Molecular testing for thyroid nodules: Where are we now?

Approximately 25% of the fine needle aspiration samples (FNAB) of thyroid nodules are classified as "indeterminate samples", that means, Bethesda III and IV categories. Until the last decade, most of these cases underwent diagnostic surgery, although only a minority (13-34%) confirmed malignancy postoperatively. In view of this, with the objective of improving the preoperative diagnosis in these cases, the molecular tests emerged, which are validated from the diagnostic point of view, presenting good performance, with good diagnostic accuracy, being able to avoid diagnostic surgeries. With the advancement of knowledge of the role of each of the mutations and gene rearrangements in thyroid oncogenesis, molecular markers have left to play only a diagnostic role and have been gaining more and more space both in defining the prognostic role of the tumor, as well as in the indication of target therapy. Thus, the objective of this review is to show how to use the tool of molecular tests, now commercially available in the world, in the management of indeterminate cytological nodules, assessing the pre-test malignancy risk of the nodule, through clinical, ultrasonographic and cytological characteristics, and decide on the benefit of molecular testing for each patient. In addition, to discuss its new and promising prognostic and therapeutic role in thyroid cancer.

Improving the TIR3B oncological stratification: try to bridge the gap through a comprehensive presurgical algorithm.

PURPOSE: Indeterminate cytology still puzzles clinicians, due to its wide range of oncological risks. According to the Italian SIAPEC-IAP classification, TIR3B cytology holds up to 30% of thyroid cancer, which justifies the surgical indication, even if more than half of cases do not result in a positive histology. The study aim is to identify potential clinical, ultrasound or cytological features able to improve the surgical indication. METHODS: Retrospective analysis. A consecutive series of TIR3B nodules referred to the Endocrine Unit of Careggi Hospital from 1st May 2014 to 31st December 2021 was considered for the exploratory analysis (Phase 1). Thereafter, a smaller confirmatory sample of consecutive TIR3B diagnosed and referred to surgery from 1st January 2022 to 31st June 2022 was considered to verify the algorithm (Phase 2). The main clinical, ultrasound and cytological features have been collected. A comprehensive stepwise logistic regression was applied to build a prediction algorithm. The histological results represented the final outcome. RESULTS: Of 599 TIR3B nodules referred to surgery, 451 cases were included in the exploratory analysis. A final score > 14.5 corresponded to an OR = 4.98 (95% CI 3.24-7.65, p < 0.0001) and showed a PPV and NPV of 57% and 79%, respectively. The Phase 2 analysis on a confirmatory sample of 58 TIR3B cytology confirmed that a threshold of 14.5 points has a comparable PPV and NPV of 53% and 80%, respectively. CONCLUSIONS: A predictive algorithm which considers the main clinical, US and cytological features can significantly improve the oncological stratification of TIR3B cytology.

Thyroglobulin is a poor predictor of differentiated thyroid cancer in patients who undergo surgery for thyroid nodular diseases.

OBJECTIVES: Thyroglobulin, produced exclusively by thyroid follicular cells, serves as a specific tumor marker for the follow-up of differentiated thyroid cancer (DTC) patients after thyroidectomy. However, its role as a predictor of malignancy in patients with thyroid nodules is controversial. We assessed the potential role of preoperative serum thyroglobulin concentration to predict DTC in patients without a preoperative diagnosis of malignancy who underwent partial or total thyroidectomy. METHODS: This retrospective study included patients with a preoperative diagnosis of benign multinodular goiter (MNG) or a thyroid nodule with indeterminate cytology (INC) (Bethesda system categories III/IV) who underwent partial or total thyroidectomy between January 2014 and May 2019. We compared the patients' demographic, clinical, imaging, and biochemical data according to their final diagnosis: DTC or benign thyroid nodular disease. Further statistical analysis included odds ratio calculation and receiver operating characteristic (ROC) curve analysis. RESULTS: Of 131 patients who met inclusion and exclusion criteria, the indication for surgery was benign MNG in 69 patients and a thyroid nodule with INC in 62 patients. A final diagnosis of DTC was reported in 18 of the 69 benign MNG patients (26%) and in 30 of the 62 thyroid nodule with INC patients (48%). The preoperative measurements of nodule diameter and serum thyroid-stimulating hormone and thyroglobulin concentrations did not significantly differ between patients with a final diagnosis of DTC and those with benign histology. CONCLUSIONS: Preoperative serum thyroglobulin alone is insufficient to differentiate between malignant and benign thyroid nodular disease.

