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Background: Recent studies have suggested that pathogenic variants of the DICER1 gene could be a driver of alterations in some pediatric thyroid nodules, but data are still limited. The aim of this study was to detect variants in the DICER1 gene in a large cohort of pediatric thyroid nodules and then correlate them with clinicopathological data, with a focus on the disease prognosis in patients with thyroid carcinoma. Methods: This retrospective cohort study consisted of 350 pediatric and young adult patients (aged 2-21 years) with thyroid nodules, from whom 275 fresh-frozen thyroid nodule samples and 92 fine-needle aspiration biopsy (FNAB) samples were collected. After an analysis of variants in major genetic alterations of thyroid tumors, variants in the DICER1 gene were identified using next-generation sequencing and multiplex ligation-dependent probe amplification methods. Peripheral blood was analyzed from patients with DICER1 -positive tumors. The results of genetic analysis were then correlated with clinicopathological data. Results: Variants in the DICER1 gene were detected in a total of 24/350 (6.9%; 95%CI [4.4;10.0]) pediatric and young adult patients, respectively, in 10/119 (8.4%; [4.1;14.9]) patients with benign fresh-frozen tissue, in 8/141 (5.7%; [1.9;9.5]) with papillary thyroid carcinoma (PTC) and in 6/86 (7.0%; [4.1;14.6]) patients with FNAB. No other gene alteration was found in DICER1 -positive samples. Germline DICER1 variants were identified in 11/24 (45.8%; [25.6;67.2]) patients. Two somatic (biallelic) variants in the DICER1 gene were found in 9/24 (37.5%; [18.8;59.4]) thyroid nodules. Somatic deletions of at least 3 Mbp long were revealed in 2/24 (8.3%; [1.0;27.0]) cases. DICER1 -positive PTCs were significantly associated with the follicular subtype of PTC ( p = 0.001), encapsulation ( p = 0.006) and were larger in size ( p = 0.035), but with no extrathyroidal extension ( p = 0.039), and less frequent lymph node metastases ( p = 0.003) compared with DICER1 -negative PTCs. Patients with DICER1- positive PTC had an excellent response to treatment in 75% of cases. Conclusions: Variants of the DICER1 gene are frequently found in the thyroid nodules of pediatric and young adult patients. In our patients, DICER1 -positive PTCs showed low invasiveness. Our findings support considering more conservative management for DICER1 -positive low-risk PTCs.

Background: Genomic profiling is now available for risk stratification of cytologically indeterminate thyroid nodules (ITNs). Mutations in RAS genes ( HRAS , NRAS , KRAS ) are found in both benign and malignant thyroid nodules, although isolated RAS mutations are rarely associated with aggressive tumors. Because the long-term behavior of RAS -mutant ITNs is not well understood, most undergo immediate surgery. In this multicenter retrospective cohort study, we characterize tumor growth kinetics of RAS -mutant ITNs followed with active surveillance (AS) using serial ultrasound (US) scans and examine the histopathologic diagnoses of those surgically resected. Methods: US and histopathologic data were analyzed retrospectively from two cohorts: (1) RAS -mutant ITNs managed with AS at three institutions (2010-2023) and (2) RAS -mutant ITNs managed with immediate surgery at two institutions (2016-2020). AS cohort subjects had ≥3 months of follow-up and two or more US scans. Cumulative incidence of nodule growth was determined by the Kaplan-Meier method and growth by ≥72% change in tumor volume. Pathological diagnoses for the immediate surgery cohort were analyzed separately. Results: Sixty-two patients with 63 RAS -mutated ITNs under AS had a median diameter of 1.7 cm (interquartile range [IQR] 1.2-2.6) at time of diagnosis. During a median AS period of 23 months (IQR 9.5-53.5 months), growth was observed in 12 of 63 nodules (19.0%), with a cumulative incidence of 1.9% (1 year), 23.0% (3 years), and 28.0% (5 years). Most nodules (81.0%) demonstrated stability. Surgery was ultimately performed in 6 nodules, of which 1 (16.7%) was malignant. In the cohort of 209 RAS -mutant ITNs triaged to immediate surgery, 33% were malignant (23.9% American Thyroid Association [ATA] low-risk cancers, 7.2% ATA intermediate-risk, and 1.9% ATA high-risk. During a median follow-up of 6.9 (IQR 4.4-7.1) years, there were no disease-specific deaths in these patients. Conclusions: We describe the behavior of RAS -mutant ITNs under AS and find that most demonstrate stability over time. Of the resected RAS -mutant nodules, most were benign; of the cancers, most were ATA low-risk. Immediate surgical resection of all RAS -mutant ITNs appears to be a low-value practice. Further research is needed to help define cases most appropriate for AS or immediate surgery.

Background: Over the last decade, the utilization of molecular testing (MT) for the evaluation of thyroid nodules has increased. Rates and patterns of adoption of MT and its effect on thyroidectomy rates nationally are unknown. Varying rates of MT adoption at the state level provide an opportunity to study the effects of MT on thyroidectomy rates using a quasiexperimental study design. Methods: We performed a retrospective analysis of American adult patients in the Merative™ MarketScan ® Research Databases who underwent thyroid fine-needle aspiration (FNA) from 2011 to 2021. MT included commercially available DNA and RNA platforms and traditional targeted mutational analysis. Interrupted time series analysis was used to evaluate the inflection of MT adoption and thyroidectomy rates after 2015. Difference-in-differences (DID) analysis was used to causally analyze the effect of MT adoption on thyroidectomy rates in high-adoption (at least a 10% increase in MT utilization) versus low-adoption states (no more than 5% increase in MT utilization) from 2015 to 2021. Results: We identified 471,364 patients who underwent thyroid FNA. The utilization of MT increased over the study period from 0.01% [confidence interval, CI: 0.00% to 0.02%] to 10.1% [CI: 9.7% to 10.5%], in 2021, with an immediate (β2 = 1.61, p  = 0.002) and deeper (β3 = 0.6, p  < 0.001) increase in MT adoption after 2015. Utilization of MT was lower in black patients, the elderly, rural areas, and patients with Medicaid ( p  < 0.05). Thyroidectomy rates were inversely correlated with MT utilization ( r  = -0.98, p  < 0.0001). From 2015 to 2021, the average MT utilization rate increased from 2.4% to 15.3% in high-adoption states and 1.6% to 5.6% in low-adoption states. In low-adoption states, thyroidectomy rates decreased more but to similar levels (18.5-13.2%) compared with high-adoption states (15.9-13.4%) with an adjusted DID rate of -3.3% [CI -5.6% to -0.8%]. Conclusions: The acceleration in adoption of MT after 2015 likely coincides with the publication of American Thyroid Association guidelines. Black, elderly, and rural patients are less likely to receive MT. Although thyroidectomy rates were inversely correlated with MT utilization, our study suggests that this correlation is not causal. The effect of MT on thyroidectomy rates may be overshadowed by decreasing aggressiveness of thyroid nodule evaluation.

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

Background: Molecular testing for cytologically indeterminate thyroid nodules (ITNs) is often reported with incomplete data on clinical assessment and ultrasound malignancy risk (USMR) stratification. This study aimed to clinically validate the diagnostic accuracy of a novel molecular test, assess the incremental preoperative malignancy risk of other clinical factors, and measure the impacts of introducing molecular testing at the population level. Methods: Comprehensive clinical data were collected prospectively for the first 615 consecutive patients with ITNs in a centralized health care system following implementation of a reflexive molecular test. Clinical data include patient history, method of nodule discovery, clinical assessment, USMR, cytology, molecular testing, and surgery or follow-up along with surgeon notes on surgical decision-making. Accuracy of molecular testing and the impact of the introduction of molecular testing were calculated. A multivariable regression model was developed to identify which clinical factors have the most diagnostic significance for ITNs. Results: A locally developed, low-cost molecular test achieved a negative predictive value (NPV) of 76-91% [confidence interval, CI 66-95%] and a positive predictive value (PPV) of 46-65% [CI 37-75%] in ITNs using only residual material from standard liquid cytology fine-needle aspiration (FNA). Sensitivity was highest (80%; [CI 63-92%]) in the American Thyroid Association (ATA) intermediate-suspicion ultrasound category, and lowest (46%; [CI 19-75%]) in the ATA high-suspicion ultrasound category. Following implementation of molecular testing, diagnostic yield increased by 14% ( p  = 0.2442) and repeat FNAs decreased by 24% ( p  = 0.05). Mutation was the primary reason for surgery in 76% of resected, mutation-positive patients. High-risk mutations were associated with a 58% ( p  = 0.0001) shorter wait for surgery. Twenty-six percent of patients with a negative molecular test result underwent surgery. Multivariable regression highlighted molecular testing and USMR as significantly associated with malignancy. Conclusions: Molecular testing improves preoperative risk stratification but requires further stratification for intermediate-risk mutations. Incorporation of clinical factors (especially USMR) with molecular testing may increase the sensitivity for detection of malignancy. Introduction of molecular testing offers some clinical benefits even in a low resection rate setting, and directly influences surgical decision-making. This study illustrates the importance of the local diagnostic pathway in ensuring appropriate integrated use of molecular testing for best outcomes.

