Comparisons between the results of tissue aspirate PTH assays and localization studies are shown in Table 2. Among the 50 lesions, three (6%) of them were triplepositive, indicating positive parathyroid scintigraphy, sonography results, and positive tissue aspirate PTH assays. Of the remaining 47 lesions that had uncertain or conflicting localization results between the parathyroid scintigraphy and sonography, 36 were assaypositive (72%) and 11 were assay-negative (22%).

**Table 2.** Correlation of the results between tissue aspirate PTH assays and localization studies.

\* One false-negative tissue aspirate PTH assay included. Abbreviation: PTH, parathyroid hormone.

#### *3.4. Surgical Intervention*

Thirty-five assay-positive (35/39, 90%) and three assay-negative lesions (3/11, 27%) in 26 patients were explored through neck incisions. All assay-positive and one assaynegative lesions (case 14) were pathologically proven to be parathyroid tissues. Explorations were performed for case 14 because of the uncertainty of the tissue aspiration procedure, which offered borderline assay results. In the other two true assay-negative lesions (case 26-1, 29), removal of the lesions was arranged due to concomitant exploration in the same operation field for other reasons. No surgery was performed for the four assay-positive lesions in the three patients because of presumed severe adhesion from previous management (4 times neck operations and 8 times alcohol injections, case 30) and controllable PTH levels (case 15, 21). The median weight of resected parathyroid glands was 878.0 mg (range 193.0 to 3674.0 mg). In the lesions for which surgery was performed, the positive predictive value of tissue aspirate PTH assays was 100%.

#### *3.5. Reoperative Findings*

The reoperative findings are shown in Table 3. Among the 36 parathyroid lesions in the 26 patients who underwent surgery, six lesions were ectopic parathyroids, nine lesions were parathyromatosis, and eight lesions were intrathyroid parathyroids. The other 13 lesions were located over the usual parathyroid areas that had already been explored during previous surgical procedures.

**Table 3.** Location of parathyroid lesions removed during reoperations.

\* Seven patients had two or more assay-positive lesions removed during the same operation. Abbreviations: LS: left superior; LI: left inferior; RS: right superior; RI: right inferior.

#### **4. Discussion**

In our series, among the patients who received reoperations, 16.7% of the lesions were ectopic, 22.2% were intrathyroid, 36.1% were located over previously explored areas, and 25.0% lesions were confirmed to be parathyromatosis in this study. In challenging medical scenarios, experienced surgeons need comprehensive pre-reoperative localization information to ensure that treatment is achieved. The elevated tissue aspirate PTH assays found with our newly proposed positive assay definitions may help surgeons identify suspicious neck lesions discovered in localization imaging studies with a high positive predictive value (100%) and effectively avoid negative neck explorations in such complicated cases.

Although tissue aspirate PTH assays can help confirm parathyroid tissue for suspicious neck lesions, not all neck lesions need this examination. We suggest this procedure be reserved for suspicious neck lesions with uncertain or discrepant localization study results, lesions with unusual locations or numbers, lesions over previously explored areas, or a combination of these findings. One of the reasons to use tissue aspirate PTH assays sparingly is the potential for seeding and causing parathyromatosis. To avoid such complications, including seeding along the needle tract, fibrotic tissue reaction, and potential reactive changes of parathyroid lesions [20], the operator should fix the needle and simply oscillate the piston with negative pressure following the needle's introduction into the center of the suspicious lesion instead of moving the needle tip back and forth, as in other studies [17,19,21].

From our series, we found that 71.8% (28/39) of the lesions with positive tissue aspirate PTH assays were considered to be uncertain (suspicious) by parathyroid sonography. Among the positive tissue aspirate PTH assays with uncertain sonographic lesions, 82% (23/28) were scintigraphy-negative or scintigraphy-suspicious lesions. Therefore, tissue aspirate PTH assays may help in identifying lesions that potentially need intervention. Moreover, without the application of tissue aspirate PTH assays, at least nine (23.1%) of the lesions with positive tissue aspirate PTH assays could have been easily missed based on the localization results (negative parathyroid scintigraphy and uncertain parathyroid sonography).

There is no established standard for the level of PTH that is considered to indicate positive evidence that the aspirated tissue represents parathyroid tissue. Theoretically, the level of PTH in nonparathyroid tissue should be undetectable, as described in a prior study [22]. There have been different positive tissue aspirate PTH values recommended in the individual studies, and the PTH values from different assays are not interchangeable. Frasoldati et al. [21] washed out the tissue aspirates with 1 mL saline and reported a 100% sensitivity and specificity with a cut-off level of 101 pg/mL. Using the same dilution formula, Maser et al. [23] reported that a value higher than the normal range (6–40 pg/mL)

indicated parathyroid tissue and a value ranged between 49~65 pg/mL was considered questionable. Stephen et al. [19] diluted a tissue aspirate in 5 mL saline and reported a 94% sensitivity, 77% negative predictive value, and 100% specificity and a positive predictive value when the level was higher than 40 pg/mL. However, by defining the positive tissue aspirate PTH value as higher than the constant or normal PTH level, the results could potentially be contaminated if the blood is aspirated from nonparathyroid tissue and the patient has a high serum PTH [22]. Since the main source of contamination of PTH comes from the aspiration of blood, it is reasonable to define the positive tissue aspirate PTH value through a comparison with the serum PTH. Abdelghani et al. [17] washed out the aspirate with 2 mL saline and reported a 91.6% sensitivity and 100% positive predictive value when the tissue aspirate PTH level was higher than that of serum PTH at the time of performing the aspiration. Nevertheless, the dilution factor of the tissue aspirate to the saline volume was not taken into consideration in Abdelghani et al.'s [17] study and would have produced more false-negative results. In our study, the tissue aspirate was washed out into 6 mL saline, and the volume of aspirate was estimated to be no more than 0.2 mL. Therefore, a positive result was considered when the tissue aspirate PTH level was higher than one-thirtieth (0.2 mL/6 mL) of the serum PTH level. Based on this, the positive predictive value was 100%, and the presumed specificity was 100% (since the possibility of a false-positive result was nearly zero) in our series. Three false-negative results using the criterion of Abdelghani et al. [17] would yield true positive results using our criterion (case 1, 3, 28) (Table S2).

However, there are limitations to this method. To determine the negative predictive value, sensitivity, and specificity, all the assay-negative lesions should be removed. However, it is important clinically to avoid operations for the potential true negative lesions while managing persistent or recurrent renal hyperparathyroidism. One may argue that our criterion maximizes specificity without providing any evidence for sensitivity; thus, this method may offer a very high positive predictive value but also produce falsenegatives. However, based on a comparison with the criteria used in other studies (Table S2), our criterion produces fewer assay-negative lesions under the absence of false positives, which justifies our definition. For the borderline assay-negative lesions (e.g., case 14, 29), repeated tissue aspiration, additional localization studies, or direct explorations should be performed. Our proposed positive assay definition is based on the assumption of the tissue aspirate volume less than 0.2 mL in the needle (0.2 mL/6 mL). Future prospective studies may be warranted to further verify this definition.

There are several strengths to this study. This is the first and largest series of tissue aspirate PTH assays performed in patients with persistent or recurrent renal hyperparathyroidism. Moreover, this is the first study to describe the complexity of pre-reoperative localization studies with uncertain or discrepant results and how tissue aspirate PTH assays could be helpful in such a difficult medical scenario. Based on the positive value of the tissue aspirate PTH assay, the relative value between the tissue aspirate PTH level and serum PTH level along with the concept of the dilution ratio was proposed to yield ideal results.

#### **5. Conclusions**

In conclusion, the selective application of a tissue aspirate PTH assay with the newly proposed positive assay definition is feasible and may help clarify the nature of suspicious neck lesions on imaging. This assay may effectively avoid negative neck explorations in patients with complicated persistent or recurrent renal hyperparathyroidism.

**Supplementary Materials:** Supplementary materials can be found at https://www.mdpi.com/2077 -0383/10/2/329/s1. Table S1. Demographic data of patients receiving tissue aspirate PTH assays. Table S2. Comparison of tissue aspirate PTH assays results using different criteria.

**Author Contributions:** Study design, S.-M.H. and C.-J.H.; data collection, C.-L.H. and C.-J.H.; data analysis, C.-L.H. and C.-J.H.; manuscript writing and figure preparation, C.-L.H., Y.-C.H. and C.-J.H. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of NCKUH (A-ER-108-167) on 29 May 2019.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

PTH parathyroid hormone

#### **References**

# *Article* **Interobserver Agreement and Plane-Dependent Intraobserver Variability of Shear Wave Sonoelastography in the Differential Diagnosis of Ectopic Thymus Tissue**

**Zbigniew Adamczewski 1,2,†, Magdalena Stasiak 1,†, Bartłomiej Stasiak 3, Magdalena Adamczewska 2 and Andrzej Lewi ´nski 1,2,\***

**Abstract:** Shear wave elastography (SWE) has been demonstrated to be a useful tool in the differential diagnosis of ectopic thymus tissues (ETs), providing quantitative values of the shear wave stiffness (SWS) of both ETs and adjacent thyroid tissue. However, no data are available on the potential influence of the imaging plane (transverse vs. longitudinal) on the obtained SWS and shear wave ratio (SWR) values in SWE of these tissues. Moreover, no reports on the interobserver repeatability of SWE were published in regard to ETs. The aim of this study has been to evaluate the potential influence of the examination plane—transverse vs. longitudinal—on the SWS and SWR results, as well as to determine whether SWE of ETs is subjected to interobserver variability. SWE was demonstrated to have high inter- and intraobserver agreement in the evaluation of ETs and adjacent thyroid tissue. Significant differences between SWS values, but not SWR values, obtained in the transverse and longitudinal planes were observed. This phenomenon is probably a result of anisotropy-related artifacts and does not reduce the reliability of the method. SWE operators should be aware of the presence of plane-dependent artifacts to properly interpret the obtained results.

**Keywords:** shear wave elastography; ectopic thymus; thyroid; ultrasound; interobserver variability

#### **1. Introduction**

Ultrasonography (US) is a readily available, noninvasive tool used in neck imaging and the first step in the diagnostic algorithm of thyroid nodules [1,2]. However, US results highly depend on the interpretation and experience of the operator, with the sensitivity and specificity values ranging between 65.3 and 81.9%, and 60.7 and 68.9%, respectively [3]. Thus, some new tools based on US have been introduced for the improvement of diagnostic capabilities. Sonoelastography provides valuable information about lesion stiffness/elasticity. Malignant thyroid lesions are known to be much stiffer than benign lesions [4,5]. The first commonly available method was strain elastography (SE), which is based on a stiffness comparison of the analyzed lesion and the adjacent healthy tissue, providing the result in the form of a strain ratio only. This tool provides semiquantitative analysis but does not allow precisely assessing the elasticity of the particular lesion. Moreover, it appeared to be researcher-dependent, as the result is associated with the degree of tissue compression [6,7]. Therefore, the use of strain elastography (SE) in US diagnosis raises much controversy. Except for supporters of this method [8], there are many opponents who do not accept the difficulties in obtaining an artifact-free result [9,10]. To avoid the limitations of SE, shear wave elastography (SWE) has been introduced. This

**Citation:** Adamczewski, Z.; Stasiak, M.; Stasiak, B.; Adamczewska, M.; Lewi ´nski, A. Interobserver Agreement and Plane-Dependent Intraobserver Variability of Shear Wave Sonoelastography in the Differential Diagnosis of Ectopic Thymus Tissue. *J. Clin. Med.* **2021**, *10*, 214. https://doi.org/10.3390/ jcm10020214

Received: 29 October 2020 Accepted: 7 January 2021 Published: 9 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

examination is conducted without manual compression, and the result is expressed as a quantitative elasticity value (kPa). These promising properties of SWE allow us to believe that this method is accurate, operator-independent and reproducible. Such an assumption requires confirmation in the studies performed on specific tissues and organs.

Most of the studies focused on SWE concern the discrimination between benign and malignant lesions or the diagnosis of diffuse organ diseases, e.g., liver fibrosis or thyroiditis [11,12]. Only limited research has been performed to determine in vivo values for healthy soft tissues [13]. None of the published studies have included ectopic thymus tissues (ETs).

Recently, young children and even infants have been frequently referred for neck US because of enlarged lymph nodes or other palpable lumps in the neck. Ultrasound is also performed as part of a population study and due to a family history of thyroid disorders. The increasing availability of the ultrasound procedure leads to a new phenomenon of the incidental detection of various types of intrathyroidal and extrathyroidal lesions in children [14].

Any thyroid lesion found in a child should raise alertness and require further evaluation, as the frequency of thyroid cancer in thyroid lesions in children is much higher than in adults [14]. The most common benign neck lesions that can mimic malignancy are ETs [15]. The incidence of ectopic thymus in the neck in children was reported from 0.99% [16] to 1.8% [17]. Intrathyroidal ETs (IETs) and extrathyroidal ETs (EETs) resemble thyroid carcinoma and metastatic lymph nodes, respectively [15]. Proper differential diagnosis of such lesions allows avoiding unnecessary invasive procedures, including surgery. Quantitative tissue assessments in normal parenchymal organs (such as thyroid or thymus) using SWE are rarely conducted [13]. Therefore, it is remarkably important for the examiner to be familiar with normal shear wave stiffness (SWS) ranges to be able to differentiate pathological (neoplastic or inflammatory) and benign lesions.