Assessment of diagnostic value of preoperative elastography in thyroid nodules having indeterminate cytology results

Background/aim: The management of nodules with indeterminate cytology [atypia of undetermined significance (AUS), follicular lesion of undetermined significance (FLUS), follicular neoplasm (FN), suspicious for a follicular neoplasm (SFN), and suspicious for malignancy (SM)] results is controversial. To assess the role of the elastography technique in the diagnosis of malignancy in the subtypes of indeterminate thyroid nodules. Materials and methods: We included 132 patients with indeterminate cytology who underwent thyroid surgery. Sensitivity, specificity, area under the curve, and optimal cut-off points were calculated with receiver operating characteristic (ROC) analysis for elastography score (ES) and strain index (SI). Results: Malignancy was observed in 27/95 (28.4%) of the AUS-FLUS cytology and 12/24 (50%) of FN, SFN cytology. All of the 13 patients (100 %) with SM are found to be malignant on histology. In the FLUS group, nodules with ES greater or equal to 3, the presence of malignancy was higher 17/41 (41.5%) when compared with nodules with ES smaller than 39/46 (19.6 %) (p = 0.023). In the SFN group, 2 of 2 nodules with an ES score of 4 and 1 of 1 nodule with an ES score of 5 were malignant. In the FLUS group, 4 of 10 nodules with an ES score of 4 and 2 of 2 nodules with an ES score of 5 were malignant. Conclusion: Thyroid elastography may reduce unnecessary surgery for both patients with AUS/FLUS and selected SFN cytology. Elastography appears to be helpful in follicular variants and other types of papillary thyroid cancer, however, not in follicular thyroid cancer.

Risk stratification of the thyroid nodule with Bethesda indeterminate cytology, category III, IV, V on the one surgeon-performed US-guided fine-needle aspiration with 27-gauge needle, verified by histopathology of thyroidectomy: the additional value of one surgeon-performed elastography.

BACKGROUND: The aim is to assess the value of strain elastography (SE) in differentiating likelihood of malignancy for the thyroid nodules, possessing the Bethesda Category III, IV, and V indeterminate cytology. METHODS: The data was obtained by ultrasonography (US)-guided fine-needle aspiration (US-g-FNA) via 27-gauge needle, with the verification of indicated thyroidectomies in a retrospective analysis, from April 2010 to April 2014, by enrolling the documents of 262 consecutive patients, with 327 thyroid nodules, subjected to one-surgeon performed neck US, SE, and US-g-FNA with 27-G needle to rule out the malignancy. RESULTS: 122 of 327 cases were Bethesda Category III, IV, and V with histopathologically benign, 110 (90.2%); PTC, 7 (5.7%); FTC, 4 (3.3%); HCC, 1 (0.8%). Tsukuba Elasticity Score (TES) 1, 2, 3, 4, and 5 were detected as 38 (31.1%), 8 (6.6%), 59 (48.4%), 4 (3.3%), and 13 (10.7%), respectively for the cases with the indeterminate cytology. No significant difference was detected between TES 4 and 5 and malign histopathology by McNemar test (p = .727) with a good level of concordance, the kappa coefficient, 0.737. CONCLUSION: SE may be a useful tool in differentiating malign from benign thyroid nodules by selecting surgery adaptation even for Bethesda indeterminate cytology on FNAC.

Diagnosis of post-surgical fine-needle aspiration biopsies of thyroid lesions with indeterminate cytology using HRMAS NMR-based metabolomics.