Background: The actual rates of suspicious thyroid nodules (TNs) and confirmed thyroid cancer (TC) in putatively "at-risk" selected populations (e.g., individuals with family history of TC) are still uncertain. Methods: Our aim was to explore the prevalence of TC and TN in a cross-sectional study of a consenting population of unaffected individuals (10 years of age or older) with a first-degree relative known to have non-medullary TC (NMTC). Enrolled subjects underwent ultrasonographic studies of the neck between 2009 and 2018. Nodules considered suspicious according to current guidelines were subjected to fine-needle aspiration biopsy (FNAB) for cytology. Results: The screenee population comprised 1176 individuals (median age 42 [26-56] years, 650 females, 55.3%) from 473 kindreds (346 with 1 established NMTC diagnosis at entry, 103 with 2 established NMTC diagnoses, and 24 with 3 or more established NMTC diagnoses at entry). Screening revealed TNs in 500 screenees (42.5%; confidence interval [CI] 39.7-45.4%). Ninety-seven of these (19.4%; CI 16.2-23.1%) underwent FNAB. Only 11 cases of TC were diagnosed in the whole population (0.9%; CI 0.5-1.7%). The prevalence of TC in screenees from kindreds with ≥3 cases (3/24, 12.5%) was higher than that for kindreds with one affected member (6/346, 1.7%; p  = 0.01, odds ratio [OR] 7.99; CI 1.21-40.75) and for those with two affected members (2/103, 1.9%; p  = 0.05, OR 7.05; CI 0.76-89.44). The prevalence of TNs was 61.8% (CI 56.6-66.8%), 75.7% (CI 66.6-83%), and 66.7% (CI 46.7-82%) in the kindreds with 1, 2, and ≥3 cases, respectively ( p  = 0.03). Conclusions : On the whole, ultrasound-based screening of unaffected relatives of individuals with established diagnoses of NMTC is likely to reveal a high prevalence of TN and a low prevalence of TC. However, a significantly higher prevalence of TC may be found among screenees from kindreds with at least three established NMTC diagnoses before screening, suggesting that closer surveillance may be warranted in kindreds with this level of familiality.

Background: The addition of genetic analysis to the evaluation of thyroid nodule fine-needle aspiration biopsy samples improves diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs) with Bethesda III or IV cytopathology. We previously reported the performance of a multiplatform molecular test, referred to in this study as MPTXv1, that includes a mutation panel (ThyGeNEXT ® ) plus an algorithmic microRNA (miRNA) risk classifier (ThyraMIR ® ). Complex interactions of growth-promoting and -suppressing miRNAs affect the phenotype. We previously demonstrated that accounting for these interactions with pairwise miRNA expression analysis improves the diagnosis of medullary thyroid carcinoma. In this study, we assess the impact of pairwise miRNA expression analysis on risk stratification of ITNs. Methods: Pairwise expression analysis of 11 miRNAs was performed on a training cohort of histopathology-proven benign nodules ( n  = 50) to define the mean and standard deviation of each pairwise analysis and create a Benign/Malignant Profiler (MPTXv2), deviations from which predicted the malignancy risk. Clinical validation of MPTXv2 was assessed using a cohort of 178 ITN (Bethesda III and IV) samples from a multicentered, blinded retrospective study, previously evaluated by MPTXv1. Results: Compared with MPTXv1, MPTXv2 significantly improved the test performance. The receiver operating characteristic (ROC) areas under the curve (AUC) increased from 0.85 to 0.97 ( p  < 0.001), and the diagnostic accuracy at the positive threshold increased significantly ( p  < 0.05) from 83% [95% confidence interval (CI) = 76-88] to 93% [CI = 89-96]. The significant improvement in the ROC AUC and the diagnostic accuracy was due to a strong statistical trend for improvement in specificity at the positive threshold. At the positive threshold, the specificity for MPTXv1 was 90% [CI = 84-95] and improved to 98% [CI = 94-99] for MPTXv2. Using the MPTXv2, the Moderate-Risk cohort decreased from 50 samples (28% of the cohort) to 24 samples (13% of the cohort). This 52% decrease is statistically significant ( p  < 0.001) and clinically meaningful. Conclusion: As compared with MPTXv1, pairwise miRNA expression analysis used in MPTXv2 significantly improved the diagnostic accuracy of ITN risk stratification and reduced the size of the Moderate-Risk group. Prospective trials are indicated to confirm these findings in a clinical practice setting.

Background: Molecular tests for thyroid nodules with indeterminate fine needle aspiration results are increasingly used in clinical practice; however, true diagnostic summaries of these tests are unknown. A systematic review and meta-analysis were completed to (1) evaluate the accuracy of commercially available molecular tests for malignancy in indeterminate thyroid nodules and (2) quantify biases and limitations in studies that validate those tests. Summary: PubMed, EMBASE, and Web of Science were systematically searched through July 2021. English language articles that reported original clinical validation attempts of molecular tests for indeterminate thyroid nodules were included if they reported counts of true-negative, true-positive, false-negative, and false-positive results. We performed screening and full-text review, followed by assessment of eight common biases and limitations, extraction of diagnostic and histopathological information, and meta-analysis of clinical validity using a bivariate linear mixed-effects model. Forty-nine studies were included. Meta-analysis of Afirma Gene expression classifiers (GEC; n  = 38 studies) revealed a sensitivity of 0.92 (confidence interval: 0.90-0.94), specificity of 0.26 (0.20-0.32), negative likelihood ratio (LR-) of 0.32 (0.23-0.44), positive LR+ of 1.24 (1.15-1.35), and area under the curve (AUC) of 0.83 (0.74-0.89). Afirma Genomic Sequencing Classifier (GSC; n  = 10) had a sensitivity of 0.94 (0.89-0.96), specificity of 0.38 (0.27-0.50), LR- of 0.18 (0.10-0.30), LR+ of 1.52 (1.28-1.87), and AUC of 0.91 (0.62-0.92). ThyroSeq v1 and v2 ( n  = 10) had a sensitivity of 0.86 (0.82-0.90), specificity of 0.74 (0.59-0.85), LR- of 0.19 (0.13-0.26), LR+ of 3.52 (2.08-5.92), and AUC of 0.86 (0.81-0.90). ThyroSeq v3 ( n  = 6) had a sensitivity of 0.92 (0.86-0.95), specificity of 0.41 (0.18-0.69), LR- of 0.24 (0.09-0.62), LR+ of 1.67 (1.09-2.98), and AUC of 0.90 (0.63-0.92). Fourteen percent of studies conducted a blinded histopathologic review of excised thyroid nodules, and 8% made the decision to go to surgery blind to molecular test results. Conclusions: Meta-analyses reveal a high diagnostic accuracy of molecular tests for thyroid nodule assessment of malignancy risk; however, these studies are subject to several limitations. Limitations and their potential clinical impacts must be addressed and, when feasible, adjusted for using valid statistical methodologies.