Our research group demonstrated the usefulness of SE in the differential diagnosis of ETs [15]. However, due to the significant limitations of the method, we recently analyzed the application of SWE in children with ETs. SWE has appeared to be a useful tool in this indication, providing quantitative values of the SWS of both ETs and adjacent thyroid tissue and computing shear wave ratio (SWR), defined as a quotient of these values [18]. However, no data are available on the potential influence of the imaging plane (transverse vs. longitudinal) on the obtained SWS and SWR values in SWE of the neck tissues. Moreover, there are few studies evaluating the interobserver repeatability of SWE, and none of them include ETs or have been performed in a pediatric population [19,20].

The aim of this study was to evaluate the potential influence of the examination technique transverse vs. longitudinal plane—on the SWS and SWR results obtained in healthy children with ETs. The aim of this research has also been to determine whether this SWE is subjected to variability between two independent researchers (interobserver variability).

#### **2. Experimental Section**

#### *2.1. Patient Selection and Diagnostic Procedures*

Fifty consecutive cytologically confirmed ETs found in 28 children who were referred to our department because of suspicion of papillary thyroid carcinoma (PTC)/neoplastic lymph nodes were included in the study. Among the 50 lesions, 33 were IETs and 17 were EETs. To exclude any thyroid disease, in all patients, laboratory tests were performed, including thyrotropin (TSH), free thyroxine (FT4), free triiodothyronine (FT3), antithyroid peroxidase antibodies (aTPO), antithyroglobulin antibodies (aTg) and TSH receptor antibodies (TRAb). All parameters were measured by electrochemiluminescence immunoassay (ECLIA) with the Cobas e601 analyzer (Roche Diagnostics, Florham Park, NJ, USA). On the basis of the laboratory results, 3 children with positive aTPO and hypothyroidism, as well as one child with Graves' disease, were excluded from the initial number of 32 children. Only children without any abnormal thyroid function and/or autoimmune disorders were included in the study. In all patients, US-guided fine-needle aspiration biopsy (FNAB) was performed under moderate sedation or general anesthesia. Cytological smears were evaluated by the same high-volume pathologist. The smears including only small lymphocytes with scattered epithelioid cells (without the presence of macrophages, plasma cells, eosinophils, histiocytes or other cell types) were diagnosed as ETs. Only lesions with cytological confirmation of ET were included in the study.

After more than 3 months from the FNAB procedure, US and SWE neck examinations were performed with a 4–15 MHz linear transducer (Aixplorer MACH30, SuperSonic Imagine, Aix en Provence, France). The neck was in a natural position to avoid hyperextension. During the SWE examination, the probe was applied gently to the skin with a gel thick coating so as to avoid direct contact between the probe and the neck. The US and SWE examinations were carried out by two independent examiners—endocrinologists with more than 10 years of experience in US and elastography of the thyroid gland (M.S.—Researcher 1; Z.A.—Researcher 2).

During the SWE examination, a single researcher performed 4 measurements in the IET group (thyroid and IET in the transverse and longitudinal planes) and 3 measurements in the EET group (EET in the transverse and longitudinal planes, thyroid in the longitudinal plane). All SWS measurements were recorded in kilopascals. SWR values were calculated for thyroid/IET pairs in both planes—longitudinal and transverse—and for thyroid/EET pairs in the longitudinal plane only, as there was no possibility to present the EET and some corresponding thyroid tissue in a single transverse plane. All children had the SWE examination carried out two times (by the two researchers) in a single session. The researchers were blinded to each other's results. No statistically significant association between patient's age and SWS of either IET or EET was previously observed [18]; thus, we did not include age into the statistical analysis.

#### *2.2. Statistical Analysis*

The analyzed variables were quantitative; they were described by the mean (as a measure of position) and standard deviation (as a measure of dispersion). Inter- and intraobserver variability of the analyzed measurements were characterized as both the absolute difference (D) and the relative difference expressed as a percentage of the absolute difference with respect to the mean value of the compared measurements (D%). Their distributions were described using the mean value together with the 95% confidence interval (95% CI). The distributions of most variables were not normal, hence the hypotheses about the equality of distributions were tested using a nonparametric method, the Wilcoxon signed-rank test. For the verification of statistical hypotheses, a value of *p* < 0.05 was considered statistically significant. To further evaluate interobserver agreement, intraclass correlation coefficients (ICC) (2, 1) were calculated, with the following interpretations: <0.40—poor; 0.40–0.59—fair; 0.60–0.74—good; 0.75–1.00—excellent [21].

All the analyses were performed using the Statistica 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA).

#### **3. Results**

#### *3.1. Interobserver Variability*

The comparison between SWS and SWR results obtained by two independent researchers revealed high similarity. No statistically significant differences were found between SWS values in ETs (in IETs, EETs and both considered jointly) as well as in adjacent thyroid tissue, separately analyzed in the transverse plane and in the longitudinal plane (Table 1, Figures 1 and 2). Similarly, no differences between the researchers were found in SWR values analyzed in both planes (Table 1, Figures 1 and 2).

The ICC values computed for SWS of ETs and SWS of the thyroid in the transverse plane were 0.76 and 0.81, respectively, and 0.86 and 0.84 for the longitudinal plane, respectively.

**Table 1.** Comparison between SWS and SWR values obtained by two independent researchers.

Abbreviations: CI, confidence interval; D, difference; EET, extrathyroidal ectopic thymuses; ET, ectopic thymuses; IET, intrathyroidal ectopic thymuses; SD, standard deviation; SWR, shear wave ratio; SWS, shear wave stiffness.

**Figure 1.** An example of the SWS and SWR results obtained by two independent researchers ((**A**) R1 and (**B**) R2—Patient 1. A high similarity is clearly visible for both the SWS and SWR values. \* Longitudinal plane imaging. SWR, shear wave ratio; SWS, shear wave stiffness.

**Figure 2.** An example of the SWS and SWR results obtained by two independent researchers ((**A**,**B**) Patient 2). A high similarity is clearly visible for both the SWS and SWR values. \* Longitudinal plane imaging.

#### *3.2. Intraobserver Variability Related to the Imaging Plane*

Highly significant differences were found in SWS values between transverse and longitudinal plane imaging. The values obtained in the longitudinal plane were higher than those observed in the transverse plane (Figure 3, Tables 2 and 3). Significant differences were found for all evaluated structures, including IETs, EETs (Table 2) and thyroid tissue adjacent to ETs (Table 3). However, no differences were found in the SWR values obtained in both imaging planes (Figures 2 and 3, Table 4).

Abbreviations: CI, confidence interval; D, difference; EETs, extrathyroidal ectopic thymuses; IET, intrathyroidal ectopic thymuses; SD, standard deviation; SWS, shear wave stiffness.

**Table 3.** Comparison of the thyroid SWS values measured in the transverse and longitudinal planes.

Abbreviations: CI, confidence interval; D, difference; SD, standard deviation; SWS, shear wave stiffness; R1, researcher 1; R2, researcher 2.

**Table 4.** Comparison of the SWR values measured in the transverse and longitudinal planes.

Abbreviations: CI, confidence interval; D, difference; SD, standard deviation; SWR, shear wave ratio; R1, researcher 1; R2, researcher 2.

**Figure 3.** Examples of the significant difference in shear wave stiffness (SWS) values of two ectopic thymuses (**A**,**B**) and adjacent thyroid tissue between the transverse and longitudinal plane imaging, simultaneously with similar shear wave ratio (SWR) values, regardless of the plane. \* Longitudinal plane imaging.

#### **4. Discussion**

The finding of a pathological lesion in a child's neck should arouse diagnostic alertness. Frequently, US examination is not sufficient to provide an unequivocal decision on whether the lesion is really suspicious. The differential diagnosis of ETs is an accurate example of such a situation, as benign ETs often resemble malignant lesions in US. Ectopic thymic tissues can be found in children only, and FNAB is possible to be performed in this group, mainly in sedation or general anesthesia. Thus, there is a need for an accurate, quick and simple diagnostic method for differentiation between actually suspicious lesions requiring immediate FNAB and IETs/EETs, which can be followed up for some time. In the latter case, FNAB can even be avoided in some children, especially those with some relative contraindications for anesthesia [15]. The utility of SWE in the differential diagnosis of ETs has recently been proven [18]. However, the potential influence of the imaging plane (transverse vs. longitudinal) and the interobserver variability on the obtained SWS and SWR values in SWE of the ETs and adjacent thyroid tissues has never been analyzed. Both factors can potentially modulate SWE results. Thus, the evaluation of their significance seems mandatory for the unequivocal recommendation of SWE as a reliable and researcherindependent method.

In the present study, a high interobserver agreement was demonstrated. We did not find significant differences between SWS measured by both the researchers regardless of the plane. Moreover, no differences between the examiners were found in the SWR values analyzed in both planes. A significant similarity of the SWS results was obtained in ETs (in IETs, EETs and both considered jointly) as well as in adjacent thyroid tissue. The highest difference between the SWS values reported by the two researchers reached 18.54% (EET, longitudinal plane) and was not statistically significant (*p* = 0.51) (Table 1). This observation advocates the reliability and repeatability of SWE in the diagnosis of neck structures. This high repeatability is what clearly promotes SWE over the SE technique, in which the interobserver agreement ranges from slight to fair [5,6]. Our results are not fully concordant with other reports, which provide inconsistent data. Lim et al. [22] provided results similar to ours and indicated significant interobserver and intraobserver agreement in SWE of thyroid nodules. Excellent interobserver reproducibility was also demonstrated by Veyrieres et al. [23], who evaluated thyroid nodules so as to find the SWE threshold for malignant lesions. On the other hand, in the paper by Swan et al. [24], the reproducibility of SWE of thyroid nodules was demonstrated as insufficient to reliably differentiate malignant nodules from benign nodules in an individual patient. Additionally, Kishimoto et al. [25] observed excellent interoperator reproducibility in phantoms, but it was insufficient for the thyroid and all other analyzed regions except for the liver in the healthy volunteers. Several important factors might generate this discrepancy. The most important factors are the influence of the surrounding tissues, including incorrect probe positioning (without an adequate gel thick coating, which should prevent pressure), anisotropy of the surrounding tissues, neck muscle tension or blood vessel pulsation [26,27].

In the present paper, significant differences between SWS assessed in the transverse and longitudinal planes were demonstrated in both ET and thyroid tissues. The same very high degree of statistical significance was achieved by both independent researchers (Tables 1 and 2). However, all of the obtained quantitative results measured in both planes were within the values that are considered typical for benign lesions. Chen et al. [3] demonstrated a strong difference between SWS of benign and malignant thyroid lesions, with mean values of 19.2 ± 7.1 kPa and 34.6 ± 14.8 kPa, respectively. In that group, the mean SWS cut-off level equal to 24 kPa had a sensitivity of 78.8% and a specificity of 84.9% [3]. A similar cut-off level of 22.3 kPa was presented by Samir et al. [4], with sensitivity, specificity, positive predictive values and negative predictive values of 82%, 88%, 75% and 91%, respectively. The highest SWS values obtained in our study in the longitudinal plane for IETs, EETs and thyroid tissue were 16.5 kPa, 16.6 and 22.5 kPa, respectively (, and the maximum (among the two researchers) mean SWS values in the longitudinal plane were 13.99 ± 4.65, 11.15 ± 2.65 and 12.74 ± 2.80 kPa, respectively

(Tables 1 and 2). Thus, only one highest result reached the value close to the cut-off levels, suggested by the authors cited above [3,4], and the mean SWS values were much lower than any of the cut-offs. At the same time, the mean SWS values measured in the transverse plane did not reach 10 kPa for any of the analyzed structures (Tables 1 and 2). The differences in SWS for malignant and benign lesions are particularly useful for the thyroid, as the most common thyroid malignancy is papillary thyroid carcinoma (PTC), which is distinctly stiffer than the normal thyroid tissue in SWE [11].

Despite the significant differences between SWS values in the transverse and longitudinal planes, the SWR values did not differ, regardless of the plane, and always indicated ETs as benign lesions whose stiffness was lower or equal to the thyroid stiffness (Table 3). These findings have proven that SWE is a useful method in the differential diagnosis of ETs, and the results are reliable despite the differences in SWS between the imaging planes. However, the question arises what the real reason for such differences is. To the best of our knowledge, no other publications analyzing this issue in regard to the neck structures are available, and thus we are not able to compare our results with those of other authors.

Quantitative elastography is based on the measurement of the shear wave propagation velocity and is sensitive to tissue anisotropy [27]. This phenomenon concerns tissues that have different microstructures in different planes. The shear wave speed is higher when the wave propagates parallel to the spatially oriented structures than perpendicularly to them. Therefore, when the ultrasound probe is placed parallel to these tissues, the shear wave propagates perpendicularly to them, and lower elasticity (higher SWS) values are observed. On the contrary, when the shear wave beam is emitted perpendicularly, it propagates at a higher speed, and higher elasticity values are generated. This property affects SWE values, as described before, particularly in studies concerning muscles, tendons and kidneys [27–32]. So far, this phenomenon has not been thoroughly described in the case of the thyroid gland, possibly because of the fact that the presence of anisotropy in thyroid tissue has not been confirmed on the basis of the apparent diffusion of the coefficient value in magnetic resonance examination [33]. Except for the present study, there is only one other published report analyzing SWS of the normal thyroid tissue in the two planes [34]. The authors of that paper mainly evaluated thyroid tissue altered in the course of Hashimoto's thyroiditis (HT), and the normal thyroid measurements served as controls. They reported the differences in SWS between transverse and longitudinal sections, and these differences were even more pronounced for control tissues than HT, reaching about a 40% increase of SWS in the longitudinal plane [34]. These results are highly similar to ours. Unfortunately, the authors did not make any attempt to analyze their findings.

