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.

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.

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.

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.

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.

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.

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.

SFE-AFCE-SFMN 2022 Consensus on the management of thyroid nodules : Role of molecular tests for cytologically indeterminate thyroid nodules.

The SFE-AFCE-SFMN 2022 consensus deals with the management of thyroid nodules, a condition that is a frequent reason for consultation in endocrinology. In more than 90% of cases, patients are euthyroid, with benign non-progressive nodules that do not warrant specific treatment. The clinician's objective is to detect malignant thyroid nodules at risk of recurrence and death, toxic nodules responsible for hyperthyroidism or compressive nodules warranting treatment. The diagnosis and treatment of thyroid nodules requires close collaboration between endocrinologists, nuclear medicine physicians and surgeons, but also involves other specialists. Therefore, this consensus statement was established jointly by 3 societies: the French Society of Endocrinology (SFE), French Association of Endocrine Surgery (AFCE) and French Society of Nuclear Medicine (SFMN); the various working groups included experts from other specialties (pathologists, radiologists, pediatricians, biologists, etc.). Because of the emerging role of molecular fine-needle cytology diagnostics, the French Endocrine Society convened a panel of experts to review the evidence for the diagnostic value of molecular tests performed on cytologically indeterminate thyroid nodules.

Clinical Use of Raman Spectroscopy Improves Diagnostic Accuracy for Indeterminate Thyroid Nodules.

BACKGROUND AND OBJECTIVE: Molecular analysis of thyroid fine-needle aspiration (FNA) specimens is believed to improve the management of indeterminate nodules. Raman spectroscopy (RS) can differentiate benign and malignant thyroid lesions in surgically removed tissues, generating distinctive structural profiles. Herein, the diagnostic performance of RS was tested on FNA biopsies of thyroid gland. DESIGN: Prospective, blinded, and single-center study. METHODS: We enrolled 123 patients with indeterminate or more ominous cytologic diagnoses (TIR3A-low-risk indeterminate lesion, TIR3B-high-risk indeterminate lesion, TIR4-suspicious of malignancy, TIR5-malignant). All subjects were surgical candidates (defined by international guidelines) and submitted to FNA procedures for RS analysis. We compared RS data, cytologic findings, and final histologic assessments (as reference standard) using various statistical techniques. RESULTS: The distribution of our study population was as follows: TIR3A:37, TIR3B:32, TIR4:16, and TIR5:38. In 30.9% of patients, histologic diagnoses were benign. For predicting thyroid malignancy in FNA samples, the overall specificity of RS was 86.8%, with 86.5% specificity in indeterminate cytologic categories. In patients with high-risk ultrasound categories, the specificity of RS increased to 87.5% for TIR3A, reaching 100% for TIR3B. Benign histologic diagnoses accounted for 72.9% of patients classified as TIR3A and 31.3% of those classified as TIR3B. Based on positive RS testing, unnecessary surgery was reduced to 7.4% overall (TIR3A-33.3%, TIR3B-6.7%). CONCLUSIONS: This premier use of RS for thyroid cytology confirms its role as a valuable diagnostic tool and a valid alternative to molecular studies, capable of improving the management of indeterminate nodules and reducing unnecessary surgery.

Preoperative Identification of Medullary Thyroid Carcinoma (MTC): Clinical Validation of the Afirma MTC RNA-Sequencing Classifier.

Background: Cytopathological evaluation of thyroid fine-needle aspiration biopsy (FNAB) specimens can fail to raise preoperative suspicion of medullary thyroid carcinoma (MTC). The Afirma RNA-sequencing MTC classifier identifies MTC among FNA samples that are cytologically indeterminate, suspicious, or malignant (Bethesda categories III-VI). In this study we report the development and clinical performance of this MTC classifier. Methods: Algorithm training was performed with a set of 483 FNAB specimens (21 MTC and 462 non-MTC). A support vector machine classifier was developed using 108 differentially expressed genes, which includes the 5 genes in the prior Afirma microarray-based MTC cassette. Results: The final MTC classifier was blindly tested on 211 preoperative FNAB specimens with subsequent surgical pathology, including 21 MTC and 190 non-MTC specimens from benign and malignant thyroid nodules independent from those used in training. The classifier had 100% sensitivity (21/21 MTC FNAB specimens correctly called positive; 95% confidence interval [CI] = 83.9-100%) and 100% specificity (190/190 non-MTC FNAs correctly called negative; CI = 98.1-100%). All positive samples had pathological confirmation of MTC, while all negative samples were negative for MTC on surgical pathology. Conclusions: The RNA-sequencing MTC classifier accurately identified MTC from preoperative thyroid nodule FNAB specimens in an independent validation cohort. This identification may facilitate an MTC-specific preoperative evaluation and resulting treatment.

