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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.)

For the first time the 2023 version of The Bethesda System for Reporting Thyroid Cytology dedicates a whole chapter (chapter 14) to ancillary studies almost exclusively represented by molecular testing. The latest data reported bring some evidence that molecular testing could help to optimize the diagnostic performance of « indeterminate » categories (AUS and NF). Other studies suggest a promising role to guide the management of suspicious of malignancy and malignant categories. Indeed, the recognition of prognostic and predictive biomarkers analyzed on cytological samples, regardless of how it is collected, has progressed thanks to advances in our knowledge of molecular abnormalities of thyroid tumors. The chapter 14 is presented here highlighting the current and emerging roles of « in-house » and commercialized molecular testing as presented by TSBRTC., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Objective To explore the diagnostic efficacy of American Thyroid Association(ATA)guidelines,American College of Radiology Thyroid Imaging Report and Data System(ACR-TIRADS),and Chinese Thyroid Imaging Reporting and Data System(C-TIRADS)alone and combined with BRAF V600E mutation in atypia of undetermined significance/follicular lesion of undetermined significance(AUS/FLUS).Methods A total of 138 patients who underwent ultrasound-guided fine needle aspiration(FNA)in the Chinese PLA General Hospital from January 2020 to May 2023 were selected.The clinicopathological and ultrasound characteristics were retrospectively analyzed for each nodule.Each nodule underwent preoperative BRAF V600E mutation testing and was diagnosed according to the ATA guidelines,ACR-TIRADS,and C-TIRADS.The diagnostic efficacy of ATA guidelines,ACR-TIRADS,and C-TIRADS alone and combined with BRAF V600E mutation was assessed based on the results of histopathological diagnosis.Results The 138 AUS/FLUS thyroid nodules included 45(32.6%)benign ones and 93(67.4%)malignant ones.The patient age( t =1.444, P =0.151),gender( χ 2 =0.259, P =0.611),and location of nodules( χ 2 =2.055, P =0.358)had no statistical significance for the differentiation between benign and malignant nodules,while nodule size( Z =2.500, P =0.012),echo( χ 2 =14.693, P <0.001),composition( χ 2 =17.075, P <0.001),aspect ratio ≥1( χ 2 =9.477, P =0.002),and microcalcification( χ 2 =6.892, P =0.009)were of significance for the differentiation.When applied alone,BRAF V600E mutation showed high specificity(95.56%)and positive predictive value(95.65%).Among the three ultrasound grading systems,ACR-TIRADS had the highest sensitivity( χ 2 =37.923, P <0.001; χ 2 =40.462, P <0.001)and accuracy( χ 2 =81.595, P <0.001; χ 2 =76.912, P <0.001),while C-TIRADS had the highest specificity( χ 2 =11.746, P <0.001; χ 2 =21.235, P <0.001).However,the three systems showed no statistically significant difference in the diagnostic efficiency when applied alone( Z =1.177, P =0.239; Z =0.213, P =0.831; Z =1.016, P =0.310).The combination of BRAF V600E mutation with ACR-TIRADS or C-TIRADS improved the diagnostic efficacy of BRAF V600E mutation in distinguishing the benign and malignant AUS/FLUS nodules( Z =2.107, P =0.035; Z =2.752, P =0.006).The combination of ATA guidelines with BRAF V600E mutation increased the diagnostic accuracy of BRAF V600E mutation( χ 2 =20.679, P <0.001),while it had no statistically significant difference in distinguishing the benign and malignant AUS/FLUS nodules( Z =1.321, P =0.186).The combination of ATA guidelines,ACR-TIRADS,or C-TIRADS with BRAF V600E mutation improved the diagnostic efficacy of ultrasound grading systems for AUS/FLUS nodules( Z =2.770, P =0.006; Z =2.770, P =0.006; Z =2.890, P =0.004).Specifically,ACR-TIRADS combined with BRAF V600E mutation showed the highest sensitivity( χ 2 =4.712, P =0.030; χ 2 =4.712, P =0.030),while C-TIRADS combined with BRAF V600E mutation showed the highest accuracy( χ 2 =77.627, P <0.001; χ 2 =85.827, P <0.001).However,there were no statistically significant differences in diagnostic performance between the combinations( Z =1.276, P =0.202; Z =0.808, P =0.419; Z =1.615, P =0.106).Conclusion ATA guidelines,ACR-TIRADS,and C-TIRADS combined with BRAF V600E mutation can improve the diagnostic efficacy of BRAF V600E mutation or ultrasound grading system alone in AUS/FLUS nodules,which can facilitate the further management and treatment of such patients.

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.)

Introduction: Chronic lymphocytic thyroiditis (CLT) may increase the likelihood of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) results in thyroid nodules by fine needle aspiration (FNA). Gene expression classifier (GEC) and Thyroid Sequencing (ThyroSeq) may better stratify rate of malignancy (ROM) of AUS/FLUS thyroid nodules. This study compares the utility of molecular tests in determining malignancy in surgical patients with concomitant AUS/FLUS thyroid nodules and CLT., Methods: A retrospective review of 1648 patients with index thyroid nodules who underwent FNA and thyroidectomy at a single institution was performed. Patients with concomitant AUS/FLUS thyroid nodules and CLT were subdivided into three diagnostic groups: FNA only, FNA with GEC, and FNA with ThyroSeq. Patients with AUS/FLUS thyroid nodules without CLT were subdivided into similar groups. Final histopathology of the cohorts was further stratified into benignity and malignancy and analyzed using Chi-squared statistics., Results: Of 463 study patients, 86 had concomitant AUS/FLUS thyroid nodules and CLT with a 52% ROM, and the difference of ROM among FNA only (48%), suspicious GEC (50%), or positive ThyroSeq (69%) was not significant. In 377 patients with AUS/FLUS thyroid nodules without CL, ROM was 59%. ROM among these patients was significantly higher when molecular testing was used (FNA only 51%, suspicious GEC 65%, and positive ThyroSeq 68%; P < 0.05)., Conclusions: Molecular tests may have limited value in predicting malignancy in surgical patients with concomitant AUS/FLUS thyroid nodules and CLT., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Although pediatric thyroid tumors have many similarities to those occurring in adults, significant differences are also recognized. For example, although thyroid nodules in children are much less common than in adults, a higher percentage is malignant. Moreover, while pediatric papillary thyroid carcinoma (PTC) is associated with more advanced disease, death due to disease in children and adolescents is very rare, even when distant metastases are present. Some subtypes of thyroid carcinoma, like diffuse sclerosing variant, are especially common in children and adolescents. Moreover, certain histologic findings, such as a tall cell morphology or increased mitotic activity, may not carry the same prognostic significance in children as in adults. Recent studies exploring the molecular underpinnings of pediatric thyroid carcinoma indicate that while driver alterations of thyroid tumorigenesis in children and adults are essentially the same, they occur at very different frequencies, with translocation-associated tumors (most commonly harboring RET and NTRK fusions) comprising a sizable and distinct group of pediatric PTC. DICER1 mutations, an infrequent mutation in adult thyroid tumors, are relatively frequent in pediatric encapsulated follicular-patterned thyroid tumors (with or without invasion or nuclear features of PTC). Additionally, tumor predisposition syndromes (most notably DICER1 syndrome and PTEN hamartoma tumor syndromes such as Cowden syndrome) should be considered in children with thyroid tumors, especially follicular-patterned thyroid tumors and poorly differentiated thyroid carcinoma. This review will explore the current state of knowledge of thyroid nodules and carcinomas in children and adolescents., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The increase in the incidence of thyroid nodules with cytological findings of TIR3b requires the identification of predictive factors of malignancy. We prospectively evaluated 2160 patients from January 2018 to June 2022 and enrolled 103 patients with indeterminate cytology TIR3b nodules who underwent total (73 patients) and hemi-thyroidectomy (30 patients). Among them, 61 had a histological diagnosis of malignancy (30 classic papillary thyroid carcinoma, 19 had follicular papillary thyroid carcinoma variant, 3 had Hurtle cell carcinoma and 9 had follicular thyroid carcinoma), while 42 had a benign histology. Clinical, ultrasonographic and cytological characteristics were recorded. In addition, BRAF mutation was analysed. Patients with a histological diagnosis of malignancy had a higher frequency of nodule diameter ≤11 mm ( p = 0.002), hypoechogenicity ( p < 0.001), irregular borders ( p < 0.001), peri- and intralesional vascular flows ( p = 0.004) and microcalcifications ( p = 0.001) compared to patients with benign histology. In contrast, patients with benign histology had more frequent nodules with a halo sign ( p = 0.012) compared to patients with histological diagnosis of malignancy. No significant differences were found in BRAF mutation between the two groups. Our study suggests that the combination of ultrasonographic and cytological data could be more accurate and reliable than cytology alone in identifying those patients with TIR3b cytology and a histology of malignancy to be referred for thyroidectomy, thus reducing the number of patients undergoing thyroidectomy for benign thyroid disease.

