Abstract
Objectives
The aim was to evaluate the clinical, ultrasound (US) and, when indicated, the cytological and histological characteristics of autonomously functioning thyroid nodules (AFTN) in consecutive patients.
Methods
A prospective, single-centre study was conducted between March 2018 and September 2021. In total, 901 consecutive patients were referred for thyroid workup and of 67 AFTN were evaluated. All enrolled patients underwent 99mTcO4 − scintigraphy, additional 123I scintigraphy only in case of normal serum TSH, evaluation of thyroid function, US examination using European Thyroid Imaging and Reporting Data System (EU-TIRADS), and US-guided fine needle aspiration (FNA) cytology when indicated. All indeterminate FNA samples were subjected to DNA sequencing analysis.
Results
More than half of the evaluated patients with AFTN were euthyroid; median serum TSH was 0.41 (IQR: 0.03–0.97) mU/L. The median AFTN size measured by US was 27.0 (IQR: 21.1–35.0) mm. 28.4% of AFTN were classified as EU-TIRADS score 3 and 71.6% as EU-TIRADS score 4, indicating that the majority of AFTN had intermediate risk for malignancy according to US. Out of the 47 AFTN subjected to cytological evaluation, 24 (51%) yielded indeterminate FNA results. DNA sequencing revealed pathogenic TSHR and GNAS mutations in 60% of cases. No malignancy was detected at final histology in surgically excised AFTN (n = 12).
Conclusions
Of the 67 AFTN evaluated in this study, 50% presented with normal serum TSH, 70% displayed ultrasound features suggesting an intermediate malignancy risk and 50% of the AFTN submitted to cytology yielded indeterminate results. No malignant AFTN was detected.
Introduction
Thyroid nodules are a very frequent problem in routine clinical practice (1, 2). In regions with past or current iodine insufficiency, such as many European countries, the prevalence of autonomously functioning thyroid nodules (AFTN) is higher than in iodine-sufficient regions, although exact epidemiological data are currently lacking (3, 4, 5, 6). AFTN are mainly caused by gain-of-function mutations of the TSH receptor (TSHR) or of the α subunit of the adenyl-cyclase stimulating G-protein (GNAS), occurring in 60–79% and 5–6% of AFTN, respectively (7, 8, 9). In consequence, due to increased TSH signalling, AFTN have increased expression of sodium–iodine symporter (NIS) and so increased capacity, in comparison to the surrounding thyroid tissue, to uptake either 99mTc or 123I. For this reason, AFTN appear as hyperfunctioning/hot nodules in thyroid scintigraphy. In the literature, AFTN are frequently synonymous to hyperfunctioning nodules; the term hyperfunctioning refers to the scintigraphic appearance of AFTN, while autonomy refers to their pathogenesis (10, 11). AFTN’s cellular density and degree of cellular activity, iodine organification capacity, patient’s iodine intake, and disease natural history are crucial determinants of serum TSH (10, 11, 12, 13, 14). When scintigraphy is routinely performed in thyroid nodule workup, it allows detection of AFTN at early stages before progression to subclinical and/or overt hyperthyroidism.
Ultrasound (US), the first-line tool to stratify malignancy risk of thyroid nodules, does not, unlike scintigraphy, evaluate the nodule’s functional characteristics. The utility to effectively diagnose AFTN relies on the generally admitted assumption that AFTN are rarely malignant (1, 2, 4, 15). Of note, AFTN malignancy rate reported in the literature varies from 0% up to 34%, suggesting possible bias and methodology limitations, since precise cancer localisation within the AFTN or not is frequently lacking (16, 17, 18).
Thyroid scintigraphy is recommended only in patients with subnormal serum TSH by the American Thyroid Association (ATA) and European Thyroid Association (ETA) and may be considered in patients with low normal TSH (0.5–1 mU/L) in iodine-deficient regions by the joint American–Italian endocrinology association American Association of Clinical Endocrinologists/American College of Endocrinology/Associazione Medici Endocrinologi (AACE/ACE/AME) guidelines (3, 15, 19). A clear TSH threshold for optimal detection of AFTN has not yet been adopted (11, 13, 14).