INTRODUCTION: Ultrasound examination coupled with fine-needle aspiration (FNA) cytology is the gold standard for the diagnosis of thyroid cancer. However, about 10-40% of these analyses cannot be conclusive on the malignancy of the lesions and lead to surgery. The cytological indeterminate FNA biopsies are mainly constituted of follicular-patterned lesions, which are benign in 80% of the cases. OBJECTIVES: The development of a FNAB classification approach based on the metabolic phenotype of the lesions, complementary to cytology and other molecular tests in order to limit the number of patients undergoing unnecessary thyroidectomy. METHODS: We explored the potential of a NMR-based metabolomics approach to improve the quality of the diagnosis from FNABs, using thyroid tissues collected post-surgically. RESULTS: The NMR-detected metabolites were used to produce a robust OPLSDA model to discriminate between benign and malignant tumours. Malignancy was correlated with amino acids such as tyrosine, serine, alanine, leucine and phenylalanine and anti-correlated with myo-inositol, scyllo-inositol and citrate. Diagnosis accuracy was of 84.8% when only indeterminate lesions were considered. CONCLUSION: These results on model FNAB indicate that there is a clear interest in exploring the possibility to export NMR metabolomics to pre-surgical diagnostics.

The impact of repeat FNA in non-diagnostic and indeterminate thyroid nodules: A 5-year single-centre experience.

INTRODUCTION: FNA is a well-established method for the preoperative diagnosis of thyroid nodules, but limitations still reside among non-diagnostic and indeterminate samples. The objective of the present study was to assess the impact of repeat FNA in thyroid nodules primarily classified as non-diagnostic and indeterminate, with the evaluation of the diagnostic resolution rate after the reassessment of the nodule. METHODS: We retrospectively collected all cases of thyroid FNA at our institution in the last 5 years that had one or more repeat aspirations of the same nodule, calculating the percentage of samples with change in the diagnostic category. Additional collected data included sex, age and interval between the repeat aspirations. RESULTS: One hundred and seventy-eight specimens from 167 patients (140 female, 27 male) with a median age of 56 years (range 11-90) were included in the study. Among the 86 cases primarily classified as non-diagnostic, 25 (29.1%) remained in the same category after the first reassessment and only 18 (20.9%) after the second repeat aspiration. Among the 40 indeterminate cases, only 10 (25%) retained their status after the second aspiration, with no change after the third assessment. CONCLUSION: Repeat aspiration of non-diagnostic and indeterminate thyroid nodules had a positive impact in both groups, with diagnostic resolution rates of 80% and 75%, respectively. The present study therefore endorses the use of such strategy for the initial follow-up of nodules with no definite diagnosis, especially in low-resource centres with limited access to modern molecular technologies.

Thyroid FNA cytology in Asian practice-Active surveillance for indeterminate thyroid nodules reduces overtreatment of thyroid carcinomas.

Although Asian thyroid practices have implemented the American Thyroid Association guidelines, significant deviations in actual risk of malignancy (ROM) have been reported. With review of the literature from Asia, the authors examine the underlining reasons for actual ROMs reported in Asia being so different from western practice based on the author's perspective. Although the most popular diagnostic system for thyroid cytology used in Asian countries is the Bethesda system, the Japan Thyroid Association published clinical guidelines, including a national reporting system for thyroid cytology, to adapt conservative clinical management (active surveillance and strict triage patients for surgery) for low-risk thyroid carcinomas. As less aggressive clinical management is favoured in Asian societies, strict triage of patients with indeterminate thyroid nodules for surgery is usually applied, which ultimately reduces overtreatment of indolent thyroid tumours. As a result, low resection rates and high ROMs for indeterminate nodules were achieved in Asian practices using the same Bethesda system. Recently, borderline thyroid tumours were introduced in the thyroid tumour classification and significant decreases in ROMs have been reported in the indeterminate categories in western practice. However, ROM of indeterminate nodules remained high in Asian practice even after borderline tumours were deemed benign. These results suggested that the diagnostic threshold of papillary thyroid carcinoma-type nuclear features varied among practices (stricter in Asia than in western practice), and diagnostic surgery was not performed for a significant number of indeterminate nodules with benign clinical features in Asian practice, resulting in low rates of borderline tumours in surgically-treated patients.

Molecular testing of BRAF, RAS and TERT on thyroid FNAs with indeterminate cytology improves diagnostic accuracy.

