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Background: Molecular triage of indeterminate thyroid aspirates offers the opportunity to stratify the risk of malignancy (ROM) more accurately. Here we examine our experience with ThyroSeq v3 testing., Methods: We analyzed 276 of 658 (42%) fine needle aspiration samples classified as indeterminate thyroid nodules using ThyroSeq v3 (Sept 2017-Dec 2019). The test provides a ROM and detects specific mutations. Surgical diagnoses were reviewed., Results: Of 276 ThyroSeq-tested cases, 42% (n = 116) harbored genetic alterations, whereas 64% (n = 74) had surgical follow-up. Notably, 79% cases within intermediate to higher risk mutations were highly associated with surgical intervention, resulting in a 77.5% ROM when including both cancer and noninvasive follicular thyroid neoplasia with papillary-like features (cancer+NIFTP) and 68% malignant diagnosis when excluding NIFTP. RAS-like alterations were most common (66%), exhibiting a 73.4% ROM and a 59% malignant diagnosis. Interestingly, this group included 24 encapsulated follicular variant papillary thyroid carcinomas (EFVPTCs), 1 infiltrative FVPTC, 9 follicular carcinomas, and 7 NIFTP. Additionally, three high-risk mutations and eight BRAF/V600E mutations had a 100% ROM, all diagnosed as classic-type papillary thyroid carcinoma (cPTC). Combined analysis of thyroid nodules from Bethesda III and IV categories revealed a 78.2% positive predictive value (PPV) and a 75.9% negative predictive value (NPV)., Conclusion: ThyroSeq v3 effectively stratifies the ROM in indeterminate thyroid nodules based on specific genetic alterations, guiding appropriate surgical management. Notably, the BRAFV600E/high-risk group and RAS-like groups exhibited ROM of 100% and 77.5%, respectively, with promising predictive accuracy (PPV of 78.2% and NPV of 75.9%)., (© 2024 Wiley Periodicals LLC.)

Introduction: Thyroid malignancy is one of the most common types of cancer in developed nations. Currently, fine-needle aspiration cytology (FNAC) is the most practical screening test for thyroid nodules. However, cytologically indeterminate samples comprise approximately 15%-30% of cases. These include cases classified as atypia of undetermined significance (AUS), follicular neoplasm (FN), and suspicious for malignancy (SFM). Indeterminate cases can be sent for molecular testing for more definitive classification to help guide management and prevent overtreatment of benign thyroid nodules. We conducted a retrospective review on molecular testing of indeterminate thyroid FNAC and reviewed subsequent histologic diagnoses in resection specimens to assess how molecular testing supported a diagnosis and its effect on clinical management of patients at our institution., Methods: A retrospective chart review was performed on all thyroid FNAC specimens, corresponding molecular testing, and subsequent surgical resection specimens over a 6-year period., Results: A total of 10,253 thyroid FNAC were performed in our hospital system during our study period, of which 10% (n = 1102/10,253) had indeterminate FNAC results. Molecular testing was performed in 16% (n = 178/1102) of indeterminate cytology cases. Genetic alterations were identified in 39% (n = 69/178) of the cases sent for molecular testing. The majority of cytologically indeterminate cases sent for molecular testing were follicular-patterned lesions and their corresponding resection specimens revealed mostly low grade follicular derived neoplasms (i.e., follicular adenoma, non-invasive follicular thyroid neoplasm with papillary-like nuclear features, and follicular variant of papillary thyroid carcinoma). Of the cases with identified genetic alterations, 75% (n = 52/69) were treated surgically. In cases with no genetic alterations identified, only 18% (n = 20/109) were treated surgically., Discussion/conclusion: Molecular testing on cytologically indeterminate thyroid nodules can help provide a more accurate risk of malignancy assessment in patients with lesions that are difficult to diagnosis based solely on FNAC morphology. The types of genetic alterations identified in the resected thyroid lesions were consistent with what has been previously described in the literature. Additionally, we found that in the patients with indeterminate thyroid FNAC with adjunct molecular testing, more than half did not undergo surgical resection. This finding emphasizes the value of adding molecular testing in patients, particularly when attempting to reduce unnecessary surgical intervention., (© 2023 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Molecular testing is an adjunct test for thyroid fine needle aspirations with indeterminate diagnoses, with certain mutations showing a greater risk of malignancy (ROM). Rat sarcoma (RAS) point mutations are the most common alterations in indeterminate thyroid nodules. While they can have a high ROM, they are also found in benign disease. This study describes the histologic outcomes of indeterminate nodules with RAS mutations. Bethesda III and IV thyroid nodules with ThyroSeq results showing RAS mutations (NRAS, KRAS, and HRAS) were identified between November 1, 2018 and February 28, 2023. Baseline patient characteristics, ThyroSeq results, and surgical diagnoses were collected. We identified 18 nodules with RAS mutations from 17 patients. Fourteen were NRAS (isolated NRAS in 6; NRAS with other abnormalities [NRAS+] in 8); one was isolated KRAS; and three were HRAS with other abnormalities (HRAS+). NRAS Q16R was the most common amino acid change. Twelve cases had follow-up. Two were malignant, a minimally invasive follicular carcinoma (NRAS+) and a papillary thyroid carcinoma, follicular variant (HRAS+). Three were noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), 2 HRAS+ and 1 NRAS+. Four were follicular adenomas, one being atypical (3 NRAS+ and one isolated NRAS). One was an oncocytic adenoma (isolated NRAS). Two were nodular hyperplasias (isolated NRAS and NRAS+, respectively). Twenty-eight percent of our RAS-mutated nodules were malignant or NIFTP. All three HRAS-mutated nodules were malignant or NIFTP. The three isolated RAS mutations with follow up were benign (adenomas or nodular hyperplasia). These findings were in line with the literature., (© 2023 Wiley Periodicals LLC.)

