Abstract
Objective
Punctate echogenic foci (PEF)/microcalcifications are thought to represent psammoma bodies (PB) in histopathology. However, there are few and contradictory data on this. Different types of sonographic echogenic microfoci (EMF) are seen in papillary thyroid carcinoma (PTC), and their histopathological equivalents are not clearly known. There is also conflicting data on the interobserver agreement between the sonographers on EMF.
Methods
We prospectively collected US video records of PTC nodules with and without EMF in two large thyroid centers. All video recordings were independently interpreted by three blinded, experienced sonographers. EMF were classified as true microcalcifications (punctate echogenic foci (PEF) ≤1 mm long), linear microechogenities (>1 mm long, posterior acoustic enhancement of the back wall of a microcystic area), comet-tail artifacts/reverberations or linear microechogenities with comet-tail artifacts/reverberations, non-shadowing coarse echogenic foci (>1 mm nonlinear areas) and unclassifiable. Histopathological evaluation was performed by two blinded, qualified pathologists.
Results
A total of 114 malignant nodules were included. The average Cohen’s kappa (κ) of three sonographers for the EMF presence was 0.775, indicating substantial agreement. A substantial agreement for PEF with 0.658 κ, only fair agreement for other types of EMF with 0.052 to 0.296 κ were detected. EMF were significantly associated with PB and papillae. PEF had an evident relationship with PB in multivariate analysis. There was a strong positive correlation between the amount of PEF and PB (r = 0.634, P < 0.001).
Conclusions
PEF in PTC mainly correspond to PB on histopathology. Although observation of EMF varies among sonographers, this inconsistency can be reduced by classifying EMF into subgroups and keeping the term ‘PEF’ only for true microcalcifications.
Introduction
Ultrasonographic (US) echogenic microfoci (EMF) are frequently seen in papillary thyroid carcinoma (PTC). European Thyroid Association, the American College of Radiology (ACR), and the Korean Society of Thyroid Radiology classify sonographic EMF with minimal differences from each other. According to European Thyroid Imaging and Reporting Data System (EU-TIRADS), EMF are divided into four classes: (i) comet-tail artifacts/reverberation, (ii) posterior acoustic enhancement of the back wall of a microcystic area, (iii) true microcalcifications, also called punctate echogenic foci (PEF), and (iv) hyperechoic spots of uncertain significance (1). PEF are highly suggestive of malignancy (2, 3). A recent study showed that PEF were seen in 58.7% PTCs (4). It has a specificity of 85–96% and a positive predictive value of 24.3–70% (5, 6). Other EMF categories have also been described in PTC. In a US-histopathology correlation study, not only PEF but also different types of EMF have been associated with malignancy (7).
Histopathologic correlations of these US findings are not well defined. Although the general belief is that PEF represent psammoma bodies (PB) in histopathology, the data are limited and conflicting. Kim et al. showed that PEF correspond to PB and stromal calcification in histopathology (8). In a Korean study, Hong et al. demonstrated that PEF correspond to psammomatous calcifications, coarse microcalcifications and inspissated colloids in histopathology and suggested that the main pathological finding of PEF were PB (4). Unlike these findings, another study with a small sample size, on pediatric cases, concluded that PEF often represent stromal calcifications or sticky colloid rather than psammomatous calcifications (9). In summary, there is no consensus on the histopathological equivalent of PEF and the other sonographic EMF in PTCs. Furthermore, echogenic foci often overlooked in papers are the presence of echogenic granules and lines within the nodule or thyroid tissue. This phenomenon, which represents connective tissue, is observed in more than 90% of cases and can also occur in normal thyroid tissue. Its occurrence is influenced by the angle between the ultrasound wave and the anatomical structure. On the other hand, thyroid US is the first and most important imaging method for the evaluation of thyroid nodules yet may show a significant interobserver variability (10, 11, 12). Solymosi et al., in their recent multicenter study, examined the agreement regarding the presence of EMF types, which they classified in a detailed and well-defined manner as punctate echogenic foci, back wall cystic figures, comet-tail artifact, unequivocal microcalcifications, and hyperechogenic spots of uncertain origin. The authors reported that the agreement was moderate for microcalcifications and fair for punctate echogenic foci (13).
Our study aimed (i)to assess the interobserver variability of the ultrasonographic interpretation of different EMF in PTC nodules and (ii)to identify the histopathological equivalent of different EMF in PTC comprehensively by using three different experienced sonographers and two different qualified pathologists.