OBJECTIVE: Liquid-based (LB)-FNA is widely recognized as a reliable diagnostic method to evaluate thyroid nodules. However, up to 30% of LB-FNA remain indeterminate according to the Bethesda system. Use of molecular biomarkers has been recommended to improve its pathological accuracy but implementation of these tests in clinical practice may be difficult. Here, we evaluated feasibility and performance of molecular profiling in routine practice by testing LB-FNA for BRAF, N/HRAS and TERT mutations. METHODS: We studied a large prospective cohort of 326 cases, including 61 atypia of undetermined significance, 124 follicular neoplasms, 72 suspicious for malignancy and 69 malignant cases. Diagnosis of malignancy was confirmed by histology on paired surgical specimen. RESULTS: Mutated LB-FNAs were significantly associated with malignancy regardless of the cytological classification. Overall sensitivity was 60% and specificity 89%. Importantly, in atypia of undetermined significance and follicular neoplasm patients undergoing surgery according to the Bethesda guidelines, negative predictive values were 85.4% and 90% respectively. TERT promoter mutation was rare but very specific for malignancy (5.5%) suggesting that it could be of interest in patients with indeterminate cytology. CONCLUSIONS: Mutation profiling can be successfully performed on thyroid LB-FNA without any dedicated sample in a pathology laboratory. It is an easy way to improve diagnostic accuracy of routine LB-FNA and may help to better select patients for surgery and to avoid unnecessary thyroidectomies.

The contemporary utility of intraoperative frozen sections in thyroid surgery.

PURPOSE: To determine the accuracy of intraoperative frozen section analysis on thyroidectomy specimens stratified by the Bethesda classification scheme and its utility for intraoperative decision-making. STUDY DESIGN: Retrospective chart review. METHODS: A retrospective review was performed on all patients who underwent thyroidectomy or thyroid lobectomy with intraoperative frozen sections at a tertiary care academic center from 2009 to 2015. RESULTS: There were 74 total patients who underwent partial or total thyroidectomy with intraoperative frozen section analysis of a thyroid nodule whom had previously undergone a thyroid fine needle aspiration of the nodule. The sensitivity, specificity, positive predictive value, and negative predictive value for a thyroid frozen section with respect to its prediction for malignancy was 81%, 95%, 98%, and 66%, respectively, with a diagnostic accuracy of 85%. For 37 patients with an indeterminate cytologic diagnosis on fine needle aspiration (Bethesda categories III-V), the sensitivity, specificity, positive predictive value, and negative predictive value for a thyroid frozen section was 81%, 91%, 95%, and 67%, respectively, with a diagnostic accuracy of 84%. False positives and false negatives resulted in 1 completion thyroidectomy for benign pathology and 3 reoperations for malignancy not discovered on frozen section. CONCLUSION: While intraoperative frozen sections on thyroid specimens may be helpful if positive, the false negative rate remains high. There appears to be limited value in routine frozen sections to guide clinical management and decision-making in the era of the Bethesda system.

Strain ratio ultrasound elastography increases the accuracy of colour-Doppler ultrasound in the evaluation of Thy-3 nodules. A bi-centre university experience.

OBJECTIVES: To assess whether ultrasound elastography (USE) with strain ratio increases diagnostic accuracy of Doppler ultrasound in further characterisation of cytologically Thy3 thyroid nodules. METHODS: In two different university diagnostic centres, 315 patients with indeterminate cytology (Thy3) in thyroid nodules aspirates were prospectively evaluated with Doppler ultrasound and strain ratio USE before surgery. Ultrasonographic features were analysed separately and together as ultrasound score, to assess sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Receiver operating characteristic (ROC) curves to identify optimal cut-off value of the strain ratio were also provided. Diagnosis on a surgical specimen was considered the standard of reference. RESULTS: Higher strain ratio values were found in malignant nodules, with an optimum strain ratio cut-off of 2.09 at ROC analysis. USE with strain ratio showed 90.6% sensitivity, 93% specificity, 82.8% PPV, 96.4% NPV, while US score yielded a sensitivity of 52.9%, specificity of 84.3%, PPV 55.6% and NPV 82.9%. The diagnostic gain with strain ratio was statistically significant as proved by ROC areas, which was 0.9182 for strain ratio and 0.6864 for US score. CONCLUSIONS: USE with strain ratio should be considered a useful additional tool to colour-Doppler US, since it improves characterisation of thyroid nodules with indeterminate cytology. KEY POINTS: • Strain ratio measurements improve differentiation of thyroid nodules with indeterminate cytology • Elastography with strain ratio is more reliable than ultrasound features and ultrasound score • Strain ratio may help to better select patients with Thy 3 nodules candidate for surgery.