Importantly, in our study, these differences, presented as absolute (D) or percentage (D%) values, did not differ much between researchers (Table 2). This fact can suggest that the observed difference in SWS values between the planes is actually an artifact related to the fact that most of the organs of the neck are known to be anisotropic. Such a conclusion is supported by the results of Lee et al. [26], who provided SWS values of reactive cervical lymph nodes with respect to US probe position (parallel or perpendicular to the muscle fiber orientation) and with respect to the presence of muscle stretch stress. This study revealed that SWS values were higher in the parallel position and thereby confirmed the effect of the anisotropic nature of muscles on the SWS values of the cervical lymph nodes. This phenomenon was exacerbated by the stretch stress of the cervical muscles [26].

The awareness of such a phenomenon should probably result in the indication for a mandatory description, of which a cross-section type was applied for the particular SWS measurement in the neck tissues. The possible impact of this finding on diagnosing thyroid cancers remains to be elucidated. Prospective studies evaluating SWE values in thyroid focal lesions in both planes are therefore necessary.

Thus, on the basis of the results presented in the current paper, we postulate that the elasticity measurements can be influenced by the tissue architecture of the neck organs, and this phenomenon should be taken into account when analyzing SWS results for thyroid and other neck tissues. We believe that this phenomenon should not be considered an important limitation of the method, but rather an artifact related to the physical properties of the tissues. SWE was demonstrated to be an easy and quick diagnostic method, characterized by high intra- and interobserver agreement. This might easily result in uncritical acceptance of the obtained results, while our results have demonstrated that the cross-section should be taken into account when interpreting the SWS values. Understanding the possible influence of artifacts, resulting from different plane imaging, provides the basis for eliminating errors in the interpretation of SWE.

The study has a limitation concerning the study sample calculation. ETs are uncommon findings, so the study sample was collected by including all the consecutive patients with lesions meeting the FNAB criteria of ETs, without the sample size calculation.

In conclusion, SWE is characterized by high inter- and intraobserver agreement in the evaluation of ETs and adjacent thyroid tissue. However, the researchers should be aware of the presence of plane-dependent artifacts, resulting in higher SWS values in the longitudinal section. These artifacts do not influence the SWR values and do not reduce the reliability of the method in ETs diagnosis. In our opinion, the data gained from this research will improve the diagnosis, follow-up and management of the thyroid and neck lesions.

**Author Contributions:** Conceptualization, Z.A., M.S. and A.L.; data curation, Z.A., M.S. and M.A.; formal analysis, Z.A., M.S. and A.L.; funding acquisition, M.S. and A.L.; investigation, Z.A. and M.S.; methodology, Z.A., M.S., B.S. and A.L.; project administration, Z.A., M.S. and A.L.; resources, Z.A. and M.S.; software, Z.A., M.S. and B.S.; supervision, A.L.; validation, Z.A., M.S. and B.S.; visualization, Z.A. and M.S.; writing—original draft, Z.A. and M.S.; writing—review and editing, M.S. and A.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by statutory funds from the Polish Mother's Memorial Hospital— Research Institute, Lodz, Poland.

**Institutional Review Board Statement:** The study was accepted by the Polish Mother's Memorial Hospital, Research Institute Bioethical Committee (Approval Code: 45/2020).

**Informed Consent Statement:** Written informed consent for all the performed procedures as well as for the publication of the results was obtained from the patients' parents.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

# *Article* **Application of Shear Wave Sonoelastography in the Di**ff**erential Diagnosis of Extra- and Intra-Thyroidal Ectopic Thymic Tissue**

**Magdalena Stasiak 1,**†**, Zbigniew Adamczewski 1,2,**†**, Renata Stawerska 1,3, Bartłomiej Stasiak 4 and Andrzej Lewi ´nski 1,2,\***

Received: 13 October 2020; Accepted: 24 November 2020; Published: 25 November 2020

**Abstract:** The ultrasound (US) pattern of intrathyroidal ectopic thymus (IET) can resemble papillary thyroid carcinoma (PTC) while the extrathyroidal ectopic thymus (EET) can mimic pathological lymph nodes. Recently, the usefulness of strain elastography (SE) was demonstrated in the differential diagnosis, however this method has several limitations. The aim of the current study was to assess the usefulness of shear wave elastography (SWE) in this field. The US, SE, and SWE were performed in 31 children with 53 ectopic thymuses (ETs) and quantitative values of SWE parameters were calculated, so as to generate potential normative values of ET elasticity and of the shear wave ratio (SWR). The mean SWRIET was 0.89 ± 0.21 and the mean shear wave stiffness (SWS) was 7.47 ± 1.93 kPa. The mean SWREET was 0.84 ± 0.15 and the mean SWSEET was 11.28 ± 2.58 kPa. The results have proven that the stiffness of ETs is lower or equal to the thyroid's. SWE was demonstrated to be a useful diagnostic method for ET evaluation. Therefore, the application of SWE in ET diagnosis allows more accurate evaluation of ET-like lesions and, in many cases, allows one to avoid invasive procedures, simultaneously providing a precise monitoring method based on combined US and SWE evaluation.

**Keywords:** ectopic thymus; thyroid; shear wave sonoelastography; strain elastography; ultrasound; thyroid cancer; metastatic lymph nodes

#### **1. Introduction**

Ultrasonography (US) is an easily accessible, non-invasive diagnostic tool used in neck imaging and a first-line method for the diagnosis of thyroid gland nodules. The increasing availability of the US procedure leads to a new phenomenon of accidental findings of many thyroid and neck lesions in children. On the basis of US and postmortem examinations, the frequency of thyroid nodules in children and young adolescents/young adults was estimated at 1–1.5% and 13%, respectively [1]. The risk of malignancy in thyroid nodules in children is high, reaching 22–26%, as compared to approximately 5% in adults [1]. Thus, it is recommended to perform a fine needle aspiration biopsy (FNAB) in every thyroid lesion found in a child, except for a pure cyst [2]. Contrary to the adult population, the size of the thyroid nodule cannot be considered as an indication for FNAB in children,

simply because their thyroid glands are smaller than an adult's. Thus, even small solid nodules should undergo FNAB. In children, thyroid cancer is often bilateral and multifocal, so none of these features can reduce diagnostic alertness [1,2]. At the time of diagnosis, lymph node metastases are present in most children with thyroid cancer. Hence, precise US examination of lymph nodes should be performed in every child with thyroid lesions and/or palpable suspicious lymph nodes.

In the US, intrathyroidal ectopic thymus (IET) can mimic papillary thyroid carcinoma (PTC), which is the most common thyroid cancer in children. After formation of the definitive thymus, it descends to the upper anterior mediastinum [3,4]. A disturbed process of the migration can result in ectopic thymus location, including an intrathyroidal one. The prevalence of ectopic neck thymus in children was reported as 0.99% [5] to 1.8% [6].

In the US, IETs are usually hypoechoic lesions, with punctate or linear bright internal echoes that look like microcalcifications [7]. In some cases, the lesion margins are irregular. Such features suggest PTC and require precise differential diagnosis [7]. The extrathyroidal ectopic thymic tissue (EET) is most often located very close to the lower pole of one of the thyroid lobes. Its US pattern may strongly suggest the presence of a pathological lymph node [7].

The usefulness of the strain elastography (SE) in the differential diagnosis of IETs was previously demonstrated by our research team [7]. This method evaluates tissue stiffness (elasticity) by measuring the degree of tissue deformation in response to mechanical compression [7–9]. The stiffness of the examined lesion must be compared to the adjacent healthy tissue to calculate the difference in relative stiffness. This difference is presented as a strain ratio (SR). This procedure helps to differentiate malignant thyroid lesions—which are stiffer than the surrounding thyroid tissue–from IETs, of which the SR was demonstrated to be low (0.95 to 1.09) [7]. However, SE has important limitations. Firstly, the SR result cannot be considered as absolutely investigator-independent because manual external compression in SE leads to operator-dependent variability. Secondly, the result does not provide quantitative data on the lesion's stiffness. Therefore, the usefulness of this method is limited to the lesions which stiffness can be compared to the surrounding healthy tissue. Thus, SE is useless in the case of some EETs and suspicious lymph nodes.

Shear wave elastography (SWE) is a further improvement of sonoelastography, providing a two-dimensional distribution map of tissue stiffness and quantitative measurement of the tissue stiffness in Young's modulus (kPa) and/or shear wave speed (m/s) [10]. Thus, SWE seems to be a great diagnostic tool for both IET and EET neck lesions.

The aim of the study was to demonstrate the usefulness of SWE in the differential diagnosis of ETs and to compare the results of SWE with SE. The additional aim of the study is to present US features of IETs and EETs in the largest group of cytologically confirmed ETs described so far.

#### **2. Materials and Methods**

#### *2.1. Patient Selection and Diagnostic Procedures*

Fifty three cytologically confirmed ETs found in 31 children who were referred to our Department because of suspicion of PTC/neoplastic lymph node were included in the study. Among the 53 lesions, 36 were IETs and 17 were EETs. In all patients, laboratory tests were performed, including thyrotropin (TSH), free thyroxine (FT4), free triiodothyronine (FT3), anti-thyroid peroxidase antibodies (aTPO), anti-thyroglobulin antibodies (aTg), and TSH receptor antibodies (TRAb). All parameters were measured by electrochemiluminescence immunoassay (ECLIA), Cobas e601 analyzer (Roche Diagnostics, Indianapolis, IN, USA). In all the children, US examination was performed in supine position using a 7–14 MHz linear transducer (AplioXG, Toshiba Medical Systems Corp., Tochigi-ken, Japan). Strain elastography was performed in all IET lesions (AplioXG, Toshiba Medical Systems Corp., Japan). Shear wave elastography was performed in all lesions (Aixplorer MACH30, Supersonic Imagine, France). The shear wave stiffness (SWS) and shear wave ratio (SWR) values were measured for thyroid/IET pairs and for normal thymus in transverse plane and for thyroid/EET pairs in

longitudinal plane as there was no possibility of simultaneously presenting the EET and corresponding thyroid tissue in a transverse plane. All SWS measurements were recorded in kilopascals. In all patients, FNAB with US guidance was performed under moderate sedation or general anesthesia. Cytological smears were evaluated by the same high-volume pathologist. The presence of only small lymphocytes with scattered epithelioid cells (without macrophages, eosinophils, plasma cells, histiocytes, or other cell types) was considered typical for thymic tissue. The absence of lymphocytes of different stages of differentiation (as well as other cells typical for lymph nodes) allowed differentiation with lymph nodes or other lymphatic tissues. The absence of oncocytic follicular cells and plasma cells allowed exclusion of lymphocytic thyroiditis.

#### *2.2. Statistical Analysis*

The basic statistical analysis of the collected data was performed using Microsoft Excel statistical functions. Excel was also used to compute Pearson correlation coefficient values *r*. Statistical library scipy.stats for Python was applied to verify the correlation results and to compute the associated *p*-values (*p)*. It was also used for the Mann-Whitney rank test of statistical significance between shear wave stiffness of IET and normal thymus. The *p*-value *p* ≥ 0.05 was considered as statistically not significant.

#### *2.3. Bioethical Procedures*

In all the cases, written informed consent for all the performed procedures and for the publication of the results were obtained from the patients' parents. The study was accepted by the Polish Mother's Memorial Hospital—Research Institute Bioethical Committee, approval code 45/2020.

#### **3. Results**

#### *3.1. Clinical Findings*

The mean age of our patients was 6.91 ± 2.44 years, ranging from 3 to 12 years. The female to male ratio was 1:1.38. None of the children had any family history of thyroid carcinoma, nor any medical history of irradiation. None of the children had signs or symptoms of any relevant disease. In all patients, results of tests of thyroid hormones and thyroid antibodies were within the normal ranges (Table 1).

**Table 1.** Clinical characteristics of the analyzed group of children.

Abbreviations: aTg, thyroglobulin antibodies; aTPO, thyroid peroxidase antibodies; FT4, free thyroxine; FT3, free triiodothyronine; TSH, thyroid stimulating hormone; TRAb, TSH receptor antibodies. Reference ranges: TSH 0.7–5.97 mIU/L for children <6 years, 0.6–4.48 mIU/L for children 6–12 years; FT4 0.97–1.67 ng/dL; FT3 2.53–5.22 pg/mL; aTPO < 34 IU/mL; aTg < 115 IU/mL; TRAb < 1.75 IU/mL.

#### *3.2. Sonographic Findings*

A total number of 53 ectopic thymic tissues were found in 31 patients. Thirty-six lesions were IETs and 17 were EETs. Ultrasound features of the analyzed lesions are presented in Table 2. The size of IETs varied from 4 to 15 mm and the mean largest dimension was 6.64 ± 2.34 mm. The size of EETs ranged from 6 to 23 mm, with the mean largest dimension of 15.59 ± 4.56 mm. Among IETs, 15 lesions were located in the right lobe and 21 in the left lobe; 22 were located in the middle part of the thyroid lobe, 13 in the lower part, and 1 in the upper part of the thyroid lobe. In six children, IET were located bilaterally, while EET were bilateral in two patients (Table 2). Coexistence of EET and IET was found in 8 children. The mean age of children with bilateral IETs was 5.83 years. The girl with the highest number of IETs (7) was 3 years old.

Abbreviations: EET, extrathyroidal ectopic thymus; IET, intrathyroidal ectopic thymus; NA, not applicable; SD, standard deviation.

#### *3.3. Elastographic Findings*

In SWE, the mean SWR of IETs (SWRIET) was 0.89 ± 0.21, ranging from 0.5 to 1.2. Thus, the stiffness of IETs was comparable to or lower than the stiffness of the adjacent thyroid tissue (Table 3). The mean SWS of IETs (SWSIET) was 7.47 ± 1.93 kPa, while the mean SWS of the adjacent thyroid tissue was 8.66 ± 2.42 kPa (Table 3). No difference between the SWSIET and SWS of the normal thymus (SWSt) was found (*p* = 0.236).