Materials and Methods
Study design and setting
US video records of PTC cases with or without EMF were prospectively collected between 2018 and 2021 from two large thyroid centers in Ankara (Ankara University, School of Medicine, Department of Endocrinology and Ankara Güven Hospital, Department of Endocrinology). Written informed consent was obtained from all the patients prior to inclusion. Ethical approval has been obtained from the ethical committee of Ankara University (project number: 07-547-19).
All video recordings were interpreted blindly and retrospectively by three experienced sonographers with at least 25 years of US experience (two endocrinologists – MFE, AG and one specialist in nuclear medicine – SI). Nonshadowing EMF were classified as (i) true microcalcifications (punctate echogenic foci (PEF) ≤1mm long), (ii) linear microechogenities (>1 mm long, posterior acoustic enhancement of the back wall of a microcystic area), (iii) comet-tail artifacts/reverberations or linear microechogenities with comet-tail artifacts/reverberations, (iv) nonshadowing coarse echogenic foci (>1 mm nonlinear areas): larger and more pronounced in size than the first three classes of microechogenicity, do not have posterior acoustic shadow, and do not comply with the definition of a coarse macrocalcification, and (v) unclassifiable: could not be attributed with certainty to one of these four categories.
The ‘hyperechoic spots of uncertain significance’ category in the ETA classification was examined in two separate categories as ‘nonshadowing coarse echogenic foci’ and ‘unclassifiable’ in our study. We observed that ‘nonshadowing coarse echogenic foci’, could potentially represent a significant different echogenicity type in PTC. Therefore, in order to understand whether they have a distinct histopathological counterpart, we chose to examine them as a separate group.
Results of sonographers’ evaluation were analyzed by an independent interpreter (ABB).
Typical examples of each category are shown in Fig. 1.
Types of echogenic microfoci. (A) Punctate echogenic foci. (B) Linear foci. (C) Comet-tail artifact. (D) Linear + comet-tail artifact (the straight arrow indicates comet-tail artifact and the curved arrow indicates linear microechogenicity). (E) Nonshadowing coarse. (F) Unclassified.
Citation: European Thyroid Journal 12, 6; 10.1530/ETJ-23-0108
US was performed with two different machines: General Electric® Acuson Antares machine equipped with a 13–10 MHz linear transducer and General Electric® Logiq S7 Expert machine equipped with 15–8 MHz linear transducer.
Histopathological examination
Histopathological evaluation was performed by two blinded pathologists (SDS and BB) with 25 years of experience in thyroid pathology. Malignant thyroid nodules were evaluated by the pathologists retrospectively for the presence and intensity (none, low, intermediate, or high) of the following five histopathological features: psammoma bodies, stromal hyalinization/fibrosis, stromal calcifications, papillae, and cystic areas. In terms of psammoma body density, groups were classified according to the number of psammoma bodies as 0 (none), 0–3 (low), 3–10 (intermediate), >10 (high) per low-power fields (×40).
The classification of these pathological findings and the grading of their intensities are decided with the agreement of both pathologists. Figure 2 shows examples of histopathological evaluation.
(A–D) Histopathological features of PTC nodules. All images depict H&E staining, Original magnification 5.0×. (A) psammoma bodies: low; stromal hyalinization/fibrosis: high; stromal calcifications: high; papillae: high; cystic areas: low. (B) psammoma bodies: high; stromal hyalinization/fibrosis: high; stromal calcifications: low; papillae: intermediate; cystic areas: none. (C) psammoma bodies: none; stromal hyalinization/fibrosis: none; stromal calcifications: low; papillae: high; cystic areas: high. (D) psammoma bodies: low; stromal hyalinization/fibrosis: high; stromal calcifications: high; papillae: high; cystic areas: low.
Citation: European Thyroid Journal 12, 6; 10.1530/ETJ-23-0108
Statistical analysis
Statistical analysis was performed using SPSS version 22.0. Descriptive statistics were given as counts and percentages for categorical variables; interquartile ranges and median were given for nonparametric continuous variables; standard deviation and mean for parametric continuous variables. Chi-square test was performed for categorical data. Kruskal–Wallis test was used to compare three groups on continuous variables. A P-value of less than 0.05 was considered statistically significant. Kappa statistics were calculated using SPSS version 22.0 to determine the proportion of interobserver agreement beyond that expected by chance. The method for estimating an overall kappa value in cases of multiple observers and categories is based on the work of Landis and Koch. A value of k.1.0 corresponds to complete agreement; 0, no agreement; and less than 0, disagreement. Landis and Koch suggested that a kappa value 0.20 indicates slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; and 0.81–1.00, almost perfect agreement (14).