Carbonic anhydrase 4 and crystallin α-B immunoreactivity may distinguish benign from malignant thyroid nodules in patients with indeterminate thyroid cytology.

BACKGROUND: Thyroid nodules are present in 19%-67% of the population and carry a 5%-10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%-30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules. METHODS: A tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone. RESULTS: Increased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99. CONCLUSIONS: Loss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.

A histological assessment of the Bethesda system for reporting thyroid cytopathology (2010) abnormal categories: a series of 219 consecutive cases.

OBJECTIVE: The purposes of this study were to establish the distribution of thyroid lesions that were seen in Hatay (a province of southern Turkey), to review the accuracy of fine needle aspiration cytology (FNAC) in the diagnosis of thyroid nodules and to correlate the FNAC results with the histopathology of the excised specimens, especially in indeterminate cases. METHODS: Data on patient cytology were retrieved by a retrospective search of all thyroid FNAC specimens that had been evaluated at the Department of Pathology, Antakya Public Hospital, Hatay, Turkey between January 2009 and February 2011; 1021 thyroid FNAC samples were reviewed and interpretations were recorded according to the Bethesda system for reporting thyroid cytopathology (TBSRTC). The results of adequate FNAC samples were compared with the histological diagnoses in the cases in which surgery was performed, and the malignancy rates, especially in indeterminate categories, were calculated. RESULTS: Of the 1021 FNAC samples, 697 (68.3%) were benign, 122 (11.9%) were non-diagnostic, 100 (9.8%) were atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), 41 (4%) were follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 36 (3.5%) were suspicious for malignancy (SM) and 25 (2.4%) were malignant. In 219 cases, there was follow-up histology. Rates of malignancy were as follows: benign, 0%; AUS/FLUS, 12.7%; FN/SFN, 35.0%; SM, 91.4%; malignant, 100%. CONCLUSIONS: In our study, the cytohistological correlation of benign and malignant lesions was 100%. In the indeterminate categories, we recommend that clinicians should evaluate both the clinical and radiological findings of patients in addition to the FNAC results.

'Indeterminate for malignancy' (Tir3/Thy3 in the Italian and British systems for classification) thyroid fine needle aspiration (FNA) cytology reporting: morphological criteria and clinical impact.

BACKGROUND: The British system (Thy1-5), the Bethesda system for reporting thyroid cytopathology (BSRTC) and the Italian Society of Anatomic Pathology and Cytology (SIAPEC) classification represent the most important international classifications for thyroid cytopathology. Irrespective of the system used, the 'indeterminate' categories are still debated among cytopathologists, particularly with regard to diagnostic criteria, clinical impact of subclassification and role of molecular techniques. AIM: We aimed to find answers to the following questions: Are there shared criteria in cytological preparations that allow the division of indeterminate follicular lesions into subcategories? What is the true clinical impact of this possible subclassification? METHODS: Among 1150 consecutive thyroid fine needle aspiration (FNA) specimens, 80 patients had nodules with a final cytological report of Tir3 (SIAPEC)/Thy3. These 80 cases were re-evaluated and subclassified according to morphological criteria into three groups: pure follicular proliferations, Hürthle cell follicular lesions and atypical proliferations. RESULTS: Sixteen (20%) cases were categorized as pure follicular proliferations, 40 (50%) as Hürthle cell follicular lesions and 24 (30%) as atypical proliferations. Surgery was performed in 57 cases (71%). Cyto-histological correlation showed that follicular adenoma was the most frequent final diagnosis in the cases treated by surgery (24/57, 42%). The overall malignancy rate in the Tir3 category was 28% (16/57). Atypical proliferations were more often malignant than either of the follicular groups (53% versus 19%, P = 0.019). CONCLUSIONS: A five-tiered classification, subdividing the 'indeterminate for malignancy' class into 'follicular proliferations' and 'atypical lesions' could be adopted. As a result of their higher risk of malignancy, surgical management of the atypical lesions would be justified. In future, the introduction of a genetic panel might contribute to their stratification, to the determination of a more accurate risk of malignancy of the atypical lesions and to the verification of follicular proliferations that are benign.