**Table 3.** Sonoelastographic data of IETs, EETs, and normal thymuses.

\* EET data were collected in the longitudinal section. Abbreviations: EET, extrathyroidal ectopic thymus; IET, intrathyroidal ectopic thymus; SD, standard deviation; SR, strain ratio; SWR, shear wave ratio; SWS, shear wave stiffness.

Examples of SWE of IETs, with quantitative assessments of SWSIET and SWRIET, are presented in Figure 1.

**Figure 1.** Examples of the application of shear wave elastography (SWE) in intrathyroidal ectopic thymuses (IETs). Quantitative assessments of shear wave stiffness (SWSIET) and shear wave ratio (SWRIET, described as "Ratio" in the Figure) allow one to confirm that the stiffness of the analyzed lesion is lower than the surrounding thyroid parenchyma and that the mean values of SWSIET are very low (6.2 and 4.9 kPa).

In SE of IET lesions, the SR ranged from 0.5 to 1.2, mean 0.99 ± 0.13 (Table 3), which is comparable to the mean SWRIET result of 0.89 ± 0.21. In SWE of EETs, the comparison with the adjacent thyroid tissue was possible in 15 lesions (longitudinal section) and the mean SWR was 0.84 ± 0.15 (Table 3). The mean SR value in SE was 0.89 ± 0.13. The mean SWS was 11.08 ± 2.57 kPa (Table 1). Examples of SWE of EETs with quantitative assessment of SWSEET and SWREET are presented in Figure 2.

**Figure 2.** Examples of application of shear wave elastography (SWE) in extrathyroidal ectopic thymuses (EETs). Quantitative assessments of shear wave stiffness (SWSEET) and shear wave ratio (SWREET, described as "Ratio" in the Figure) allow one to confirm that the stiffness of the analyzed lesion is lower (**A**) or similar (**B**) to the surrounding thyroid parenchyma and that the mean values of SWSIET are low in both of the cases—8.9 kPa (**A**) and 13.1 kPa (**B**).

No statistically significant correlation between patient's age and SWSIET (*r* = −0.225, *p* = 0.250) or SWSEET (*r* = −0.029, *p* = 0.917) was observed.

The mean SWS of the normal thymus (SWSt) in the analyzed group of children was 6.96 ± 1.58 kPa (Table 3). No statistically significant correlation between SWSt and patient age was observed (*r* = 0.225, *p* = 0.250). Examples of thymus SWE with quantitative assessment of SWSt are presented in Figure 3.

#### **4. Discussion**

The presence of a pathological lesion in the thyroid or in other cervical locations in children always arouses diagnostic alertness. IETs and EETs are frequently difficult to differentiate from potentially malignant lesions. Ectopic thymic tissues are found in children only and most of the patients are younger than 10 years of age. In this group of patients, FNAB is possible to be performed mainly in sedation or general anesthesia, which requires hospitalization. Moreover, finding a suspicious thyroid/neck lesion in a child generates significant stress for the patient and his/her parents, who need

to know promptly whether the lesion may be malignant. For all of these reasons, it is necessary to find an accurate diagnostic method for a quick and simple differentiation between really suspicious lesions that require immediate FNAB and IETs/EETs, which may be observed for some time and in some cases, FNAB may even be avoided, especially if there are some relative contraindications for anesthesia.

We have recently demonstrated the usefulness of SE in the differential diagnosis of IETs [7]. Except for that paper, there is only one other published report on SE application in IETs [11]. To our best knowledge, the current study is the only one in the literature focusing on SWE in ET. Thus, comparison with other authors' results is not possible.

In the present study, we have confirmed the great usefulness of sonoelastography in ET diagnosis. SWE was demonstrated to be an accurate tool, providing a quantitative value of SWS (kPa) which is independent of any additional manual pressure required in SE. The mean SWSIET and SWSEET were 7.47 ± 1.93 kPa and 11.08 ± 2.57 kPa, respectively. The mean SWSIET value was very similar to the SWSt (6.96 ± 1.58 kPa). The differences in SWSIET and SWSEET and between SWSEET and SWSt result from the fact that SWSIET and SWSt were assessed in the transverse section, while SWSEET were assessed in the longitudinal section. Therefore, SWSEET values should not be directly compared with SWSIET or SWSt. The only clinical parameter that can potentially influence SWS of ETs is age as thymus tissue involution and fibrosis progresses with age. However, in our study, neither in IETs/EETs nor in normal thymus was any statistically significant association between SWS and the patients' age found. The lack of such a correlation probably resulted from the prepubertal status of all our patients. The results of SWR in IETs and in the selected EETs were 0.89 ± 0.21 and 0.84 ± 0.15, respectively. They were similar to the obtained SR values, which were 0.99 ± 0.13 and 0.89 ± 0.13, respectively. The values of SWR and SR observed in the present study confirmed our previous results based on SE, which demonstrated the SR mean value of 1.02 [7]. The only other paper published on this issue reported mean SR values in 22 IETs of 0.99 (ranging from 0.96 to 1.05) [11]. In that group, seven IETs were confirmed cytologically and 15 were diagnosed on the basis of US features. Their results together with ours—both the previous ones and the ones reported in the present paper—proved that the crucial value of sonoelastography in differential diagnosis of ETs is the fact that the stiffness of thymic tissue is lower or similar to the thyroid. This phenomenon is clearly visible on the basis of SWS values (kPa) for ET, which are lower or similar to those of the thyroid. This low stiffness is comparable to that of the normal thymus. On the contrary, the stiffness of malignant lesions is much higher than the stiffness of the adjacent thyroid and the presence of microcalcifications further increases the SWS values [12]. This is of great importance in the differentiation of IET and PTC since PTC is known to be significantly stiffer than thyroid tissue [13]. Chen et al. [14] observed the statistically significant difference between SWS of benign and malignant thyroid lesions, with mean values of 19.2 ± 7.1 kPa and 34.6 ± 14.8 kPa, respectively. The authors demonstrated that the mean SWS cut-off level equal to 24 kPa attained a sensitivity of 78.8% and a specificity of 84.9% [14]. Similar results were presented by Samir et al. [15], who reported a cut-off value for suspicion of malignancy of 22.3 kPa, having a sensitivity, specificity, positive predictive value, and negative predictive value of 82%, 88%, 75%, and 91%, respectively. The highest SWS values obtained in our study for IETs and EETs were 12.7 kPa and 16.6 kPa, respectively. These differences in SWS for malignant and benign thyroid lesions are particularly useful in the case of PTC, which covers over 90% of all thyroid malignancies in children [1]. Follicular cancer, which is often soft in elastography and different in US pattern, is extremely rare in this age group. Therefore, the usefulness of sonoelastography is of particular importance in children because it can be assumed that all lesions resembling IETs in US of which the SWS is similar to or lower than the thyroid tissue, are actually IETs. On the basis of our results and the studies by Chen et al. [14] and Samir et al. [15], we postulate to consider the ET-like lesion suspected of malignancy in the cases with the SWS value greater than 22.3 kPa (the cut-off value proposed by Samir et al. for thyroid nodules [15]).

Bayramo ˘glu et al. [16] have recently published a paper which provides normative values of SWSt in regard to age and gender. In that study, the mean value of SWSt was 6.76 ± 1.04 kPa, ranging from 4.50 kPa to 10.40 kPa [16]. Our results provide nearly identical values, with the mean SWSt 6.96 ± 1.58, ranging from 4.7 kPa to 10.9 kPa. This high consistency of the SWSt values supports that the obtained results are researcher-independent. Bayramo ˘glu et al. found significant negative correlations of thymus elasticity and velocity values with age [16]. However, the differences of mean SWSt between individual age groups were found to be statistically insignificant [16]. In our present study, we have not found a significant correlation between SWSt and age, although the obtained value of the Pearson correlation coefficient itself was not much lower. This probably results from the smaller analyzed number of thymuses in our study. Bayramo ˘glu et al. analyzed SWS values for normal thymuses only, so they could collect a much larger group of children (146 healthy children) [16].

The US-based differential diagnosis between IET and suspicious thyroid nodules is often challenging. Children with IETs are often referred for surgery as hypoechoic nodules with microcalcification-like echoes strongly resemble lesions suspicious of malignancy and cytology frequently reveals a non-diagnostic result (Bethesda category I). The application of SWE together with the detailed knowledge of the US features of ETs can highly facilitate differential diagnosis and can allow one to avoid invasive procedures in a group of children with the values of SWS, SWR, and US features typical for ETs.

Ultrasound characteristics of ETs were reported in only a few large groups of children and the IETs were diagnosed in most of them on the basis of US image only, without cytological evaluation [4,5,11,17–21]. Up to now, the biggest group with cytologically confirmed ETs was described in our previous report and included 16 lesions [7]. In most reports on the US characteristics of ETs, cytological evaluation was not performed despite the small number of lesions [3,20,21]. Such an approach carries the potential risk of misdiagnosis. Therefore, we believe that studies analyzing particular characteristic features of ETs should include lesions with cytologically confirmed ETs only.

Similarly to our previous report [7] and other studies [4,21], in the present study, the analyzed IETs were located mostly in the middle part of the thyroid lobe and less frequently, in the lower part. Having analyzed more than a three times larger number of patients than in our previous report, we found a less frequent occurrence of bilateral IETs (17% vs. 50%) [7]. This observation is more consistent with other authors' studies [3,4,21], however, on the other hand, the complete absence of bilateral lesions reported in some papers still seems confusing [6,21]. In the US, most of the IET lesions analyzed in our study were oval (58%), fusiform (14%), triangular (14%), or longitudinal (11%) in shape. The predominance of oval and fusiform IETs was also observed in other reports [7]. Oval and triangular shapes also dominated in our EET group. All the ETs were solid, hypoechoic with internal linear or, less often, punctual bright echoes. Linear echoes are easier to differentiate from microcalcifications than the punctual ones. Thus, in the cases with such punctual echoes, the application of SWE can be particularly useful as real microcalcifications substantially increase the SWS value, while the SWS of ETs remains low. Therefore, SWE can serve as a quick and easy tool to differentiate lesions suspicious for PTC or metastatic lymph nodes from ETs.

Most ETs typically have clear margins, but in some cases, the margins can be blurred, which can additionally suggest malignancy. In our present study, as many as 13 (36%) IETs and 2 (12%) EETs had blurred margins. In such US images, SWE can again clarify whether the lesion is actually suspicious.

Blood flow is known to be decreased or absent in ETs [7] and our observations are similar to other authors' findings [3–6,17–21]. In 19 (53%) of the IETs and in 7 (41%) of the EETs, no blood flow was detected. However, one should always remember that in a significant portion of PTC, vascularization can be absent and the lack of blood flow in thyroid tumors cannot be considered a benign feature [22]. Thus, once again, the application of SWE can allow us to distinguish ETs and malignancy-suspicious lesions.

It is well known that the US pattern of IETs can mimic PTCs and EETs can resemble metastatic lymph nodes. High volume experience is mandatory to distinguish the suspicious thyroid nodule from IETs in US examination. Punctual bright internal echoes in ETs are actually impossible to differentiate from microcalcifications in US. Moreover, ETs can demonstrate other suspicious US features, such as blurred margins or lack of vascularity. The awareness of typical location and US pattern of ETs is required to start with the US examination of ETs. However, in most cases, the US features do not provide unequivocal differential diagnosis. The introduction of SWE can change the quality of ET imaging as it provides quantitative assessment of the tissue elasticity and, simultaneously, it enables us to compare it with the elasticity of the thyroid. The combination of US pattern and SWE results highly improves the quality of US-based evaluation. In the first step of the diagnostic procedures, this results in the avoidance of unnecessary alert and parents' stress. In the next steps, with such an accurate diagnostic tool in the hands of an experienced ultrasonographer, this gives a chance to avoid invasive diagnostic procedures or even surgery. A typical US pattern of an ET with low SWS values may empower an experienced specialist to temporarily abandon FNAB, particularly in young children with contraindications to even short-term sedation. Regular US and SWE monitoring is mandatory in such cases.

Moreover, one should remember that rare cases of thymoma, thymic carcinoma, and lymphoblastic lymphoma arising from ET were reported [7,23–26]. Therefore, the appearance of any suspicious US feature or an increase in SWS with higher SWR always require further diagnosis.

In conclusion, SWE has been demonstrated to be a useful diagnostic method in the differential diagnosis of ETs and malignant lesions. It provides a quantitative assessment of lesion elasticity and calculates the useful SWR value by comparing this elasticity with that of the adjacent thyroid tissue. In the present study, we have provided the SWS and SWR/SR values that should be considered normative for ET. The SWS values for IETs are usually below 10 kPa and the cut-off value of below 22.3 kPa may be considered as typical for benign lesions, including ETs, irrespective of the US plane (transverse or longitudinal), provided that the SWR value is close to 1.0 or lower. Taking into account this SWR criterion is especially important if borderline SWS results are observed. SWE complements the US diagnostics and helps elucidate the suspicious US features of ETs. Therefore, the application of SWE in ET diagnosis allows for more accurate evaluation of ET-like lesions and, in many cases, helps to avoid invasive procedures, simultaneously providing the precise monitoring method based on combined US and SWE evaluation.