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: Evidence guiding the management of cytologically indeterminate thyroid nodules with nondiagnostic (ND) or benign cytology on repeat fine-needle aspiration (FNA) is limited. This study evaluates the utility of molecular testing and estimates the risk of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and cancer among such nodules., Methods: This was a retrospective single-institution review of thyroid nodules from adults that were classified as atypia of undetermined significance (AUS) or follicular neoplasm (FN) on initial FNA and underwent repeat FNA for cytology and Afirma testing (June 2013-July 2021). The association between repeat FNA cytology and RNA yield for Afirma was determined. Histologic outcomes were integrated with Afirma results to define end points for each nodule., Results: A total of 691 AUS and FN nodules underwent repeat FNA and Afirma testing. Diagnostic Afirma results were obtained in 98% of cases overall and in 91% of nodules with ND cytology on repeat FNA. Using combined molecular and histologic end points, the NIFTP and/or cancer prevalence for nodules with ND cytology on repeat FNA was 9% (95% confidence interval [CI], 0.042-0.182), falling between those nodules classified as benign (5%; 95% CI, 0.029-0.094) and those classified as AUS or FN (18%; 95% CI, 0.140-0.218) on repeat FNA, although not reaching statistical significance from either subgroup (p = .38 and .10, respectively)., Conclusions: AUS and FN nodules that are ND on repeat FNA have low but nonnegligible risk of NIFTP and/or cancer and may benefit from molecular testing, given the low test failure rate in this subgroup. Conversely, AUS and FN nodules reclassified as benign on repeat FNA have a very low risk of NIFTP and/or cancer and are unlikely to benefit from molecular testing., (© 2023 American Cancer Society.)

Purpose: To explore the utility of the BRAF V600E mutation in combination with the Chinese Thyroid Imaging Reporting and Data System (C-TIRADS) in the management of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) thyroid nodule (TN)., Methods: 138 AUS/FLUS TNs in 129 patients were included. Each TN underwent preoperative BRAF V600E mutation analysis and was classified using the C-TIRADS. Histopathologic diagnosis served as reference standard., Results: 46 benign TNs and 92 malignant TNs were identified. The C-TIRADS 4C and 5 (OR = 10.409, P = 0.000), BRAF V600E mutation (OR = 36.493, P = 0.000) were independent predictors of malignant nodules. There were significant differences in malignancy rate among the different C-TIRADS TNs (P = 0.000), and these TNs with higher C-TIRADS were associated with increased malignancy rate (P for trend = 0.000). The rate of the nodule with BRAF V600E mutation increased with the increase of C-TIRADS (P for trend = 0.001). For AUS/FLUS TNs without BRAF V600E mutation, the malignancy rates of the C-TIRADS 3, 4A, 4B, 4C, and 5 were 0%, 21.4%, 20.8%, 70.8%, and 100%, respectively (P = 0.000), and the malignancy rate increased from C-TIRADS 3 to C-TIRADS 5 (P for trend = 0.000). C-TIRADS and BRAF V600E mutation had similar diagnostic efficacy (P > 0.05), and the sensitivity, negative predictive value, and accuracy of the combination were significantly higher than BRAF V600E gene or C-TIRADS alone (P < 0.05)., Conclusions: C-TIRADS can effectively provide risk stratification for AUS/FLUS nodules. The combination is helpful in selecting appropriate management for AUS/FLUS patients., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Data on Afirma's genomic sequencing classifier (GSC) performance in atypia of undetermined significance (AUS) subcategories is limited. This study investigated GSC performance in AUS nodules with architectural atypia (AUS-A), cytological atypia (AUS-C), architectural and cytological atypia (AUS-AC), and predominantly Hürthle cells (AUS-HC)., Methods: This study retrieved consecutive thyroid nodules having a recurrent cytologic diagnosis of AUS with qualifiers and a concurrent GSC diagnostic result. All nodules were followed by either surgical intervention or clinical and/or ultrasound monitoring (≥6 months). GSC benign call rate (BCR), rate of histology-proven malignancy, and diagnostic parameters of GSC were calculated for individual AUS subcategories. Statistical analysis was performed using the Fisher exact test., Results: A total of 135 AUS nodules fulfilled inclusion criteria, including 79 AUS-A, 9 AUS-C, 29 AUS-AC, and 18 AUS-HC. BCR was 72.2%, 66.7%, 44.8%, and 77.8% in AUS-A, AUS-C, AUS-AC, and AUS-HC, respectively. AUS-A showed a greater BCR than AUS-AC (p &lt; .05). All GSC-benign nodules were considered benign on clinical or surgical follow-up. Among GSC-suspicious nodules, histology-proven malignancies represented 4.5% of AUS-A, 0% of AUS-C, 56.3% of AUS-AC, and 25.0% of AUS-HC cases. AUS-AC demonstrated a higher malignant rate compared with AUS-A (p &lt; .05). GSC offers 100% NPV and a wide range (5%-56%) of PPV across all AUS subcategories. AUS-AC demonstrated a greater PPV compared with AUS-A (p &lt; .05)., Conclusion: BCR of GSC and malignant rates associated with suspicious GSC may differ in various AUS subcategories. GSC-suspicious nodules with both architectural and cytologic atypia are more likely to be malignant. These findings may improve clinical triage and/or management of patients with AUS thyroid nodules., (© 2022 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

The incidence of cancer in thyroid nodules carrying germline or somatic phosphatase and tensin homolog (PTEN) mutations is not well-defined. This study characterizes the clinical and histopathologic features of thyroid nodules with preoperatively detected PTEN mutations and their impact on management. Thyroid nodules with PTEN mutations on molecular testing of fine-needle aspiration (FNA) specimens from November 2017 to July 2020 at our institution were included. Demographic and clinicopathologic data were obtained through retrospective chart review. We identified 49 PTEN mutation-positive nodules from 48 patients. Surveillance was pursued for 28 patients and surgery for 20 patients. There were 14 follicular adenomas (FA), 4 oncocytic adenomas, 1 oncocytic hyperplastic nodule, and 1 encapsulated follicular variant papillary thyroid carcinoma (EFVPTC). The EFVPTC had two somatic PTEN mutations, an NRAS mutation, and was a low-risk tumor with capsular but no angiolymphatic invasion. Four patients, all with multiple nodules, had PTEN hamartoma syndrome (PHTS) with germline mutations or a clinical diagnosis of Cowden syndrome (CS); two had surgery finding FAs, and one previously had follicular carcinoma removed. Among surveillance patients, 1/20 had a significant increase in the size of the thyroid nodule and underwent repeat FNA, and no thyroid malignancy was found with a mean of 1.77 years of follow-up (range 1.00-2.78). Thyroid nodules with isolated somatic PTEN mutations are primarily benign and unlikely to grow at a high rate, at least on short-term follow-up. About 8% of patients with PTEN mutations may have PHTS or CS, which should be suspected in younger patients with multiple thyroid nodules.

EIF1AX gene mutations are reported in both benign and malignant thyroid tumors, with unclear outcomes when detected preoperatively. The aim of this study was to determine the features and outcomes of thyroid nodules with various types of mutation identified in cytologic (fine-needle aspiration) samples on preoperative ThyroSeq testing and with surgical outcomes. In this single-institution retrospective study of 31 consecutive patients, 77% were female and nodule size ranged from 1.5 to 9.4 cm with widely varying cytologic and TI-RADS ultrasound categorizations. Among two main mutational hotspots, 55% were located in exon 2 and 45% at the intron 5/exon 6 splice site. On histology, 45% of -positive nodules were cancer/noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) including 19% encapsulated follicular variant papillary thyroid carcinoma, 10% follicular carcinoma, 10% anaplastic carcinoma (ATC), and 7% NIFTP. Almost half (48%) of patients had one or more coexisting mutations, most frequently RAS. The prevalence of cancer/NIFTP was 80% for mutation with coexisting molecular alteration vs 13% with an isolated mutation (P = 0.0002). Cancer probability was associated with mutation type and was 64% for splice-site mutation and 29% for non-splice mutation (P = 0.075). All 3 nodules with EIF1AX+RAS+TERT+TP53 mutations were ATC. In summary, in this study, all nodules with an isolated non-splice mutation were benign, one-third of those with an isolated splice mutation were cancer, and most nodules with coexisting with RAS or other alterations were malignant. These findings suggest that clinical management decisions for patients with EIF1AX-mutant nodules should consider both the type of mutation and its co-occurrence with other genetic alterations.