Results
Characteristics of the study group
Of the videos of 160 PTC patients, videos of 114 malignant nodules with sufficient detailed images were included in the study. Videos deemed inadequate by any of the sonographers in terms of recording time, resolution, and technique were excluded. Thirty percent (n = 34) were from Ankara University and 70% (n = 80) were from Güven Hospital. Seventy-five percent (n = 85) of the patients were female. Mean age of patients was 38.3 ± 10.2. Surgical procedures were total thyroidectomy and central lymph node dissection (CLND) in 75 (66%), total thyroidectomy central and right LND in 13 (11%), lobectomy and ipsilateral CLND in 9 (8%), total thyroidectomy central and left LND in 7 (6%), and other in 10 (9%). Fifty-two (46%) of tumors was located in the right lobe, 46 (40%) in the left lobe, and 16 (14%) in the isthmus. The mean size (i.e. maximal diameter) of tumors was 12.2 ± 9.6 mm. Based on the histopathological subtype, there were 88 (77%) classic, 18 (16%) follicular variant, and 8 (7%) PTCs of other subtypes (i.e. three diffuse sclerosing , two tall cell, three warthin-like).
Interobserver variation analyses
Interobserver agreement of three sonographers were presented in Table 1. The sonographers revealed 0.775 average Cohen’s kappa indicating substantial agreement EMF presence. They had also substantial agreement for PEF with 0.658 Cohen’s kappa. Interobserver agreements of other types of microfoci reached only fair agreement (Table 1).
Interobserver agreement of three sonographers for echogenic microfoci (EMF) presence and types.
| Cohen’s kappa | ||
|---|---|---|
| Minimum–maximum | Average | |
| EMF | 0.698–0.860 | 0.775 |
| Linear and comet tail | 0.086–0.562 | 0.296 |
| Lineara | -0.047–0.113 | 0.052 |
| PEF (microcalcification)b | 0.590–0.711 | 0.658 |
| Nonshadowing coarse calcifications | 0.015–0.490 | 0.283 |
| Unclassifiable | 0.065–0.105 | 0.097 |
aposterior acoustic enhancement of the back-wall of a microcystic area; bpunctate echogenic foci.
Bold values indicate statistically significant (P < 0.05) values.
While 84 of 114 malignant nodules had one or more type of EMF (group 1), 30 had none (group 2); 25% (n = 21) of microfoci were centrally, whilst 31% (n = 26) were peripherally localized, and 44% (n = 37) were diffusely spread in the nodule. Group 1 and group 2 did not differ regarding nodule size (max diameter 12.3 mm vs 11.8 mm, P =0.830) and gender (female: 72.6% vs 76.7% P =0.758), but group 1 was younger than group 2 (mean: 36.8 vs 42.6, P = 0.007) years.
Correlations between histopathological features and echogenic microfoci in papillary thyroid carcinoma
Histopathological comparison of group 1 (with EMF) and group 2 (without EMF) was presented in Table 2. Both univariate and multivariate analysis showed significant relationships between microfoci with PB (Table 2).
The relationship between echogenic microfoci (EMF) presence and histopathological features.
| Univariate analysis, n (%) | Multivariate analysis | ||||||
|---|---|---|---|---|---|---|---|
| Group 1 | Group 2 | P | OR | 95% CI | P | ||
| EMF (+) | EMF (–) | Lower | Upper | ||||
| n | 84 | 30 | |||||
| Psammoma bodies | 58 (69) | 2 (7) | <0.001 | 19.145 | 4.082 | 89.796 | <0.001 |
| Hyalinization | 74 (88) | 25 (83) | 0.508 | 1.277 | 0.274 | 5.947 | 0.756 |
| Stromal calcifications | 30 (36) | 1 (3) | 0.001 | NA | NA | NA | |
| Papillae | 71 (85) | 12 (40) | <0.001 | 9.014 | 0.902 | 90.134 | 0.061 |
| Cystic areas | 50 (60) | 11 (38) | 0.04 | 0.362 | 0.037 | 3.505 | 0.380 |
When we compare histopathological features of PEF with group 2, only PB revealed an evident relationship with PEF in multivariate analysis, although stromal calcification, papillae, and cystic areas reached significant difference in the univariate analysis (Table 3).
The relationship between punctate echogenic foci (PEF) and histopathological features.
| Univariate analysis | Multivariate analysis | ||||||
|---|---|---|---|---|---|---|---|
| PEF ( +) | PEF (–) | P | OR | 95% CI | P | ||
| Lower | Upper | ||||||
| n (total = 104) | 74 | 30 | |||||
| Psammoma bodies | 54 (73) | 2 (7) | <0.001 | 0.045 | 0.009 | 0.214 | <0.001 |
| Hyalinization | 66 (89) | 25 (83) | 0.514 | 0.742 | 0.131 | 4.206 | 0.736 |
| Stromal calcifications | 26 (35) | 1 (3) | 0.001 | NA | NA | NA | |
| Papillae | 64 (87) | 12 (40) | <0.001 | 0.111 | 0.010 | 1.220 | 0.072 |
| Cystic areas | 45 (61) | 11 (38) | 0.036 | 0.390 | 0.037 | 4.117 | 0.434 |
There was a strong positive correlation between the amount of PEF and PB (r = 0.634, P < 0.001). The amount of papillae and stromal calcification have weak positive correlations between the amount of PEF (r = 0.296, r = 0.227; P < 0.001, P = 0.015, respectively). Hyalinization and cystic areas had no correlations.