**Author Contributions:** Conceptualization, M.S., Z.A., and A.L.; data curation, M.S., Z.A., and R.S.; formal analysis, M.S., Z.A., and A.L.; funding acquisition, M.S. and A.L.; investigation, M.S. and Z.A.; methodology, M.S., Z.A., R.S., and A.L.; project administration, M.S., Z.A., and A.L.; resources, M.S. and Z.A.; software, M.S., Z.A., and B.S.; supervision, A.L.; validation, M.S., Z.A., and B.S.; visualization, M.S. and Z.A.; writing—original draft, M.S., Z.A., and B.S.; writing—review & editing, M.S. and A.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by statutory funds from the POLISH MOTHER'S MEMORIAL HOSPITAL—RESEARCH INSTITUTE, Lodz, Poland.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

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# *Article* **Surgical Management of Primary Hyperparathyroidism—Clinicopathologic Study of 1019 Cases from a Single Institution**

#### **Jacek Gawrychowski \*, Grzegorz J. Kowalski, Grzegorz Buła, Adam Bednarczyk, Dominika Z ˛ ˙ adło, Zbigniew Niedzielski, Agata Gawrychowska and Henryk Koziołek**

Department of General and Endocrine Surgery, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland<EMAIL_ADDRESS>(G.J.K<EMAIL_ADDRESS>(G.B<EMAIL_ADDRESS>(A.B<EMAIL_ADDRESS>(D.Z<EMAIL_ADDRESS>(Z.N.); ˙<EMAIL_ADDRESS>(A.G<EMAIL_ADDRESS>(H.K.)

**\*** Correspondence<EMAIL_ADDRESS>
Received: 24 September 2020; Accepted: 28 October 2020; Published: 2 November 2020

**Abstract:** Background: Primary hyperparathyroidism (pHPT) is an endocrine disorder characterized by hypercalcemia and caused by the presence of disordered parathyroid glands. Parathyroidectomy is the only curative therapy for pHPT, but despite its high cure rate of 95–98%, there are still cases where hypercalcemia persists after this surgical procedure. The aim of this study was to present the results of a surgical treatment of patients due to primary hyperparathyroidism and failures related to the thoracic location of the affected glands. Methods: We present a retrospective analysis of 1019 patients who underwent parathyroidectomy in our department in the period 1983–2018. Results: Among the group of 1019 operated-on patients, treatment failed in 19 cases (1.9%). In 16 (84.2%) of them, the repeated operation was successful. In total, 1016 patients returned to normocalcemia. Conclusions: Our results confirm that parathyreoidectomy is the treatment of choice for patients with primary hyperparathyroidism. The ectopic position of the parathyroid gland in the mediastinum is associated with an increased risk of surgical failure. Most parathyroid lesions in the mediastinum can be safely removed from the cervical access.

**Keywords:** primary hyperparathyroidism; parathyroidectomy; remedial surgery; ectopic mediastinal localization; persistent hypercalcemia

#### **1. Introduction**

Primary hyperparathyroidism (pHPT) is a type of endocrine disorder resulting in increased secretion of the parathyroid hormone from abnormal parathyroid glands. It leads to serious disturbances in calcium and phosphate metabolism leading to elevated levels of calcium in blood serum which may be a risk factor for developing a hypercalcemic crisis [1,2]. Primary hyperparathyroidism is a relatively common disorder that is diagnosed in 0.1 to 1% of the general population, and more often in postmenopausal women [2]. In patients with pHPT, the parathormone (PTH) excess originates from neoplastic or hyperplastic cells of parathyroid parenchyma. Most cases involved a single abnormal parathyroid gland located in an usual neck site, but many of such changes occur in other sites including the thymus gland, the retroesophageal space or the thyroid gland. Sometimes, parathyroid lesions have been reported in the pericardium or soft and adipose tissues of the mediastinum up to the angle of the jaw [3,4]. The differentiation between hyperplasia and adenoma may not be easy even for an experienced surgeon. In case of hyperplasia, it is essential to the establish not only the size, but also the symmetry of affected glands. In many cases, even affected glands can appear normal in the gross appearance. This points to the importance of a biopsy taken from the tissue and its examination by a pathologist. Parathyroidectomy (PTX) is the procedure of choice for patients suffering from pHPT with a cure rate of 95–98%. Such an operation is now the procedure of choice at experienced surgical centers, with the use of an intraoperative PTH level (iPTH). However, even then the PTX for ectopic mediastinal lesions is a challenging procedure [4]. We reported a group of 1019 patients operated on for pHPT between the years 1983 and 2018.

#### **2. Materials and Methods**

This is a retrospective study of 1019 patients operated on between 1983 and 2018 (between 1983 and 2004 318 patients were operated on and from 2005 to 2018 701 patients were operated on) (Figure 1) for primary hyperparathyroidism (pHPT). The sample consisted of 759 women (74.5%) and 260 men (25.5%) between the ages of 19 and 81. We evaluated 953 operated-on patients during the same time period for parathyroid lesions located in the neck and carried out comparisons with a group of 66 patients with ectopic mediastinal lesions. Patients were identified based on operation protocols and histopathological findings regarding the number of lesions, their localization, and biochemical and surgical results. The diagnostic management consisted of a careful case history and physical examination, and routine biochemical examinations. The biochemical examinations included measurements of the serum calcium levels and intact PTH. Parathyroid localization studies included up until 2004 the ultrasound and subtraction technique (also known as dual-isotope imaging); from 2005 sestamibi technetium-99 m scintigraphy was used and from 2010, the single-photon emission computed tomography (SPECT) technique has usually been utilized, and rarely the CT scan. Two positive imaging studies were always required. Upon their discharge from the hospital, the patients were seen at least four times a year—i.e., every three months within the first two years and every six months thereafter. Physical and biochemical examinations were also performed. We arbitrarily divided cases of hypercalcemia after prior surgery into persistent (defined as hypercalcemia recurring within six months of the initial operation) or recurrent (hypercalcemia recurring after six months of normocalcemia).

**Figure 1.** Number of operations in years 1983–2018.

#### **3. Methods of Statistical Analysis**

All of the collected data were included in a spreadsheet in Microsoft Office Excel 2019. The statistical analysis was performed in Statistica 12.5. After establishing and classifying the data using a Kolmogorow–Smirnow test, we used a Student's *t*-test to compare data with a normal distribution and for the data non-normal distribution we used the Mann–Whitney U test. The results are presented as percentage changes, full numbers, means and standard deviations. The numerical data were compared with that of the Pearson chi-square test. The level of significance was calculated at *p* < 0.05. Effect size—ES—was determined using Youle's Phi coefficient and Cramer's V.

#### **4. Results**

Parathyroid lesions were identified in 1019 patients. Fatigue, muscle or bone weakness, and loss of appetite were the most commonly observed symptoms in 923 symptomatic patients with normal or ectopic localizations of dysfunctional parathyroid glands. No symptoms were recorded among the remaining 96 patients (Table 1).

**Table 1.** Symptoms of 1019 patients operated on for Primary hyperparathyroidism (pHPT).

The vast majority (93.6%) of parathyroid lesions were localized in the neck (predominantly within the left and right superior glands), whereas a minority (6.4%) were found in the mediastinum. We observed a significant difference in the occurrence of arterial hypertension among the patients with neck parathyroid glands when compared to mediastinal localization. Dermopathies were significantly more frequent in the group of patients operated on for mediastinal glands, but both effect sizes were weak.

Histological findings demonstrated benign lesions in 990 patients while malignancy was reported in the remaining 29 cases. Among the 1019 patients, we investigated 1226 lesions, with 856 of them related to a single gland and 163 impacting two or more glands (Table 2). Among multiple lesions, the affection of two glands was the most prominent, while affection of four glands was sporadic. The benign neoplasms have been further differentiated into hyperplasia—which were identified most often (827 lesions)—and adenomas, which made up the remaining 370 lesions.

**Table 2.** Patient's characteristics.

The analysis of biochemical examination results indicated elevated serum calcium and parathyroid hormone levels within the entire group prior to operation without distinction of etiologies or localizations (Figures 2 and 3).

**Figure 3.** Blood serum parathormone (PTH) concentration before and after operation (\*—*p* < 0.001).

Histopathological findings imply that hyperplasia and adenoma are the most common types of single and multiple lesions (Table 3). In the case of multiple lesions, the intercurrence of adenoma and hyperplasia of several glands occurred within the neck area, but it was not seen in mediastinal localization.

**Table 3.** Histopathological findings.

During performing operations, we discovered that the parathyroid gland most commonly affected within the entire group of patients was the inferior right, with 436 cases. The inferior left parathyroid gland was affected in 351 cases, superior left in 220 cases, and superior right in 219 cases. We also performed a comparison of the occurrence of neoplastic gland in patients with single-gland changes and those with multiple abnormal glands. In the case of patients with single-gland neoplasm, the most commonly affected was the inferior right parathyroid gland (347 cases) and subsequently the superior left (142 cases), the superior right (140 cases), and the inferior left (27 cases). Within the patients with more than one affected gland, abnormalities were found most often within the inferior right parathyroid gland (89 cases) and subsequently the superior right (79 cases), the superior left (78 cases), and rarely the inferior left parathyroid gland (24 cases). Double adenomas were situated most commonly in the lower right location (53%), subsequently the right (49%) and the upper left (30%) locations, and sporadically in the lower left location (17%), implying that double adenomas are usually found in the right part of the neck when parathyroid glands are not ectopic. The mean size of double adenomas was approximately 1.5 cm. After a parathyroidectomy was performed, no symptoms were prominent among almost all of the patients and both PTH and calcium levels decreased. Calcium levels has normalized postoperatively and remained within the normal range for 12 months or longer.

A total of 19 patients required remedial surgery due to persistent or recurrent hypercalcemia. Out of the group of 1019 patients, failures in the surgical treatment of pHPT were reported in 19 cases—in eight cases, the lesions were located on the neck, and in 11 cases the lesions were in the mediastinum (Table 4).

**Table 4.** Treatment failures.

Persistent hyperfunction was the cause of repeated operation in nine of them and relapse in 10. However, imaging studies failed to find the cause of the hyperfunction in three of them. The remaining 16 were operated on successfully (Table 5).

**Table 5.** Persistent and recurrent hypercalcemia.

There were two (0.2%) cases of persistent hypercalcemia caused by lesions of the neck and some cases (10.6%) in the mediastinum. Recurrent hypercalcemia was found in four patients (0.4%) due to a neoplastic gland remaining in the neck area and three (4.5%) within the mediastinum. We did not find any changes in three cases, where two cases were related to a neck localization and one case to the mediastinum. The main cause of failure of the surgical treatment were parathyroid carcinomas (twice—the cause of persistent activity and four—recurrent).

#### **5. Discussion**

Parathyroidectomy is the treatment of choice in patients with primary hyperparathyroidism. The goal of PTX is to achieve normocalcemia by removal of the hyperfunctioning tissue that causes excessive secretion of parathyroid hormones. Surgical failures may result from an incomplete excision or parathyromatosis. Reoperative parathyroid surgery can be required in approximately 10% of patients [5]. Out of the 1019 patients, 19 (1.9%) received a surgery that failed. This mainly applies to patients with ectopic mediastinal lesions (16.6%).

We want to emphasize that 15% of our patients have a multiple gland disease and that the heterogenic group includes patients with multiple hyperplasia, double adenoma, hyperplasia as well as cancer. We believe that a visual indication of all parathyroid glands and excision or biopsy of the rest of them is an effective procedure for most patients operated on for pHPT. Moreover, in our opinion, an intraoperative measurement of PTH (iPTH) by a quick assay (QPTH) in predicting operative outcomes of parathyroidectomy is the procedure of choice. For the last 15 years we have measured QPTH in all operated-on patients. The goal of the surgical treatment is to remove hyperfunctioning glands. If hyperplasia is diagnosed, we remove three and a half parathyroid glands, or hyperfunctioning adenomas. Collar incision is a feasible approach to the mediastinal glands even at re-exploration. Among our 66 patients treated for a mediastinal ectopic gland, only four required thoracotomy. We must emphasize that about one-third of normal lower parathyroid glands are found below the lower thyroid pole, in the thyroid-thymic tract of fat or cervical tongue of the thymus. In our experience of reoperation for a "missing" gland, the thymus is its most probable location. Among seven of our patients reoperated on for persistent hypercalcemia, four glands were found within the thymus, one between the pericardial sac and the mediastinal pleura, close to the main left bronchus, one in the aorto-pulmonary window and one close to the right innominate vein and the superior vena cava.

The analysis of surgical treatment failures for various parathyroid changes implies several explanations. These include the variable anatomy of parathyroid glands among different patients as well as ectopic locations of adenomas [6]. There are various opinions that all glands inferior to the superior border of the manubrium tend to be mediastinal [6,7]. Others consider that mediastinal parathyroid glands are those which are unreachable by cervical incisions [7,8]. On the other hand, we noted an extreme case where the changed parathyroid gland appeared in the soft palate of a 57-year old patient who suffered from persistent hypercalcemia even after initial parathyroidectomy. The investigation of this patient also shows that there is still a possibility that parathyroid adenomas can be missed even with previous use of advanced imaging methods if they occur in various anatomical

positions [9]. Moreover, ectopic parathyroid glands can be so small that they may be invisible, or a single removed gland is one when all function abnormally [5,7]. In our opinion, the best surgical solution for the latter is a removal of three and a half of the parathyroid glands. Re-operation is also required when one of two affected glands is more active than the other. In such a situation, when the dominant parathyroid gland has been removed, the other becomes the cause of hyperfunction. A second operation is also needed when the altered parathyroid gland has not been completely removed—for example, when undergoing disintegration—and the rest of the parathyroid gland develops again causing symptoms of hyperactivity.

Most of the reasons for the failure of initial parathyroidectomy may be eliminated by the right diagnostic measures in the form of imaging possibilities [10,11]. Radiological examination methods are particularly precise and helpful to properly localize most of hyperfunctioning parathyroid glands, thus allowing for shorter surgeries, a decrease in ineffective surgeries and fewer complications [12, 13]. Some studies show that in primary hyperparathyroidism the results of preoperative imaging examinations do not allow to differentiate between a uniglandular and multiglandular disease in [14,15]. Finding the parathyroid gland facilitates the intraoperative examination of parathyroid hormone concentration, as well as the use of radio-guidance [16–18].