Background: The American Thyroid Association recommends either repeat fine-needle aspiration biopsy (FNAB) or molecular testing (eg, ThyroSeq) of Bethesda category III (atypia of undetermined significance/follicular lesion of undetermined significance [AUS/FLUS]) nodules to provide further risk stratification. How a testing algorithm that uses ancillary molecular tests performs as a reflex test for repeat sampling of indeterminant nodules remains unclear., Methods: Thyroid FNABs performed over a 24-month period that received a diagnosis of AUS/FLUS and underwent subsequent FNAB were analyzed., Results: In total, 187 patients were identified who received an FNAB diagnosis of AUS/FLUS and had repeat sampling. Of these patients, 64% received a subsequent indeterminant diagnosis on repeat biopsy: 7 (3.7%) repeat biopsies were diagnosed as nondiagnostic/unsatisfactory, 104 (55.6%) were diagnosed as AUS/FLUS, and 8 (4.3%) were diagnosed as follicular neoplasm/suspicious for follicular neoplasm. Of the repeat biopsied nodules, 63% underwent subsequent testing with ThyroSeq version 3. The diagnostic performance was calculated using only surgically confirmed nodules (sensitivity, 100%; specificity, 30%; positive predictive value, 41%; negative predictive value, 100%) and by assigning nonresected nodules with negative ThyroSeq or benign cytology results as benign (sensitivity, 100%; specificity, 88%; positive predictive value, 41%; negative predictive value, 100%)., Conclusions: In the majority of patients, repeat FNAB for AUS/FLUS did not preclude subsequent molecular ancillary testing because of the high rate of indeterminant results on repeat biopsy. The diagnostic performance of the testing algorithm reported here was very similar to other reports using either repeat biopsy or molecular testing alone. Ultimately, the algorithm of performing molecular testing on repeat indeterminant nodules increased the number of biopsies performed and lengthened the time to definitive risk stratification without a disproportionate decrease in the use of molecular testing or an appreciable improvement in diagnostic performance., (© 2022 American Cancer Society.)

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: Molecular testing (MT) of thyroid fine-needle aspiration (FNA)-derived genetic material is commonly used to assess malignancy risk for indeterminate cases. The Bethesda System for Reporting Thyroid Cytopathology (TBS) provides limited guidance for the appropriate use of category III (atypia of undetermined significance [AUS]). The authors combined MT with cytomorphology to monitor AUS diagnoses in a cytopathology laboratory., Methods: Neoplasia-associated genetic alterations (NGAs) were determined by MT of preoperative FNA biopsies or resected malignancies and were categorized as BRAF V600E mutations, RAS-like mutations (HRAS, NRAS, or KRAS mutations or non-V600E BRAF mutations), or other mutations., Results: Among 7382 thyroid FNA biopsies, the AUS rate was 9.3% overall and ranged from 4.3% to 24.2% among 6 cytopathologists (CPs) who evaluated >150 cases. The ratio of specimens falling into TBS category III to specimens falling into category VI (malignant) (the III:VI ratio) was 2.4 overall (range, 1.1-8.1), and the ratio of specimens falling into TBS categories III and IV (follicular neoplasm or suspicious for follicular neoplasm) combined (III+IV) to specimens falling into category VI (the [III+IV]:VI ratio) was 2.9 overall (range, 1.4-9.5). MT was performed on 588 cases from 560 patients (79% women) with a median age of 56 years (range, 8-89 years). BRAF V600E mutation was the most common (76% of cases) in TBS category VI and was rare (3%) in category III. RAS-like mutations were most common in TBS categories III (13%), IV (25%), and V (suspicious for malignancy) (17.5%). The NGA rate in AUS cases fell between 5% and 20% for 5 of 6 CPs and did not correlate with the III:VI ratio or the (III+IV):VI ratio., Conclusions: Lack of correlation between the NGA rate and easily calculable diagnostic ratios enables the calibration of diagnostic thresholds, even for CPs who have normal metrics. Specifically, calculation of the NGA rate and the III:VI ratio may allow individual CPs to determine whether they are overcalling or undercalling cases that other CPs might otherwise recategorize., (© 2021 American Cancer Society.)

Objective: American Thyroid Association (ATAn) 2015 guidelines recommend repeat fine-needle aspiration with molecular marker profiling (MMP) or diagnostic lobectomy in thyroid nodules yielding atypia of unknown significance/follicular lesion of unknown significance (AUS/FLUS) or follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) cytology. Our objective is to describe the molecular profiles and histological correlates of these cytologically indeterminate nodules (CIN) to aid risk stratification., Design: Retrospective chart review., Patients: Adults with CIN that underwent MMP from 2017 to 2020., Measurements: Pearsons' χ 2 , Fisher's exact test, nonparametric testing and multiple regression analysis were performed., Results: A total of 89 CIN underwent mutational analysis. Of 55% (n = 49) were Bethesda class III AUS/FLUS and 45% (n = 40) were Bethesda class IV FN/SFN. The US phenotype of a CIN was isoechoic (53%) or hypoechoic (32%) with well-defined margins (98%), absence of calcifications (75%) and mildly increased internal vascularity (70%). A total of 84% and 87% of nodules were classified as mild/moderate or low/intermediate risk per the Thyroid Imaging Reporting and Data System and ATA classifications, respectively. Based on the Thyroseq patient management resource, 6.7% (n = 6) of nodules had a high predicted probability of cancer (≥95%), 41.6% (n = 37) were intermediate probability (40%-94%) and 51.7% (n = 46) were low probability (<40%). MMP revealed positive mutations in 45% (n = 40) of nodules, with 71% demonstrating RAS mutations. Of the nodules that underwent resection (n = 38), 39% (n = 15) had malignant pathology. Increasing the threshold to recommend surgical resection to a Thyroseq predicted probability of cancer to ≥50%, had a 100% sensitivity and 65% specificity for detecting malignant nodules (area under the ROC curve: 0.86). The positive predictive value was 37% and the negative predictive value was 100%., Conclusion: US phenotypes of CIN nodules were variable and did not aid in differentiating malignant from benign nodules. Of the CIN nodules with a positive MMP, most were RAS and had a benign pathology. With the exception of high-risk genetic markers for malignancy, the threshold to recommend surgical resection should be raised for CIN. Further studies to improve risk stratification in these nodules are required., (© 2021 John Wiley & Sons Ltd.)

Background: The Afirma Genomic Sequencing Classifier uses whole transcriptome RNA sequencing to identify thyroid nodules as benign or suspicious. The Afirma Xpression Atlas became available in 2018 and reports findings across 593 genes, including 905 variants and 235 fusions. When an alteration is identified, its risk of malignancy and associated neoplasm type is listed. We report the results of Afirma Xpression Atlas testing at our institution during its first 2 years of clinical use., Methods: All patient charts with indeterminate thyroid nodules and Afirma Xpression Atlas results at our institution were reviewed. Thyroid nodule characteristics, cytology, Afirma Genomic Sequencing Classifier results, Afirma Xpression Atlas results, and final histopathology were reported., Results: Afirma Xpression Atlas was performed on 136 indeterminate nodules since May 2018, and 103 met inclusion criteria. Forty-three nodules had positive Afirma Xpression Atlas results, and of these, 83.7% were follicular cell-derived thyroid cancer on surgical histopathology. This is similar to the overall 82.5% positive predictive value among Afirma Genomic Sequencing Classifier-suspicious indeterminate nodules during the same time period. Of the 60 nodules with negative Afirma Xpression Atlas, 73.3% were follicular cell-derived thyroid cancer on surgical histopathology., Conclusion: Afirma Xpression Atlas positivity is predictive of follicular cell-derived thyroid cancer, but its positive predictive value is similar to that of Genomic Sequencing Classifier-suspicious results alone at our institution, which is higher than previously published. Specific mutations likely predict follicular cell-derived thyroid cancer with higher accuracy, but our current sample size of any given mutation is too small to evaluate this further. Larger studies are needed to determine whether Afirma Xpression Atlas results predictably inform the risk of malignancy and tumor characteristics in thyroid nodules., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 American Cancer Society.)