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

Background: Thyroid nodules with indeterminate cytology are increasingly subjected to molecular testing. We evaluated the diagnostic performances of Afirma Genomic Sequencing Classifier (GSC) and ThyroSeq v3 in thyroid nodules with high versus low/intermediate suspicion ultrasound classification. Methods: In this prospective cohort study, we analyzed all Bethesda III and IV thyroid nodules that underwent fine-needle aspiration biopsies in the University of California Los Angeles Health System from July 2017 to April 2020. All patients underwent molecular testing with Afirma GSC or ThyroSeq v3 as part of an institutional randomized trial (NCT02681328). Nodules were categorized according to the American Thyroid Association (ATA) ultrasound risk classification. The benign call rate and the positive predictive value of molecular testing were compared between ATA high suspicion versus all other categories. Results: A total of 343 patients with 375 indeterminate thyroid nodules were included. The malignancy rate in ATA high suspicion nodules was not significantly increased by a suspicious Afirma GSC result (77.8% for all ATA high suspicion nodules vs. 87.5% for nodules with ATA high suspicion and suspicious Afirma GSC results, positive likelihood ratio [LR] = 2.0, 95% confidence interval [CI 0.5-8.0], p  = 1.0) or by a positive ThyroSeq v3 result (80.0% vs. 80.0%, positive LR = 1.0 [CI 1.0-1.0], p  = 1.0). The rate of malignancy in ATA low/intermediate suspicion nodules increased from 21.0% to 56.3% with a suspicious Afirma GSC result (positive LR = 4.8 [CI 3.4-6.9], p  < 0.0001) and decreased to 3.8% with a benign Afirma GSC result (negative LR = 0.1 [CI 0.07-0.3], p  < 0.0001). Similarly, the rate of malignancy in ATA low/intermediate suspicion nodules increased from 24.3% to 66.7% with a positive ThyroSeq v3 result (positive LR = 6.2 [CI 4.0-9.7], p  < 0.0001) and decreased to 2.1% with a negative ThyroSeq v3 result (negative LR = 0.07 [CI 0.02-0.3], p  < 0.0001). Conclusions: Afirma GSC and ThyroSeq v3 performed well in ruling out malignancy in sonographically low/intermediate suspicion thyroid nodules but has limited diagnostic value in sonographically high suspicion nodules. Molecular testing can prognosticate more aggressive thyroid cancers, which can inform treatment decisions.

Background: Thyroid nodules are a very common often incidental finding on physical examination or imaging. Of those who undergo fine needle aspiration, cytology is indeterminate in up to 15%. Molecular testing is increasingly being used to help identify which nodules may be high risk for malignancy and guide management with regard to clinical follow-up or surgical intervention. Recently there has been an increase in publication of independent studies assessing the performance of these molecular tests and comparing "real-world" data with the validation studies. Methods: This retrospective study identified all thyroid nodules at our institution that had Afirma gene expression classifier (GEC), genomic sequencing classifier (GSC), or Thyroseq v3 molecular testing from January 2014 to January 2020 and compared measurements of test performance between them at our institution, and then with the original validation studies and other published institutional data. Results: Overall, the benign call rate was highest in the Afirma GSC group (78%) compared with the GEC group (60%) and Thyroseq group (66%). Surgical histopathology revealed malignancy in 6 of 31of biopsied nodules in the GEC group, 8 of 13 in the GSC group, and 3 of 16 in the Thyroseq v3 group. Based on our data, the GSC specificity (73.7%) and positive predictive value (PPV) (61.5%) were higher than the GEC specificity (60.4%) and PPV (22.2%) as well as Thyroseq v3 specificity (55.2%) and PPV (18.8%). Conclusions: From our short-term institutional experience, we found that the GSC classified more cytologically indeterminate nodules as benign compared with the Afirma GEC, and had improved specificity and PPV, which is similar to the validation study and other institutions' reported experiences. We also found that the Thyroseq v3 was similar to the Afirma GEC in terms of specificity and PPV, both of which are much lower than the validation studies.

Background: Thyroid adenoma-associated (THADA)-IGF2BP3 fusions have been identified as an oncogenic event in thyroid neoplasms. However, the prevalence of this gene fusion and associated phenotypical and clinical features are not well defined. The aim of this study was to characterize thyroid nodules positive for THADA-IGF2BP3 fusions on preoperative molecular analysis, review surgical outcomes, and explore potential impact of the fusion detection on patient management. Methods: Thyroid nodules positive for THADA-IGF2BP3 fusion on ThyroSeq v3 genomic classifier (GC) testing of fine needle aspiration (FNA) ( n  = 30) samples from November 2017 to August 2019 were identified. Demographic and clinical data were obtained by retrospective chart review; pathology slides were re-examined. Results: Thirty nodules positive for THADA-IGF2BP3 fusion on FNA were identified, representing ∼2% of 1280 nodules that underwent molecular analysis. Of the 27 nodules with available cytology diagnosis data, 22 (81%) were diagnosed as atypia of undetermined significance, 3 (11%) as follicular neoplasm, and 1 (4%) each were benign, and suspicious for malignancy. No additional mutations or gene fusions were identified in any of the nodules. Of the 24 cases with available clinical data, 22 (92%) THADA-IGF2BP3 -positive nodules were managed surgically, 14 (64%) by thyroid lobectomy, and 8 (36%) by total thyroidectomy. Of the patients who had initial lobectomy, 3 (21%) had completion surgery. On surgical pathology, 7 (32%) THADA-IGF2BP3 -positive nodules were malignant (six encapsulated follicular variant papillary thyroid carcinomas (EFVPTC), one minimally infiltrative FVPTC), 10 (45%) noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), and 5 (23%) follicular adenomas (FA). THADA-IGF2BP3- positive malignancies were intrathyroidal, without aggressive histology. Nodule size was similar between malignant nodules, NIFTP, and FA (2.6, 2.7, and 2.3 cm, respectively; p  = 0.77). On limited follow-up (mean, 18 months) available for six patients with malignant fusion-positive nodule and 4 patients with NIFTP, no tumor recurrences were found. Conclusions: In this series of patients, 77% of THADA-IGF2BP3 fusion-positive thyroid nodules were thyroid tumors requiring surgery, either papillary carcinoma or NIFTP. However, all cancers were low risk, predominantly encapsulated FVPTCs and thus can likely be adequately treated with lobectomy.

Background: Molecular testing (MT) refines risk stratification for thyroid nodules that are indeterminate for cancer by fine needle aspiration (FNA) cytology. Criteria for selecting nodules for MT vary and remain largely untested, raising questions about the best strategy for maximizing the usefulness of MT while minimizing the harms of overtesting. We used a unique data set to examine the effects of repeat FNA cytology-based criteria for MT on management decisions and nodule outcomes. Methods: This was a study of adults (age 25-90 years; 281 women and 72 men) with cytologically indeterminate (Bethesda III/IV) thyroid nodules who underwent repeat FNA biopsy and Afirma Gene Expression Classifier (GEC) testing ( N  = 363 nodules from 353 patients) between June 2013 and October 2017 at a single institution, with follow-up data collected until December 2019. Subgroup analysis was performed based on classification of repeat FNA cytology. Outcomes of GEC testing, clinical/sonographic surveillance of unresected nodules, and histopathologic diagnoses of thyroidectomies were compared between three testing approaches: (i) Reflex (MT sent on the basis of the initial Bethesda III/IV FNA), (ii) SemiRestrictive (MT sent if repeat FNA is Bethesda I-IV), and (iii) Restrictive (MT sent only if repeat FNA is Bethesda III/IV) testing approaches. Results: Restricting MT to nodules that remain Bethesda III/IV on repeat FNA would have missed 4 low-risk cancers and 3 noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) (collectively 2% of the test population) but would have avoided diagnostic surgery for 42 benign nodules (12% of the test population). The Restrictive testing strategy was more specific (delta 0.126 confidence interval [CI 0.093 to 0.159] and 0.129 [CI 0.097 to 0.161], respectively) but less sensitive (delta -0.339 [CI -0.424 to -0.253] and -0.340 [CI -0.425 to -0.255], respectively) than the Reflex and SemiRestrictive approaches for detecting NIFTP or cancer. Conclusions: Repeat FNA cytology can guide the selection of cytologically indeterminate thyroid nodules that warrant MT. The Restrictive model of performing Afirma GEC only on nodules with two separate biopsies showing Bethesda III/IV cytology would reduce the rate of diagnostic surgery for histologically benign nodules while missing only rare low-risk tumors. Given the low but nontrivial risks of thyroidectomy, the higher specificity of the Restrictive testing approach disproportionately outweighs the potential harms.