Background: There is limited data comparing the performance of Afirma Genomic Sequencing Classifier (GSC) in thyroid nodules carrying an initial versus a repeat diagnosis of atypia of undetermined significance (AUS). This study reported an institutional experience in this regard., Materials and Methods: This retrospective study included consecutive thyroid nodules that had an initial or a repeat AUS diagnosis and had a subsequent GSC diagnostic result (benign or suspicious) from 2017 to 2021. All nodules were followed by surgical intervention or by clinical and/or ultrasound monitoring. GSC's benign call rate (BCR), rate of histology-proven malignancy associated with a suspicious GSC result, and diagnostic parameters of GSC were calculated and compared between the two cohorts (initial versus repeat AUS). Statistical significance was defined with a p-value of <.05 for all analysis., Results: A total of 202 cases fulfilled inclusion criteria, including 67 and 135 thyroid nodules with an initial and a repeat AUS diagnosis, respectively. BCR was 67% and 66% in initial and repeat AUS cohorts, respectively. Rate of histology-proven malignancy associated with a suspicious GSC result were 22% and 24% in initial and repeat AUS cohorts, respectively. Compared with the repeat AUS cohort, the initial AUS cohort showed slightly lower sensitivity (83% vs. 100%), specificity (70% vs. 73%), PPV (23% vs. 24%), NPV (98% vs. 100%), and diagnostic accuracy (72% vs. 75%). Nevertheless, these differences did not reach statistical significance., Conclusion: GSC demonstrated comparable performance in thyroid nodules with a repeat AUS diagnosis versus nodules with an initial AUS diagnosis., (© 2023 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Background: A preoperative method is desired to discriminate benign from malignant thyroid nodules. This retrospective study evaluated the diagnostic performance of BRAF (B-Raf proto-oncogene) mutation (BRAF V600E ) positivity and fine-needle aspiration cytology (FNAC) relative to intraoperative frozen section pathology., Methods: Patients underwent preoperative FNAC of thyroid nodules. Cytology specimens were classified according to the Bethesda System for Reporting Thyroid Cytopathology (BSRTC), and analyzed for BRAF V600E using an amplification-refractory mutation system (ARMS). Thyroid tissue was surgically removed and frozen sections processed for histology. The sensitivities and specificities of each analysis were compared, alone and in combination., Results: Among 346 patients, 333/358 FNACs (93%) showed malignant nodules; 322 (93%) patients received a pathological diagnosis of papillary thyroid carcinoma (PTC). The sensitivity and specificity of BSRTC VI for malignancy was the highest among the BSRTC categories. Compared with FNAC, the BRAF V600E analysis had significantly higher sensitivity and specificity. The diagnostic efficacy of frozen section pathology was significantly higher than that of either BSRTC category or BRAF V600E analysis alone. Combining methods variably improved diagnostic performance. BRAF V600E was not associated with capsule infiltration, neurovascular infiltration, mono- or multifocal PTC, lymph node metastasis, or clinical stage., Conclusion: The diagnostic performance of preoperative BRAF V600E determination was better than that of the BSRTC-FNAC category; combining both improved sensitivity and specificity. Patients with positive malignancy scores from both should be recommended for surgery; those with negative scores require close monitoring. Surgical treatment should include comprehensive intraoperative frozen section assessment. BRAF mutations cannot indicate aggressive treatment., (© 2023 Wiley Periodicals LLC.)

Thyroblastoma is a rare, aggressive embryonal thyroid neoplasm associated with DICER1 mutation. It usually presents as a rapidly growing thyroid mass diffusely infiltrating the thyroid lobes and extending into perithyroidal tissue. Most thyroblastomas were initially diagnosed as malignant teratoma or carcinosarcoma. The cytologic features of thyroblastoma have not been well documented. Here, we present the cytological findings of a case of thyroblastoma in a 19-year-old female with a dominant solid left thyroid nodule. A fine needle aspiration biopsy of the mass revealed a highly cellular aspirate composed of crowded, atypical, high nuclear to cytoplasmic ratio epithelial cells, arranged in a variety of architectural patterns including rosette-like microfollicular, solid, and morular. In addition, the background contains a minor population of atypical mesenchymal cells. The cytologic differential diagnosis of thyroblastoma includes primary thyroid neoplasms such as adenomatous nodule, follicular adenoma, follicular carcinoma, and poorly differentiated thyroid carcinoma as well as metastatic carcinoma., (© 2023 Wiley Periodicals LLC.)

Background: Thyroid nodules are commonly faced by clinicians as palpable nodules or incidentally identified on imaging. Nodules that are found to be suspicious by imaging can be biopsied by fine needle aspiration, which can yield material for molecular testing to refine the diagnosis., Methods: The current literature concerning molecular testing in thyroid nodules including available commercial assays was reviewed and summarized., Results/conclusions: Commonly encountered alterations include mutations in RAS, BRAF, TERT promoter, PTEN, and DICER1 as well as fusions of RET, ALK, PAX8-PPARγ, and NTRK. This article provides a summary of these molecular alterations, commercially available molecular assays, and general considerations for thyroid epithelial malignancies and benign thyroid nodules., (© 2022 Wiley Periodicals LLC.)