Very recently, one prospective study from Germany and two subsequent retrospective studies from Switzerland and Northern Italy reported that AFTN may present suspicious ultrasound features according to various classification systems (5, 20, 21).
The goal of this study was to obtain ultrasound characterisation of consecutive AFTN according to European Thyroid Imaging Reporting and Data System (EU-TIRADS) classification and to evaluate their cytological and molecular aspect.
Materials and Methods
Patients and study design
This is a single-centre, observational, prospective study performed between March 2018 and September 2021, on 901 patients aged ≥18 years consecutively referred for workup to the Thyroid Nodule Diagnostic Unit (TNDU) within the Nuclear Medicine Department of H.U.B. Hôpital Erasme, ULB, Brussels. All referred patients to TNDU routinely undergo US, and those presenting at least one nodule of ≥1 cm in US undergo 99mTcO4 scintigraphy, unless the latter has been previously performed or the presence of confirmed hypothyroidism has been established. The study was approved by the Hôpital Erasme, ULB Ethics Committee (P2018/226), and all enrolled patients provided a written informed consent.
The strategy of patients’ enrolment to the study protocol is shown in Fig. 1. Patients were included in the study when they presented a well-defined AFTN with a perfect anatomic match to a nodule of ≥1 cm, identified by US performed the same day. The diagnostic criterion of AFTN was based on 99mTcO4 scintigraphy: that is, a distinct nodule with increased radionuclide intake (hyperfunctioning = autonomously functioning) in comparison to the surrounding thyroid tissue, as previously described (11). AFTN that were mainly cystic, displaying only a small peripheral hyperfunctioning area were not included in the study because of the difficulty of the US characterisation of these minority solid areas. The other exclusion criteria were pregnancy or lactation, active malignancy, uncontrolled concomitant disease requiring further clinical investigation, previous radioiodine therapy, recent administration of iodine-contrast media, current glucocorticoid, levothyroxine, amiodarone, or thyrostatic drug use.
Methodology flowchart for patients’ enrolment in this study.
Citation: European Thyroid Journal 12, 6; 10.1530/ETJ-23-0135
The routinely recorded data for all evaluated patients were the following: age, sex, weight, body mass index (BMI), cervical irradiation history, familial history of thyroid disease, hypertension, diabetes, smoking habits, serum TSH and serum FT4, FT3, and thyroid antibody status when available. Grade 2 subclinical hyperthyroidism (SCH) was defined as TSH: < 0.1 mU/L and grade 1 SCH as TSH: 0.1–0.39 mU/L (22). In patients with normal serum TSH (0.4 mU/L ≤ TSH ≤ 4 mU/L), we additionally performed 123I thyroid scintigraphy to ensure that patients did not present rare trapping-only thyroid nodules as previously described (23). US-guided FNA was performed when indicated, according to the current guidelines taking into account EU-TIRADS score and nodule size (15). No patient received anti-thyroid drugs before FNA. Cytological results were reported according to the Bethesda classification system (24). DNA was obtained from all Bethesda III, IV, and V cytological samples and was subjected to targeted next-generation sequencing using Ion Gene Studio S5, Ion Torrent Platform with AmpliSeqTM Kit (Life Technologies) as previously published (25). Of note, only exons 9 and 10 of the TSHR gene are included in the routine NGS analysis for cytologically indeterminate thyroid nodules in our centre. Thyroidectomy, as a treatment option, was discussed with the patient and with the local multidisciplinary team. Histological results were recorded when available.
Laboratory evaluation
TSH, FT4, FT3, anti-TPO Abs, and anti-Tg Abs were measured by electrochemiluminescence immunoassay using the cobas e801 module (Roche Diagnostics). Anti-TSH receptor-stimulating antibodies were measured by 2000/2000 Immulite Xpi immunoassay (Siemens Medical Solutions, negative threshold <0.55 U/L).