Health-related quality of life following FDG-PET/CT for cytological indeterminate thyroid nodules.

Objective: This study assessed the health-related quality of life (HRQoL) in patients undergoing 2-[18F]fluoro-2-deoxy-D-glucose (FDG)-PET/CT for an indeterminate (Bethesda III/IV) thyroid nodule. FDG-PET/CT accurately rules out malignancy and prevents 40% of futile diagnostic surgeries in these nodules. Design: Secondary analyses of HRQoL data from a randomised controlled multicentre trial (NCT02208544) in 126 patients from 15 hospitals in the Netherlands were done. Methods: Longitudinal HRQoL assessment was performed using the EuroQol 5-dimension 5-level (EQ-5D-5L), the RAND 36-item Health Survey v2.0 (RAND-36), and the Thyroid Patient-Reported Outcome (ThyPRO) questionnaire on baseline, 3, 6, and 12 months, relative to the date of the FDG-PET/CT scan. Results: Patients who were randomised to active surveillance following an FDG-negative nodule instead of diagnostic surgery reported stable HRQoL scores throughout the year. Univariate analysis indicated better HRQoL for patients undergoing surveillance than surgical patients with benign histopathology on multiple physical and psychosocial domains. Univariate within-group analysis suggested both temporary and continued HRQoL deteriorations in patients with benign histopathology over time. Multivariate within-group analysis demonstrated no significant longitudinal HRQoL changes in patients undergoing active surveillance. In contrast, in patients with benign histopathology, worse HRQoL was observed with regard to ThyPRO cognitive impairment (P = 0.01) and cosmetic complaints (P = 0.02), whereas goitre symptoms (P < 0.001) and anxiety (P = 0.04) improved over time. In patients with malignant histopathology, anxiety also decreased (P = 0.05). Conclusions: The reassurance of a negative FDG-PET/CT resulted in sustained HRQoL throughout the first year of active surveillance. Diagnostic surgery for a nodule with benign histopathology resulted in more cognitive impairment and physical problems including cosmetic complaints, but improved goitre symptoms and anxiety. Anxiety was also reduced in patients with malignant histopathology.

The Autoantibodies against Tumor-Associated Antigens as Potential Blood-Based Biomarkers in Thyroid Neoplasia: Rationales, Opportunities and Challenges.

The Autoantibodies targeting Tumor-Associated Antigens (TAA-AAbs) emerge as a result of a variety of tumor-related immunogenic stimuli and may be regarded as the eyewitnesses to the anti-tumor immune response. TAA-AAbs may be readily detected in peripheral blood to unveil the presence of a particular TAA-expressing tumor, and a fair number of TAAs eliciting the tumor-associated autoantibody response have been identified. The potential of TAA-AAbs as tumor biomarkers has been extensively studied in many human malignancies with a major influence on public health; however, tumors of the endocrine system, and, in particular, the well-differentiated follicular cell-derived thyroid neoplasms, remain understudied in this context. This review provides a detailed perspective on and legitimate rationales for the potential use of TAA-AAbs in thyroid neoplasia, with particular reference to the already established diagnostic implications of the TAA-AAbs in human cancer, to the windows for improvement and diagnostic niches in the current workup strategies in nodular thyroid disease and differentiated thyroid cancer that TAA-AAbs may successfully occupy, as well as to the proof-of-concept studies demonstrating the usefulness of TAA-AAbs in thyroid oncology, particularly for the pre-surgical discrimination between tumors of different malignant potential in the context of the indeterminate results of the fine-needle aspiration cytology.

Performance of Afirma Gene Sequencing Classifier versus Gene Expression Classifier in thyroid nodules with indeterminate cytology.

