and to the overactivation of hexokinases that phosphorylate [¹⁸F]FDG to [¹⁸F]FDG-6-phosphate and trap the tracer in the cell. Interestingly, a visually [¹⁸F]FDG-negative indeterminate thyroid nodule has a negligible risk of malignancy, making [¹⁸F]FDG PET/CT a suitable ruling-out test (as robustly demonstrated by meta-analyses [20,21,22]). Moreover, [¹⁸F]FDG PET/CT radiomics analysis preliminarily proved to increase specificity and PPV [23].

Table 1 gives an overview of the different tracers and imaging methods to further evaluate thyroid nodules in a clinical setup.

When DTC patients receive ¹³¹I therapy based on clinical-pathological risk stratification a [¹³¹I] post-treatment whole-body scintigraphy (TxWBS) is obtained 3–10 days after treatment. TxWBS is a highly sensitive and specific diagnostic tool useful to determine the location and extent of iodine-avid thyroid tissue. Consequently, it permits accurate disease restaging by detecting unknown loco-regional and/or distant metastases thus changing the initial risk stratification and customizing additional therapies and follow-up strategies. The diagnostic performance of TxWBS can be significantly improved using additional single-photon emission computed tomography/computed tomography (SPECT/CT). Compared to planar imaging, SPECT/CT images (i) reveal more foci of pathological radioiodine uptake located either in the cervical region or at distance (i.e., higher sensitivity), (ii) distinguish physiological uptakes from the foci of the disease (i.e., higher specificity), and (iii) detect metastatic lesions in unexpected sites [28,29].

Figure 1 illustrates a patient that received both pre-ablational WBS with a diagnostic dose and its correlative after therapeutic dose (TxWBS).

When the functional-imaging guided approach is preferred by the local team, postoperative whole-body scintigraphy with diagnostic activities of different radioactive iodine isotopes ([¹³¹I], [¹²³I], [¹²⁴I]) is performed. Theoretically, postoperative DxWBS leads to a significant improvement of risk stratification and staging of DTC patients and informs subsequent [¹³¹I] therapy [30]. As an example, visualization of metastatic lesions prompts risk re-stratification and, potentially, adjustment of [¹³¹I] administered activity. Indeed, the suspicion of noniodine-avid disease (i.e., negative radioiodine WBS with elevated thyroglobulin (Tg) or Tg levels out of proportion to the WBS findings) may require additional studies (i.e., neck/chest computed tomography, CT; [¹⁸F]FDG PET/CT). Finally, negative WBS results with undetectable Tg levels may rule out ¹³¹I therapy in low-risk DTC patients. Some authors, however, argue that TxWBS is more sensitive in identifying metastatic lesions (not initially seen on DxWBS), avoiding the so-called “stunning effect” of iodine-avid tissue [31]. Notably, recent improvements in technology (i.e., SPECT/CT), image acquisition, and reconstruction protocols enabled the use of lower [¹³¹I] activities [32]. Accordingly, a systematic review of 14 original research articles describing the incremental value of [¹³¹I] SPECT/CT demonstrated significant clinical benefit in terms of staging, risk stratification, and follow-up of DTC [33]. Figure 2 and Figure 3 show examples of [¹³¹I] TxWBS in patients with lymphogenous and/or distant spread.

In addition, [¹²³I] and/or [¹²⁴I] can be used instead of ¹³¹I to minimize the risk of stunning. Pre-ablation [¹²³I] WBS provided additional critical information in 25% of 122 patients, by revealing unsuspected regional or distant metastases and thus guiding the administration of higher ¹³¹I therapeutic activities, or revealing unexpected large thyroid remnants [34].

Other authors also demonstrated that the information gained by [¹²³I] DxWBS changed the applied [¹³¹I] activity in 49% of cases [35]. Santhanam and colleagues, in a recent meta-analysis, described a 94% sensitivity of [¹²⁴I] PET/CT in postoperative detection of metastatic lesions amenable to RAI [36]. In particular, it allows lesion-based evaluation of iodine uptake and clearance that is especially advisable to tailor [¹³¹I] therapy in patients with advanced disease [37,38]. Finally, new perspectives in the field are represented by NIS-imaging via [¹⁸F]tetrafluoroborate ([¹⁸F]TFB) and [¹⁸F]fluorosulfate ([¹⁸F]FSO₃). The visualization of the expression of NIS through in vivo molecular imaging has been based for a number of years on diagnostic or post-therapeutic [¹³¹I] WBS. The limitations of scintigraphic methods, including resolution and the possibility of target quantification, are well known. The technique of choice in the diagnostic setting could be [¹²⁴I] PET/CT, as it shows higher sensitivity compared to the [¹³¹I] WBS, but also allows for dosimetric estimation in therapeutic cases. However, it is not easily available and for its intrinsic characteristic requires a long time to scan with (presently) suboptimal quality and resolution of images. Recently, new tracers such as [¹⁸F]TFB or [¹⁸F]FSO₃ have shown promise as potential candidates for NIS visualization in preclinical studies and first preliminary human applications [39]. From a technical point of view, the development of fluorinate tracers has the advantages of easier labeling, higher image quality, and high tumor to background contrast in animal and human studies. From a biological point of view [¹⁸F]TFB and [¹⁸F]FSO₃ are iodine analogues, but with a main difference, the formers do not go into the iodine organification process in thyroid cells. These radiopharmaceuticals have been tested in healthy volunteers with promising biodistribution, in particular, low background uptake in the liver, muscle, and brain and high uptake in organs that normally express NIS (thyroid, salivary gland, and stomach) [40]. Furthermore, a rapid blood clearance (20 min after injection) and stability up to 4 h after injection have been observed. In breast cancer mice models [¹⁸F]TFB compared to [¹²³I] whole-body scan showed superior imaging characteristics related to a faster blood clearance, higher tumor/blood ratio, and higher sensitivity related to PET imaging [41]. O’Doherty et al. evaluated [¹⁸F]TFB uptake in 5 patients with intrathyroidal thyroid cancer while Samnik et al. compared [¹⁸F]TFB to [¹²⁴I] PET in 9 patients after total thyroidectomy [42,43]. In the first study, intra-thyroid tumor nodules showed lower uptake compared to normal thyroid tissue, while in the second the two tracers showed almost comparable performance. In only 2 patients, [¹⁸F]TFB showed two more lesions compared to [¹²⁴I] PET and in all cases lower retention in thyroid tissue, probably due to the absence of the organification phase. Their applications in thyroid cancer patients still require further exploration and at this moment are under investigation in pilot studies (NCT03196518). Of note, any therapeutic [¹³¹I] administration should be followed by a TxWBS to assess therapeutic [¹³¹I] localization, which is routinely used together with a preablation Tg measurement, to complete post-operative staging and predict the patient’s outcome.