Ultrasound
All US examinations were performed in Hôpital Erasme by a single radiologist (N.T., with 20 years of experience in thyroid US). Conventional 2D greyscale US images were obtained on the ACUSON S2000 system (Siemens, France) by using a linear-array transducer (14L5) with a frequency range of 14–6 MHz in 2D mode and 7.5–5.5 MHz for Doppler analysis. During greyscale US examination, thyroid nodules were evaluated for location (side, superior, medial, and inferior), size (width, depth, length, and volume), composition, echogenicity, shape, margins, and presence or absence of macro- and micro-calcifications, using EU-TIRADS classification system guidelines (15). Next, the colour Doppler blood flow imaging technology was used to observe the vascularisation of the nodules, which was rated as absent or perinodular (type I), perinodular and/or slight intranodular (type II), and marked intranodular and slight perinodular (type III) (15). Each examination included the study of lymph nodes (levels II, III, IV, V, VI) and of the thyroglossal duct. All images were reviewed in a double-blind manner by another expert (G.R.) with more than 30 years of experience in thyroid US imaging. A final single EU-TIRADS score (EU-TIRADS 2, 3, 4, 5) and vascularity type were assigned to each thyroid nodule approved by both radiologists.
Scintigraphy
Scintigraphy was performed 20 min after an intravenous injection of 22.2 MBq of 99mTcO4 −, using a gamma camera SOPHA MEDICAL DSX (SMV International, BUC, France) equipped with a pinhole collimator with a 205 mm height, a 295 mm diameter and a 5 mm aperture.123I scintigraphy after injection of 26 MBq of Na 123I, using the same gamma camera was performed when indicated. Acquisition time was set at 10 min, or when 250,000 counts were obtained. Based on 99mTcO4 − scintigraphy, thyroid nodules were classified as autonomously functioning = hyperfunctioning (nodule’s radionuclide uptake greater than the surrounding normal thyroid tissue), isofunctioning/scintigraphically indifferent (nodule’s radionuclide uptake equal to the surrounding normal thyroid tissue), and hypofunctioning (nodule’s radionuclide uptake less than the surrounding normal thyroid tissue). A systematic anatomic correlation between planar images, SPECT-CT and US images was performed to ensure perfect anatomic match for each scintigraphically classified nodule. Areas that displayed increased radionuclide uptake greater than the surrounding normal thyroid tissue but did not correspond to US-identified nodules were classified as hyperfunctioning areas. In this study, we evaluated only AFTN with a perfect anatomic match to a nodule of ≥1 cm, identified by US performed the same day.
Statistical analysis
Statistical analysis was carried out with GraphPad prism, version 9.2.0. Data are reported as mean ± s.d. or median, 25–75th range as appropriate. Results were considered to be significant when P < 0.05.
Results
Of the patients referred to our Thyroid Nodule Diagnostic Unit, 81 patients were diagnosed with a distinct, well-defined AFTN in 99mTcO4− scintigraphy having a perfect anatomic match to a ≥1 cm nodule identified by US (Fig. 1). One patient had active extra-thyroidal malignancy, three had almost entirely cystic AFTN, two patients refused participation and seven eluded recruitment procedure, and so were excluded. Twenty-seven out of the 36 euthyroid patients with AFTN underwent 123I scintigraphy which was positive and concordant with 99mTcO4 − for the presence of an AFTN for all cases but 2, so the latter were excluded from the final analysis. Finally, a total of 66 patients, harbouring 67 AFTN (1 patient had 2 distinct and well-defined AFTN) were evaluated (Fig. 1).
Clinical characteristics of the study population
The majority of patients were female (F/M = 55/11) with a mean age of 56.3 ± 14.9 years and family history of thyroid diseases in one-third of them (Table 1). The median serum TSH was 0.41 mU/L (IQR: 0.03–0.97), 51.5% of patients were euthyroid, 31.8% had SCH grade 2 (TSH: <0.1 mU/L), and 16.6% of patients had SCH grade 1 (TSH: 0.1–0.39 mU/L), no patient had overt hyperthyroidism (Table 1, Fig. 2). Notably, 15 of the enrolled patients had TSH ≥1 mU/L, 5 patients had TSH ≥1.5 mU/L, and only 3 patients had TSH >2 mU/L. No patient was positive for anti-TSH receptor antibodies, the latter were measured only patients with subnormal TSH (<0.4 mU/L, data not shown).
