Edited by: Alex Friedlaender, Geneva University Hospitals (HUG), Switzerland
Reviewed by: Trevor Edmund Angell, University of Southern California, United States; Liborio Torregrossa, University of Pisa, Italy
*Correspondence: Maria Grazia Castagna,
This article was submitted to Thyroid Endocrinology, a section of the journal Frontiers in Endocrinology
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The management of patients with indeterminate thyroid nodules, which account for 10–25% of thyroid fine needle aspiration biopsies (FNABs), is still very challenging.
To verify the utility of the seven-gene panel in combination with ultrasound features in the clinical management of indeterminate thyroid nodules.
The study group included 188 indeterminate thyroid nodules, divided into TIR3A (56.4%) and TIR3B (43.6%). A significant correlation between US categories and both cytological and molecular results was observed. In detail, TIR3B cytology was more frequent in EU-TIRADS 4 and 5 nodules (54.7 and 50%, respectively) than in EU-TIRADS 2 and 3 nodules (31%,
US score seems able to correctly discriminate between TIR3A nodules in which a conservative approach may be used, and those in which additional test, such as molecular test, may be indicated. On the contrary, in TIR3B nodules both US risk stratification and seven-gene panel seem to be of little use, because the risk of thyroid cancer remains high regardless of US score and mutational status.
The management of thyroid nodules cytologically defined as “indeterminate” is still very challenging. Therefore, it has been advocated to apply individualized strategies based on more accurate anatomo-pathological classifications and the use of immunocytochemical or molecular markers (
Thus, we aimed to establish whether US features combined with the seven-gene panel results may significantly contribute to the pre-surgical diagnosis of nodules with indeterminate cytology.
A retrospective analysis was conducted on 503 indeterminate thyroid nodules belonging to 467 patients with uni- or multinodular thyroid disease, who underwent fine-needle aspiration cytology (FNAC) at the Section of Endocrinology of the University of Siena, Italy, from 2009 to 2019. The study cohort included 119 males (25.5%) and 348 females (74.5%), with a mean age at diagnosis of 54.1 ± 14.1 years (range 16–93 years). In 73/188 (38.8%) indeterminate thyroid nodules, surgery was performed. Criteria for surgery were: 1) indeterminate nodule diameter >3 cm; 2) large size of uni- or multinodular goiter; 3) young age at diagnosis; 4) positive seven-gene panel.
All thyroid nodules were assessed by using a high-resolution ultrasound color Doppler apparatus (My Lab 40 HD, Esaote Biomedica, Firenze, Italy) with a 12 MHz linear transducer by two dedicated endocrinologists, who usually perform more than 1,000 neck ultrasound examinations per year. US reports and images of each thyroid nodule included in the study were recorded in the database by the endocrinologist who performed the examination. We classified the ultrasound reports based on the European Thyroid Association Guidelines for Ultrasound Malignancy Risk Stratification of Thyroid Nodules in Adults (EU-TIRADS) (
FNAC was performed by the same endocrinologists upon ultrasound guidance using a 23/25-gauge needle. The material was air dried, stained with May-Grunwald Giemsa and interpreted by one experienced cytologist who was blinded to the US risk of thyroid nodules. All cytological smears were reviewed by the same pathologist (DA). Thyroid nodules were cytologically defined as low-risk indeterminate (TIR3A) or as high-risk indeterminate (TIR3B) according to the Italian Society of Anatomic Pathology and Diagnostic Cytology (SIAPEC-IAP) (