Very promising results of [¹⁸F]FDG PET/CT performed at the time of first postoperative staging were reported in patients with high-risk DTC and Hürthle cell and poorly differentiated histotypes. In particular, [¹⁸F]FDG PET/CT might be very useful in correctly addressing disease aggressiveness and in detecting distant metastases (especially bone) in such cases [44].

There is no consensus regarding the routine use of DxWBS during the follow-up of patients with DTC [47]. Indeed, DxWBS remains a useful tool in selected patients with (i) higher risk of persistent/recurrent disease, (ii) extra-thyroid radioiodine uptake at TxWBS (i.e., loco-regional and/or distant metastases), (iii) poorly informative TxWBS (i.e., large remnants), or (iv) positive thyroglobulin antibody values. Gonzalez Carvalho and colleagues [48] evaluated a very large cohort of DTC patients (n = 1420) in all TNM categories in whom a life-long follow-up (up to 25 years) was regularly performed also using DxWBS. They concluded that in DTC patient follow-up, the use of DxWBS can still be justified at least until stimulated thyroglobulin is below functional sensitivity (in the absence of thyroglobulin antibodies) and DxWBS is negative (i.e., no evidence of radioiodine loco-regional and/or distant uptake). Importantly, the results of a large SEER database (28,220 patients diagnosed with DTC between 1998 and 2011) showed that follow-up DxWBS performed after primary treatment of DTC are the only imaging studies associated with improved disease-specific survival [49].

The main indication for [¹⁸F]FDG PET/CT in DTC is during DTC follow-up in the case of high or increasing thyroglobulin levels, but negative or doubtful ultrasound, and negative diagnostic and post-therapeutic RAI imaging. In such a context, evidence supports the use of [¹⁸F]FDG PET/CT, with reported pooled sensitivity and specificity of 80% and 90%, respectively [50,51,52]. Figure 4 demonstrates a patient with this flip flop phenomenon (i.e., WBS negative and FDG positive). The current ATA guidelines suggest that [¹⁸F]FDG PET/CT should be performed when stimulated thyroglobulin levels are >10 ng/mL. Further, although the positivity rate of [¹⁸F]FDG PET/CT increases with higher serum thyroglobulin level, true-positive results have also been reported in 10–20% of DTC patients with serum thyroglobulin levels <10 ng/mL [53,54]. Giovanella et al. suggested that shortened thyroglobulin doubling time (i.e., <1 year) independently predicted a positive [¹⁸F]FDG PET/CT scan in patients with biochemical recurrence [55]. Considering the relatively low sensitivity of diagnostic ¹³¹I WBS, one of the most important issues is the comparison of post-therapeutic ¹³¹I WBS with [¹⁸F]FDG PET/CT. Leboulleux et al. evaluated the sensitivity of post-therapeutic [¹³¹I] WBS versus [¹⁸F]FDG PET/CT in patients with elevated serum thyroglobulin levels. The sensitivity in detecting DTC relapse was 88% for [¹⁸F]FDG PET/CT and 16% for TxWBS (p < 0.01), respectively. [¹⁸F]FDG-PET/CT was abnormal in 22 patients, five of whom also had an abnormal TxWBS. The authors concluded that in patients with suspicion of recurrence based on thyroglobulin levels after a normal TxWBS, [¹⁸F]FDG PET/CT is better able to localize disease than TxWBS [56]. Similarly, Kim et al. reported that second empiric ¹³¹I therapy and TxWBS were neither diagnostically nor therapeutically useful in 39 patients with elevated stimulated thyroglobulin and negative [¹⁸F]FDG PET/CT after initial treatment. These data suggest that the correct integration of radioiodine imaging and [¹⁸F]FDG PET/CT may optimize additional administrations of high [¹³¹I] activities and inform alternative strategies (i.e., surgical procedures or external beam radiotherapy) [57].