Serum TSH levels (mU/L) of the 66 included patients measured within 3 months prior to AFTN diagnosis. Each column (marked with a tick in the x-axis) represents a single TSH measurement of one individual patient. Median serum TSH was 0.41 mU/L and 51.5% (n = 34) of patients were euthyroid (TSH ≤0.4–4 mU/L, columns within the light grey box). Fifteen patients of our studied population had serum TSH ≥1 mU/L (columns within the dash-lined box), threshold proposed in consensus/guidelines for AFTN diagnosis. Five patients had serum TSH ≥1.5 mU/L and only three had serum TSH ≥2 mU/L.
Citation: European Thyroid Journal 12, 6; 10.1530/ETJ-23-0135
Clinical characteristics of patients (n = 66) harbouring 67 autonomously functioning thyroid nodules (AFTN). Data are represented as mean ± s.d., median (IQR: 25th–75th range) or n (%).
| Characteristics | Values |
|---|---|
| Sex | |
| Female | 55 |
| Male | 11 |
| Age (years) | 56.3 ± 14.9 |
| BMI | 26.1 ± 4.2 |
| Hypertension | 26 (39.3%) |
| Diabetes mellitus | 9 (13.6%) |
| Current smoking | 13 (19.6%) |
| Personal history | |
| Non-active malignancy | 3 (4.5%) |
| Cervical radiation | 1 (1.5%) |
| Family history of thyroid disease | 21 (31.8%) |
| TSH (mU/L) | 0.41 (0.03–0.97) |
| Euthyroid patients (TSH ≤0.4–≤4) | 34 (51.5%) |
| Subclinical hyperthyroidism | |
| Grade 2 (TSH <0.1) | 21 (31.8%) |
| Grade 1 (TSH ≤0.1–<0.4) | 11 (16.6%) |
| FT4 (pmol/L) (n = 59) | 14.6 (12.6–16.1) |
| FT3 (pmol/L) (n = 31) | 5.6 (4.9–7.4) |
| Anti-TPO Abs positive (n = 53) | 2 (3.8%) |
| Anti-Tg Abs positive (n = 34) | 1 (2.9%) |
| Thyroid gland US volume (cm3) | 18.2 (14.5–26.2) |
| Females (n = 55) | 17.8 (14.2–24.5) |
| Goitre (>16 cm3) | 35 (63.6%) |
| Males (n = 11) | 19.6 (18–30.8) |
| Goitre (>20 cm3) | 5 (45.5%) |
| Treatment received following diagnostic evaluation | 22 (33.3%) |
| 131I therapy | 10 (15.1%) |
| Thyroidectomy | 12 (18.2%) |
| Partial | 5 |
| Total | 7 |
| Malignancy in final histological analysis | 0 |
Normal assay ranges for TSH: 0.27–4.20 mU/L, for FT4: 12–22 pmol/L, for FT3: 3.1–6.8 pmol/L, negative threshold for anti-TPO Abs <34 kUI/L, and for anti-Tg Abs <115 kUI/L.
Abs, antibodies; BMI, body mass index; FT4, FT3, free serum T4, T3 levels; Tg, thyroglobulin; TPO, thyroperoxidase; US, ultrasound.
Ultrasound and scintigraphy characteristics
The median greatest dimension of the AFTN evaluated was 27 (IQR: 21.1–35.0) mm, and median volume was 4.2 (IQR: 2.4–8.1) cm3. AFTN presented as unique clinically significant nodule in 26 out of 66 (39.4%) patients. The majority of AFTN (n = 42, 62.7%) had a mixed (solid and partially cystic) composition at ultrasound (Table 2). According to EU-TIRADS, 19 AFTN (28.4%) were classified as low malignancy risk score 3 and 48 (71.6%) as intermediate malignancy risk score 4 (Fig. 3A) at US. No AFTN was classified as EU-TIRADS score 2 or 5. Nodular vascularity was classified into type I in 2 (2.9%) AFTN, type II in 14 (20.9%) and type III in 51 AFTN (76.1%) (Fig. 3B). Representative US and scintigraphy appearance (99mTcO4 −, 123I images) of AFTN with normal TSH (panel C, a) and subnormal TSH (panel C, b) in our study is shown in Fig. 3.