Histopathology of telomerase reverse transcriptase promoter (TERT) mutated indeterminate thyroid nodules.

Objective: The objective of this study was to analyze the risk of malignancy and the histopathology of telomerase reverse transcriptase promoter (TERT) mutated cytologically indeterminate thyroid nodules (ITN). Methods: A PUBMED search of molecularly tested ITN was conducted and data on TERT mutated ITN with histopathology correlation were extracted. Results: Twenty-six manuscripts (published between 2014 and 2022) reported on 77 TERT mutated ITN. Sixty-five nodules were malignant (84 %), with 16 (25 %) described with high-risk histopathology, 5 (8 %) described as low-risk, and most without any description. Isolated TERT mutations were malignant in 26/30 ITNs (87 %) with 9 (35 %) described as high risk and none described as low risk. TERT + RAS mutated ITNs were malignant in 29/34 ITNs (85 %) with 3 (10 %) described as high risk and 4 (14 %) described as low risk. Finally, all 5 TERT + BRAFV600E mutated nodules were malignant and 3/5 (60 %) were described as high risk. Conclusion: TERT mutated ITNs have a high risk of malignancy (84 %), and the current data does not show a difference in malignancy rate between isolated TERT mutations and TERT + RAS co-mutated ITNs. When described, TERT + RAS co-mutated ITNs did not have a higher rate of high-risk histopathology as compared to isolated TERT mutated lesions. Most TERT mutated ITNs did not have a description of histopathology risk and the oncologic outcomes, including rate of recurrence, metastases, and disease specific survival, are unknown. Further data is needed to determine if TERT mutated ITNs should be subjected to aggressive initial treatment.

Molecular Diagnostics and [18F]FDG-PET/CT in Indeterminate Thyroid Nodules: Complementing Techniques or Waste of Valuable Resources?

Background: An accurate preoperative workup of cytologically indeterminate thyroid nodules (ITN) may rule out malignancy and avoid diagnostic surgery for benign nodules. This study assessed the performance of molecular diagnostics (MD) and 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG)-positron emission tomography/computed tomography (PET/CT) in ITN, including their combined use, and explored whether molecular alterations drive the differences in [18F]FDG uptake among benign nodules. Methods: Adult, euthyroid patients with a Bethesda III or IV thyroid nodule were prospectively included in this multicenter study. They all underwent MD and an [18F]FDG-PET/CT scan of the neck. MD was performed using custom next-generation sequencing panels for somatic mutations, gene fusions, and copy number alterations and loss of heterozygosity. Sensitivity, specificity, negative and positive predictive value (NPV, PPV), and benign call rate (BCR) were assessed for MD and [18F]FDG-PET/CT separately and for a combined approach using both techniques. Results: In 115 of the 132 (87%) included patients, MD yielded a diagnostic result on cytology. Sensitivity, specificity, NPV, PPV, and BCR were 80%, 69%, 91%, 48%, and 57% for MD, and 93%, 41%, 95%, 36%, and 32% for [18F]FDG-PET/CT, respectively. When combined, sensitivity and specificity were 95% and 44% for a double-negative test (i.e., negative MD plus negative [18F]FDG-PET/CT) and 68% and 86% for a double-positive test, respectively. Concordance was 63% (82/130) between MD and [18F]FDG-PET/CT. There were more MD-positive nodules among the [18F]FDG-positive benign nodules (25/59, 42%, including 11 (44%) isolated RAS mutations) than among the [18F]FDG-negative benign nodules (7/30, 19%, p = 0.02). In oncocytic ITN, the BCR of [18F]FDG-PET/CT was mere 3% and MD was the superior technique. Conclusions: MD and [18F]FDG-PET/CT are both accurate rule-out tests when unresected nodules that remain unchanged on ultrasound follow-up are considered benign. It may vary worldwide which test is considered most suitable, depending on local availability of diagnostics, expertise, and cost-effectiveness considerations. Although complementary, the benefits of their combined use may be confined when therapeutic consequences are considered, and should therefore not routinely be recommended. In nononcocytic ITN, sequential testing may be considered in case of a first-step MD negative test to confirm that withholding diagnostic surgery is oncologically safe. In oncocytic ITN, after further validation studies, MD might be considered. Clinical Trial Registration: This trial is registered with ClinicalTrials.gov: NCT02208544 (August 5, 2014), https://clinicaltrials.gov/ct2/show/NCT02208544.