Background: Clinical management of follicular neoplasms (FNs) using molecular testing of thyroid-aspirated materials is not routinely performed in Japan. This article aims to identify low-risk FN nodules that can be followed up without molecular testing., Methods: The relationship between preoperative findings, factors influencing surgical decision, and the risk of malignancy (ROM) was examined in 356 thyroid nodules with cytological diagnosis of FN at Kuma Hospital from January to December 2020., Results: ROMs of FN with cytology results favouring malignancy (41.2%) were significantly higher than those favouring benign (7.7%) or borderline (8.2%) (p < .001). Moreover, ROMs of FN with ultrasonography results of high suspicion (54.5%) were significantly higher than those with low (4.5%) or intermediate suspicion (0%) (p < .0001). There was a large difference in overall ROM in tumours bordering 30 mm in size (<30 mm; 3.6%, ≥30 mm; 20.0%). ROMs of FNs with a tumour volume doubling rate (TVDR) of 1.0/year or more (28.6%) were higher than those of FNs with a lower TVDR (9.9%) (p < .05). The ROMs of FNs with or without one or more of the following four findings suggestive of malignancy: cytological findings favouring malignancy, ultrasonography findings of high suspicion, tumour size ≥30 mm, and TV-DR ≥1.0/year, were 14.6% and 1.0%, respectively., Conclusion: FNs with no cytological findings favouring malignancy, no ultrasonography findings of high suspicion, tumour size <30 mm and TV-DR <1.0/year, are considered low risk and can be followed up without the need for molecular testing., (© 2022 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Background: Afirma gene expression classifier (GEC) is an adjunct to thyroid fine needle aspiration shown to improve pre-operative risk assessment and reduce unnecessary surgery of indeterminate thyroid nodules. Genomic sequencing classifier (GSC) is a newer version aiming to improve specificity and positive predictive value (PPV) of Afirma testing. There are limited studies comparing GSC vs GEC. This study was undertaken to compare these classifiers in terms of diagnostic performance and effect on clinical management of indeterminate thyroid nodules., Methods: The study cohort consisted of patients with thyroid nodules that had a recurrent cytologic diagnosis of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) and were tested by either GEC or GSC. Patient demographics, nodule size, and clinical follow-up were recorded. Benign call rate (BCR) of Afirma testing, rate of subsequent surgery (RSS), rate of histology-confirmed malignancy (RHM), as well as diagnostic sensitivity, specificity, PPV, negative predicative value (NPV), and accuracy were calculated and compared between GSC and GEC cohorts., Results: Among 264 AUS/FLUS thyroid nodules, 127 and 137 were tested with GEC and GSC, respectively. Compared to GEC, GSC demonstrated increased BCR (77.3% vs 52%), decreased RSS (31.4% vs 51.2%), greater RHM (29% vs 9.8%) associated with a suspicious Afirma result, as well as improved specificity (82.8% vs 54.5%), PPV (29% vs 9.8%), and diagnostic accuracy (83.9% vs 56.7%), while maintaining high sensitivity and NPV., Conclusion: Afirma GSC substantially improved BCR, RSS, RHM, and diagnostic performance, enhancing appropriate triage and thereby helped avoid unnecessary surgery in AUS/FLUS thyroid nodules., (© 2021 Wiley Periodicals LLC.)

Background: The multigene genomic classifier ThyroSeq V3 has proven to be an effective triage modality in the management of indeterminate thyroid nodules. This study reports on the clinical management of patients with indeterminate thyroid nodules., Methods: ThyroSeq V3 testing at our institution was implemented April 1, 2019. Over a 17-month period (1 April 2019 through 31 August 2020). Thirty five indeterminate thyroid nodules were sent for testing; diagnoses included FLUS, suspicious for a follicular/Hurthle cell neoplasm (SFN/SHCN) and suspicious for papillary thyroid carcinoma (SPTC). There were 26 females, nine males; mean age 56 years. Aspirated nodules, mean size of 2 cm, were evaluated by rapid on site evaluation with adequacy assessed by the cytopathologist via telecytology., Results: Of the 35 nodules, 17 (49%) were positive and 18 (51%) were negative on ThyroSeq Testing. All of the 17 positive nodules (four FLUS, 10 SFN/SHCN, three SPTC) showed neoplastic lesions; five adenomas, one NIFTP and 11 carcinomas on surgical resection. Only 4 (22%) of the 18 nodules that were negative were resected and showed two colloid/adenomatous nodules, one NIFTP and one follicular variant of papillary thyroid carcinoma. As FLUS FNAs were the majority of the indeterminate nodules, a comparison was made pre-and post ThyroSeq testing. Prior to ThyroSeq testing the majority (69%) of the nodules were resected as compared to 36% post implementation; a statistically significant value (P < .005). Surgical resection yielded a majority of benign nodules pre-ThyroSeq testing, 68%, as compared to post-ThyroSeq testing, 25%., Conclusion: Implementation of ThyroSeq V3 testing has led to improvements in clinical management of patients with indeterminate thyroid nodules., (© 2021 Wiley Periodicals LLC.)

Risk assessment is critical to determine the timing of elective surgeries and preserve valuable resources in time of pandemic. This study was undertaken to better understand the potential value of molecular testing to risk-stratify thyroid nodules with malignant cytology (Bethesda VI). Systematic review of the literature contributed 21 studies representing 2036 preoperative specimens. The BRAF p.V600E substitution was detected in 46% to 90% of cases with a pooled positivity rate of 70% (95% confidence intervals: 64%-76%). None of the studies used comprehensive oncogene panels. Retrospective analysis of 531 clinical specimens evaluated with the next-generation sequencing ThyGeNEXT Thyroid Oncogene Panel identified a total of 436 gene alterations. BRAF mutation rate was 64% in specimens tested as part of standard clinical care and 75% in specimens from cross-sectional research studies (P = .022). Testing for additional actionable gene alterations such as TERT promoter mutations or RET and NTRK gene rearrangements further increased the diagnostic yield to 78%-85% and up to 95% when including the ThyraMIR Thyroid miRNA Classifier. These data support the role of molecular cytopathology in surgical and therapeutic decision-making and warrant additional studies., (© 2020 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Thyroid cancer is the most common endocrine malignancy. Approximately 70% of cases of papillary thyroid carcinoma and 50% of poorly differentiated and anaplastic thyroid carcinoma harbor well-characterized driver mutations and chromosomal rearrangements that drive tumorigenesis. Molecular profiling has been helpful in identifying and informing follow-up strategies in tumors with more aggressive trajectories. Here, we report a case of papillary thyroid cancer (PTC) discovered in a patient with thyroid nodules with relatively benign ultrasound and fine needle aspiration (FNA) findings. Molecular testing in this patient identified a rare STRN-ALK fusion in two thyroid nodules with indeterminate and/or benign cytology. This led to the patient undergoing a thyroid lobectomy and a subsequent confirmation of papillary thyroid carcinoma upon resection. The report highlights the role of comprehensive molecular testing in thyroid lesions of indeterminate cytology., (© 2020 Wiley Periodicals LLC.)