Background: The Bethesda System for Reporting Thyroid Cytopathology contains an atypia of undetermined significance (AUS) category with heterogeneous and distinct inclusion criteria. The purpose of this study was to investigate differences in malignancy rates and molecular alterations based on the presence of different criteria., Methods: A laboratory information search was conducted to identify thyroid fine-needle aspiration specimens signed out as AUS. The cases were reclassified as architectural atypia (3A), cytologic atypia (3C), both architectural and cytologic atypia (3B), or Hürthle cell aspirate (3H). Surgical follow-up and concurrent molecular test results, if available, were collected., Results: Five hundred ten specimens, including 258 reclassified as 3A, 40 reclassified as 3B, 119 reclassified as 3C, and 86 reclassified as 3H, were identified. The risks of malignancy for the subcategories were 13.4%, 26.3%, 44.1%, and 13.8%, respectively. Additionally, BRAF V600E mutations were more prevalent in specimens with cytologic atypia (3B/3C), whereas low-risk alterations, including KRAS, PTEN, and PAX8-PPARy2, were more prevalent in those with architectural atypia (3A)., Conclusions: Subdividing AUS specimens on the basis of the type of atypia can yield categories associated with distinct molecular alterations and risks of malignancy., (© 2021 American Cancer Society.)

Background: The Bethesda System for Reporting Thyroid Cytopathology has 6 diagnostic categories, each with an implied cancer risk of malignancy (ROM). Bethesda III, defined as atypia or follicular lesions of undetermined significance (AUS/FLUS) on fine needle aspiration (FNA), has an indeterminate ROM. This study investigates the utility of Afirma Gene Expression Classifier (GEC) and Thyroid Sequencing (ThyroSeq) molecular testing to predict malignancy in AUS/FLUS thyroid nodules., Methods: A retrospective review of prospectively collected data of 1457 patients with index thyroid nodules who underwent FNA and thyroidectomy at a single academic institution was performed. Use of GEC or ThyroSeq for AUS/FLUS thyroid nodules was examined. GEC testing was reported benign or suspicious for malignancy whereas ThyroSeq testing was reported on a spectrum of low, intermediate or high ROM. Descriptive statistics were utilized to compare the ROM among AUS/FLUS thyroid nodules., Results: Of 1457 patients with FNA thyroid cytology, 359 (25%) corresponded to AUS/FLUS results. There were 132 (37%) patients with GEC testing and 88 (24%) had ThyroSeq testing. ROM without GEC or ThyroSeq testing was 49%, whereas ROM with suspicious GEC was 55%. ROM with positive ThyroSeq was 73%. Among ThyroSeq patients, 43 had intermediate-risk mutations with 60% malignancy, and 23 had high-risk mutations with 96% malignancy (P < 0.01)., Conclusion: Surgical patients with AUS/FLUS thyroid nodules have a high ROM. High-risk ThyroSeq testing may have some utility in predicting malignancy, but GEC and intermediate-risk TGC results have limited value. Surgeons should carefully consider the utility of molecular tests to determine surgical resection., (Copyright © 2021. Published by Elsevier Inc.)

Objective: Mutations in the EIF1AX gene have been recently detected in a small percentage of benign and malignant thyroid lesions. We sought to investigate the prevalence and clinical significance of EIF1AX mutations and co-mutations in cytologically indeterminate thyroid nodules at our institution., Materials and Methods: A 5-year retrospective analysis was performed on thyroid nodules with a cytologic diagnosis of Bethesda category III or IV, which had undergone testing by our in-house next generation sequencing panel. Surgically resected nodules with EIF1AX mutations were identified, and mutation type and presence of co-mutations were correlated with histopathologic diagnosis., Results: 41/904 (4.5%) cases overall and 26/229 (11.4%) surgically resected nodules harbored an EIF1AX mutation. The most common histologic diagnoses were follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. 11/26 (42.3%) of nodules had isolated EIF1AX mutation. Comutations were found in RAS (12/26; 46.2%), TERT (5/26; 19.2%) and TP53 (2/26; 7.7%). EIF1AX mutation alone conferred a 36.4% risk of malignancy (ROM) and 54.5% ROM or noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), while the ROM was significantly higher in nodules with concurrent RAS (71.4%), TERT, TP53 and RAS+TERT (100%) mutations., Conclusion: EIF1AX mutations occur in benign and malignant follicular thyroid neoplasms. In our cohort, the majority of mutations occurred at the splice acceptor site between exons 5 and 6. Importantly, the coexistence of EIF1AX mutations with other driver pathogenic mutations in RAS, TERT and TP53 conferred a 100% ROM or NIFTP, indicating that such nodules require surgical removal., (Copyright © 2021 by Academy of Sciences and Arts of Bosnia and Herzegovina.)

Atypia of undetermined significance/follicular lesions of undetermined significance (AUS/FLUS) refers to an intermediate histologic category of thyroid nodules in The Bethesda System for Reporting Thyroid Cytopathology. Although the risk of malignancy in this category was originally cited as 5-15%, recent literature has suggested higher rates of related malignancy ranging from 38% to 55%. Malignant nodules warrant surgery with total thyroidectomy or thyroid lobectomy, whereas benign nodules can be observed or followed with serial ultrasounds (US) based on their imaging characteristics. The management of nodules with a cytopathologic diagnosis of AUS/FLUS can be difficult because theses nodules lie between the extremes of benign and malignant. The management options for such nodules include observation, repeat fine-needle aspiration, and surgery. The use of molecular genetics, the identification of suspicious US characteristics, and the recognition of additional clinical factors are all important in the development of an appropriate, tailored management approach. Institutional factors also play a crucial role.

An increasing number of children are diagnosed with thyroid cancer. Most patients do not have an identifiable cause; however, tumor predisposition syndromes may be associated with development of both differentiated and medullary thyroid cancer. With an excellent prognosis for most patients, the goal of therapy is to optimize outcome and reduce complications. The increased knowledge of the oncogenic drivers provides opportunities to improve the accuracy of diagnosis, stratify surgery, and select systemic therapy that may be considered for neoadjuvant and adjuvant treatment. Treatment complications can be reduced by referral to regional, high-volume pediatric thyroid centers., Competing Interests: Disclosure The author has no financial disclosures that affect the content of this article. The author has received an honorarium from Hexal AG to review abstracts for EndoToday (2019)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To analyze the malignant probability of thyroid nodules with the diagnosis of atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) determined by fine-needle aspiration (FNA) and to explore the value of the combined application of BRAF V600E gene detection for the diagnosis of benign and malignant thyroid nodules. Methods: A total of 114 patients including 20 males and 94 females, aged 16-76 years old with thyroid nodules underwent FNA examination and surgical treatment in the Affiliated Cancer Hospital of Zhengzhou University from October 2018 to November 2019 were retrospectively analyzed. Postoperative histopathological results were used as the gold standard for the diagnosis of malignant thyroid nodules. The malignant rate of thyroid nodules with the diagnosis of AUS/FLUS was evaluated. Differential diagnostic efficacy of preoperative FNA combined with BRAF V600E gene detection for papillary thyroid carcinoma (PTC) was analyzed by McNemer test and diagnostic test evaluation method. Results: The mutation rate of BRAF V600E gene was 84.76% (89/105) in PTC. PTC accounted for 57.14% (12/21) of the patients with the diagnoses of AUS/FLUS determined by FNA. The specificity, sensitivity, positive predictive value and negative predictive value of BRAF V600E mutation examination for the diagnosis of malignant thyroid nodules determined preoperatively as AUS/FLUS were 9/9, 5/12, 5/5 and 9/16, respectively. BRAF V600E mutation examination could improve the detection rate of PTC in patients with AUS/FLUS ( OR =0.438, 95% CI =0.251-0.763, P =0.016). Conclusion: FNA combined with BRAFV600E mutation examination can significantly improve the detection rate of malignant thyroid nodules diagnosed preoperatively as AUS/FLUS.