Discussion
We hereby demonstrated that psammoma bodies in histopathology are the main cause of PEF in PTC nodules in a systematic, controlled, prospective study. Our findings suggest that stromal calcification, papilla and cystic areas may also contribute to the sonographic image of EMF in PTC. Psammoma bodies are 50–70 μm in size, concentric lamellar calcified structures thought to arise from dystrophic calcification of the necrotic papilla (15, 16). Although only PB have long been thought to be associated with sonographic microcalcifications/PEF, there are few studies in the literature that criticize this assumption (4, 9, 17). Tahvildari et al. suggested that more than half of the cases with ultrasonographic PEF in PTC were not correlated with psammomatous calcifications on pathological examination. They showed that coarse/dystrophic calcifications and sticky colloid may also give rise to PEF in US (18). Hong et al. reported the incidence of PB slightly higher than us, approximately 75%, in cases with PEF. They showed that the pathological equivalent of PEFs are psammomatous calcifications, coarse microcalcifications, and inspissated colloids (74.6%, 42.3%, and 46.5%, respectively) (4). In a similar study, it was stated that pathological calcification (psammomatous and dystrophic calcification) was not observed in most of the nodules evaluated as having microcalcification on US and echogenic hyalinized areas or colloid material has been reported to cause echogenic foci in US (17). Conflicting results in the literature may be explained by high interobserver differences in defining ultrasonographic echogenic foci. One of the strengths of our study is the blind evaluation of videos by three experienced sonographers and the analysis of EMF in five different categories. In this way, PEF, formerly known as true microcalcifications, were examined by separating them from other echogenicities. Although it was observed that stromal calcification, papillae, and cystic areas may contribute to the EMF and PEF, the only histopathological finding that independently, predicted PEF were PB. Additionally, it has been shown that the number of PEF on US image is in straight correlation with number of PB defined in the histopathology.
Thyroid ultrasound is the first and most important diagnostic tool for thyroid nodules. However, it is a highly operator dependent method and a learning curve is necessary to reach only moderate interobserver agreement rates (10, 12). The higher interobserver variability is reported for the evaluation of sonographic echogenic foci or calcifications comparing nodule composition and echogenicity. However, recent multicenter study reported higher agreement values than the general literature data on EMF by better definition of the subtypes of EMF, as done in our study (13). Agreement between sonographers on the presence of microechogenicity was substantial in the current study (κ coefficient: 0.775). Agreement between sonographers was the highest in PEF group (κ coefficient: 0.658). Therefore, we suggest that ultrasonographic EMF in thyroid ultrasound should be evaluated under different well-defined categories.
Splitting the ‘Hyperechoic spots of uncertain significance’ category in the ETA classification into two subgroups, resulting in five subgroups in our study, became a factor that reduced interobserver agreement. Therefore, we still believe that the four class-tiered ETA classification remains more appropriate and advantageous in terms of interobserver agreement.
Conclusion
In conclusion, PEF reported in ultrasound of papillary thyroid carcinoma mainly corresponds to psammoma bodies on histopathology. Although ultrasonographic EMF show high variability among sonographers, this inconsistency can be reduced by classifying EMF to subgroups and keep the term ‘punctate echogenic foci’ only for true microcalcifications .
Declaration of interest
Faik Murat is on the editorial board of European Thyroid Journal. Murat was not involved in the review or editorial process for this paper, on which he is listed as an author. The other authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the study reported.
Funding
This study was funded partly by Society of Endocrinology and Metabolism of Turkey (SEMT).
Author contribution statement
ABB: conception and design of the work; collecting data, analysis and interpretation of data; and article writing. AG: collecting data; design of the work; and revising the article. SDS: performing histopathological analyses; design of the work; and revising the article. SI: collecting data; design of the work; and revising the article. BB: performing histopathological analyses; design of the work; and revising the article. MFE: conception and design of the work; acquiring funds; collecting data; article writing; and revising the article.
Acknowledgements
This work was accepted to the 45th Annual Meeting of the European Thyroid Association (ETA) in 2023.
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