Background: RAS gene family mutations are the most prevalent in thyroid nodules with indeterminate cytology and are present in a wide spectrum of histological diagnoses. We evaluated differentially expressed genes and signaling pathways across the histological/clinical spectrum of RAS -mutant nodules to determine key molecular determinants associated with a high risk of malignancy. Methods: Sixty-one thyroid nodules with RAS mutations were identified. Based on the histological diagnosis and biological behavior, the nodules were grouped into five categories indicating their degree of malignancy: non-neoplastic appearance, benign neoplasm, indeterminate malignant potential, low-risk cancer, or high-risk cancer. Gene expression profiles of these nodules were determined using the NanoString PanCancer Pathways and IO 360 Panels, and Angiopoietin-2 level was determined by immunohistochemical staining. Results: The analysis of differentially expressed genes using the five categories as supervising parameters unearthed a significant correlation between the degree of malignancy and genes involved in cell cycle and apoptosis ( BAX , CCNE2 , CDKN2A , CDKN2B , CHEK1 , E2F1 , GSK3B , NFKB1 , and PRKAR2A ), PI3K pathway ( CCNE2 , CSF3 , GSKB3 , NFKB1 , PPP2R2C , and SGK2 ), and stromal factors ( ANGPT2 and DLL4 ). The expression of Angiopoietin-2 by immunohistochemistry also showed the same trend of increasing expression from non-neoplastic appearance to high-risk cancer ( p  < 0.0001). Conclusions: The gene expression analysis of RAS -mutant thyroid nodules suggests increasing upregulation of key oncogenic pathways depending on their degree of malignancy and supports the concept of a stepwise progression. The utility of ANGPT2 expression as a potential diagnostic biomarker warrants further evaluation.

Background: The Afirma gene expression classifier (GEC) has been used to aid in the diagnosis and management of thyroid nodules having Bethesda category III fine-needle aspiration cytologic diagnosis (B3 nodules). The American Thyroid Association sonographic risk stratification system for thyroid nodules (ATA-US) may stratify B3 nodules and aid in the decision to order a molecular test. The aim of this study was to assess the association between ATA-US and GEC as well as to determine their individual and combined diagnostic performances when applied to B3 nodules. Methods: A retrospective single-center study included B3 nodules that had undergone evaluation by GEC. Each ultrasound was reviewed by three radiologists, and nodules were classified using the 2015 ATA sonographic risk categories. Nodules were determined to be benign or malignant based on surgical pathology or minimum 11 months of follow-up. Positive predictive values (PPV) and negative predictive values (NPV) were calculated for GEC, ATA-US, and GEC across all ATA-US categories. Results: One hundred twenty-six B3 nodules with GEC results were included and deemed benign or malignant based on final pathology or follow-up. Prevalence of malignancy was 32%. The rate of malignancy was similar in the ATA-US high suspicion (HS) and intermediate suspicion (IS) categories at 42% and 38%, respectively; and lower in nodules with low suspicion sonography (LS) and very low suspicion sonography (VLS) at 23% and 11%, respectively. The PPV and NPV of ATA-US was calculated by designating HS or IS sonography as a "positive" test and the lower risk categories as "negative." ATA-US had a PPV of 40% and NPV of 79%. The GEC PPV was 40% and NPV was 83%. The PPV of GEC was 50% in nodules with HS or IS ATA-US and lower at 28% and 20%, respectively, in LS and VLS nodules. The NPV of GEC was 80% in HS, 77% in IS, 84% in LS, and 100% in VLS sonography categories. Conclusions: In B3 nodules, ATA-US and GEC have similar diagnostic performance. The PPV of GEC varies across ATA-US categories, while the NPV remains similar. These data support the need for future prospective studies.

Background: Constitutively activating mutations in the thyrotropin receptor ( TSHR ) and the guanine nucleotide-binding protein G subunit alpha ( GNAS ) are the primary cause of hot thyroid nodules (HTNs). The reported prevalence of TSHR and GNAS mutations in HTNs varies. Previous studies show TSHR mutations in 8-82% of HTNs and GNAS mutations in 8-75% of HTNs. With sensitive and comprehensive targeted next-generation sequencing (tNGS), we re-evaluated the prevalence of TSHR and GNAS mutations in HTNs. Methods: Samples from three previous studies found to be TSHR and GNAS mutation negative were selected and re-evaluated using high-resolution melting (HRM) PCR. Remaining mutation negative samples were further reanalyzed by tNGS with a sequencing depth between 3000 × and 10,000 × . Our tNGS panel covered the entire TSHR coding sequence along with mutation hot spots in GNAS . Sequencing reads were aligned to reference and variants were called using Torrent Suite software v5.8. Results: In total, 154 of 182 previously mutation negative HTNs were positive for TSHR or GNAS mutations, resulting in an 85% prevalence of TSHR and GNAS mutations in HTNs, 79% and 6%, respectively. In a subset of 25 HTNs with multiple samples per nodule, and analyzed by tNGS at high sequencing depth, TSHR mutations were detected in 23 (92%) HTNs and 1 GNAS mutation was detected in 1 (4%) HTN, 96% mutation positive HTNs in this subset. Conclusions: Owing to the higher sensitivity of tNGS as compared with denaturing gradient gel electrophoresis and HRM-PCR, TSHR or GNAS mutations could be detected in 85% of HTNs. The detection of TSHR and GNAS mutations occurred in 96% of HTNs in a sample set with multiple samples per nodule analyzed by tNGS. Taken together with the fact that no other driver mutations could be identified by whole exome sequencing, our study strongly supports the hypothesis that TSHR and GNAS mutations are the main somatic mutations leading to HTNs.

Background: Novel molecular tests (MTs), such as ThyroSeq, may improve the management of thyroid nodules with indeterminate cytologic diagnoses; however, the impact of these tests on cost and outcome of management is unknown. Here, we evaluated the impact of ThyroSeq testing on the cytopathologic diagnosis, management, and cost of care in patients with thyroid nodules. Methods: In a retrospective study, using actual patient cohorts, the outcome and cost of management of patients with thyroid nodules seen before the introduction of ThyroSeq v2 at our institution (standard of care [StC] cohort) were compared with those seen after the introduction of this test (MT cohort). Results: A total of 773 consecutive patients entered the study (393 StC, 380 MT). The incidence of cytologically benign nodules decreased from 71.0% (StC) to 53.2% (MT) and those of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) increased from 8.9% (StC) to 21.3% (MT) and from 3.1% (StC) to 6.3% (MT), respectively. The overall rate of surgery did not change significantly (23.4% in StC vs. 23.2% in MT). Among patients who underwent surgery, the rate of overtreatment (surgeries performed on histologic benign nodules without clinical indication: compressive symptoms, hyperthyroidism resistant to medication, and nodule size >4 cm) slightly decreased from 18.8% (StC) to 16.7% (MT). The rate of malignancy decreased from 45.5% (StC) to 37.9% (MT) in AUS/FLUS and increased from 40.0% to 53.8% in FN/SFN. However, the overall rate of malignancy remained equal (47.8% in StC vs. 47.7% in MT). The average cost of care per patient in the AUS/FLUS and FN/SFN categories increased from $6,566 (StC) to $8,444 (MT) and from $9,313 (StC) to $10,253 (MT), respectively. Similarly, the overall average cost of care of a patient who underwent thyroid fine-needle aspiration increased from $3,088 (StC) to $4,282 (MT). Finally, the average cost per thyroid cancer detected increased from $26,312 (StC) to $38,746 (MT). Conclusions: Introduction of ThyroSeq v2 resulted in a shift toward indeterminate cytology results. The institutional rate of surgery, overtreatment, and malignancy did not change significantly. Lack of decrease in the rate of surgery along with the additional cost of ThyroSeq v2 increased the overall cost of care of patients including those with indeterminate cytology results.