A Prospective Study of Publicly Funded Molecular Testing of Indeterminate Thyroid Nodules: Canada's Experience.

CONTEXT: Indeterminate thyroid nodules (ITNs) lead to diagnostic surgeries in many countries. Use of molecular testing (MT) is endorsed by several guidelines, but costs are limitative, especially in public healthcare systems like in Canada. OBJECTIVES: Primary objective: evaluate the clinical value of Thyroseq® v3 (TSv3) using benign call rate (BCR) in a real-world practice. Secondary objective: assess cost-effectiveness of MT. DESIGN: This is a multicentric prospective study. SETTING: This study was conducted in 5 academic centers in Quebec, Canada. PATIENTS OR OTHER PARTICIPANTS: 500 consecutive patients with Bethesda III (on 2 consecutive cytopathologies) or IV and TIRADS 3 or 4 nodules measuring 1 to 4 cm were included. INTERVENTION: MT was performed between November 2021 and November 2022. Patients with a positive TSv3 were referred to surgery. Patients with a negative TSv3 were planned for follow-up by ultrasonography for a minimum of 2 years. MAIN OUTCOME MEASURE: The BCR, corresponding to the proportion of ITNs with negative TSv3 results, was assessed. RESULTS: 500 patients underwent TSv3 testing, with a BCR of 72.6% (95% CI: 68.5-76.5; p<0.001). 99.7% of patients with a negative result avoided surgery. The positive predictive value of TSv3 was 68.2% (95% CI: 58.5-76.9). The cost-benefit analysis identified that the implementation of MT would yield cost savings of $6.1 million over the next 10 years. CONCLUSIONS: Use of MT (TSv3) in a well-selected population with ITNs led to a BCR of 72.6%. It is cost-effective and prevents unnecessary surgeries in a public healthcare setting.

Artificial neural network analysis for evaluating cancer risk in multinodular goiter.

BACKGROUND: The aim of this study was to create a diagnostic model using the artificial neural networks (ANNs) to predict malignancy in multinodular goiter patients with an indeterminate cytology. MATERIALS AND METHODS: Out of 623 patients, 411 evaluated for multinodular goiter between July 2004 and March 2010 had a fine-needle aspiration biopsy. All patients underwent total thyroidectomy. The interpretation was consistent with an indeterminate lesion in 116 (18.6%) patients. Patient's medical records including age, sex, dominant nodule size, pre-operative serum thyroid-stimulating hormone level, thyroid hormone therapy and final pathologic diagnosis were collected retrospectively. RESULTS: The mean age of the patients was 44.6 years (range, 17-78 years). About 104 (89.7%) were female and 12 (10.3%) were male patients. Final pathology revealed 24 malignant diseases (20.7%) and 92 (79.3%) benign diseases. After the completion of training, the ANN model was able to predict diagnosis of malignancy with a high degree of accuracy. The area under the curve of ANNs was 0.824. CONCLUSION: The ANNs technique is a useful aid in diagnosing malignancy and may help reduce unnecessary thyroidectomies in multinodular goiter patients with an indeterminate cytology. Further studies are needed to construct the optimal diagnostic model and to apply it in the clinical practice.