(A) Classification score by ultrasound of the 67 autonomously functioning thyroid nodules (AFTN) evaluated in this study according to European Thyroid Imaging Reporting and Data System (EU-TIRADS). EU-TIRADS score 4, that is intermediate risk for malignancy was attributed to 71.6% of AFTN studied. (B) Type of vascularity of AFTN as evaluated by US colour Doppler imaging and classified as absent or perinodular (type I), perinodular and/or slight intranodular (type II), marked intranodular and slight perinodular (type III). The great majority (76.1%) of AFTN were markedly vascularised. (C, a) Representative US (greyscale and colour Doppler) and 99mTcO4− and 123I scintigraphy images of an AFTN in a patient with normal serum TSH. The nodule had mixed (solid and cystic) composition, was classified as EU-TIRADS score 4, with vascularity type III and the extra-nodular parenchyma on both radionuclide scans was not completely extinct. (C, b) Representative US and 99mTcO4− scintigraphy images of an AFTN in a patient with subnormal serum TSH. The nodule had solid composition, EU-TIRADS score 4, vascularity type III and extinction of the extra-nodular parenchyma was observed on 99mTcO4− scan.
Citation: European Thyroid Journal 12, 6; 10.1530/ETJ-23-0135
Ultrasound and molecular characterisation of autonomously functioning thyroid nodules (AFTN) (n = 67) in 66 patients. Data are represented as mean ± s.d., median (IQR 25th–75th range) or n (%).
| Characteristics | Values |
|---|---|
| AFTN US greatest dimension (mm) | 27.0 (21.1–35.0) |
| AFTN US volume (cm3) | 4.2 (2.4–8.0) |
| AFTN US localisation (right/left lobe) | 43/24 |
| AFTN as solitary nodulea | 26 (39.4%) |
| AFTN US composition | |
| Solid | 25 (37.3%) |
| Mixed (not entirely solid, cystic areas) | 42 (62.7%) |
| % of nodule surface corresponding to cystic areas | |
| <10% | 12 |
| ≥10–<50% | 20 |
| ≥50% | 10 |
| DNA sequencing of AFTN with Bethesda III–V cytology results (n = 25) | |
| TSHR mutations | 14 (56%) |
| GNAS mutations | 1 (4%) |
| Otherb | 2 (8%) |
| Negative | 8 (32%) |
Bethesda III: atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), Bethesda IV: follicular neoplasm/suspicious for follicular neoplasm, Bethesda V: suspicious for malignancy.
aNodules <5 mm were considered as clinically non-significant and were not described in US protocol; b1 HRAS gene mutation found in a nodule with Bethesda IV cytology and 1 KRAS mutation found in a nodule with Bethesda III cytology.
US, ultrasound.
Cytological analysis
Overall, FNA indication was present for 58 (86.6%) AFTN studied hereby, 30 AFTN in euthyroid patients and 28 AFTN in SCH patients. FNA was performed in all euthyroid patients and in more than half (n = 17) SCH patients. Thus, a total of 47 (70.1%) AFTN (32 nodules with EU-TIRADS score 4 and >15 mm size and 15 nodules with EU-TIRADS score 3 and >20 mm) were subjected to FNA.
Only one FNA sample was unsatisfactory, no sample was malignant; the other samples were diagnosed as benign (n = 21; 44.7%), Bethesda III (n = 16; 34%), Bethesda IV (n = 8; 17%), and Bethesda V (n = 1; 2.1%) (Fig. 4).
Cytological evaluation by fine needle aspiration (FNA) of 46 autonomously functioning thyroid nodules (AFTN). Bethesda categories II, III, IV, and V correspond to benign, atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), follicular neoplasm/suspicious for follicular neoplasm, and suspicious for malignancy, respectively. Fifty-one percent of AFTN had indeterminate cytology results, that is, Bethesda III (34%) and IV (17%). Only one sample was non-diagnostic and only one yielded Bethesda V results. No malignancy was found in cytological examination.
Citation: European Thyroid Journal 12, 6; 10.1530/ETJ-23-0135
DNA sequencing
DNA sequencing of all AFTN with Bethesda III–V cytology results (n = 25) are illustrated in Table 2.