Background: Although most thyroid nodules with indeterminate cytology are benign, in most of the world, surgery remains as the most frequent diagnostic approach. We have previously reported a 10-gene thyroid genetic classifier, which accurately predicts benign thyroid nodules. The assay is a prototype diagnostic kit suitable for reference laboratory testing and could potentially avoid unnecessary diagnostic surgery in patients with indeterminate thyroid cytology. Methods: Classifier performance was tested in two independent, ethnically diverse, prospective multicenter trials (TGCT-1/Chile and TGCT-2/USA). A total of 4061 fine-needle aspirations were collected from 15 institutions, of which 897 (22%) were called indeterminate. The clinical site was blind to the classifier score and the clinical laboratory blind to the pathology report. A matched surgical pathology and valid classifier score was available for 270 samples. Results: Cohorts showed significant differences, including (i) clinical site patient source (academic, 43% and 97% for TGCT-1 and -2, respectively); (ii) ethnic diversity, with a greater proportion of the Hispanic population (40% vs. 3%) for TGCT-1 and a greater proportion of African American (11% vs. 0%) and Asian (10% vs. 1%) populations for TGCT-2; and (iii) tumor size (mean of 1.7 and 2.5 cm for TGCT-1 and -2, respectively). Overall, there were no differences in the histopathological profile between cohorts. Forty-one of 155 and 45 of 115 nodules were malignant (cancer prevalence of 26% and 39% for TGCT-1 and -2, respectively). The classifier predicted 37 of 41 and 41 of 45 malignant nodules, yielding a sensitivity of 90% [95% confidence interval; CI 77-97] and 91% [95% CI 79-98] for TGCT-1 and -2, respectively. One hundred one of 114 and 61 of 70 nodules were correctly predicted as benign, yielding a specificity of 89% [95% CI 82-94] and 87% [95% CI 77-94], respectively. The negative predictive values for TGCT-1 and TGCT-2 were 96% and 94%, respectively, whereas the positive predictive values were 74% and 82%, respectively. The overall accuracy for both cohorts was 89%. Conclusions: Clinical validation of the classifier demonstrates equivalent performance in two independent and ethnically diverse cohorts, accurately predicting benign thyroid nodules that can undergo surveillance as an alternative to diagnostic surgery.

Background: It has been advocated to apply individualized strategies to evaluate thyroid nodules due to the growing awareness that the pathogenesis of thyroid cancer is not uniform. Molecular markers in fine needle biopsies (FNBs) may be helpful for the diagnosis and management decisions. Unlike the detection of BRAF mutations, the clinical utility of rat sarcoma viral oncogene homolog ( RAS ) mutations has not been fully elucidated. This study aimed at presenting a real-world performance of RAS mutations in identifying thyroid malignancies, at investigating the nature of thyroid tumors carrying RAS mutations, and at providing an additional reference for interpreting how to utilize the presence of RAS mutations in the decision-making process of thyroid nodule management. Methods: Between February 2015 and December 2017, 1400 sequential thyroid biopsies were performed at Boston Medical Center. Of these, 546 FNBs were evaluated for RAS mutations by using a ThyroSeq next-generation sequencing panel. Nodules carrying RAS mutations were prospectively followed, and medical records were collected. Results: ThyroSeq successfully provided molecular information in 504 nodules; 173 with molecular alteration(s); and 80 positive for mutations in the Kirsten- , Neuroblastoma- , or Harvey-RAS genes. RAS gene mutations constituted up to 46.2% of the total molecular alterations found in the study. Fifty-six of the 80 RAS -positive nodules underwent surgery, 33 (58.9%) were confirmed to be benign, 7 (12.5%) were noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), and 16 (28.6%) were thyroid carcinomas. The positive predictive value, negative predictive value, and accuracy of RAS mutations for identifying malignancies among cytologically indeterminate nodules were 25.5%, 89.7%, and 54.0% when NIFTP was not counted as cancer. A combination of RAS and other mutations increased the risk of malignancy. Twelve histopathologically proved RAS -only-positive malignant nodules all showed low-risk features and favorable prognosis. RAS isoforms added little assistance for predicting a malignancy and the response to therapy in our series. Conclusions: RAS mutations represent the most frequently detected genetic alterations in our series. RAS mutations, when occurring alone, are not helpful markers to identify malignancy among Bethesda III/IV cytologies, but may predict favorable behavior, and hence should be considered to guide initial management.

Background: Gene panels are routinely used to assess predisposition to hereditary cancers by simultaneously testing multiple susceptibility genes and/or variants. More recently, genetic panels have been implemented as part of solid tumor malignancy testing assessing somatic alterations. One example is targeted variant panels for thyroid nodules that are not conclusively malignant or benign upon fine-needle aspiration (FNA). We systematically reviewed published studies from 2009 to 2018 that contained genetic data from preoperative FNA specimens on cytologically indeterminate thyroid nodules (ITNs) that subsequently underwent surgical resection. Pooled prevalence estimates per gene and variant, along with their respective positive predictive values (PPVs) for malignancy, were calculated. Summary: Our systematic search identified 540 studies that were supplemented by 18 studies from bibliographies or personal files. Sixty-one studies met all inclusion criteria and included >4600 ITNs. Overall, 26% of nodules contained at least 1 variant or fusion. However, half of them did not include details on the specific gene, variant, and/or complete fusion pair reported for inclusion toward PPV calculations. The PPVs of genomic alterations reported at least 10 times were limited to BRAF V600E (98%, 95% confidence interval [CI 96-99%]), PAX8/PPARG (55% [CI 34-78%]), HRAS Q61R (45% [CI 22-72%]), BRAF K601E (42% [CI 19-68%]), and NRAS Q61R (38% [CI 23-55%]). Excluding BRAF V600E , the pooled PPV for all other specified variants and fusions was 47%. Multiple variants within the same nodule were identified in ∼1% of ITN and carried a cumulative PPV of 77%. Conclusions: The chance that a genomic alteration predicts malignancy depends on the individual variant or fusion detected. Only five alterations were reported at least 10 times; BRAF V600E had a PPV of 98%, while the remaining four had individual PPVs ranging from 38% to 55%. The small sample size of most variants and fusion pairs found among ITNs, however, limits confidence in their individual PPV point estimates. Better specific reporting of genomic alterations with cytological category, histological subtype, and cancer staging would facilitate better understanding of cancer prediction, and the independent contribution of the genomic profile to prognosis.

Background: The Afirma Gene Expression Classifier (GEC) has been used to further characterize cytologically indeterminate (cyto-I) thyroid nodules into either benign or suspicious categories. However, its relatively low positive predictive value (PPV) limited its use as a classifier for patients with suspicious results. The Afirma Gene Sequencing Classifier (GSC) was developed to improve PPV while maintaining a high negative predictive value (NPV), yet real-world assessment of its performance is lacking. Methods: We analyzed all patients who had cyto-I nodules and molecular testing with either GEC or GSC between 2011 and 2018 at a single academic medical center. Clinical information was obtained for 343 GEC-tested nodules and 164 GSC-tested nodules. Results: The GSC had a statistically significant higher benign call rate (76.2% vs. 48.1%, p  < 0.001), PPV (60.0% vs. 33.3%, p  = 0.01), and specificity (94.3% vs. 61.4%, p  < 0.001) than the GEC. Improvement was statistically significant in both Bethesda III and Bethesda IV nodules. In particular, the benign call rate of GSC was significantly higher in nodules with Hürthle cell changes (88.8% vs. 25.7%, p  < 0.01). The rate of surgical intervention in the indeterminate nodule cohort has decreased by 66.4% since switching to the GSC; 52.5% of indeterminate nodules went to surgery while using the GEC compared with 17.6% with the GSC ( p  < 0.001). This reduction was statistically significant in nodules with Bethesda III diagnoses, demonstrating a 70.9% decrease (GEC 51.3% vs. GSC 14.9%, p  < 0.001), and in nodules with Bethesda IV cytology, a 39.2% decrease was noted (GEC 54.8% vs. GSC 33.3%, p  = 0.003). Conclusions: Data from a single academic tertiary center show an improved specificity and PPV while maintaining high sensitivity and NPV for GSC compared with GEC. A statistically significant increase in benign call rates was observed in GSC compared with GEC, likely indicating fewer false positive results. After implementation of GSC, surgical interventions have been reduced by 68%.