Cases with multiple tumours are particularly more complicated, as a fall in the intraoperative PTH to the lower end of a normal range cannot guarantee that all tumours have been removed [19–21]. A reduction in intraoperative PTH by more than 50% after the procedure is a predictive factor [22,23] and may be prevented by preoperative supplementation with bisphosphonates [24,25]. Age is not a relevant contributing factor for the occurrence of complications [26,27], while anesthesia can often cause sudden elevation of PTH levels in patients suffering from hyperparathyroidism [28,29] and can worsen renal functions [30,31]. The latest works shows that minimally invasive radio-guided parathyroidectomy using a very low dose of Tc-99 MIBI, even without an intraoperative assay or a frozen section analysis, resulted in an excellent cure rate [32,33]. Based on our results, we also wanted to emphasize that PTX is a safe procedure, with only a small number of complications, the most serious of which seems to be recurrent laryngeal nerve palsy. Based on our observations, we believe that patients should be examined every three months after PTX for the first two years, and then twice a year afterwards. Some authors indicate that this should last for even more than 10 years after parathyroidectomy [34].

#### **6. Conclusions**

**Author Contributions:** Conceptualization, J.G.; Methodology, G.B., A.B., D.Z., Z.N., A.G. and H.K.; ˙ Writing—original draft, G.J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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# *Article Snail-1* **Overexpression Correlates with Metastatic Phenotype in BRAFV600E Positive Papillary Thyroid Carcinoma**

**Katarzyna Wieczorek-Szukala 1, Janusz Kopczynski 2, Aldona Kowalska 3,4 and Andrzej Lewinski 1,\***

Received: 24 July 2020; Accepted: 18 August 2020; Published: 21 August 2020

**Abstract:** The ability of cancer to metastasize is regulated by various signaling pathways, including transforming growth factor β (TGFβ), also implicated in the upregulation of Snail-1 transcription factor in malignant neoplasms. B-type Raf kinase gene (BRAF)V600E, the most common driving mutation in papillary thyroid carcinoma (PTC), induces epithelial to mesenchymal transition (EMT) in thyroid cancer cells through changes in the Snail-1 level, increasing cell migration and invasion. However, little is known about the mechanism of Snail-1 and BRAFV600E relations in humans. Our study included 61 PTC patients with evaluated BRAFV600E mutation status. A total of 18 of those patients had lymph node metastases—of whom 10 were BRAFV600E positive, and 8 negative. Our findings indicate that the expression of Snail-1, but not TGFβ1, correlates with the metastatic phenotype in PTC. This is the first piece of evidence that the upregulation of Snail-1 corresponds with the presence of BRAFV600E mutation and increased expression of Snail-1 in metastatic PTC samples is dependent on BRAFV600E mutation status.

**Keywords:** PTC; thyroid; metastasis; Snail-1; BRAFV600E

#### **1. Introduction**

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer accounting for 80–85% of all thyroid cancer cases. Although its incidence is gradually growing, the cancer-related mortality rate is relatively stable, and PTC has a good prognosis with an average 5-year survival rate of above 90% [1]. However, above 50% of PTC case often have performed local lymph node metastasis and approximately 10% of PTC cases represent aggressive metastatic neoplasm with distant metastases to lungs or bones [2]. In addition to an increased rate of primary tumors, there has been also growing incidence of regional and distant PTC metastases, which obviously increases the global need for improving our understanding of pathogenesis process and identifying effective, targeted treatments.

The fundamental mechanism by which epithelial-derived tumor cells may become malignant and obtain an invasive phenotype is the epithelial to mesenchymal transition (EMT). EMT is essential to both physiological developmental processes, as well as metastatic cancer. Numerous studies have demonstrated that this process is aberrantly activated during thyroid cancer development [3,4]. Moreover, it was discovered both in vitro and in vivo that thyroid tumour cells from PTCs and anaplastic cancer (ATCs) may constitutively display an active EMT process when compared to normal

thyrocytes. This results in the loss of cells' polarity, decreased expression of particular epithelial markers and increased expression of mesenchymal markers [5,6].

It is currently known that EMT may be initialized and maintained by various molecular factors, including an inflammatory cytokine-transformation growth factor-β (TGF-β) [3]. Transforming growth factor β (TGFβ) is one of the most abundant cytokines that regulates many biological processes such as cell differentiation, apoptosis, proliferation, and other physiological and pathological conditions. TGFβ1 is thought to be the primary isoform which is overexpressed in many tumor types and may promote tumor invasion and metastasis through multiple Smad-independent or non-canonical signaling pathways [7,8]. The TGFβ family of signaling molecules has been implicated in the upregulation of key transcription factors such as Twist, Zeb or Snail-1 [8,9]. Importantly, it has been also discovered that such transcription factors may initiate the EMT process itself [3,10].

Snail-1 belongs to a family of zinc-finger transcription factors and its primary function is the repression of E-cadherin, which results in reduced cell adhesion and promotes migratory capacity [9,11]. In addition, Snail-1 activation induces fibrosis in various organs such as the kidneys, liver and lungs. The pathological activation of Snail-1 probably contributes to organ fibrosis which may further lead to chronic inflammation and cancer [12]. Snail-1 has been also recently implicated in the regulation of drug resistance and the induction of the cancer stem cell (CSC) phenotype [9,13].

However, the induction of the EMT process in the context of cancer invasion remains Snail-1 s most studied function. A large number of studies have found that metastatic tumors of the colon, breast, ovary and prostate carcinomas overexpress the Snail-1 protein [9,14]. Its overexpression is generally correlated with aggressiveness, lymph node metastases (LNM), tumor recurrence and poor prognosis [15,16]. Our previous experiments concerning colon adenocarcinoma HT29 cells also indicated that Snail-1 overexpression reduced cell adhesion and increased migratory properties, which coincides with the above observations [17]. Furthermore, it was shown that Snail-1 may also play a key role in cancer cell cytoskeleton reorganization, while a positive correlation between Snail-1 presence and Tubulin–β3, (TUBB3) (one of the main proteins building microtubules, a major component of the eukaryotic cytoskeleton) upregulation in adenocarcinoma cell lines has been observed [18]. Although Snail-1 is not expressed in normal thyroid tissue, it was described to be overexpressed in different thyroid cancer cell lines [19].

PTC oncogenesis is associated with the occurrence of the mutations within the B-type Raf kinase gene–BRAF [20]. Although numerous different variants of BRAF mutations have been discovered so far, over 90% are the BRAFV600E mutation. This genetic aberration influences and constantly activates the mitogen-activated protein kinase (MAPK) signaling pathway and leads to uncontrolled cell proliferation and differentiation [21]. Among other genetic alternations involving the MAPK pathway (such as RET/PTC or RAS) BRAFV600E is present in more than 50% of PTC, but is rare in follicular variants, and not found in follicular thyroid cancer. Therefore, BRAFV600E has been postulated as a good prognostic marker to assist in risk stratification for patients with PTC [22].

The tumorigenic role of BRAFV600E in PTC development was well documented before in BRAFV600E transgenic mice models. Studies performed on rat thyroid cells overexpressing BRAFV600E suggested that BRAFV600E is an initiator of tumorigenesis and is required for tumor progression in PTC [23]. A large number of clinical studies have demonstrated an association of BRAFV600E mutation with aggressive clinicopathologic characteristics and high tumor recurrence, although the results are controversial [24].What is important, it has also been shown that BRAFV600E plays a crucial role in direct EMT induction. Baquero et al. have studied this phenomenon in thyroid cancer cells and reported that BRAFV600E induces EMT through changes in Snail-1 and E-cadherin protein expression levels, which in turn, increase cell migration and invasion [25]. The interaction of BRAFV600E and Snail-1 has been observed also in other cancer types. Interestingly, Massoumi et al. have reported that hyperactivation of the BRAFV600Emutant in melanoma cells resulted in Snail-1 overexpression and increased their metastatic potential. Introducing mutated BRAF into primary melanocytes led

to increased Snail-1 expression after malignant transformation, constitutively high ERK activity and resulted in acquisition of a more aggressive cell phenotype [26].

An association has been suggested between BRAFV600E and TGFβ in aggressiveness induction of PTCs. It has been shown in rat thyroid cells overexpressing BRAFV600E that this oncogene stimulates TGFβ secretion, and both proteins exert the same effect on both E-cadherin expression and cell invasion [27]. However, the above studies, based on animal or cell line models (mostly anaplastic thyroid cancer model), may not fully represent the interactions between Snail-1 and BRAFV600E mutation in PTC in vivo.

The aim of our study was to determine whether there is any correlation between *Snail-1* and *TGF*β*1* gene expression and the existence of BRAFV600E mutation in patients with metastatic or non-metastatic PTC. To our knowledge, this study is the first attempt assessing the expression of the above EMT markers and the metastatic potential of PTC in paraffin slides containing human PTC collected during surgery.

#### **2. Materials and Methods**

#### *2.1. Patients*

The study group was recruited from PTC patients diagnosed and recruited to the trial during routine follow-up from 2012 at the Endocrinology Clinic of the Holy Cross Cancer Center (Kielce, Poland). Thin-section paraffin-embedded slides of thyroid tissues were prepared by a Holycross Cancer Centre pathologist at the time of diagnosis as decribed earlier [28,29]. One of our pathologists (J. K.) marked the area containing primary tumour foci on one haematoxylin and eosin-stained slide (red line, Figure 1).

**Figure 1.** Representative cancer foci images. Hematoxylin and eosin staining of 2 different papillary thyroid carcinoma (PTC) samples-the primary tumour foci have been marked by pathologist (red line) (**A**,**C**). The images (**B**,**D**) show magnification ×400.

A total of 61 PTC specimens were chosen for further analysis. All the study procedures were approved by the Bioethical Committee at the Holy Cross Medical Chamber, Kielce (no. 2/2013). Patients provided their written consent for molecular tests.The patient characteristics are described in Table 1.

The presence of the V600E mutation in the BRAF gene was evaluated by three genotyping methods: allele-specific amplification PCR (ASA-PCR), qPCR, and Seq as described previously by Kowalik et al. [29]. Within the 61 patients in this study, 30 samples were BRAFV600E positive and 31 BRAFV600E negative. Within 18 patients with lymph node metastasis–10 were BRAFV600E positive, and 8 negative. Controls were obtained from surrounding normal tissue after pathologist supervision.

**Table 1.** Characteristics of papillary thyroid cancer patients from whom PTC tissue has been collected.

#### *2.2. Molecular Methods*

The pathologist marked the area containing PTC tumor cells on a hematoxylin and eosin-stained slide. Then, 4 to 5 unstained slides were deparaffinized by soaking twice in xylene for 15 min, twice in 96% alcohol for 5 min and once in distilled water. Tissue was scraped from thepathologist-selected area with a scalpel and transferred to a test tube for further RNA isolation.

RNA isolation has been performed using a High Pure FFPE RNA Isolation Kit (Roche, Switzerland), according to the manufacturer's protocol. The purity and integrity of total RNA was assessed by an Agilent 2100 Bioanalyzer. The degradation rate of total RNA was determined using RIN values. Only the samples with RIN > 7 were further analyzed. Total RNA was used in the first strand cDNA synthesis with a Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Scientific, Waltham, MA, USA) according to manufacturer's instructions. RT-qPCR was performed on the ABI PRISM® 7500 Sequence Detection System (Applied Biosystem, CA, USA) by using TaqMan Gene Expression Assays and TaqMan™ Fast Advanced Master Mix according to the manufacturer's specification. The Assays Identification numbers were: SNAI1-Hs00195591\_m1; TGFβ1-Hs00998133\_m1; GAPDH Hs99999905\_m1. Thermal cycler conditions were as follows: 2 min at 50 ◦C, hold for 20 s at 95 ◦C, followed by two-step PCR for 40 cycles of 95 ◦C for 3 s followed by 60 ◦C for 30 s. Amplification reactions, in triplicate for each sample, were performed and the results were normalized to the GAPDH gene expression level. An analysis of relative gene expression data was performed, using the 2–ΔΔCT method. The calibrator was prepared as mean gene expression values for normal tissue from 12 patients.

#### *2.3. Statistics*

The statistical analysis was carried out using the Statistica 12 software (StatsoftPolska, Kraków, Poland). A graphical representation of the results was prepared using the SigmaPlot 11 software (Systat Software Inc., San Jose, CA, USA). The results are presented as the mean of three independent experiments ± SD. The normality of the distribution was assessed using the Shapiro–Wilk test. The Student's *t*-test was used for normally distributed parameters. Chi-square tests and *t*-test were used in the medical history analysis. In all the analyses, results were considered statistically significant when *p* < 0.05.

#### **3. Results**

#### *3.1. Snail-1, but not TGF*β*1 Expression is Correlated with Metastatic Phenotype in PTC*

The EMT process may be induced both through transcription factors such as Snail-1 or growth factor TGFβ1. Altered expression of those genes has been observed previously in thyroid cancer cell line models [4,25]. We analyzed the expression of those two genes in 61 FFPE tissues from PTC patients. A total of 18 PTC samples were representing metastases (18 lymph node, with three distant—as described in Table 1)—described later as "Metastatic PTC", and 43 PTC samples had no diagnosed metastases ("Non-metastatic PTC"). RT-qPCR was performed using TaqMan Gene Expression Assays. We observed a statistically significant (*p* < 0.001), more than 2.5-fold increase in *Snail-1* gene expression in metastatic PTC samples as compared to non-metastatic samples (Figure 2, grey bars). The expression of the *TGF*β*1* gene in non-metastatic PTC patients was slightly higher, however, no statistical significance for this tendency between the studied groups was noted.