Overall, TSHR and GNAS mutations were found in 60% of them. Of note the only Bethesda V sample found in this study displayed a TSHR (F631L) mutation.
Treatment and follow-up
All patients of the study are alive and actively being followed up till now; ten patients received an outpatient-administered therapeutic 131I activity, four patients underwent thyroid lobectomy, and eight patients underwent total thyroidectomy (Table 1).
Histological analysis
Histological analysis of the 12 (18.2%) patients submitted to surgery showed no malignancy within the AFTN (Table 1). The only Bethesda V cytology result corresponded to a 23 mm follicular adenoma with lymphocytic thyroiditis in the surrounding thyroid parenchyma. We observed one non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) in a thyroid nodule of 31 mm, EU-TIRADS score 3, Bethesda III cytology displaying a PAX8/PPARG gene fusion, which was adjacent to a 32 mm AFTN, both located in the left lobe, in a patient with normal TSH. In four patients undergoing total thyroidectomy an incidental papillary thyroid microcarcinoma (PTMC) was identified outside of the AFTN: in two cases (PTMC of 8 mm and 9 mm size respectively, both BRAFV600E mutated) located in the contralateral lobe, in one case (PTMC of 3 mm) in the same lobe and in one case (PTMC of 2 mm) in the thyroid isthmus.
Discussion
The present study of 67 consecutive AFTN showed that 70% of them had intermediate malignancy risk at US according to EU-TIRADS, yielding a 50% rate of indeterminate cytology results.
The ultrasound appearance of AFTN remained largely unknown till recently. In the present study 71.6% of the AFTN evaluated, were classified as EU-TIRADS score 4 and 86.6% of them had an ultrasound-based FNA indication according to EU-TIRADS score and nodule size. Our results are in accordance with the only other available prospective study, performed by Schenke et al., in 2019. The authors classified 54.2% of 615 AFTN as TIRADS 4B or 4C (5). In two retrospective studies in 2020, about ~30% of AFTN were classified as intermediate–high risk according to different US classification systems and ~27−90% of AFTN had an FNA indication depending on the system used (20, 21). Thus, our data draw attention that these generally assumed benign nodules may appear suspicious in US and so may warrant further investigation such as FNA if the scintigraphy is not performed.
Furthermore in the present study, 51.5% of the patients with AFTN were euthyroid, corroborating knowledge that subnormal serum TSH alone cannot effectively rule out the presence of AFTN, especially in geographic regions with previous or current iodine deficiency (11, 13, 14). Fifteen of the enrolled patients had TSH ≥1 mU/L, five patients had TSH ≥1.5 mU/L, and only three patients had TSH >2 mU/L. If the 1 mU/L TSH threshold as proposed by AACE/ACE/AAME guidelines had been applied to perform scintigraphy, a substantial proportion (22.7%) of patients would have been missed (19). An optimal TSH threshold in iodine-insufficient populations in order to efficiently diagnose AFTN remains to be determined in larger studies.
In our study population, 51% of AFTN submitted to FNA yielded indeterminate cytology results, that is Bethesda III–IV diagnostic categories. Of note only one FNA sample was non-diagnostic, displaying a high diagnostic accuracy. In retrospective studies, with a higher proportion of non-diagnostic samples (15.8–22%), a rate varying from 4.6% to 31.6% of indeterminate cytology resulting in AFTN/hot nodules has been reported (26, 27). Our data point out that if undiagnosed AFTN are submitted to FNA, the confirmation of the benign character of AFTN by cytology may be challenging.
Regarding cancer risk, there is a discordance between the presumed benign nature of AFTN that is generally accepted by expert societies and the actual AFTN malignancy rate reported in the literature, ranging from 0% up to 34%, pointing to a methodological bias (16, 17, 18). There is only one prospective study, by Rosario et al., carrying out preoperative 123I scintigraphy in a target population of n = 84 cytologically indeterminate nodules with serum TSH <2 mU/L, all submitted to surgery (28). The authors reported a high prevalence of 25% of AFTN among indeterminate nodules. They observed no malignancy but one tumor of undetermined malignancy potential in resected AFTN. In our study, we did not find thyroid cancer within AFTN at histology. The observed non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and all four papillary microcancers were located clearly outside the AFTN. These data suggest low cancer risk for AFTN and underlie the difficulty of drawing conclusions from retrospective studies without exhaustive diagnostic nodule workup and precise localisation in the preoperative period.