Thyroid nodules can be detected by ultrasonography in up to 68% of the general population. They are typically benign and are often discovered incidentally. The primary goal of thyroid nodule evaluation is to determine whether it is malignant. After thyroid ultrasonography has been performed, the next step is measurement of serum thyroid-stimulating hormone. If levels are low, a radionuclide thyroid uptake scan is indicated. Hyperfunctioning nodules are rarely malignant and do not require tissue sampling. Nonfunctioning nodules and nodules in a patient with a normal or high thyroid-stimulating hormone level may require fine-needle aspiration based on ultrasound characteristics and size. Nodules with suspicious features and solid hypoechoic nodules 1 cm or larger require aspiration. The Bethesda System (categories 1 through 6) is used to classify samples. Molecular testing can be used to guide treatment when aspiration yields an indeterminate result. Molecular testing detects mutations associated with thyroid cancer and can help inform decisions about surgical excision vs. continued ultrasound monitoring. Treatment of pregnant women with nonfunctioning thyroid nodules and of children with thyroid nodules is similar to that for nonpregnant adults, with the exception of molecular testing, which has not been validated in these populations.

Introduction: We evaluated the clinical performance of an expanded mutation panel in combination with microRNA classification (MPTX) for the management of indeterminate thyroid nodules., Materials and Methods: MPTX included testing of fine-needle aspirates from multiple centers with a combination of ThyGeNEXT mutation panel for strong and weak driver oncogenic changes and ThyraMIR microRNA risk classifier (both from Interpace Diagnostics; Pittsburgh, PA). MPTX test status (positive or negative) and MPTX clinical risk classifications (low, moderate, or high risk) were determined blind to patient outcomes. Surgical pathology and clinical follow-up records of patients from multiple centers were used to determine patient outcomes. MPTX performance was assessed by Kaplan Meier analysis for cancer-free survival of patients, with risk of malignancy determined by hazard ratio (HR)., Results: Our study included 140 patients with AUS/FLUS or FN/SFN nodules, of which 13% had malignancy. MPTX negative test status and MPTX low risk results conferred a high probability (94%) that patients would remain cancer-free. MPTX positive test status (HR 11.2, P < 0.001) and MPTX moderate-risk results (HR 8.5, P = 0.001) were significant risk factors for malignancy, each conferring a 53% probability of malignancy. MPTX high-risk results elevated risk of malignancy even more so, conferring a 70% probability of malignancy (HR 38.5, P < 0.001)., Conclusions: MPTX test status accurately stratifies patients for risk of malignancy. Further classification using MPTX clinical risk categories enhances utility by accurately identifying patients at low, moderate, or high risk of malignancy at the low rate of malignancy encountered when clinically managing patients with indeterminate thyroid nodules., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Distinctive rounded structures termed spherules have been observed in thyroid fine-needle aspiration (FNA) samples. Although they are often small and interpreted as microfollicles, spherules are notable for the even spacing of the follicular cells along the perimeter of the structure. Because they have an orderly architectural arrangement, it has been hypothesized that spherules are benign and do not carry the same risk of malignancy that traditional microfollicles do. The aim of this study was to identify the clinical significance and histopathologic correlate of spherules., Methods: Thyroid FNAs (n = 637) with an interpretation of atypia of undetermined significance (AUS) were reviewed for the presence of spherules, which were defined as small follicles with a rounded, smooth, sharply defined outer contour, evenly spaced nuclei, and a 3-dimensional appearance. Cases were included if spherules accounted for >50% of follicular cell arrangements. Clinical, histopathologic, molecular, and sonographic follow-up data were obtained., Results: Twenty-five spherule cases were identified, and they represented 3.9% of all AUS cases reviewed. All 24 cases with follow-up had a benign outcome. Eleven were tested with the Afirma gene expression classifier; 8 were benign according to the gene expression classifier, and 3 were suspicious but histologically benign after surgical resection. Four additional cases were histologically benign after surgery. Five patients had repeat benign cytology. Four patients had repeat stable ultrasound findings., Conclusions: Thyroid nodules diagnosed as AUS, if composed predominantly of spherules, are associated with a benign outcome. Recognizing this distinctive cytomorphologic finding may help to reduce the number of FNAs interpreted as AUS and save patients from unnecessary additional testing and surgery., (© 2019 American Cancer Society.)

Introduction: Inconclusive cytologic results of thyroid fine‑needle aspiration biopsy (FNAB) include atypia or follicular lesion of undetermined significance (FLUS) and follicular neoplasm or suspicious for follicular neoplasm (SFN)., Objectives: We aimed to assess the genetic background of indeterminate thyroid nodules and to identify new genetic pathways potentially involved in the development of follicular thyroid cancer., Patients and Methods: Genomic DNA was isolated from FNAB samples from 25 white patients (2 men; 23 women) diagnosed preoperatively with FLUS (n = 16) and SFN (n = 9). Next‑generation sequencing (NGS) was performed. The results were compared with clinical data, including final postsurgical diagnoses., Results: The malignancy rate was 28%. KDR, RET, and TP53 gene mutations were most frequent in FLUS and SFN samples finally diagnosed as cancers, whereas alterations in RET, TP53, FLT3, APC, and PDGFRA predominated in benign tumors. KDR tended to be more common in malignant samples (75% vs 20%, P = 0.1). A total number of mutated genes was higher in patients with benign tumors (17 vs 11, P = 0.02), but there was no difference between groups in the mean number of mutations per patient (4.9 [range, 1-9])., Conclusions: We showed that the heterogeneity in the genetic background of indeterminate thyroid nodules corresponds to their histopathologic diversity. The role of KDR as a possible malignancy marker needs to be confirmed. Glass slides with FNAB samples may constitute a reliable source of genetic material for NGS studies, providing a better insight into the molecular profile of thyroid nodules.

We investigated whether use of American College of Radiology thyroid imaging report and data system (ACR TIRADS) in combination with K-RAS mutation status may facilitate risk stratification of patients with cytological Bethesda Category III and IV thyroid nodules. Ultrasonographic, cytological, and histopathological diagnoses were retrospectively correlated with K-RAS mutation status in a series of 43 cytologically indeterminate thyroid nodules (CITNs) that were referred for surgical excision. K-RAS mutations were detected in 8/43 (18.6%) fine-needle aspiration (FNA) samples as against 11/43 (25.6%) surgical specimens. ACR TIRADS level (TR) TR3 lesions had a malignancy risk of 40%; the K-RAS mutation rate in FNA samples and surgical specimens of category TR3 lesions was 40% and 60%, respectively. K-RAS mutation-positive malignancy was significantly more frequently detected in follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) lesions than that in atypia or follicular lesion of undetermined significance (AUS/FLUS) (P<0.01). Combined use of ACR TIRADS (TR5 as the diagnostic threshold) and K-RAS mutation status helped identify 83.3% (10/12) malignant nodules (58.6% specificity, 45.5% positive predictive value, 89.5% negative predictive value, and 65.9% accuracy). CITNs with ACR TIRADS category TR3 showed an unexpectedly high risk of malignancy. K-RAS mutation-positive FN/SFN nodules have a 50% risk of malignancy and surgery should be recommended. Combined use of ACR TIRADS and K-RAS mutation may facilitate risk-stratification of patients with CITNs. The high negative predictive value (NPV) for malignancy seems sufficient to allow conservative management of patients with active surveillance., Competing Interests: The authors have declared that no competing interests exist.

Metastatic follicular thyroid carcinoma (FTC) is rare. The aim of this study was to determine the clinical, histologic, and molecular differences between patients with metastatic FTC who present with distant metastatic (DM) disease versus those who present with a primary thyroid nodule (PT). Clinical and pathologic information was extracted from the medical record and surgical pathology report. When available, slides were reviewed. Molecular testing was performed on available primary and/or metastatic lesions. Thirty-six patients with metastatic FTC were identified: 15 DM and 21 PT. DM patients were significantly older than those with PT (P=0.0001). In DM patients, bone was the most common site of initial metastasis (P=0.03), compared with lung in PT patients (P=0.03). Unique to primary carcinomas in DM patients was extensive intratumoral fibrosis (50%), occasionally reaching such a degree as to obscure histologic features of malignancy (2 cases). Oncocytic features were more common in those who presented with PT (P=0.03). Pathogenic mutations were identified in 85% of cases, most commonly in RAS (55%) and TERT promoter (45%); of these, combined RAS and TERT was present in 30%. Pathogenic PTEN, NF1, RET, and BRCA2 mutations were also identified. The prevalence and type of pathogenic mutations did not differ between DM and PT patients. The acquisition of a pathogenic mutation in the metastatic focus that was not present in the primary carcinoma was rare (1 case). In summary, FTC presenting with DM compared with PT was more likely to be present in an older age group, to metastasize to bone, and to demonstrate extensive fibrosis possibly representing histologic regression.