**Figure 2.** The expression of *Snail-1* but not *TGF*β*1* was correlated with the metastatic phenotype in PTC samples. The gene expression was evaluated by quantitative RT-qPCR in metastatic (*n* = 18) and non-metastatic (*n* = 43) PTC groups. The relative mRNA level was normalized to GAPDH. The results are presented as the mean of three independent experiments ± SD. \*\* *p* < 0.001.

#### *3.2. The Presence of BRAFV600E Mutation Associates with Snail-1 Expression in PTC*

As described before, Snail-1 protein expression may be regulated by BRAFV600E mutation in human thyroid cancer cells [25]. Therefore, we divided our studied group into BRAFV600E positive (30 patients) and BRAFV600E negative samples (31 patients). The presence of the V600E mutation in BRAF gene was evaluated previously [14]. *Snail-1* mRNA expression was significantly higher (ca. 2-fold) in PTC with BRAFV600E mutation in comparison to BRAFV600E negative samples (*p* < 0.05) (Figure 3). However, the expression *TGF*β*1* did not differ between those two groups.

**Figure 3.** The expression of *Snail-1* is correlated with the presence of B-type Raf kinase gene (BRAF)V600E mutation in PTC samples. The gene expression was evaluated by quantitative RT-qPCR in BRAFV600E (+) (*n* = 30) and BRAFV600E (−) (*n* = 31) PTC groups. The relative mRNA level was normalized to GAPDH. The results are presented as the mean of three independent experiments ± SD. \* *p* < 0.05.

#### *3.3. The Upregulation of Snail-1 in Metastatic PTC Samples is Dependent on BRAFV600E Mutation Status*

Furthermore, we aimed to evaluate the role of BRAFV600E mutation on the expression of *Snail-1* and *TGF*β*1* within metastatic PTC samples. Among 18 PTC patients with lymph node metastasis–10 samples harboured BRAFV600E mutation (described later as "Metastatic- BRAF V600E (+)"), and 8 were BRAFV600E negative ("Metastatic–BRAF V600E (−)"). Among 43 PTC patients with no diagnosed metastases—20 were BRAFV600E positive ("Non-metastatic–BRAF V600E (+)"), and 23 BRAFV600E negative ("Non-metastatic–BRAF V600E (−)").

*Snail-1* expression level was nearly 3-fold higher in metastatic PTC that harbored the BRAFV600E mutation when compared to the metastatic but BRAFV600E negative group (*p* < 0.05) (Figure 4A, grey bars). Interestingly, the expression level of the *Snail-1* gene in metastatic but BRAFV600E negative PTC samples (Figure 4A) remained on a similar level as in the non-metastatic PTC patients, regardless of BRAFV600E mutation status (Figure 4B).

**Figure 4.** The upregulation of *Snail-1* in metastatic PTC samples is dependent on BRAFV600E mutation status. The gene expression was evaluated by quantitative RT-qPCR in both BRAF V600E (+) (*n* = 10) and BRAF V600E (−) metastatic (*n* = 8) (**A**) and non-metastatic BRAF V600E (+) (*n* = 20) and BRAF V600E (−) (*n* = 23) PTC groups (**B**). The relative mRNA level was normalized to GAPDH. The results are presented as the mean of three independent experiments ± SD. \* *p* < 0.05.

On the other hand, in both metastatic and non-metastatic PTC groups: BRAF V600E (+) or (−) (Figure 4A,B, white bars), no significant differences in *TGF*β*1* gene expression levels were found (Student's *t*-test, *p* > 0.05 in each case).

Table 2 summarizes some vital clinical features of the studied metastatic and non-metastatic PTC patient groups. Statistical analysis showed no significant statistical correlation between BRAFV600E mutation status in PTC tissue and gender, age at diagnosis, or T-classification of the primary tumor. However, it was observed that significantly more samples of non-metastatic PTC tumors with T3–T4 classification harbored the BRAFV600E mutation. Furthermore, it can be observed that more patients with non-metastatic and BRAFV600E negative PTC had been diagnosed as less advanced T1–T2 tumor stage (*p* = 0.0002). Nevertheless, due to the limited number of patients in the studied groups evaluation of such analysis requires further research.

**Table 2.** Clinical features of metastatic and non-metastatic PTC tumors with or without BRAFV600E mutation, \*\* *p* < 0.001.

#### **4. Discussion**

PTC is the most common of all thyroid cancers, with a gradually growing incidence. The majority of PTC cases generally present good prognosis with conventional therapies. Nevertheless, approximately 10% of PTC cases progress to more aggressive forms associated with local invasion, distant metastases and poorer clinical outcome [1,2]. Despite metastasis remaining the key cause of failure in cancer treatment and mortality, its molecular mechanisms remain poorly examined.

Metastatic process is associated with change of phenotype due to the epithelial–mesenchymal transition (EMT), an essential physiological mechanism in embryonic development and tissue repair. However, EMT may also contribute to the progression of disease, such as organ fibrosis or cancer [3]. This process results from the induction of transcription factors that change gene expression, therefore promoting the loss of cell–cell adhesion, modifying cytoskeletal dynamics and leading to a change from epithelial morphology and physiology to the mesenchymal phenotype. EMT can be induced by various signaling pathways, for example mediated by transforming growth factor β (TGFβ), Wnt–β-catenin, Notch, Hedgehog receptor tyrosine kinases (RTK) [4,5].

The role of TGFβ in cancer is complex: this pathway may function both in tumor suppression and in tumor promotion. TGFβ also functions as a tumor suppressor in early tumor development. In a number of human cancers, TGFβ inhibits cell cycle progression, increases apoptosis, and suppresses the expression of growth factors, cytokines, and chemokines. The function of *TGF*β as a tumor suppressor or a tumor promoter depends on the context and stage of tumor progression. Its shift to a pro-metastatic role at later tumor stages has been shown to be mediated by various signaling pathways [7,10].

The thyroid gland expresses the *TGF*β*1* gene mRNA and synthesizes the protein, which under physiologic conditions regulates thyroid growth and function. Interestingly, it was concluded that the alterations in the signaling pathway of *TGF*β*1* are not the same in tumors from different species [7]. It seems it is not entirely possible to apply the results obtained in animal or cell culture studies to normal or pathological human thyroid tissue. It should be noted that using a single experimental research model usually entails some limitations. For example, employing exclusively tissue material collected from patients, may lead to a somehow less comprehensive scientific reasoning.

The studies of the expression of *TGF*β*1* performed before in human PTC revealed that, when compared to the central areas, the peripheral invasive areas of the tumors had increased expression of *TGF*β*1*, *NFk*β, and CDC42 as well as showing a decrease in cell–cell adhesion proteins. Moreover, these regions presented also an overexpression of vimentin, a characteristic marker of EMT. The authors concluded that these changes might be related to increased tumor invasiveness and suggest an important role of TGFβ as inductor of EMT and metastasis [5,7]. Other studies of Brace et al. have shown that *TGF*β*1*, but not *TGF*β*2*, is significantly increased in PTC compared to benign thyroid nodules and therefore may serve as a potential diagnostic marker [30].

However, in our studies, no statistically significant differences were found in *TGF*β*1* gene expression in BRAF V600E (+) and BRAF V600E (−) PTC groups, as well as in PTC metastatic samples with known BRAFV600E mutation status. Possibly, this may result from the fact that TGFβ has been found overexpressed locally in the invasive front of cancer cells, also in different cancer types, for example in the breast cancer [31]. Moreover, in ovarian cancer Wang et al. have demonstrated that the expression of TGF-β1 is not associated with lymphatic metastasis [32]. Moreover, it has been discovered that BRAFV600E increases exclusively *TGF*β*1* secretion through the MEK/ERK pathway in thyroid tumour cells [27]. The observations of Riesco-Eizaguirre et al. have led to the concept that the action of TGFβ in PTC is very important locally as a modulator of the tumor microenvironment, however, the total *TGF*β*1* expression may not be altered [33].

It has been widely described that the TGFβ cascade also induces the expression of Snail-1-a zinc finger transcription factor, the main E-cadherin repressor and one of the most potent EMT inducers [34]. Snail-1 was found to be overexpressed in the invasive fronts of several human tumors derived from epithelial cells [4,14] and proved to play a key role in PTC, as well. Although Snail-1 is not expressed in normal thyroid tissue, histopathological analyses have proved that the expression of Snail-1 protein was mainly located in the cytoplasm and nuclei of PTC cells [19]. The overexpression of this gene and the upregulated protein level have been discovered in numerous cancer types such as ovarian and breast carcinomas, melanomas, oral squamous carcinomas, human thyroid carcinomas and their metastases. In the context of thyroid cancers, the effects of Snail-1 interactions have not been thoroughly studied or understood. It has been suggested that Snail-1 expression, together with TGFβ1, is associated with lymph node metastasis in PTC and may be a potential biomarker of tumor diagnosis and prognosis in PTC [8].

Interestingly, Baquero et al. have discovered that Snail-1 expression may also be regulated by BRAFV600E—the most common mutation found in PTCs [25]. In the thyroid carcinoma cell model, BRAFV600E induced EMT via changes in Snail-1 and E-cadherin protein expression levels, which in turn increased the migration and invasiveness of these cells. Moreover, inhibition of BRAFV600E significantly decreased invasion of thyroid cancer cells, tumor volume and metastases in a mouse model of anaplastic thyroid carcinoma [25,27].

In our studies, it was observed that BRAFV600E mutation was present in significantly more cases of non-metastatic aggressive tumors (with T3–T4 classification). However, due to the limited number of patients in the studied groups, such statistics should be evaluated with some perspective.

Most clinical research has demonstrated an association between BRAFV600E mutation status, aggressive clinicopathologic characteristics of PTC and high tumor recurrence, although the results remain controversial [24]. Contradictory results from other groups have stressed that, although BRAFV600E mutation may show very high prevalence of the *BRAF* mutation in PTC in the population, the presence of the mutation itself was not statistically associated with a metastasis, patient age, completeness of resection, local invasion or tumor size score. Although BRAFV600E analysis may have some value in distinguishing malignant and benign thyroid tumors, it is currently believed that alone it is not a good predictor of more the aggressive clinical course of PTC [28].

Therefore, we have aimed to evaluate if *Snail-1* and *TGF*β*1* genes expression correlate with one another in human PTC tissue samples or whether they are related to BRAFV600E mutation status or LNM tumor staging. We demonstrate that *Snail-1* gene expression is significantly increased in metastatic PTC patients group (based on LNM scale) in comparison to non-metastatic samples. Moreover, we show that *Snail-1* elevated expression also correlates with BRAFV600E positive status. Our results clearly correspond with the data obtained by Baquero et al. on the cellular model [25]. On the contrary, Mitchell et al., utilizing immunohistochemical techniques, suggested that Snail-1 protein expression did not correlate with BRAF status and E-cadherin expression in PTC [35]. Such discrepancies may result from different molecular techniques, or the relatively small number of studied patient samples. It must be also emphasized that Snail-1 protein is relatively unstable, and in the cytoplasm, it may be prone to proteasomal degradation, with a half-life of only twenty-five minutes. GSK3β is a Snail kinase

can bind to and phosphorylate this transcription factor, facilitating its degradation. Phosphorylation determines Snail-1 s subcellular location, as GSK-3β-mediated phosphorylation induces Snail-1 export to the cytoplasm [9,36]. Therefore, every study concerning Snail-1 protein expression should also consider its localization.

Interestingly, for the first time using human PTC tissue model, our findings reveal that *Snail-1* gene expression was significantly higher exclusively in metastatic PTC that harbored the BRAFV600E mutation, but not in metastatic BRAFV600E-negative PTC tissue. In contrast, we did not observe such differences in *Snail-1* gene expression in non-metastatic PTC patients, regardless of BRAFV600E mutation status. This may suggest that in cells altered by metastatic processes, such as EMT, BRAFV600E mutation facilitates the mechanism of *Snail-1* overexpression. It has previously been proposed that BRAFV600E can increase Snail-1 expression both at transcriptional level and via GSK3β inhibition [27]. However, the mechanism of transcription regulation in the presence of BRAF mutation remains to be further elucidated.

Despite some limitations of our study resulting from the relatively small sample size due to the selection of metastasizing PTC cases, caused by only one mutation of individual gene (BRAFV600E), the presented data confirm that BRAFV600E participates in mechanisms involved in the increase in *Snail-1* expression in metastatic PTC. Possibly, the interplay between BRAFV600E and *Snail-1* may play a key role in the thyroid gland neoplastic transformation and cancer cell invasiveness. The further elucidation of the mutual interactions between *Snail-1* and BRAFV600E mutation in PTC in vivo may shed new light on the issue of metastatic thyroid cancer development. Better understanding of its complexity would help to identify specific targets for modern molecular therapies and develop more accurate diagnostic strategies.