Background: Approximately 25% of thyroid nodule fine-needle aspirates (FNAs) have cytology that is indeterminate for malignant disease. Accurate risk stratification of these FNAs with ancillary testing would reduce unnecessary thyroid surgery., Methods: We evaluated the performance of an ancillary multiplatform test (MPTX) that has three diagnostic categories (negative, moderate, and positive). MPTX includes the combination of a mutation panel (ThyGeNEXT®) and a microRNA risk classifier (ThyraMIR®). A blinded, multicenter study was performed using consensus histopathology diagnosis among three pathologists to validate test performance., Results: Unanimous consensus diagnosis was reached in 197 subjects with indeterminate thyroid nodules; 36% had disease. MPTX had 95% sensitivity (95% CI,86%-99%) and 90% specificity (95% CI,84%-95%) for disease in prevalence adjusted nodules with Bethesda III and IV cytology. Negative MPTX results ruledout disease with 97% negative predictive value (NPV; 95% CI,91%-99%) at a 30% disease prevalence, while positive MPTX results ruledin high risk disease with 75% positive predictive value (PPV; 95% CI,60%-86%). Such results are expected in four out of five Bethesda III and IV nodules tested, including RAS positive nodules in which the microRNA classifier was useful in rulingin disease. 90% of mutation panel false positives were due to analytically verified RAS mutations detected in benign adenomas. Moderate MPTX results had a moderate rate of disease (39%, 95% CI,23%-54%), primarily due to RAS mutations, wherein the possibility of disease could not be excluded., Conclusions: Our results emphasize that decisions for surgery should not solely be based on RAS or RAS-like mutations. MPTX informs management decisions while accounting for these challenges., (© 2020 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Extracardiac rhabdomyoma is an uncommon benign striated muscle tumor with a predilection for the head and neck region. However, it is extremely rare for extracardiac rhabdomyoma to present as a thyroid nodule. We report a case of rhabdomyoma diagnosed by thyroid fine-needle aspiration (FNA) in a patient with Birt-Hogg-Dubé (BHD) syndrome. A 60-year-old man with BHD syndrome presented for recurrent pneumothorax. Chest CT incidentally identified a thyroid nodule. Subsequent sonography confirmed a 4.44 × 2.28 × 2.82 cm solid, hypoechoic nodule with smooth margins in the right upper pole. Ultrasound-guided FNA revealed many clusters and scattered isolated large polygonal cells with abundant granular cytoplasm and small peripherally located nuclei. Vague striations in the cytoplasm were focally identified. No follicular cells or colloid was present. Immunocytochemistry on one direct smear slide demonstrated diffuse positivity for desmin, supporting muscular differentiation. Subsequent surgery identified an adult rhabdomyoma originating from the inferior constrictor muscle of the neck and anteriorly displacing the thyroid. Because the mass was intimately associated with the thyroid gland, it was initially mistaken for a thyroid nodule on ultrasound. Diagnosis of rhabdomyoma on FNA is challenging, especially when rhabdomyoma mimics a thyroid nodule on imaging. The differential diagnosis includes Hurthle cell neoplasm, granular cell tumor, colloid nodule, and normal striated skeletal muscle. Adequate radiologic data and familiarity with the cytologic features of rhabdomyoma are critical for an accurate diagnosis., (© 2020 Wiley Periodicals, Inc.)

Introduction: Focused and expanded mutation panels were assessed for the incremental utility of using an expanded panel in combination with microRNA risk classification., Methods: Molecular results were reviewed for patients who underwent either a focused mutation panel (ThyGenX®) or an expanded mutation panel (ThyGeNEXT®) for strong and weak oncogenic driver mutations and fusions. microRNA results (ThyraMIR®) predictive of malignancy, including strong positive results highly specific for malignancy, were examined., Results: Results of 12 993 consecutive patients were reviewed (focused panel = 8619, expanded panel = 4374). The expanded panel increased detection of strong drivers by 8% (P < .001), with BRAFV600E and TERT promoters being the most common. Strong drivers were highly correlated with positive microRNA results of which 90% were strongly positive. The expanded panel increased detection of coexisting drivers by 4% (P < .001), with TERT being the most common partner often paired with RAS. It increased the detection of weak drivers, with RAS and GNAS being the most common. 49% of nodules with weak drivers had positive microRNA results of which 33% were strongly positive. The expanded panel also decreased the number of nodules lacking mutations and fusions by 15% (P < .001), with 8% of nodules having positive microRNA results of which 22% were strongly positive., Conclusions: Using expanded mutation panels that include less common mutations and fusions can offer increased utility when used in combination with microRNA classification, which helps to identify high risk of malignancy in the cases where risk is otherwise uncertain due to the presence of only weak drivers or the absence of all drivers., (© 2019 The Authors. Diagnostic Cytopathology published by Wiley Periodicals, Inc.)