Background: Fine-needle aspiration (FNA) is the most reliable method for diagnosing thyroid nodules; however, some features such as atypia of undetermined significance or follicular lesion of undetermined significance can confound efforts to identify malignancies. Similar to BRAF, cyclin D1 may be a strong marker of cell proliferation., Methods: One hundred two patients with thyroidal nodule were enrolled in this prospective study. Expression of cyclin D1 in thyroid nodules was determined by immunohistochemistry using both surgical specimens and their cytological specimens. The identification of the optimal cut off points for the diagnosis of malignancy were evaluated using the receiver operating characteristic (ROC) curves and the assessment of the area under the ROC curve (AUC). The specificity, sensitivity, positive predictive value (PPV) of markers were evaluated from crosstabs based on cut off points and significance were calculated. We also analyzed genetic variants by target NGS for thyroid nodule samples., Results: The positive predictive value (PPV) and median stain ratio (MSR) of cyclin D1 nuclear staining was determined in papillary thyroid carcinoma (PPV = 91.5%, MSR = 48.5%), follicular adenoma (PPV = 66.7%, MSR = 13.1%), and adenomatous goiter and inflammation controls (MSR = 3.4%). In FNA samples, a threshold of 46% of immunolabelled cells allows to discriminate malignant lesions from benign ones (P < 0.0001), with 81% sensitivity and 100% specificity. A 46% cutoff value for positive cyclin D1 immunostaining in thyroid cells demonstrated 81% sensitivity and 100% specificity. In surgical specimens, ROC curve analysis showed a 5.8% cyclin D1 immunostaining score predicted thyroid neoplasms at 94.4% sensitivity and 92.3% specificity (P = 0.003), while a 15.7% score predicted malignancy at 86.4% sensitivity and 80.5% specificity (P < 0.0001). Finally, three tested clinico-pathological variables (extra thyroidal extension, intraglandular metastasis, and lymph node metastasis) were significant predictors of cyclin D1 immunostaining (P < 0.001)., Conclusion: Our cytological cyclin D1 screening system provides a simple, accurate, and convenient diagnostic method in precision medicine enabling ready determination of personalized treatment strategies for patients by next generation sequencing using cytological sample.

Background: The ThyroSeq mutational panel and Afirma gene expression classifier (GEC) are used to risk stratify cytologically indeterminate thyroid nodules. In the current study, the authors evaluated the performance of these tests within the context of ultrasonographic features and with the incorporation of the noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) nomenclature., Methods: The authors reviewed nodules using ThyroSeq or Afirma GEC testing. For nodules that were surgically resected, both tests were studied within the context of ultrasound findings, comparing performance stratified by the 2015 American Thyroid Association guideline (ATA 2015) sonographic patterns and assessing the positive predictive value (PPV) of these tests both including and excluding NIFTP in the malignant category., Results: A total of 304 cases were identified, 119 of which were resected. All cases that met the criteria for NIFTP on excision demonstrated either high-risk mutations on ThyroSeq or a "suspicious" result on Afirma GEC. When NIFTP cases were shifted from the malignant to nonmalignant category, the PPV of "positive" tests for both ThyroSeq and Afirma GEC decreased from 42.9% to 14.3% (an absolute decrease of 28.6%) and 30.1% to 25.3% (an absolute decrease of 4.8%), respectively. No cases of malignancy were found in the ATA 2015 "very low suspicion" group, even with a "suspicious" Afirma GEC result., Conclusions: Both the ThyroSeq and Afirma GEC tests demonstrated decreases in the PPV when NIFTP was considered nonmalignant. In the era of NIFTP, a "positive" test result for either the Afirma GEC or ThyroSeq should be interpreted in light of clinical factors and should not exclude conservative (ie, lobectomy) surgical management. ATA 2015 "very low suspicion" nodules, even with "suspicious" Afirma GEC results, were not found to demonstrate malignancy in this series. Cancer Cytopathol 2018. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

Objective: Circulating tumor DNA (ctDNA), a subset of cell-free DNA (cfDNA), is a potential biomarker for thyroid cancer. We determined the performance of a ctDNA panel for detecting thyroid malignancy in patients with thyroid nodules., Methods: Sixty-six patients with thyroid nodules without a prior history of cancer enrolled in a prospective, 1-year study in which blood was drawn for ctDNA analysis prior to undergoing fine-needle aspiration biopsy (FNAB) of thyroid nodules. The ctDNA panel consisted of 96-mutations in 9 cancer driver genes. The primary outcome measures were the sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of our ctDNA panel for the diagnosis of thyroid malignancy as determined by pathologic and/or molecular tissue examination., Results: Results from 10 subjects could not be determined due to inadequate volume or technical issues. The final classifications of the thyroid nodules were 13 malignant and 43 benign lesions. A KRAS G12V mutation was detected in the plasma of 1 patient with stage IVA papillary carcinoma whose tissue contained the same mutation. Two of the 43 patients with benign lesions also had ctDNA detected, giving a sensitivity of 7.7%, specificity of 95.35%, PPV of 33.33%, and NPV of 77.35%. There were no significant differences between benign or malignant lesions in cfDNA levels., Conclusion: Neither cfDNA measurements nor our panel of ctDNA mutations are sensitive or specific enough to provide valuable information over FNAB. An expanded panel and the inclusion of proteomics may improve sensitivity and specificity for thyroid cancer detection., Abbreviations: cfDNA = cell-free DNA; ctDNA = circulating tumor DNA; FNAB = fine-needle aspiration biopsy; NIFTP = noninvasive follicular thyroid neoplasm with papillary-like nuclear features.

Purpose: The incidence of thyroid cancer (TC) is increasing. Cytology by itself cannot distinguish TC from some benign nodules especially in certain subtypes of TC. Our immediate goal is to identify DNA methylation markers for early detection of TC and to molecularly differentiate TC subtypes from benign nodules., Methods: Promoter methylation status of 21 candidate genes was examined on formalin-fixed paraffin-embedded tissue (FFPE) utilizing quantitative methylation-specific polymerase chain reaction (QMSP) in a retrospective cohort of 329 patients (56% white, 29% African American, 61% female) comprising 71 normal thyroid, 83 benign nodules [follicular adenomas (FA)], 90 follicular TC (FTC) and 85 papillary TC (PTC). All genes were analyzed individually (Kruskal-Wallis and Wilcoxon rank sum tests) and in combination (logistic regression models) to identify genes whose methylation levels might best separate groups., Results: Combination gene panels TPO and UCHL1 (ROC = 0.607, sensitivity 78%) discriminated FTC from FA, and RASSF1 and TPO (ROC = 0.881, sensitivity 78%) discriminated FTC from normal. Methylation of TSHR distinguished PTC from FTC (ROC = 0.701, sensitivity 84%) and PTC from FA (ROC = 0.685, sensitivity 70%). The six gene panel of TIMP3, RARB2, SERPINB5, RASSF1, TPO and TSHR, which differentiates PTC from normal thyroid, had the best combination sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue., Conclusions: Aberrant gene methylation used in combination panels may be useful clinically in differentiating FTC and PTC from benign nodules. If confirmed in additional studies, these findings could help reduce the over diagnosis of thyroid cancer and surgeries related to over diagnosis.

Background: Current thyroid molecular tests are specifically designed for the differential diagnosis of nodules with indeterminate or suspicious fine-needle aspiration (FNA) cytology., Summary: However, their clinical validity faces challenges from both variation among institutions in cancer prevalence and, most recently, the new category of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). The latter diagnosis was previously classified as malignant. Relevant to this, all molecular panels on the market today were originally tested and validated within the context of these entities being considered malignant., Conclusion: This review examines possible effects of the NIFTP reclassification as a precancerous lesion on the original validation studies and, investigates the effect of the significant reported variability in thyroid cancer prevalence on the performance of these tests.