**Author Contributions:** Conceptualization, K.W.-S. and A.L.; methodology, K.W.-S. and J.K.; resources, A.K.; writing—original draft preparation, K.W.-S.; writing—review and editing, A.L.; supervision, A.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by the Medical University of Lodz, project No 502-03/1-107-03/502-14-316 and statutory funds 503/1-107-03/503-11-001-19-00.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

# **S-Detect Software vs. EU-TIRADS Classification: A Dual-Center Validation of Diagnostic Performance in Di**ff**erentiation of Thyroid Nodules**

**Ewelina Szczepanek-Parulska 1,\*,**†**, Kosma Wolinski 1,\*,**†**, Katarzyna Dobruch-Sobczak 2, Patrycja Antosik 1, Anna Ostalowska 1, Agnieszka Krauze 3, Bartosz Migda 3, Agnieszka Zylka 4, Malgorzata Lange-Ratajczak 5, Tomasz Banasiewicz 5, Marek Dedecjus 4, Zbigniew Adamczewski 6,7, Rafal Z. Slapa 3, Robert K. Mlosek 3, Andrzej Lewinski 6,7 and Marek Ruchala 1**

Received: 11 June 2020; Accepted: 30 July 2020; Published: 3 August 2020

**Abstract:** Computer-aided diagnosis (CAD) and other risk stratification systems may improve ultrasound image interpretation. This prospective study aimed to compare the diagnostic performance of CAD and the European Thyroid Imaging Reporting and Data System (EU-TIRADS) classification applied by physicians with S-Detect 2 software CAD based on Korean Thyroid Imaging Reporting and Data System (K-TIRADS) and combinations of both methods (MODELs 1 to 5). In all, 133 nodules from 88 patients referred to thyroidectomy with available histopathology or with unambiguous results of cytology were included. The S-Detect system, EU-TIRADS, and mixed MODELs 1–5 for the diagnosis of thyroid cancer showed a sensitivity of 89.4%, 90.9%, 84.9%, 95.5%, 93.9%, 78.9% and 93.9%; a specificity of 80.6%, 61.2%, 88.1%, 53.7%, 73.1%, 89.6% and 80.6%; a positive predictive value of 81.9%, 69.8%, 87.5%, 67%, 77.5%, 88.1% and 82.7%; a negative predictive value of 88.5%, 87.2%, 85.5%, 92.3%, 92.5%, 81.1% and 93.1%; and an accuracy of 85%, 75.9%, 86.5%, 74.4%, 83.5%, 84.2%, and 87.2%, respectively. Comparison showed superiority of the similar MODELs 1 and 5 over other mixed models as well as EU-TIRADS and S-Detect used alone (*p*-value < 0.05). S-Detect software is characterized with high sensitivity and good specificity, whereas EU-TIRADS has high sensitivity, but rather low specificity. The best diagnostic performance in malignant thyroid nodule (TN) risk stratification was obtained for the combined model of S-Detect ("possibly malignant" nodule) and simultaneously obtaining 4 or 5 points (MODEL 1) or exactly 5 points (MODEL 5) on the EU-TIRADS scale.

**Keywords:** thyroid nodules; thyroid cancer; ultrasound; computer-aided diagnosis; S-Detect; EU-TIRADS

#### **1. Introduction**

Thyroid nodules (TNs) are the most frequent endocrine disorder and occur in 10–70% of the general population, with a relatively low malignancy rate of 3–10% [1]. The most challenging issue for medical practitioners is the differentiation between benign and malignant TNs. Ultrasonography (US) of the thyroid gland is commonly used by physicians and is crucial to decide on further management, such as qualification for fine-needle aspiration biopsy (FNAB) as well as the decision on a conservative or surgical approach [2,3]. However, the analysis of nodules' composition, echogenicity, shape, orientation, margin, calcifications, presence of halo, type of vascularization, and elasticity [4] is time consuming and subject to diverse inter-observer and intra-observer variability in the multi-ultrasound descriptor assessment of thyroid nodules, with slight to substantial agreement (κ-values ranging from 0.33 to 0.61) [5]. Moreover, no single US feature or combination of features can reliably predict the malignant character of TNs [6,7]. Furthermore, the results of US strongly depend on the operator's interpretation and experience, which implies US sensitivity and specificity varying from 52% to 81% and 54% to 83%, respectively [8,9]. In some thyroid pathologies, additional imaging methods may be applied together with US to increase its diagnostic value [10,11]. Difficulties with the interpretation of US findings often lead to redundant FNAB or even diagnostic surgery. Recently, the European Thyroid Imaging Reporting and Data System (EU-TIRADS) was established to facilitate standardization and provide a simple lexicon for distinguishing between benign and malignant TNs and to reduce the number of unnecessary invasive interventions (FNAB, thyroidectomy) [12]. A meta-analysis of seven studies, evaluating 5672 thyroid nodules, indicated that stratifying the risk of thyroid nodules by EU-TIRADS showed high performance, while the prevalence of malignancy in EU-TIRADS class 5 was equal to 76.1% [13]. Nonetheless, even this classification might be intricate for inexperienced physicians [8]. To overcome this difficulty, computer-aided diagnosis (CAD) systems are gaining interest for US image analysis and are developed on the basis of statistical data mining algorithms, collected from medical center databases. It enables non-invasive judgement on benignity or malignancy of TNs on the basis of US image analysis [14,15]. The purpose of CAD is to increase the diagnostic confidence, achieve interpretation constancy of US features, and eliminate the inter-observer variability in order to increase diagnostic accuracy, especially when the examination is performed by ultrasonographers outside referenced centers for thyroid cancer diagnostics. The practical potential of CAD has been suggested in a few recent studies [8,9,14,16]. Kwak TIRADS has high sensitivity and low specificity. Thus, it is very useful for discarding the benign cases and reducing the number of biopsies [17]. CAD is supposed to be an applicable tool in TN diagnostics and clinical decision-making for medical practitioners with basic US skills [15,16]. It may be useful especially for less experienced operators, increasing the specificity of the examination [18]. However, there is an unmet need for validation of the method on a large cohort of patients in highly referenced centers to provide reliable information on its real clinical utility and identify a target group of physicians who may benefit from the CAD-supported evaluation of TNs. Thus, the aim of our study was to compare CAD diagnostic performance with the state-of-the-art thyroid nodule classification EU-TIRADS applied by physicians based on US morphological features, in order to support decision-making regarding the further management of TNs.

#### **2. Experimental Section**

#### *2.1. Patients*

The studied group consisted of 88 patients with thyroid nodular disease. Lesions included in the study comprised nodules whose character was verified by the unambiguous diagnostic result of a fine-needle aspiration biopsy (concerning nodules presenting category II, V, or VI according to the Bethesda classification, unless the histopathological verification was performed) or nodules in patients subjected to a thyroidectomy, for whom the result of histopathological examination was available. The **exclusion criteria** for the study were as follows:

The diagram depicting excluding factors and the process of recruitment of the patients for the study is presented in Figure 1.

**Figure 1.** Diagram presenting the process of recruitment of the patients for the study.

#### *2.2. Methods*

This was a prospective study. Patients eligible for the study were recruited from those admitted to the tertiary reference endocrine or surgical centers for urgent evaluation of the indications for thyroidectomy due to the following results of the Bethesda System for Reporting Thyroid Cytopathology (BSRTC): (a) suspicion of malignancy or thyroid cancer (BSRTC category IV-VI or (b) nodular goiter with clinical symptoms (BSRTC category II). Nodules with category IV, V, or VI were ultimately subjected to thyroidectomy and the final result of histopathological examination served as a source

of final diagnosis on the character of the lesions. In the case of patients for whom the result of the biopsy was consistent with benign lesions and who did not present with local symptoms (large goiter, compressive symptoms, dysphagia, dysphonia), they were not subjected to surgical procedure and the result of the biopsy served as the final verification of the character of the lesions.

#### 2.2.1. Ultrasound Examination

The ultrasound examination of the thyroid was performed once more on admission to the department of surgery just before the thyroidectomy or during visit in an outpatient clinic after the results of the biopsy were obtained or during the qualification for thyroidectomy. The ultrasound examinations were carried out using a Samsung RS80 EVO device (Samsung Medison, Seoul, Korea) with a 3–12 MHz linear array probe (L3-12A). CAD in our study was based on K-TIRADS classification and using S-Detect 2 software (Samsung Medison Co. Ltd., Seoul, South Korea). The following ultrasound features were assessed automatically: composition (partially cystic, solid, or cystic), echogenity (hyper/isoechoic or hypoechoic), orientation (parallel or non-parallel), margin (ill-defined, well-defined smooth, or microlobulated/spiculated), spongiform (appearance or nonappearance), shape (ovoid to round or irregular), and calcifications (microcalcification, macrocalcification, or no calcification), which were automatically assessed by the US device. Elasticity and vascularity were not assessed by CAD and have not been taken into consideration for risk stratification, because they had to be introduced manually, while for our study we intended to check the performance of CAD without being distracted by any features that were operator-dependent. Each nodule was eventually classified by S-Detect into two categories: possibly benign and possibly malignant.

The ultrasound examinations were conducted at the Department of Endocrinology, Metabolism, and Internal Medicine of Poznan University of Medical Sciences in Poznan, Poland, and at the Department of Oncological Endocrinology and Nuclear Medicine at Maria Sklodowska-Curie National Research Institute of Oncology in Warsaw, Poland. During the ultrasound examination, the transverse and longitudinal planes for both the gland and the nodules were obtained while the patient was in the supine position. The anteroposterior, transverse, and longitudinal diameters of the nodules were measured on frozen images during examination and then archived.

The examinations were performed by four physicians with at least 10 years of experience in thyroid ultrasound examination and working at the departments specialized in diagnostics and treatment of thyroid cancer. The nodules were classified with an EU-TIRADS score independently by two physicians on the basis of recorded examinations. In seven cases, there was disagreement with the score by one point. The consensus on the final grade was made together after discussion. Physicians evaluating the EU-TIRADS score were blinded to the biopsy results and/or histopathological examination. All nodules were scored according to the European Thyroid Association Guidelines for Ultrasound Malignancy Risk Stratification of Thyroid Nodules in Adults [7]. EU-TIRADS was used in a pattern-based model, where we assessed only the above-mentioned ultrasound features.

#### 2.2.2. Statistical Analysis

The calculations were performed with Statistica 12 (TIBCO Software Inc., Palo Alto, CA 94304, USA). The *p*-value < 0.05 was considered significant. Sensitivities, specificities, positive and negative predictive values, and diagnostic accuracies were calculated to assess S-Detect and the EU-TIRADS scale. Comparisons of the diagnostic values of the tests were done using McNemar's test. EU-TIRADS classes 4 and 5 were considered as positive, whereas classes 1 to 3 were considered as negative (MODELS 1–3); in MODELS 4 and 5, only EU-TIRADS class 5 was considered as positive. Moreover, "possible malignant" by S-Detect corresponded to a positive result, whereas "possibly benign" corresponded to a negative result. The histopathological and cytological examinations served as a reference for a true or a false result.

#### 2.2.3. Ethical Approval

The study was approved by the institutional bioethical review board of each participating institution where the patients were examined, namely Poznan University of Medical Sciences and Maria Sklodowska-Curie National Research Institute of Oncology, both in Poland. All procedures were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all patients participating in the study to publish this report.

#### **3. Results**

Eighty-eight patients (70 women, 18 men) aged 49.4 ± 15.5 (17–80) years with 133 thyroid lesions were included in the study; of these, fifty-eight patients were diagnosed with 66 malignant lesions (including 57 papillary thyroid cancers (PTCs), four follicular variants of PTC, two medullary thyroid cancers (MTCs), one follicular thyroid cancer (FTC), two nodules in one patient described as poorly differentiated thyroid cancer). Benign lesions were colloid or hyperplastic nodules. The results of the diagnostic value of CAD-based S-Detect and physician-based EU-TIRADS (lesions with the score 4 or 5 were regarded as suspected for malignancy) are presented in Table 1. There was a statistically significant difference in the performance of CAD-based S-Detect and physician-based EU-TIRADS (*p* = 0.009).

confidence interval; PPV, positive predictive value; NPV, negative predictive value; EU-TIRADS, European Thyroid Imaging Reporting and Data System.

 5

Next, we structured five models using both CAD-based S-Detect and physician-based EU-TIRADS (Table 2).

**Table 2.** Description of the mixed models. The S-Detect classification was based on computer-aided diagnosis (CAD). The nodules were classified with an European Thyroid Imaging Reporting and Data System (EU-TIRADS) score independently by two physicians on the basis of recorded examinations.

#### **MODEL 1** assumes that:

#### **and**

A significant difference between the diagnostic efficacy of MODEL 1 and EU-TIRADS (*p* < 0.0001) as well as MODEL 1 and S-Detect (*p* = 0.013) was detected. The diagnostic effectiveness of MODEL 1 is presented in Table 1 (row 4).

**MODEL 2** was based on the assumption that:

**or**

There was no statistically significant difference between MODEL 2 and the EU-TIRADS scale alone (*p* = 0.13), whereas MODEL 2 performed significantly worse than S-Detect itself (*p* < 0.001).

**MODEL 3** was based on the assumption that:

**or**

**MODEL 4** assumes that:

**and**

There was no significant difference (*p* > 0.05) between the accuracy of MODEL 4 and the similar MODEL 1, however the sensitivity was slightly lower with better specificity.

**MODEL 5** was based on the assumption that:

**or**

There was no significant difference between the results of MODEL 5 and the similarly constructed MODEL 2 (*p* > 0.05).

The overall diagnostic performance of MODEL 3 is also not statistically significantly different from the EU-TIRADS scale alone (*p* = 0.26) but is slightly worse (*p* < 0.027) than S-Detect alone (Table 1, row 5). The obtained results suggest the superiority of MODEL 1 over the other two models as well as over the EU-TIRADS scale or S-Detect used separately. Table 2 summarizes the details of the approach used for each MODEL. Figure 2 presents an example of two lesions that were diagnosed

in one patient and whose character was correctly predicted by S-Detect. The first lesion was given 4 points on the EU-TIRADS scale and was diagnosed as "possibly malignant" by S-Detect, whereas the final histopathological examination revealed the malignant character of the lesion (medullary thyroid cancer). The second lesion, although it may be attributed as suspected due to its taller-than-wide shape, was diagnosed as "possibly benign" by S-Detect, whereas it eventually turned out to be a benign hyperplastic nodule.