Background: Molecular tests such as the Afirma gene expression classifier (GEC) and mutational panels (such as ThyroSeq) have been introduced to help risk stratify cytologically indeterminate thyroid nodules with the aim to reduce the number of unnecessary thyroidectomies. Some reports have suggested that samples with Hurthle cell predominance have higher false-positive rates on GEC testing, but data are limited., Methods: We reviewed thyroid nodules with indeterminate (Bethesda III/IV) cytology at our institution. Patient demographics, cytologic and histologic diagnoses (where available), and molecular test results were collected., Results: GEC was performed on 202 nodules, and ThyroSeq was performed on 81 nodules. In the GEC cohort, 66% of nodules with Hurthle cell predominance yielded "suspicious" result vs 46% of nodules without Hurthle cell predominance, with risk of malignancy (ROM) for surgically resected nodules of 16% and 33%, respectively. In ThyroSeq cohort, 8% of nodules with Hurthle cell predominance yielded a high-risk mutation vs 19% of nodules without Hurthle cell predominance, with ROM of 50% and 33%, respectively., Conclusions: For ThyroSeq molecular panel, while it did not appear that there was an increase in rate of high-risk mutations detected in the samples with Hurthle cell predominance, small numbers limit the generalizability of these results. For the GEC cohort, indeterminate thyroid nodules with predominance of Hurthle cells showed an increased rate of "suspicious" results compared to samples without Hurthle cell predominance. The ROM for GEC "suspicious" nodules with Hurthle cell predominance on surgical resection was lower in our study. Repeat FNA may be of use in patients with these types of nodules. In the context of a Hurthle cell predominant lesion, positive results on molecular testing may not carry a high rate of malignancy., (© 2019 Wiley Periodicals, Inc.)

Background: The management of thyroid nodules with indeterminate cytology is challenging. Recently, molecular testing on fine-needle aspirates (FNAs) has been advocated to determine whether clinical follow-up or surgery is warranted for patients. Three different testing platforms were performed on aspirates from our institution (Afirma Thyroid FNA Analysis, RosettaGX Reveal, and Interpace ThyGenX/ThyraMIR). This study compares their diagnostic efficacy., Methods: We conducted a retrospective analysis of indeterminate thyroid FNAs with correlating molecular testing over 4 years (2015-2018). The aspirates included diagnoses of follicular lesion of undetermined significance, follicular neoplasm, or suspicious for malignancy (SM). Based on cases that underwent surgical resection (Afirma, n = 37; Rosetta, n = 19; Interpace, n = 14), we calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for risk of malignancy and neoplasia., Results: The three tests performed similarly when predicting risk of malignancy. They showed high sensitivity (80-100%) and NPV (90-100%) but lower specificity (10-64%) and PPV (21-44%). When assessing their value to predict neoplasia, each test had a high PPV (76-89%) but low NPV (20-33%). The sensitivity for neoplasm was intermediate to high (50-93%), and the specificity remained extremely variable (11-67%)., Conclusion: Overall, these molecular platforms performed similarly, displaying high NPV but low to intermediate PPV for malignancy and low NPV but high PPV for neoplasm. The risk of neoplasm is a good index for surgery, and we argue that many of the neoplasms are low-risk tumors. We endorse conservative treatment with lobectomy for cases that are indeterminate at FNA but suspicious by molecular testing., (© 2019 Wiley Periodicals, Inc.)

Background: We report results of a multicenter clinical experience study examining the likelihood of patients with indeterminate thyroid nodules to undergo surgery or have malignant outcome based on multiplatform combination mutation and microRNA testing (MPT)., Methods: MPT assessed mutations in BRAF, HRAS, KRAS, NRAS, and PIK3CA genes, PAX8/PPARγ, RET/PTC1, and RET/PTC3 gene rearrangements, and the expression of 10 microRNAs. Baseline clinical information at the time of MPT and clinical follow-up records were reviewed for 337 patients, of which 80% had negative MPT results. Kaplan Meier analysis for cumulative probability of survival without having a surgical procedure or malignant diagnosis over the course of patient follow-up was determined for MPT results of 180 patients, among which only 14% had malignancy., Results: A negative MPT result in nodules with Bethesda III or IV cytology (2009) conferred a high probability of non-surgical treatment, with only 11% expected to undergo surgery and a high probability of survival without malignancy (92%) for up to 2 years follow up. A positive MPT result conferred a 57% probability of malignancy and was an independent risk factor for undergoing surgical treatment (Hazard Ratio [HR] 9.2, 95% confidence intervals 5.4-15.9, P < .0001) and for malignancy (HR 13.4, 95% confidence intervals 4.8-37.2, P < .0001). For nodules with weak driver mutations, positive microRNA test results supported high risk of cancer while negative results downgraded cancer risk., Conclusion: MPT results are predictive of real-world decisions to surgically treat indeterminate thyroid nodules, with those decisions being appropriately aligned with a patient's risk of malignancy over time., (© 2019 The Authors. Diagnostic Cytopathology published by Wiley Periodicals, Inc.)