Objective: To describe the evolution and clinical utility of molecular testing for thyroid nodules and cancer achieved over the last 2 decades., Methods: Scientific reports on thyroid cancer genetics and molecular diagnostics in thyroid nodules., Results: Over the last 2 decades, our understanding of the genetic mechanisms of thyroid cancer has dramatically expanded, such that most thyroid cancers now have known gene driver events. This knowledge provides the basis for establishing and further improving molecular tests for thyroid nodules and cancer and for the introduction of new entities such as noninvasive follicular thyroid neoplasm with papillary-like nuclear features. The progress with molecular tests for thyroid nodules started in the 1990s from demonstrating feasibility of detecting various molecular alterations in fine-needle aspiration (FNA) material collected from thyroid nodules. It was followed by the introduction of the first single-gene mutational markers, such as BRAF, and a small mutational panel into clinical practice in the mid 2000s. Currently, several more advanced molecular tests are available for clinical use. They are based on multiple molecular markers and have increasing impact on the clinical management of patients with thyroid nodules., Conclusion: The evolution of molecular tests for thyroid nodules followed the discovery of various diagnostic and prognostic molecular markers of thyroid cancer that can be applied to thyroid FNA samples to inform more individualized management of these patients., Abbreviations: FNA = fine-needle aspiration miRNA = micro RNA NGS = next-generation sequencing NIFTP = noninvasive follicular thyroid neoplasm with papillary-like nuclear features NPV = negative predictive value PPV = positive predictive value PTC = papillary thyroid carcinoma RAI = radioactive iodine.

Fine-needle aspiration (FNA) plays a key role in the early evaluation of patients with thyroid nodules; however, from 15% to 30% of FNA specimens are cytologically indeterminate. Molecular testing has proven useful when applied to indeterminate thyroid FNAs, and its use has been endorsed in the American Thyroid Association guidelines. In addition to the noncommercial ("in-house") application of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), rat sarcoma (RAS), rearranged in transformation/papillary thyroid carcinoma (RET/PTC), and peroxisome proliferator-activated receptor γ/paired box gene 8 (PPARγ/PAX8) testing, there are currently 3 commercially available molecular panels that vary in their relative reported performances, strengths, and limitations. Here, we discuss the role of molecular testing for indeterminate thyroid aspirates, taking into consideration the recent reclassification of the encapsulated follicular variant of papillary thyroid carcinoma (PTC) as "noninvasive follicular neoplasm with papillary-like nuclear features (NIFTP)." Cancer Cytopathol 2017;125(6 suppl):477-85. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Background: Thyroid nodules with atypia of undetermined significance (AUS) on fine-needle aspiration (FNA) have a low risk of malignancy that appears to vary based on specific features described in the AUS diagnosis. The Afirma gene expression classifier (GEC) is a molecular test designed to improve preoperative risk stratification of thyroid nodules, but its performance for different patterns of AUS has not been defined. The objective of this study was to assess GEC results and clinical outcomes in AUS nodules with architectural atypia (AUS-A), cytologic atypia (AUS-C) or both (AUS-C/A)., Methods: This was a retrospective review of all thyroid nodules with AUS cytopathology that underwent GEC testing at the authors' institution over a period of >4 years., Results: In 227 nodules that had AUS cytology results and Afirma GEC testing, the rate of benign GEC results was higher in AUS-A nodules (70 of 107; 65%) than in AUS-C/A nodules (25 of 65; 38%; P = .0008), and AUS-C nodules exhibited an intermediate rate of benign results (27 of 55 nodules; 59%). The risk of cancer among patients who had GEC-suspicious nodules, 86% of whom underwent resection, was 19% (6 of 25) for AUS-A nodules compared with 57% (21 of 37) for AUS-C/A nodules (P = .003) and 45% (10 of 22) for AUS-C nodules (P = .07). In nodules that had an indeterminate repeat cytology result, no difference was observed in the rate of benign GEC results or in the malignancy rate compared with nodules that had a single cytology result., Conclusions: The performance characteristics of Afirma GEC testing vary, depending on qualifiers of cytologic atypia. Recognition of these differences may enable clinicians to provide improved counseling and treatment recommendations to patients. Cancer Cytopathol 2017;125:313-322. © 2017 American Cancer Society., (© 2017 The Authors. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.)

Objective: To determine if patients elect molecular testing over diagnostic surgery or repeat fine needle aspiration for indeterminate thyroid nodules. Can ThyroSeq v2.1 molecular testing reduce diagnostic thyroid surgery and rule out cancer?, Study Design: Retrospective review Setting: Single institution, single-practice surgeon., Subjects and Methods: Fifteen month retrospective review of indeterminate thyroid nodules that went on to ThyroSeq v2.1 testing., Results: 286 patients met American Thyroid Association guideline criteria for surgeon- performed, ultrasound-guided fine needle aspiration for a thyroid nodule with on-site cytopathology. The indeterminate (Bethesda III or IV) fine needle aspiration cytology rate was 9.1 percent. Prevalence of malignancy in indeterminate nodules was 19 percent. 26/26 (100 percent) patients with indeterminate thyroid nodules elected molecular testing. 16 patients had no mutation, 9 had one or more mutations, and I had no result. 16 of 25 (64 percent) patients with no mutation elected not to undergo diagnostic surgery for indeterminate thyroid nodules., Conclusions: Patients demonstrated a strong preference for molecular testing instead of diagnostic thyroid surgery for indeterminate thyroid nodules. All patients in this series, 25/25 (100 percent) with indeterminate thyroid nodules elected molecular testing instead of repeat biopsy or diagnostic thyroid surgery. 16 of 25 (64 percent) patients tested had no mutation. All 16/16 (100 percent) patients with no mutation on ThyroSeq "rule out" testing elected active surveillance rather than surgery or biopsy, reducing diagnostic surgery. The risk of malignancy among mutation negative patients was not definitively established. There are a number of factors currently that may mute the power of "rule in" testing.

Objective: The purpose of our prospective longitudinal study was to evaluate the predictive efficacy of genetic testing for malignancies in fine-needle aspiration biopsy samples that are cytologically benign at the time of biopsy., Methods: A total of 779 aspirated cytological samples collected from thyroid nodules of 626 patients were included in a 3-year follow-up study. Consecutive patients with cytologically benign thyroid nodules by the Bethesda System for Reporting Thyroid Cytopathology were enrolled in the study. At enrollment, somatic 1-point nucleotide polymorphisms of BRAF and RAS family genes were tested by melting-point analysis, while RET/PTC and PAX8/PPAR-gamma rearrangements were examined by real-time polymerase chain reaction. The genetic test was considered to be positive if a somatic mutation was found. Malignant cytopathologic diagnoses were confirmed by histopathology., Results: In samples collected from 779 thyroid nodules, there were 39 BRAF, 33 RAS mutations, and 1 RET/PTC rearrangements found at the beginning of the study. No PAX8/PPAR-gamma rearrangement was identified. There were 52 malignant thyroid tumors removed during follow-up, out of which 24 contained a somatic mutation. The specificity of the presence of somatic mutations for malignancies was as high as 93.3%, and sensitivity was 46.2%. The negative predictive value of genetic testing reached 96.0%., Conclusion: Our results show that our set of genetic tests can predict the appearance of malignancy in benign thyroid nodules (at the beginning of follow-up) with high specificity and strong negative predictive value., Abbreviations: BRAF = v-raf murine sarcoma viral oncogene homolog B1 FLUS = follicular lesion of undetermined significance FNAB = fine-needle aspiration biopsy FTC = follicular thyroid carcinoma HRAS = homologous to the oncogene from the Harvey rat sarcoma virus KRAS = homologous to the oncogene from the Kirsten rat sarcoma virus NRAS = first isolated from a human neuroblastoma/neuroblastoma RAS = viral oncogene homolog PAX8 = paired box 8 PCR = polymerase chain reaction PPAR-gamma = peroxisome proliferator-activated receptor gamma PTC = papillary thyroid carcinoma RAS = rat sarcoma RET = rearranged during transfection tyrosine-kinase proto-oncogene SM = somatic mutation SNP = single-nucleotide polymorphism.