INTRODUCTION: About 15% to 30% of thyroid fine-needle aspiration (FNA) nodules have indeterminate cytology. The Afirma (Veracyte Inc, South San Francisco, CA) Gene Expression Classifier (GEC)/Gene Sequencing Classifier (GSC) tests were designed to improve risk stratification of the indeterminate thyroid nodules. This study aimed to evaluate and compare the performance of the Afirma GEC and GSC tests in the indeterminate thyroid lesions. METHODS: Thyroid FNA cases with indeterminate cytology were searched in the pathology database and only those with available Afirma results were selected for this study. Each patient's demographic, sonographic, cytologic, molecular, and subsequent surgical follow-up results were collected and analyzed. RESULTS: There were 100 cases with indeterminate thyroid FNA results, including 49 cases tested by GEC and 51 cases by GSC. In the GEC group, benign call rate (BCR) was 53% (26 of 49) and the calculated negative predictive value (NPV) and positive predictive value (PPV) were 88% and 47% respectively. In the GSC group, the BCR was 63% (32 of 51) and the calculated NPV and PPV were 100% and 64%, respectively. Whereas only 17% (1 of 6) of benign oncocytic lesions were tested benign by the GEC, 60% (3 of 5) of benign oncocytic nodules were tested benign by the GSC. CONCLUSION: We demonstrated in this study that a little more than half of the indeterminate thyroid nodules had negative Afirma GEC/GSC results and the BCR using the Afirma GSC test was higher than GEC. The Afirma GSC showed higher NPV and PPV than GEC. In addition, the Afirma GSC appeared to be superior for differentiating benign and malignant oncocytic thyroid lesions.

Significance of RAS Mutations in Thyroid Benign Nodules and Non-Medullary Thyroid Cancer.

Thyroid nodules are detected in up to 60% of people by ultrasound examination. Most of them are benign nodules requiring only follow up, while about 4% are carcinomas and require surgery. Malignant nodules can be diagnosed by the fine-needle aspiration cytology (FNAC), which however yields an indeterminate result in about 30% of the cases. Testing for RAS mutations has been proposed to refine indeterminate cytology. However, the new entity of non-invasive follicular thyroid neoplasm, considered as having a benign evolution and frequently carrying RAS mutations, is expected to lower the specificity of this mutation. The aggressive behavior of thyroid cancer with RAS mutations, initially reported, has been overturned by the recent finding of the cooperative role of TERT mutations. Although some animal models support the carcinogenic role of RAS mutations in the thyroid, evidence that adenomas harboring these mutations evolve in carcinomas is lacking. Their poor specificity and sensitivity make the clinical impact of RAS mutations on the management of thyroid nodules with indeterminate cytology unsatisfactory. Evidence suggests that RAS mutation-positive benign nodules demand a conservative treatment. To have a clinical impact, RAS mutations in thyroid malignancies need not to be considered alone but rather together with other genetic abnormalities in a more general context.

Do Ultrasound Patterns and Clinical Parameters Inform the Probability of Thyroid Cancer Predicted by Molecular Testing in Nodules with Indeterminate Cytology?

Background: Molecular testing (MT) is commonly used to refine cancer probability in thyroid nodules with indeterminate cytology. Whether or not ultrasound (US) patterns and clinical parameters can further inform the risk of thyroid cancer in nodules predicted to be positive or negative by MT remains unknown. The aim of this study was to test if clinical parameters, including patient age, sex, nodule size (by US), Bethesda category (III, IV, V), US pattern (American Thyroid Association [ATA] vs. American College of Radiology Thyroid Image Reporting and Data System [TI-RADS] systems), radiation exposure, or family history of thyroid cancer can modify the probability of thyroid cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) predicted by MT. Methods: We studied 257 thyroid nodules in 232 patients from 10 study centers with indeterminate fine needle aspiration cytology and informative MT results using the ThyroSeq v3 genomic classifier (TSv3). Univariate and multivariate logistic regression was used for data analysis. Results: The presence of cancer/NIFTP was associated with positive TSv3 results (odds ratio 61.39, p < 0.0001). On univariate regression, patient sex, age, and Bethesda category were associated with cancer/NIFTP probability (p < 0.05 for each). Although ATA (p = 0.1211) and TI-RADS (p = 0.1359) US categories demonstrated positive trends, neither was significantly associated with cancer/NIFTP probability. A multivariate regression model incorporating the four most informative non-MT covariates (sex, age, Bethesda category, and ATA US pattern; Model No. 1) yielded a C index of 0.653; R2 = 0.108. When TSv3 was added to Model number 1, the C index increased to 0.888; R2 = 0.572. However, age (p = 0.341), Bethesda category (p = 0.272), and ATA US pattern (p = 0.264) were nonsignificant, and other than TSv3 (p < 0.0001), male sex was the only non-MT parameter that potentially contributed to cancer/NIFTP risk (p = 0.095). The simplest and most efficient clinical model (No. 3) incorporated TSv3 and sex (C index = 0.889; R2 = 0.588). Conclusions: In this multicenter study of thyroid nodules with indeterminate cytology and MT, neither the ATA nor TI-RADS US scoring systems further informed the risk of cancer/NIFTP beyond that predicted by TSv3. Although age and Bethesda category were associated with cancer/NIFTP probability on univariate analysis, in sequential nomograms they provided limited incremental value above the high predictive ability of TSv3. Patient sex may contribute to cancer/NIFTP risk in thyroid nodules with indeterminate cytology.