Objectives: Real-world clinical results of (1) Bethesda categorization, (2) mutation analysis, and (3) a microRNA classifier were correlated to show the utility of molecular analysis in assessing malignancy risk of indeterminate thyroid nodules., Methods: Cytology and molecular results of clinically tested thyroid nodules were compared. An additional microRNA threshold was determined based on nodules with known disease status, establishing a 3-tiered microRNA approach to clinical risk assessments. Expected rate of malignancy given mutation panel and 3-tiered microRNA approach was validated in an independent cohort of atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm or suspicious for follicular neoplasm (FN/SFN) nodules with surgically derived outcomes., Results: In 2685 patients clinically tested, PIK3CA, PAX8/PPARγ, and RET/PTC mutations occurred in less than 1%. Of note, 2% had BRAFV600E mutation and 82% lacked mutations. The maximum expected risk of malignancy in nodules lacking mutations was 9% and 17% for AUS/FLUS and FN/SFN nodules, respectively. Positive microRNA status further increased risk, with the most worrisome status (level-3) elevating risk to 36% and 54%, respectively. RAS mutations occurred in 15% of nodules tested clinically, including in 8% of those that were cytologically benign. The maximum expected risk of malignancy in nodules with RAS or PAX8/PPARγ mutations was 49% and 65% for AUS/FLUS and FN/SFN nodules, respectively. Positive microRNA status further increased risk, with the most worrisome microRNA status (level-3) elevating risk to 85% and 91%, respectively., Conclusions: Mutation panels alone do not sufficiently risk stratify thyroid nodular disease. microRNA classification complements cytology and mutation analysis with the capacity to better differentiate nodules at high risk of malignancy., (© 2018 Wiley Periodicals, Inc.)

Introduction: Currently, molecular studies are widely used as a guiding tool in further management of cytologically indeterminate thyroid nodules. At our institution, clinicians have recently expressed concern over receiving "less positive molecular results" upon switching from an extended 14 gene mutation panel (EGMP) to a 7 gene mutation panel (GMP). Our goal is to compare outcomes of these two tests in regards to the performance characteristics and clinical impact., Materials and Methods: All thyroid fine-needle aspiration (FNA) biopsy specimens sent for molecular studies from 2016 to 2017 were retrospectively studied. Cytopathology diagnosis, pertinent clinical findings, molecular results, and follow-up (F/U) surgical and cytology diagnoses were recorded., Results: Of the total 165 cases sent for molecular tests 86 (52%) were GMP and 79 (47%) EGMP. There were 21 (24%) and 40 (50%) cases with positive GMP and EGMP results, respectively. Within these positive cases (n = 61), there were a total of 33 (54%) patients who underwent surgical resection and 28 (45%) patients had no follow-up. The molecular findings and surgical pathologic diagnoses obtained are illustrated in Figures 1 through 4 for GMP and EGMP, respectively., Conclusions: The selection of molecular testing should be directed toward optimizing patient care and facilitate clinical management. This quality assurance study helped in understanding the complexities associated with test selection best suited for our institution and in educating clinicians., (© 2018 Wiley Periodicals, Inc.)

Background: Molecular tests are increasingly used to triage cytologically indeterminate thyroid nodules for surgery and/or follow-up. We retrospectively compared the performance of the Afirma Gene Expression Classifier (AGEC) with that of the more recently developed RosettaGX ® Reveal™ miRNA Classifier (Reveal) in a cohort of Bethesda III-V thyroid FNAs with surgical follow-up., Design: Eighty-one samples (54 Bethesda III, 26 Bethesda IV, 1 Bethesda V) with available AGEC (74 AGEC-SUSP and 7 AGEC-BENIGN) and surgical pathology results were studied from three academic centers. Reveal was performed in a blinded fashion., Results: The final diagnoses were benign/NIFTP (n = 63) and malignant (n = 18). The overall "correct" rate was 64.2% for Reveal and 28.4% for AGEC (P = 1.4e-6). The specificity of Reveal was 60.3%, compared with 9.5% for AGEC (P = 2.1e-9). Among the 18 malignant cases, 77.8% and 94.4% were correctly classified as suspicious by Reveal and AGEC, respectively (P = 0.2). In the FLUS and the FN group, the specificity of AGEC was lower than the specificity of Reveal. Whether the 7 NIFTP in our study were considered benign or malignant, specificity and PPV of Reveal were higher than those of AGEC. Reveal also outperformed AGEC in correctly classifying the 26 benign Hürthle lesions studied (P = 7.6e-5)., Conclusion: Reveal outperformed AGEC in this cohort, whether NIFTP is considered benign or malignant, and in Hürthle lesions. Reveal has the potential to reduce the number of unnecessary resections in patients with indeterminate thyroid cytology. Based on our findings and the practical advantages offered by Reveal methodology, large prospective studies are warranted. Diagn. Cytopathol., (© 2018 Wiley Periodicals, Inc.)

Background: Thyroid-stimulating hormone receptor (TSHR) gene mutations play a critical role in thyroid cell proliferation and function. They are found in 20%-82% of hyperfunctioning nodules, hyperfunctioning follicular thyroid cancers (FTC), and papillary thyroid cancers (PTC). The diagnostic importance of TSHR mutation testing in fine needle aspiration (FNA) specimens remains unstudied., Methods: To examine the association of TSHR mutations with the functional status and surgical outcomes of thyroid nodules, we evaluated 703 consecutive thyroid FNA samples with indeterminate cytology for TSHR mutations using next-generation sequencing. Testing for EZH1 mutations was performed in selected cases. The molecular diagnostic testing was done as part of standard of care treatment, and did not require informed consent., Results: TSHR mutations were detected in 31 (4.4%) nodules and were located in exons 281-640, with codon 486 being the most common. Allelic frequency ranged from 3% to 45%. Of 16 cases (12 benign, 3 FTC, 1 PTC) with surgical correlation, 15 had solitary TSHR mutations and 1 PTC had comutation with BRAF V600E. Hyperthyroidism was confirmed in all 3 FTC (2 overt, 1 subclinical). Of 5 nodules with solitary TSHR mutations detected at high allelic frequency, 3 (60%) were FTC. Those at low allelic frequency (3%-22%) were benign. EZH1 mutations were detected in 2 of 4 TSHR-mutant malignant nodules and neither of 2 benign nodules., Conclusion: We report that TSHR mutations occur in ∼5% thyroid nodules in a large consecutive series with indeterminate cytology. TSHR mutations may be associated with an increased cancer risk when present at high allelic frequency, even when the nodule is hyperfunctioning. Benign nodules were however most strongly correlated with TSHR mutations at low allelic frequency., (© 2018 Wiley Periodicals, Inc.)