Background: Indeterminate thyroid fine-needle aspiration (FNA) cytology, including atypia of undetermined significance (AUS/FLUS) and suspicious for follicular neoplasm (SFN), continues to generate uncertainty about the presence of malignancy, resulting in repeated follow-up, repeat FNA, or diagnostic surgery. Mutational panel testing may improve the malignancy risk prediction in indeterminate nodules, but the general application of such testing has not been investigated extensively., Methods: A retrospective review was performed of all patients undergoing thyroidectomy at a tertiary care facility over a two-year period. Mutational panel test results, when present, were analyzed relative to FNA cytologic result and surgical histopathologic diagnosis. Malignancy rates, sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV) and positive and negative likelihood ratios (LR) were calculated., Results: A total of 261 operated thyroid nodules had the following initial FNA cytology results: 2% non-diagnostic, 23% benign, 28% AUS/FLUS, 11% SFN, 9% suspicious for malignancy (SUSP), and 27% malignant. The histopathologic malignancy rate was 48%, subcategorized by cytology into benign 7%, AUS/FLUS 30%, SFN 38%, and SUSP 83%. Mutations were more frequent in indeterminate nodules that were histologically malignant versus benign (p < 0.0001) or versus adenoma (p = 0.001). Mutational analysis in 44 AUS/FLUS nodules resulted in a malignancy detection sensitivity of 85%, a specificity of 65%, a PPV of 50%, a NPV of 91%, and a positive LR of 2.4. In 12 SFN nodules analyzed with ThyroSeq(®) testing, sensitivity was 100%, specificity 57%, PPV 63%, NPV 100%, and LR 2.3. Performance of the seven-gene mutational panel was not significantly different from the ThyroSeq(®) panel in the AUS/FLUS group. The malignancy yield, comparing the mutation positive AUS/FLUS group with the untested AUS/FLUS surgical cohort, did not reach statistical significance (p = 0.17)., Conclusions: In a surgical cohort, a similar NPV but a lower PPV was found with the use of mutational panel testing compared to the published literature. Following the identification of a mutation, the prevalence of malignancy in the AUS/FLUS or SFN category was increased by nearly 15% to 45% and 53%, respectively. Further study is needed to confirm these results and to analyze clinical outcome subcategories relative to the utility of mutational testing.

Background: Molecular diagnostic testing is increasingly used in the management of indeterminate thyroid nodules. Limited data exist regarding the influence of clinical factors on gene expression classifier (GEC) test performance. This study examined the positive and negative predictive value of GEC as stratified by nodule size., Methods: A prospectively maintained pathology database from a single tertiary referral center was queried from 2012 to 2015 for indeterminate thyroid nodules that underwent GEC testing. Nodule size, patient demographics, Bethesda classification, and Hürthle cell-predominant nodules (HCNs) were evaluated as predictors of GEC performance., Results: Two hundred and thirty-one patients with 245 indeterminate nodules were examined. Assuming all nodules to be benign unless proven malignant on histopathology, the sensitivity and specificity of GEC testing were 95.2% and 60.1%, respectively. The malignancy rate among resected nodules was 25.3%. The positive predictive value was consistent across nodule sizes: 45.5% for nodules <1 cm, 42.9% for nodules 1-1.9 cm, 36.0% for nodules 2-2.9 cm, 54.2% for nodules 3-3.9 cm, and 50.0% for nodules ≥4 cm. The negative predictive value ranged from 93.3% to 100% and was not affected by nodule size. HCNs had a high rate of GEC suspicious results (77.4% vs. 50.5% for nodules without Hürthle cell predominance, p < 0.01), though this did not correspond to a difference in the rate of malignancy (25.8% vs. 25.3%)., Conclusions: Nodule size did not affect GEC test performance in the present cohort. GEC benign results remain reliable in large nodules. GEC suspicious nodules >3 cm carry a similar risk of malignancy compared to smaller nodules, and do not warrant more aggressive treatment. GEC testing has limited clinical utility for HCNs due to the high rate of false-positive results.

Background: It is now recognized that noninvasive follicular variant of papillary thyroid carcinoma (NFVPTC) is a distinct subset of FVPTC with an exceedingly indolent clinical course. The Afirma gene-expression classifier (GEC) helps guide clinicians in the management of thyroid nodules with indeterminate fine-needle aspiration (FNA) results. Thyroid surgery is recommended for nodules with a suspicious Afirma result, whereas observation is deemed reasonable for most nodules with a benign result. The aim of this study was to confirm that the Afirma test detects NFVPTCs and to determine how many carcinomas detected by the Afirma GEC represent NFVPTCs., Methods: From a database of 249 FNAs sent for Afirma testing between January 2012 and October 2014, a search was conducted for cases with a preceding FNA diagnosis of atypia/follicular lesion of undetermined significance (AUS/FLUS) or suspicious for a follicular neoplasm (SFN), a suspicious Afirma result, and a corresponding resection specimen reviewed at Brigham and Women's Hospital. The diagnoses of the prior FNAs and subsequent resection specimens were recorded. Slides for all resection specimens with a diagnosis of FVPTC were reviewed to identify NFVPTCs., Results: Sixty-three cases met the inclusion criteria. The preceding FNA diagnosis was AUS/FLUS in 34 (54%) cases and SFN in 29 (46%) cases. The surgical resection specimen demonstrated 16 (25%) FVPTCs, five (8%) follicular thyroid carcinomas, one (2%) classical type PTC, and 41 (65%) benign tumors/nodules. Of the 16 FVPTCs, 14 (88%) were NFVPTCs. Thus, NFVPTCs accounted for 64% of the carcinomas in the cohort., Conclusion: These results indicate that the Afirma GEC detects NFVPTCs and that many of the carcinomas detected by Afirma are NFVPTCs. While all care should be individualized and include clinical and sonographic assessment, these results suggest lobectomy as opposed to total thyroidectomy should be considered for nodules with a preceding AUS/FLUS or SFN on cytology and a suspicious Afirma result.

Purpose: The role of thyroid-specific transcription factors in thyroid malignancy is still poorly understood, so we investigate thyroid-specific transcription factors gene expression both in benign and in malignant thyroid nodules, aiming to study a possible clinical utility of these molecules., Methods: We quantified TTF-1, FOXE1 and PAX8 mRNA levels, relating their expression to diagnostic and prognostic features of thyroid tumors. RNA was extracted from 4 normal thyroid tissues, 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesion tissues [49 goiter and 50 follicular adenomas (FA)]., Results: Levels of mRNA of both FOXE1 (P < 0.0001) and PAX8 (P < 0.0001) genes, but not TTF-1 (P = 0.7056), were higher in benign than in malignant thyroid lesions. FOXE1 was able to identify malignant nodules with 75.8 % sensitivity, 76.1 % specificity, 75.8 % positive predictive value, 76.1 % negative predictive value and 75.9 % accuracy. PAX8 was able to identify malignancy with 60.6 % sensitivity, 81.1 % specificity, 76.9 % positive predictive value, 66.4 % negative predictive value and 70.6 % accuracy. Both FOXE1 and PAX8 gene expression patterns were also able to differentiate FA from the follicular variant of PTC-FVPTC. However, the investigated gene expression was neither associated with any clinical feature of tumor aggressiveness nor associated with recurrence or survival., Conclusions: We suggest that FOXE1 and PAX8 gene expression patterns may help to diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions, but are not determinants of thyroid tumor progression.

Background: Thyroid carcinomas are known to harbor oncogenic driver mutations and advances in sequencing technology now allow the detection of these in fine needle aspiration biopsies (FNA). Recent work by The Cancer Genome Atlas (TCGA) Research Network has expanded the number of genetic alterations detected in papillary thyroid carcinomas (PTC). We sought to investigate the prevalence of these and other genetic alterations in diverse subtypes of thyroid nodules beyond PTC, including a variety of samples with benign histopathology. This is the first clinical evaluation of a large panel of TCGA-reported genomic alterations in thyroid FNAs., Results: In FNAs, genetic alterations were detected in 19/44 malignant samples (43% sensitivity) and in 7/44 histopathology benign samples (84% specificity). Overall, after adding a cohort of tissue samples, 38/76 (50%) of histopathology malignant samples were found to harbor a genetic alteration, while 15/75 (20%) of benign samples were also mutated. The most frequently mutated malignant subtypes were medullary thyroid carcinoma (9/12, 75%) and PTC (14/30, 47%). Additionally, follicular adenoma, a benign subtype of thyroid neoplasm, was also found to harbor mutations (12/29, 41%). Frequently mutated genes in malignant samples included BRAF (20/76, 26%) and RAS (9/76, 12%). Of the TSHR variants detected, (6/7, 86%) were in benign nodules. In a direct comparison of the same FNA also tested by an RNA-based gene expression classifier (GEC), the sensitivity of genetic alterations alone was 42%, compared to the 91% sensitivity achieved by the GEC. The specificity based only on genetic alterations was 84%, compared to 77% specificity with the GEC., Conclusions: While the genomic landscape of all thyroid neoplasm subtypes will inevitably be elucidated, caution should be used in the early adoption of published mutations as the sole predictor of malignancy in thyroid. The largest set of such mutations known to date detects only a portion of thyroid carcinomas in preoperative FNAs in our cohort and thus is not sufficient to rule out cancer. Due to the finding that variants are also found in benign nodules, testing only GEC suspicious nodules may be helpful in avoiding false positives and altering the extent of treatment when selected mutations are found.

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.)