Background: For thyroid nodules with indeterminate cytology, the Afirma Gene Expression Classifier (GEC) identified benign nodules to reduce diagnostic surgery, though many nodules classified as suspicious still proved histopathologically benign. The current Afirma Genomic Sequencing Classifier (GSC) demonstrates improved specificity, suggesting more nodules will have a benign result (benign call rate [BCR]), but independent data are needed to confirm this in clinical practice. Methods: Retrospective analysis was performed of all Bethesda III or IV cytology thyroid nodules ≥1 cm tested with GEC (between January 1, 2011, and July 19, 2017) or GSC (between July 20, 2017, and August 27, 2018) at the authors' institution. Afirma testing was not performed reflectively for all nodules with Bethesda III or IV cytology, but rather was applied based on physician-patient decision making. Demographic, sonographic, and cytologic data were collected. The BCR for GEC- versus GSC-tested nodules was compared and further stratified by Bethesda classifications. Results: The study evaluated 600 nodules in 563 patients tested with either GEC ( n  = 486) or GSC ( n  = 114). The BCR was 233/486 (47.9%) for the GEC compared to 75/114 (65.8%) for the GSC ( p  = 0.0006). Hürthle-cell cytology was present in 99/486 (20.4%) nodules in the GEC group compared to 31/114 (27.2%) nodules in the GSC group ( p  = 0.28). The GSC BCR was significantly higher than the GEC BCR for Bethesda III nodules characterized by Hürthle cells ( p  = 0.006), but the BCRs were similar for nodules with architectural or cytologic atypia. In Bethesda IV nodules suspicious for follicular neoplasm, BCR for the GEC and GSC were similar ( p  = 0.68), but for cytology suspicious for Hürthle-cell neoplasm, the GSC BCR was 68.2% (15/22) compared to the GEC BCR of 16.4% (10/61; p  < 0.0001). Positive predictive value in resected nodules with a suspicious result was 16/32 (50%) for GSC nodules and 75/221 (33.9%) for GEC nodules ( p  = 0.1). Conclusions: The higher BCR for the GSC compared to the GEC for indeterminate thyroid nodules, predominantly among nodules with Hürthle-cell cytology, will likely lead to further reduction in surgical management.

Background: Thyroid nodules can be identified in up to 68% of the population. Fine-needle aspiration (FNA) cytopathology classifies 20%-30% of nodules as indeterminate, and these are often referred for surgery due to the risk of malignancy. However, histological postsurgical reports indicate that up to 84% of cases are benign, highlighting a high rate of unnecessary surgeries. We sought to develop and validate a microRNA (miRNA)-based thyroid molecular classifier for precision endocrinology (mir-THYpe) with both high sensitivity and high specificity, to be performed on the FNA cytology smear slide with no additional FNA. Methods: The expression of 96 miRNA candidates from 39 benign/39 malignant thyroid samples, (indeterminate on FNA) was analyzed to develop and train the mir-THYpe algorithm. For validation, an independent set of 58 benign/37 malignant FNA smear slides (also classified as indeterminate) was used. Results: In the training set, with a 10-fold cross-validation using only 11 miRNAs, the mir-THYpe test reached 89.7% sensitivity, 92.3% specificity, 90.0% negative predictive value and 92.1% positive predictive value. In the FNA smear slide validation set, the mir-THYpe test reached 94.6% sensitivity, 81.0% specificity, 95.9% negative predictive value, and 76.1% positive predictive value. Bayes' theorem shows that the mir-THYpe test performs satisfactorily in a wide range of cancer prevalences. Conclusions: The presented data and comparison with other commercially available tests suggest that the mir-THYpe test can be considered for use in clinical practice to support a more informed clinical decision for patients with indeterminate thyroid nodules and potentially reduce the rates of unnecessary thyroid surgeries.

Background: RAS mutations are common in the available mutational analysis of cytologically indeterminate (Cyto-I) thyroid nodules. However, their reported positive predictive value (PPV) for cancer is widely variable. The reason for this variability is unknown, and it causes clinical management uncertainty. A systematic review was performed, evaluating the PPV for cancer in RAS mutation positive Cyto-I nodules, and variables that might affect residual heterogeneity across the different studies were considered., Methods: PubMed was searched through February 22, 2017, including studies that evaluated at least one type of RAS mutation in Cyto-I nodules, including any (or all) of the Bethesda III/IV/V categories or their equivalents and where the histological diagnosis was available. The PPV residual heterogeneity was investigated after accounting for Bethesda classification, blindedness of the histopathologist to the RAS mutational status, Bethesda category-specific cancer prevalence for each study, and which RAS genes and codons were tested. This was studied using five meta-regression models fit to different sets of Bethesda classification categories: Bethesda III, IV, or V (III/IV/V); Bethesda III or IV (III/IV); Bethesda III only; Bethesda IV only; and Bethesda V only., Results: Of 1831 studies, 23 were eligible for data inclusion. Wide ranges of PPV were found at 0-100%, 28-100%, and 0-100% in Bethesda III, IV, and V, respectively. Residual heterogeneity remained moderately high for PPV after accounting for the above moderators for Bethesda III/IV/V (21 studies; I 2  = 59.5%) and Bethesda III/IV (19 studies; I 2  = 66.0%), with significant Cochran's Q-test for residual heterogeneity (p < 0.001). Among individual Bethesda categories, residual heterogeneity was: Bethesda III (eight studies; I 2  = 89.0%), IV (12 studies; I 2  = 53.5%), and V (10 studies; I 2  = 34.4%), with significant Cochran's Q-test for Bethesda III (p < 0.001) and IV (p = 0.04)., Conclusion: The PPV of RAS mutations in Bethesda III and IV categories is quite heterogeneous across different studies, creating low confidence in the accuracy of a single estimate of PPV. Clinicians must appreciate this wide variability when managing a RAS-mutated Cyto-I nodule. Future studies should seek to resolve this unexplained variability.

Background: Thyroid nodules are less common in pediatric patients (i.e., those ≤18 years) than they are in adults. The Bethesda System for Reporting Thyroid Cytopathology allows for individual risk stratification, but a significant number of nodules are indeterminate. Incorporating gene mutation panels and gene expression classifiers may aid in preoperative diagnosis. The overall aim of this study was to assess the prevalence of oncogene alterations in a representative pediatric population and across a broad-spectrum of thyroid tumor diagnoses., Methods: This was a retrospective cross-sectional evaluation of 115 archived samples, including: 47 benign (29 follicular adenoma, 11 diffuse hyperplasia, four thyroiditis, and three multinodular goiter), six follicular thyroid carcinomas (FTC), 24 follicular variant of papillary thyroid carcinomas (fvPTC), 27 classic variant of PTC (cPTC), eight diffuse sclerosing variant of PTC (dsvPTC), and three other PTC. Molecular testing was performed by multiplex qualitative polymerase chain reaction followed by bead array cytometry. Oncogene results were analyzed for association with age, sex, histology, lymph node metastasis, and intrathyroidal spread., Results: A mutation in one of the 17 molecular markers evaluated was found in: 2/6 (33%) FTC, 8/24 (33%) fvPTC, 17/27 (63%) cPTC, and 4/8 (50%) dsvPTC. Mutations in RAS or PAX8/PPARG were exclusive to FTC and fvPTC. BRAF was the most common mutation in cPTC (12/17; 71%), and RET/PTC was the only mutation associated with dsvPTC. Overall, a mutation was found in 32/68 (47%) malignant specimens, with a single follicular adenoma positive for PAX8/PPARG. The relative distribution of gene alterations in pediatric lesions was similar to adults. The presence of a BRAF mutation in pediatric cPTC did not predict a more invasive phenotype., Conclusions: Of 33 nodules with genetic alterations, 32 were malignant. Mutations in RAS were most frequently associated with FTC, RET/PTC rearrangements with dsvPTC, and invasive fvPTC, and BRAF with cPTC. These results suggest a clinical role for mutational analysis of pediatric nodules to guide the surgical approach.

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.

Background: The Afirma ® Gene Expression Classifier (GEC) risk stratifies The Bethesda System for the Reporting of Thyroid Cytopathology class III/IV (indeterminate) thyroid nodules (ITNs) as suspicious for malignancy or benign. Several authors have published studies describing the diagnostic accuracy of the GEC. However, the quality of these methods has not been rigorously examined., Summary: In this study, MEDLINE and EMBASE were searched for studies published between January 1, 2010, and June 30, 2016, examining the sensitivity, specificity, negative predictive value, and positive predictive value of the GEC. The Quality of Diagnostic Accuracy Studies 2 was customized to evaluate the methods of included studies in each of four domains: nodule selection, index test execution, reference standard assignment, and flow and timing. Signaling questions were used to identify sources of potential bias in calculation of diagnostic accuracy, and issues of applicability were assessed. Three panelists applied the Quality of Diagnostic Accuracy Studies 2 tool to each study included, and divergence was resolved in conference. In 12 studies evaluated, the most common methodologic flaw was lack of reference standard diagnosis assignment to un-excised GEC-benign ITNs. Exclusion of these ITNs from the analyses resulted in unreliable estimates of specificity and negative predictive value. Other flaws identified included restriction to ITNs that had already been selected for referral for thyroidectomy or lobectomy., Conclusions: Future studies should define and assign a "true negative" label to GEC-benign nodules that do not develop malignant signs or symptoms during a pre-specified period of follow-up, and these nodules should be included in calculations of diagnostic accuracy.