Background: In fine needle aspiration biopsy (FNAB) of thyroid nodules, LBC is adopted in most of the hospitals and clinics in Korea for its convenience. BRAF mutation test has been introduced as an important ancillary test, but its applicability has not been completely proven with LBC samples., Methods: Five aspirates from thyroidectomy specimens were simultaneously processed into LBC and CS slides, in which BRAF mutation tests were performed using three primer sets with PCR products of 72, 164, and 226 base pairs (bp) at 6, 9, and 12 months after processing. In addition, BRAF mutation tests were performed in nine clinical samples that had been prepared by LBC or CS and stored for 3-5 years after processing., Results: At 9 months after processing, LBC failed to provide DNA of sufficient quality for PCR, whereas CS succeeded with primers for amplifying a 226 bp fragment. Furthermore, CS had successful amplification of DNA despite a delay of more than 1 year. The failure of DNA amplification in LBC was overcome by using primers to amplify shorter PCR products, suggesting that DNA degradation occurred in LBC. However, false positive or negative results were observed in primers for amplifying shorter size. The kind of preservative solutions used in LBC did not affect test results., Conclusion: LBC may have disadvantages in long-term DNA preservation because of its accelerated DNA degradation compared with alcohol-fixed CS. Using primers to amplify shorter size fragments might be helpful in mitigating loss of signal due to DNA degradation in LBC., (© 2016 Wiley Periodicals, Inc.)

Background: The measurement of calcitonin in washout fluids of thyroid nodule aspirate (FNA-calcitonin) has been reported as accurate to detect medullary thyroid carcinoma (MTC). The results from these studies have been promising and the most updated version of ATA guidelines quoted for the first time that "FNA findings that are inconclusive or suggestive of MTC should have calcitonin measured in the FNA washout fluid." Here we aimed to systematically review published data on this topic to provide more robust estimates., Research Design and Methods: A comprehensive computer literature search of the medical databases was conducted by searching for the terms "calcitonin" AND "washout." The search was updated until April 2015., Results: Twelve relevant studies, published between 2007 and 2014, were found. Overall, 413 thyroid nodules or neck lymph nodes underwent FNA-calcitonin, 95 were MTC lesions and 93 (97.9%) of these were correctly detected by this measurement regardless of their cytologic report., Conclusions: The present study shows that the above ATA recommendation is well supported. Almost all MTC lesions are correctly detected by FNA-calcitonin and this technique should be used to avoid false negative or inconclusive results from cytology. The routine determination of serum calcitonin in patients undergoing FNA should improve the selection of patients at risk for MTC, guiding the use of FNA-calcitonin in the same FNA sample and providing useful information to the cytopathologist for the morphological assessment and the application of tailored ancillary tests., (© 2015 Wiley Periodicals, Inc.)

TROP-2 is a type I transmembrane glycoprotein which is over-expressed in various malignancies, and is related to epithelial cell adhesion molecule (EpCAM), also called TROP-1, gp40, and KSA. In this study, we evaluated TROP-2 expression in papillary thyroid carcinoma (PTC) and compared it to other thyroid neoplastic and non-neoplastic lesions. Immunohistochemical (IHC) evaluation for TROP-2 was performed on 137 thyroid fine needle aspiration (FNA) cell blocks (CB) which included classic PTC (64), follicular variant PTC (FVPTC) (10), anaplastic thyroid carcinoma (AC) (2), medullary carcinoma (MC) (8), follicular neoplasms (FN) (8), Hurthle cell neoplasms (HCN) (9), follicular lesion of uncertain significance (FLUS) (12), and benign thyroid nodule (BTN) (24). IHC for TROP-2 expression was also performed on 331 BTN and malignant tumor tissue sections in tissue microarray (TMA). Membranous staining in >5% of tumor cells was considered positive. TROP-2 stained 61 of 64 PTC CB, 7 of 10 FVPTC CB, and 9 of 12 FLUS CB. All other cases were negative for TROP-2. TROP-2 showed a sensitivity of 95.31% and specificity of 89% for classic PTC in FNA CB. In TMA samples, TROP-2 stained 54 of 60 classic PTC cases and hence showed a high sensitivity and specificity. All BTN in CB and TMA were negative. We conclude that TROP-2 is a highly sensitive and specific IHC marker for identifying classic PTC. TROP-2 may play an important role in diagnosing classic PTC, especially in equivocal cases. This study also identifies a strong role for TROP-2 in separating PTC from BTN., (© 2015 Wiley Periodicals, Inc.)

Lung adenocarcinoma and papillary thyroid carcinoma (PTC) share a number of microscopic and immunophenotypical features. Thus, patients presenting with thyroid and lung synchronous neoplasms may be difficult on fine-needle aspiration (FNA) samples to define the site of origin of the malignancy. In the case reported here, inherent to a 57-years-old man presenting with a right lung mass and a large (44 mm) thyroid nodule, an integrated cytological, immunocytochemical and molecular approach enabled to clarify the primary nature of the neoplasm. FNA cytology showed in both sites papillary structures and nuclear changes reminiscent of PTC. The lung origin of the neoplasm was suggested on cell-block immunocytochemistry showing thyroid transcription factor-1 positive and PAX8 and TGB negative neoplastic cells. Next generation sequencing performed on the Ion Torrent platforms by the Ion Ampliseq Colon and Lung Cancer panel showed a similar genomic profile in both neoplastic sites with a concurrent KRAS G12C mutation. An integrated approach on FNA biospecimen is safe, cost effective, and may be coupled effectively with modern ancillary molecular techniques that may be useful, besides their predictive value, as a adjunctive diagnostic tool when the synchronous occurrence of lesions featuring overlapping morphologies challenge the cytopathologist., (© 2015 Wiley Periodicals, Inc.)