Since 2009, the Section of Endocrinology of the University of Siena has represented the Reference Center for the mutational analysis of thyroid nodules in the South-East of the Tuscany Region. The protocol for TIR3 nodules, adopted since then at our Institute, provides for the application of a seven-gene molecular test. Patients whose FNAC results were indeterminate (TIR3A/TIR3B) were subjected to a second FNA procedure, performed twice consecutively in the same session. All patients who submitted to FNA procedure signed informed consent. One sample was used for repeating cytological examination, and the other was dispersed into TRI Reagent buffer (Sigma) and stored at −20°C until DNA and RNA extraction. A specimen was qualified as satisfactory if there were six groups of epithelial cells with at least 10 cells per group. Smears were reviewed by the same pathologist and analyzed by a molecular biologist with the seven-gene panel searching for mutations. Samples were screened for the presence of BRAF (V600E and K601E), H- K-NRAS and hTERT mutations point mutations and for RET/PTC-1 and -3 and PAX8-PPARgamma rearrangements. For BRAF point mutations (V600E and K601E), exon 11 and exon 15 were amplified in a mixture containing 2× PCR Master Mix (AmpliTaq Gold® PCR Master Mix, Applied Biosystems) and a final primer concentration of 200 nM at 52.5°C. To identify RAS mutations (H-, K-, and N-RAS) codons 12, 13 and 61 were amplified with 200 nM primer final concentration at 64.9°C for H-RAS and 58°C for K-RAS and N-RAS. hTERT was evaluated using a Fast PCR procedure at 63°C and in the presence of a GCrich solution (Roche). PCR products were subjected to direct sequencing. Rearrangements were evaluated with real time RT-PCR. For PAX8/PPAR
Epidemiological data are presented as the mean ± SD and median when needed. To evaluate significant differences in data frequency we analyzed contingency tables. Tables with size larger than 2 × 2 were examined by the Chi-squared test or a numerical approximation of the Fisher exact test when all cell frequencies were greater than 4 or not, respectively. “Test for trend” (X square for trend or Harmitage test) was also performed. Thyroid nodules were retrospectively classified according to EU-TIRADS system. For the statistical analysis, nodules classified as EU-TIRADS 2 and 3 or EU-TIRADS 4 and 5 were grouped separately, and thyroid nodules classified at histology as non-invasive follicular thyroid neoplasm with papillary like nuclear features (
Statistical analysis was performed using the software StatView for Windows version 5.0.1 (SAS Institute, Cary, NC) and the
Among the 503 indeterminate thyroid nodules, cytological diagnosis of TIR3 was confirmed in 386/503 (76.7%) nodules. In the remaining 117/503 (23.3%) nodules, benign cytology (TIR2) was found at FNAC repetition, and they were excluded from the study. In the confirmed TIR3 nodules (n = 386), the seven-gene panel test was performed (point mutations within
As shown in
Demographic and clinical-pathological features in patients with indeterminate thyroid nodules (n = 188).
Parameters | TIR3 nodules (n=188) |
---|---|
|
|
Male | 42 (22.3) |
Female | 146 (77.7) |
|
|
Mean ± SD | 54.5 ± 14.0 |
Range | 18–88 |
Median | 55 |
|
|
Mean ± SD | 23.9 ± 12.1 |
Range | 7–83 |
Median | 20 |
|
|
Multinodular goiter | 105 (55.9) |
Uninodular goiter | 83 (44.1) |
|
|
EU-TIRADS 2/3 | 84 (44.7) |
EU-TIRADS 4 | 86 (45.7) |
EU-TIRADS 5 | 18 (9.6) |
|
|
TIR3A | 106 (56.4) |
TIR3B | 82 (43.6) |
|
|
yes | 18 (9.6) |
no | 170 (90.4) |
|
|
BRAF | 2 (11.1) |
H-K-NRAS | 10 (55.6) |
RET/PTC | 6 (33.3) |
A significant correlation between EU-TIRADS categories and both cytological and molecular results was observed. In detail, TIR3B cytology was more frequent in EU-TIRADS 4 and 5 nodules (54.7 and 50%, respectively) than in EU-TIRADS 2–3 nodules (31%,
Relationship between thyroid ultrasound and cytological categories and seven-gene panel results in 188 indeterminate thyroid nodules (TIR3).
Surgical treatment was performed in 73/188 (38.8%) indeterminate thyroid nodules. Among thyroid nodules submitted to surgery, final histology was benign in 44/73 (60.2%), while differentiated thyroid carcinoma was diagnosed in 29/73 (30.8%) thyroid nodules (
Demographic and clinical-pathological features in patients with indeterminate thyroid nodules submitted to surgery (n = 73).