Malignancy risk in Bethesda class IV thyroid nodules in an iodine deficient region.

Background: According to the latest guidelines, in patients with high-risk nodules with indeterminate cytology, diagnostic lobectomy should be the preferable surgical approach in the absence of factors that suggest a total thyroidectomy. Methods: This retrospective observational study has as its main aim the evaluation of the cases that underwent surgery, for Bethesda class IV nodules in our iodocarent geographical area. Particular attention was paid to carcinoma incidence, preoperative nodule size, histological characteristics of the neoplasm, surgical approach and eventual need of radiometabolic treatment. A total of 320 patients were included that underwent surgery for Bethesda IV nodules, between January 2010 and December 2020, at the General Surgical Clinic of the University Hospital of Parma, Italy. Results: A total of 230 total thyroidectomies (71.9%) and 90 lobectomies (28.1%) were performed. Our data showed a strong impact of the 2015 ATA Guidelines on the surgical approach choice, with a progressive propensity towards a conservative approach and an increase of lobectomies from 7.2% to 41.5% after the new guidelines introduction. However, in our sample the percentage of lobectomies remains below 50%; this data is certainly influenced by the number of cases of multinodular pathology, often bilateral, in our geographical area. The nodules malignancy rate resulted 28.8%. Our data showed that increasing size correlated with an increasing malignancy rate (P<0.01), and follicular carcinomas were found to be larger than papillary carcinomas (P<0.001). A statistically significant correlation also emerged between nodule size increase and local/lymphovascular invasion (P<0.05). On the other hand, there was no statistically significant correlation between nodule size and multifocality, and between nodule size and presence of lymph node metastases. Out of the patients where it was possible to find this data, 66% underwent radioiodiometabolic treatment: 59% with papillary carcinoma, and 85% with follicular carcinoma. Conclusions: In patients with Bethesda IV thyroid nodules, diagnostic lobectomy should be the preferable surgical approach in absence of factors that suggest total thyroidectomy. In our opinion, total thyroidectomy remains the first choice in large nodules (≥4 cm) as these nodules have a high malignancy rate, greater local/lymphovascular invasion and a consequent frequent indication for post-operative radiometabolic treatment.

Performance of multigene testing in cytologically indeterminate thyroid nodules and molecular risk stratification.

Objective: Thyroid cancer is the third most prevalent cancer among females. Genetic testing based on next-generation sequencing may provide an auxiliary diagnosis to reduce cytologically diagnostic uncertainty. However, commercial multigene tests are not widely available and are not well-tested in the Chinese population. Methods: In this study, we designed a multigene testing panel and evaluated its performance in 529 cytologically indeterminate thyroid nodules (Bethesda III, IV and V). The molecular data of the DNA mutations and RNA fusions of fine needle aspiration samples were reviewed in conjunction with a clinical diagnosis, pathological reports, and definitive surgery for retrospective analysis. Then, the molecular risk stratification was investigated for its accuracy in malignant risk prediction. Results: The overall combined consistency revealed substantial agreement (Kappa = 0.726) with the sensitivity, specificity, positive predictive value, and negative predictive values of 97.80%, 82.14%, 98.99%, and 67.65%, respectively. The most common aberration was BRAFV600E (82.59%), followed by NRAS mutants (4.07%), RET fusions (3.70%), and KRAS mutants (3.15%). Two cases (0.44%) were categorized into a high-risk group, 426 cases (94.67%) were categorized into a BRAF-like group with totally histopathologic papillary patterned tumors, and 22 cases (4.89%) were categorized into a RAS-like group with 14 papillary and eight follicular patterned tumors when the cohort concurrent aberrations were excluded. Potentially aggressive features may be related to concurrent molecular alterations of BRAFV600E with TERTQ302R, and AKT1L52R, NRASG12C, NRASQ61R, and CCDC6-RET fusions. Conclusions: This study provided a multigene panel for identifying benign nodules from cytologically indeterminate thyroid nodules to avoid unnecessary surgery. We provide further evidence for using molecular risk stratification as a promising predictor of disease outcomes. The results of this study may be limited by the extremely high prevalence of cancer in the cohort for clinical reference.