A genetic link between cutaneous melanoma and thyroid cancer (TC) has been identified. A high percentage of both melanomas and papillary carcinomas of the thyroid harbors a recurrent mutation (i.e., BRAF(V600E) ) in the BRAF oncogene. Herein, we report the case of a 65-year-old man with papillary TC and cutaneous malignant melanoma metastatic to masseter muscle, both characterized by BRAF mutation. This is one of the rare reports in which a complete molecular characterization has been performed. As the patients with papillary thyroid carcinoma have a higher risk of malignant melanoma and vice versa, continuous monitoring of such patients, with either of these tumors is necessary. Fine-needle aspiration cytology is useful as shown in the present case., (© 2013 Wiley Periodicals, Inc.)

Fine-needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, approximately 5-20% of thyroid nodules are considered indeterminate or suspicious cases that do not meet clinical standards. The B-RAF(V600E) mutation has been reported in FNA specimens. We conducted a systematic review to evaluate the diagnostic value of testing for B-RAF(V600E) in thyroid nodules that are difficult to diagnose by FNA. A systematic literature search was performed from January 1, 2002 to June 30, 2012. Articles were obtained by searching two electronic databases (MEDLINE and EMBASE), hand searching selected journals, and contacting authors. Article quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool. Sensitivity, specificity, and other measures of accuracy were pooled using random effects models. Summary receiver operating characteristic (SROC) curves were used to summarize overall diagnostic accuracy. A total of 16 studies incorporating 1131 patients were included in a meta-analysis on diagnostic accuracy of B-RAF(V600E) tests. Pooled sensitivity was 0.60 (95% confidence interval [CI]: 0.556-0.634), pooled specificity was 0.99 (95% CI 0.976-0.997), and the area under the curve of the SROC curve was 0.8376. Q index value was 0.7696. Our data suggest a potentially useful adjunct to evaluating thyroid nodules that are difficult to diagnose. The B-RAF(V600E) test has a high positive predictive value and could help clinicians formulate a more individualized treatment schedule. When supplemented with other noninvasive test methods, the B-RAF(V600E) test could be a powerful adjunct with extensive clinical applications., (Copyright © 2013 Wiley Periodicals, Inc.)

V600E BRAF mutation is emerging as an independent marker of papillary thyroid carcinoma aggressive behavior. Papillary thyroid carcinomas harboring this mutation should be extensively resected. However, this requires an unquestionable cytological diagnosis of malignancy. Thus, cytological specimens should be properly handled to provide both morphological and molecular information. Here, we assessed whether our method of preparation of fine-needle aspiration material is suitable for both tests. A series of 128, routinely performed, fine-needle aspirations was analyzed. Each nodule was punctured three times. A representative Diff-Quik smear prepared from the first two passages was evaluated onsite. When microscopy was diagnostic (n = 44), the third needle pass was dedicated to harvest material for BRAF testing; in the remaining cases (n = 84), additional direct smears for cytology were prepared and the remaining material in the needle plus the needle rinsing was collected for BRAF testing. Cellularity was adequate in 126/128 (98%) cases. Cytological diagnoses were inadequate (2%), benign (85%), follicular lesion of undetermined significance (5%), follicular neoplasms (2%), suspicious for malignancy (2%), and malignant (4%). Higher average of extracted DNA concentration was observed in the dedicated pass group (25.9 vs 7.95 ng/microl). However, the rate of successful exon 15 BRAF amplification was similar with (43/44; 97.7%) or without (79/84; 94%) the dedicated pass. Thus, our protocol is suitable for both tests. Whenever necessary BRAF testing may also be performed on the residual samples of thyroid nodules, without interfering with routine cytology.

Thyroid nodules are frequent in clinical practice and fine-needle aspiration biopsy (FNAB) is widely used for its evaluation, but approximately 20% of the cases are diagnosed as indeterminate for malignancy. Aspirates from thyroid nodules can be used for ancillary methods, but molecular techniques are not routinely applied to these specimens. Forty-six consecutive, routinely performed, FNAB of thyroid nodules were evaluated for the feasibility of applying RT-PCR method. RNA was extracted from 1 of 3 fresh residual samples and analyzed to determine its pureness, integrity, and concentration. Cellularity was adequate in all 46, except one, specimens analyzed, scored as 0, 1+, 2+, 3+, and 4+ in 1, 10, 14, 9, and 8 cases, respectively. Thirty-three nodules measured less than 1.5 cm. Cytological diagnosis was positive for malignancy in 3 cases, indeterminate for malignancy in 3, most probably benign follicular lesion in 7, negative for malignancy in 32, and suggestive of benign follicular lesion in 1. Good quality RNA was successfully isolated in 45/46 (97.8%) samples, with an average RNA concentration of 14 ng/microl and detection of B2M mRNA in 97.7% (44/45). There was no significant correlation between RNA concentration and nodule size or specimen cellularity. In conclusion, molecular analysis using individual, residual samples of thyroid nodules aspirates is feasible and could be employed for molecular preoperative studies in the future, adding elements for final cytological diagnosis of indeterminate cases, without altering the routine procedure., ((c) 2008 Wiley-Liss, Inc.)