Parameters | Malignant histology (n = 29) | Benign histology (n = 44) |
|
---|---|---|---|
|
0.8** | ||
Male | 8 (27.6) | 11 (25) | |
Female | 21(72.4) | 33 (75) | |
|
0.1* | ||
Mean ± SD | 49.2 ± 14.6 | 53.9 ± 13.4 | |
Range | 18–81 | 20–79 | |
Median | 52 | 55 | |
|
0.8* | ||
Mean ± SD | 31.5 ± 17.5 | 29.1 ± 12.6 | |
Range | 9–82 | 8–58 | |
Median | 28 | 29 | |
|
0.8** | ||
Multinodular goiter | 17 (41.4) | 24 (58.6) | |
Uninodular goiter | 12 (37.5) | 20 (62.5) | |
|
|
||
TIR 3A | 3 (9.7) | 28 (90.3) | |
TIR 3B | 26 (66.6) | 16 (33.4) | |
|
0.10** | ||
EUTIRADS 2-3 | 10 (29.4) | 24 (70.6) | |
EUTIRADS 4-5 | 19 (48.7) | 20 (50.3) | |
|
|
||
positive | 12 (66.6) | 6 (33.4) | |
negative | 17 (30.9) | 38 (69.1) | |
|
0.40** |
||
BRAF (n = 2) | 1 (50%) | 1 (50%) | |
RAS (n = 10) | 8 (80%) | 2 (10%) | |
RET/PTC (n = 6) | 3 (50%) | 3 (50%) |
*By Mann–Whitney U test; **By X2 test.
In bold significant p values (<0.05).
Using only US score as criterion for decision-making in the TIR3A subgroup of indeterminate nodules, we correctly classified 20/31 (64.5%) of thyroid nodules. In detail, 100% of low risk US nodules were benign at histology, while the rate of malignancy in high risk US nodules was only 21.4%, with a trend toward significance (
US and molecular features as criteria for decision-making in TIR3A nodules (n = 31 nodules).
Using only the seven-gene panel as criterion for decision-making in TIR3B nodules, we correctly classified 26/42 (61.9%) indeterminate nodules. In detail, all but one of the nodules with a mutation were malignant at histology (91.6%) while only 50% of non-mutated nodules were benign at histology (
US and molecular features as criteria for decision-making in TIR3B nodules (n = 42 nodules).
Cytologically indeterminate thyroid nodules currently represent a challenge for clinical decision-making. Since the overall rate of malignancy is low, a tool that helps to rule out thyroid cancer preoperatively might reduce the number of diagnostic surgeries. To spare patients from unnecessary surgery, molecular testing as a diagnostic adjunct to FNA cytopathology were developed (
We, also, compared histological results with US features in a subgroup of indeterminate thyroid nodules submitted to surgery (n = 73). The cancer rate was higher in US high risk nodules (48.7
The main limitation of our study was the small size of our cohort mainly due to the difficulty of retrieving thyroid US images and histological results of all patients. The other limitation was not having access to the NGS multi-gene panel such as Thyroseq v3 which allows the analysis of more than hundred genes and detection of different classes of genetic mutations. Finally, due to the retrospective design, patient selection bias may have occurred in our study, especially in the subgroup of patients submitted to surgery. Indeed, after cytological and molecular results, the decision-making process was defined by the care provider of the patients. Therefore, we acknowledge that the results could be different in other Centers. For this reason we believe that, although promising, our results should be confirmed in a prospective study including a larger series of indeterminate thyroid nodules. The strengths of our study are as follows. First, the confirmation of indeterminate cytology by a second FNAC in all patients included in the study. Second, to the best of our knowledge, this is the first study to evaluate the performance of US score and the seven-gene panel exclusively on indeterminate thyroid nodules using a series approach.
In conclusion, US score seems able to correctly discriminate between benign TIR3A nodules in which a conservative approach may be valid and those in which additional test, such as molecular test, may be indicated. On the contrary, in TIR3B nodules, the US risk stratification as well as the seven-gene panel, appears to be of little utility because the risk of thyroid cancer remains high regardless of US score and mutational status.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by the Comitato Etico regione Toscana-Area Vasta sud-est. The patients/participants provided their written informed consent to participate in this study.
MC and MGC contributed to the conception, design, and interpretation of the project. SC, AnS, and AlS contributed to the experimental part and data analysis of the project. TP, CC, FM, RF, and AC contributed to drafting or revising intellectual content of the manuscript. MC and MGC had primary responsibility for final content. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.