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.

Background: The use of calcitonin screening for the rare medullary thyroid cancer (MTC) is controversial due to questions of efficacy, accuracy, and cost-effectiveness. This study reports the results of a large prospective validation using a machine-trained algorithm (MTC Classifier) to preoperatively identify MTC in fine-needle aspiration biopsies in lieu of calcitonin measurements., Methods: Cytology analysis on a prospective consecutive series of 50,430 thyroid nodule biopsies yielded a total of 7815 indeterminate (Bethesda categories III/IV) cases, which were tested with the MTC classifier. A prospective, consecutively submitted series of 2673 Bethesda III-VI cases with cytology determined locally was also evaluated. RNA was isolated and tested for the MTC Classifier using microarrays., Results: Forty-three cases were positive by the MTC Classifier among 10,488 tested nodules (0.4%), consistent with the low prevalence of MTC. Of these, all but one was histologically or biochemically confirmed as MTC, yielding a positive predictive value (PPV) of 98%. Of the positive cases, only 19 (44%) had been specifically suspected of MTC by cytology, highlighting the limitations of light microscopy to detect this disease. Three surgically confirmed MTC cases that were detected by the MTC Classifier had low basal serum calcitonin values, indicating these would have been missed by traditional calcitonin screening methods. A pooled analysis of three independent validation sets demonstrates high test sensitivity (97.9%), specificity (99.8%), PPV (97.9%), and negative predictive value (99.8%)., Conclusions: A clinical paradigm is proposed, whereby cytologically indeterminate thyroid nodules being tested for common malignancies using gene expression can be simultaneously tested for MTC using the same genomic assay at no added cost.

Background: Patients labeled as having indeterminate thyroid nodular disease following fine-needle aspiration cytology are at risk of non-optimal initial surgery: an overly radical total thyroidectomy, or an unnecessary two-stage operation. The objective of this study was to assess the impact of combining mutational markers and ultrasonographic (US) features preoperatively on predicting the risk of malignancy in patients with indeterminate nodules, thereby offering them a tailored initial surgical intervention., Methods: The records of 258 patients who underwent conventional total thyroidectomy for single nodules reported as suspicious for a follicular neoplasm (Bethesda category IV) in a four-year period were reviewed. Main issues addressed included: certain US findings (individually and in combination), mutational markers (BRAF and NRAS), and combinations of both. Correlation of these with malignancy was assessed, as was their ability to predict malignancy. The usefulness of combining the absence of suspicious sonographic features and the absence of mutational markers was also evaluated., Results: Among the 258 patients with an indeterminate diagnosis, only 90 lesions were found to be malignant. The sonographic features that correlated significantly with malignancy were irregular margins, microcalcifications, and a "taller than wide" shape. The presence of irregular margins was the feature with the highest positive predictive value. Combinations of two or more features were always associated with predictivity in excess of 90%, and at times at 100%. NRAS mutation was the most common gene alteration. Both BRAF and NRAS mutations were mutually exclusive and correlated significantly with malignancy. Their predictivity of malignancy was high, particularly when combined with suspicious sonographic features (100%). The major limitation of both suspicious sonographic features and/or mutational markers was their low occurrence in malignancy. The absence of both mutational markers and suspicious sonographic features proved extremely useful in tailoring surgical strategy, as it could have ultimately spared 143/258 patients (55%) an overly radical thyroidectomy., Conclusion: The preoperative utility of mutational markers and sonographic features in combination has a synergistic impact. It can predict the risk of malignancy with high accuracy, properly triaging patients to appropriate surgery.

Background: Multiple gene mutations in thyroid nodules are rare. The presence of several oncogenic mutations could be associated with aggressive biological behavior of tumors., Patient Findings: A 60-year-old female presented to her physician after she felt a lump in her neck. On ultrasound, she was found to have a 1.4 cm × 0.8 cm × 1.3 cm nodule in the isthmus and a 0.5 cm × 0.6 cm × 0.6 cm nodule with irregular margins and hypoechogenicity in the right thyroid lobe, warranting fine-needle aspiration (FNA). Cytological examination of the smaller nodule yielded a diagnosis of atypia of undetermined significance (AUS/FLUS, Bethesda Category III). The aspirate was submitted for molecular testing using the next-generation sequencing ThyroSeq(®) v2 panel. The test revealed four distinct mutations: BRAF (p.V600E), TERT (C228T), PIK3CA (p.H1047R), and AKT1 (p.E17K). Presence of multiple oncogenic mutations in the FNA specimen was highly indicative of cancer, and suggestive of a cancer with propensity toward more aggressive biological behavior. Four weeks after the FNA results were available, the patient underwent total thyroidectomy. This was followed by radioactive iodine ablation after the final pathology revealed a 0.5 cm papillary thyroid carcinoma (PTC) with extrathyroidal extension and positive resection margins (pT3 stage)., Summary: Herein, the first case of four mutations preoperatively detected in a subcentimeter thyroid nodule that was confirmed to be a PTC with aggressive biological behavior is reported., Conclusions: The judicious indication of FNA and use of molecular screening can potentially help in predicting aggressive behavior of small-sized thyroid cancers and in identifying patients who may benefit from early and more extensive therapy.

Background: Fine-needle aspiration (FNA) cytology is a common approach to evaluate thyroid nodules. It offers definitive diagnosis of a benign or malignant nodule in the majority of cases. However, 10-25% of nodules yield one of three indeterminate cytologic diagnoses, leading to suboptimal management of these patients. Atypia of undetermined significance/follicular lesion of undermined significance (AUS/FLUS) is a common indeterminate diagnosis, with the cancer risk ranging from 6% to 48%. This study assessed whether a multi-gene next-generation sequencing (NGS) assay can offer significant improvement in diagnosis in AUS/FLUS nodules., Methods: From May 2014 to March 2015, 465 consecutive FNA samples with the cytologic diagnosis of AUS/FLUS underwent prospective molecular testing using the ThyroSeq v2.1 panel. The panel included 14 genes analyzed for point mutations and 42 types of gene fusions occurring in thyroid cancer. In addition, eight genes were assessed for expression in order to evaluate the cell composition of FNA samples. Ninety-eight (21%) of these nodules had definitive surgical (n = 96) or nonsurgical (n = 2) follow-up and were used to determine the assay performance., Results: Among 465 AUS/FLUS nodules, three were found to be composed of parathyroid cells and 462 of thyroid follicular cells. Of the latter, 31 (6.7%) were positive for mutations. The most frequently mutated genes were NRAS and HRAS, and overall point mutations in seven different genes and five types of gene fusions were identified in these nodules. Among 98 nodules with known outcome, histologic analysis revealed 22 (22.5%) cancers. ThyroSeq v2.1 was able to classify 20/22 cancers correctly, showing a sensitivity of 90.9% [confidence interval (CI) 78.8-100], specificity of 92.1% [CI 86.0-98.2], positive predictive value of 76.9% [CI 60.7-93.1], and negative predictive value of 97.2% [CI 78.8-100], with an overall accuracy of 91.8% [CI 86.4-97.3]., Conclusions: The results of the study demonstrate that the ThyroSeq v2.1 multi-gene NGS panel of molecular markers provides both high sensitivity and high specificity for cancer detection in thyroid nodules with AUS/FLUS cytology, which should allow improved management for these patients.