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.

Combined molecular and histologic end points inform cancer risk estimates for thyroid nodules classified as atypia of undetermined significance.

BACKGROUND: Thyroid nodules classified as atypia of uncertain significance (AUS) on fine-needle aspiration cytology are heterogeneous. Prior studies reported a higher risk of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)/cancer among AUS nodules that had cytologic (AUS-C) versus architectural (AUS-A) atypia; however, such studies were generally confined to resected cohorts, introducing bias into risk calculations. The authors hypothesized that combined histologic and molecular end points would permit clinically meaningful calculations of NIFTP/malignancy risk among AUS nodules. METHODS: The study consisted of 279 thyroid nodules classified as AUS on initial fine-needle aspiration and tested by the Afirma Gene Expression Classifier (GEC) between June 2013 and October 2017. Results of GEC testing and histopathologic diagnoses were stratified by AUS classifiers. The AUS-A category was further subclassified as 1) hypocellular microfollicular or 2) cellular with mixed but predominantly microfollicular architecture. NIFTP/cancer risk was calculated for each subgroup, with the inclusion of unresected nodules that had benign GEC results as low-risk end points comparable to histologically benign nodules. RESULTS: When only histologic end points were considered, there was no difference in NIFTP/cancer risk (25% vs 23%; P = .82). By using molecular and histologic end points, AUS cases with cytologic atypia trended toward higher NIFTP/cancer risk than AUS-A cases (14% vs 6%; P = .06). Furthermore, AUS-A cases showed a trend toward lower NIFTP/cancer risk for hypocellular microfollicular aspirates (3%) compared with cellular samples that had mixed/predominantly microfollicular architecture (13%; P = .18). CONCLUSIONS: The inclusion of unresected benign GEC nodules in risk-of-malignancy calculations provides more accurate results, which may be helpful for informing patient management as well as quality improvement in the cytopathology laboratory.

Retrospective analysis of cancer-specific gene expression panel for thyroid fine needle aspiration specimens.

BACKGROUND: While molecular testing is a promising strategy for preoperative assessment of cytologically indeterminate thyroid nodules, thyroid fine needle aspiration biopsy (FNA) presents unique challenges for molecular assays, including contaminating peripheral blood mononuclear cells (PBMC) and variable numbers of evaluable epithelial thyroid cells. Moreover, the newly recognized entity, noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), has added an additional challenge to the currently available molecular diagnostic platforms. New diagnostic tools are still needed to correctly distinguish benign and malignant thyroid nodules preoperatively. METHODS: Twenty-two transcript splice variants from 12 genes we previously identified as discriminating benign from malignant thyroid nodules were characterized in 80 frozen thyroid tumors from 8 histological subtypes. Isoforms detectable in PBMC were excluded, and the 5 most discriminating isoforms were further validated by real-time quantitative PCR (qPCR) on intraoperative FNA samples from 59 malignant tumors, 55 benign nodules, and 23 NIFTP samples. The qPCR threshold cycle values for each transcript were normalized to the thyrocyte-specific thyroid peroxidase isoform 1 (TPO1) and z-transformed. Receiver operating characteristic (ROC) analyses of the composite transcript scores were used to evaluate classification of thyroid FNAs by the 5-gene isoform expression panel. RESULTS: A molecular signature was developed by combining expression levels of specific isoforms of CDH3, FNDC4, HMGA2, KLK7, and PLAG1. FNAs containing at least 12-36 thyrocytes were sufficient for this assay. The 5-gene composite score achieved an area under the ROC curve (AUC) of 0.86 for distinguishing malignant from benign nodules, with a specificity of 91%, sensitivity of 75%, negative predictive value of 91%, and positive predictive value of 74%. CONCLUSION: Our newly developed 5-gene isoform expression panel distinguishes benign from malignant thyroid tumors and, may help distinguish benign from malignant thyroid nodules in the context of the new NIFTP subtype.