In our study, DNA sequencing, carried out as part of routine diagnostic procedure, confirmed the presence of a pathogenic mutation of TSHR (n = 14) or GNAS (n = 1) gene in 60% of cytologically indeterminate AFTN. Importantly, we did not detect any BRAFV600E mutations, RET/PTC, PAX8/PPARγ, NTRK fusions in AFTN with indeterminate cytology. Diagnostic rate could be improved with sequencing of the entire TSHR gene. In the only retrospective study on the subject, by Mon et al., 4.4% of TSHR gene mutations in 703 FNA samples with indeterminate results were reported (29). As a future perspective, the inclusion of TSHR, GNAS gene mutations in sequencing panels for cytologically indeterminate thyroid nodules, at least in centres that do not routinely perform scintigraphy, could be relevant for the accurate diagnosis of AFTN.
The actual cost–benefit ratio of scintigraphy, the only method to detect AFTN, as a first-line diagnostic tool to stratify thyroid nodule malignancy risk remains yet to be studied. Most probably, scintigraphy might not be cost-effective in an unselected population, given the unknown exact prevalence of AFTN. However, given these data, scintigraphy can still be relevant in the initial thyroid workup in populations where AFTN prevalence is expected to be high, that is patients with TSH <1.5 mU/L in regions with past or current iodine deficiency. In this setting, scintigraphy could help to avoid unnecessary FNA, better guide management decisions and avoid unnecessary thyroidectomies in cytologically indeterminate nodules. Ideally, the cost–benefit ratio of molecular sequencing versus scintigraphy in cancer risk stratification of cytologically indeterminate thyroid nodules should be addressed in future studies.
Our study has some limitations. It was a single-centre study, the number of patients was relatively small and cytology was not performed for the whole study population. However, functional and anatomic matching in the preoperative imaging studies and in final histology, was performed meticulously for all evaluated AFTN, DNA sequencing was carried out for all cytologically indeterminate nodules, offering the advantage of one of the rare prospective studies with a thoroughly described AFTN population.
In conclusion, in this study the majority of evaluated AFTN displayed ultrasound features of intermediate malignancy risk according to EU-TIRADS, requiring an FNA. Of the cytologically evaluated AFTN, 51% yielded indeterminate results, potentially leading to unnecessary thyroidectomy. Molecular diagnosis was possible with routine-practice DNA sequencing for 60% of cytologically indeterminate AFTN. None of the AFTN was malignant at histology, suggesting an overall low cancer risk in our AFTN population. Last but not least, presence of normal TSH alone did not effectively rule out AFTN diagnosis because half of them occurred in euthyroid patients. Cost–benefit studies evaluating scintigraphy as the first-line tool to stratify cancer risk of thyroid nodules are still needed.
Declaration of interest
The authors of this article declare no relationships with any companies, whose products or services may be related to the subject matter of the article.
Funding
The authors state that this work has not received any funding.
Author contribution statement
AK participated in study design, patient recruitment, data collection; performed all data analysis; and wrote the manuscript. NT, LL and IS participated in data collection. GR provided a detailed double-blind lecture of all AFTN US images and provided manuscript corrections. RM-R participated in study design, patient recruitment, and provided manuscript corrections. BC participated in study design, patient recruitment, and supervised data analysis and manuscript writing. All authors read and approved the final version of the manuscript.
Acknowledgements
We thank all our patients for participating in this study, the paramedical team of Nuclear Imaging Department for their collaboration, Prof Nicky D’Haene for helping with FNA sample sequencing and Prof Matteo Cappello and Dr Maria Ruiz for their long-standing devotion to our thyroid patients’ management. Aglaia Kyrilli and Bernard Corvilain are members of European Reference Network on Rare Endocrine Diseases (aka ENDO-ERN).
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