Background: The accurate diagnosis of thyroid nodules is important for making management decisions. The purpose of this study is to evaluate the clinical usefulness of BRAF(V600E) mutation analysis with core-needle biopsy (CNB+BRAF(V600E)) in thyroid nodules with previous atypia of undetermined significance (AUS) or follicular lesions of undetermined significance (FLUS) results., Materials and Methods: From January 2011 to December 2012, 590 CNB+BRAF(V600E) mutation analyses were performed. We analyzed 200 nodules from 200 patients with previous AUS/FLUS results (22 men, 178 women; mean age, 48.6 years). The clinical usefulness of CNB+BRAF(V600E) was assessed by comparing the rates of conclusive results, the additional value of BRAF(V600E) mutation analysis, diagnostic performances, and therapeutic/diagnostic surgery results with those of CNB alone. For the subgroup analysis, the study patients were divided into those with nodules with previous AUS results and those with previous FLUS results., Results: All CNB+BRAF(V600E) procedures were well-tolerated. CNB+BRAF(V600E) did not show significantly better diagnostic performance than CNB alone in thyroid nodules with previous AUS/FLUS results. However, the conclusive result rate of CNB+BRAF(V600E) was improved in thyroid nodules with previous AUS/FLUS results (76.5% vs. 73.0%, p=0.016), especially with previous AUS results (81.1% vs. 76.4%, p=0.031). Of the 56 previous AUS result thyroid nodules with surgical management, BRAF(V600E) mutation analysis led to therapeutic surgery in 5.4% by decreasing unnecessary diagnostic surgery., Conclusions: In general, CNB+BRAF(V600E) did not show significantly higher diagnostic accuracy than CNB alone. Although CNB+BRAF(V600E) may add additional value in nodules with previous AUS results, routinely adding BRAF(V600E) mutation analysis to CNB is not recommended.

Background: The recently introduced Afirma gene expression classifier (AGEC) provides binary results (benign or suspicious) to guide management of cytologically indeterminate thyroid nodules. The AGEC is intended to reduce unnecessary surgeries for benign nodules, and management algorithms favor surgery for suspicious results. Limited data are available on the performance of this test for Hürthle cell nodules (HCNs). This study hypothesized that a predominance of Hürthle cells leads to an increased rate of suspicious AGEC results with a potential for overtreatment, despite a relatively low risk of malignancy., Methods: The pathology databases from three tertiary care facilities were queried from 2010 to 2014 for fine-needle aspirates (FNAs) diagnosed as suspicious for Hürthle cell neoplasm (SHCN) or atypia of undetermined significance/follicular lesion of undetermined significance concerning for Hürthle cell neoplasm (AFHCN). Cytology diagnoses were rendered internally prior to AGEC testing. The patient demographics, FNA diagnosis, AGEC result, surgical procedure, and pathologic outcomes were recorded., Results: The cohort consisted of 134 patients with HCNs. Prior to AGEC availability, 62 patients underwent surgery: 81% (50/62) of patients had surgery, and 34% (17/50) of the resected index nodules were malignant. After introduction of the AGEC, 72 patients underwent AGEC testing: 65% (47/72) of patients had surgery, and 13% (6/46) of the resected nodules were malignant. Thirty-two percent (23/72) of patients had a benign AGEC result and did not undergo surgery, and 4% (3/72) had surgery despite a benign AGEC result with benign final pathology, whereas 63% (45/72) of patients had suspicious AGEC results, with 96% of these patients (43/45) undergoing surgery, and 14% (6/43) of these index nodules were malignant., Conclusions: While 32% of tested patients declined surgery based on a benign AGEC, 86% of patients with suspicious AGEC findings had unnecessary surgery, reflecting a substantially lower rate of malignancy from what was previously reported for all indeterminate nodules. Given the approximate pretest malignancy risk of 25-35% for an FNA diagnosis of SHCN or AFHCN, a suspicious AGEC diagnosis does not increase the probability of malignancy in an HCN, and patients should be counseled accordingly.

Background: Recent advances in research on thyroid carcinogenesis have yielded applications of diagnostic molecular biomarkers and profiling panels in the management of thyroid nodules. The specific utility of these novel, clinically available molecular tests is becoming widely appreciated, especially in perioperative decision making by the surgeon regarding the need for surgery and the extent of initial resection., Methods: A task force was convened by the Surgical Affairs Committee of the American Thyroid Association and was charged with writing this article., Results/conclusions: This review covers the clinical scenarios by cytologic category for which the thyroid surgeon may find molecular profiling results useful, particularly for cases with indeterminate fine-needle aspiration cytology. Distinct strengths of each ancillary test are highlighted to convey the current status of this evolving field, which has already demonstrated the potential to streamline decision making and reduce unnecessary surgery, with the accompanying benefits. However, the performance of any diagnostic test, that is, its positive predictive value and negative predictive value, are exquisitely influenced by the prevalence of cancer in that cytologic category, which is known to vary widely at different medical centers. Thus, it is crucial for the clinician to know the prevalence of malignancy within each indeterminate cytologic category, at one's own institution. Without this information, the performance of the diagnostic tests discussed below may vary substantially.

Background: Thyroid nodules are less common in children than adults, but the risk of malignancy in thyroid nodules is much higher in children. The ability to characterize pediatric thyroid nodules has improved with the use of ultrasound-guided fine-needle aspiration, the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) classification system, and expanded molecular testing. Nevertheless, stratification criteria to predict thyroid malignancy in children are poorly defined. Our objective was to determine if clinical presentation and molecular genetics could predict malignancy in pediatric thyroid nodules., Methods: Retrospective chart review of patients ≤18 years of age at the Children's Hospital of Pittsburgh of the University of Pittsburgh Medical Center with the diagnosis of a thyroid nodule from January 2007 to January 2012 was conducted. Eighty-nine subjects fulfilled the inclusion criteria: 1) thyroid nodule ≥0.8 cm and biopsy (n=76), or 2) thyroid nodule ≥0.8 cm, no biopsy, and ultrasound follow-up for at least 2 years (n=13)., Results: Twenty-four (27%) of 89 patients were diagnosed with thyroid cancer (50% papillary thyroid carcinoma [PTC], 50% follicular variant of papillary thyroid carcinoma [FVPTC]). Features associated with malignancy included larger nodule size, palpable nodule, or palpable lymphadenopathy. There were no differences in presenting features between patients with PTC and those with FVPTC. Thyroid malignancy was diagnosed in all nine patients with a molecular abnormality (BRAF, RAS, RET/PTC, PAX8/PPARγ)., Conclusions: Clinical features, FNA cytology, and molecular genetics are valuable tools to discriminate benign from malignant nodules in pediatric patients. This information is important to direct subsequent clinical management.

Background: Molecular testing for oncogenic gene mutations and chromosomal rearrangements plays a growing role in the optimal management of thyroid nodules, yet lacks standardized testing modalities and systematic validation data. Our objective was to assess the performance of molecular cytology on preoperative thyroid nodule fine-needle aspirates (FNAs) across a broad range of variables, including independent collection sites, clinical practices, and anatomic pathology interpretations., Methods: Single-pass FNAs were prospectively collected from 806 nodules 1 cm or larger by ultrasonography at five independent sites across the United States. Specimens were shipped in a nucleic acid stabilization solution and tested at a centralized clinical laboratory. Seventeen genetic alterations (BRAF, KRAS, HRAS, and NRAS mutations, PAX8-PPARG and RET-PTC rearrangements) were evaluated by multiplex polymerase chain reaction and liquid bead array cytometry in 769 FNAs that met inclusion criteria. Cytology, histology, and clinical care followed local procedures and practices. All results were double-blinded., Results: Thirty-two specimens (4.2%) failed to yield sufficient nucleic acid to generate molecular data. A single genetic alteration was detected in 80% of cytology malignant cases, 21% of indeterminate, 7.8% of nondiagnostic, and 3.5% of benign cases. Among 109 nodules with surgical histology reference standard, oncogenic mutations were present in 50% of malignant nodules missed by cytology. There were 14 cancers not identified by cytology or molecular tests, including 5 carcinomas with histologic sizes less than 1 cm (3 multifocal) and 8 noninvasive follicular variants of papillary carcinoma (4 encapsulated). No mutations were detected in 89% of the nodules benign by histopathology with 6 false-positive molecular results in 5 adenomas (2-5.5 cm) and 1 cystic nodule with an incidental papillary microcarcinoma (0.15 cm). The posttest probability of thyroid cancer was 100% for nodules positive for BRAF or RET-PTC, 70% for RAS or PAX8-PPARG, and 88% for molecular cytology overall., Conclusions: Centralized and standardized molecular testing for genetic alterations associated with a high risk of malignancy efficiently complements the local cytopathologic diagnosis of thyroid nodule aspirates in the clinical setting. Actionable molecular cytology can improve the personalized surgical and medical management of patients with thyroid cancers, facilitating one-stage total thyroidectomy and reducing the number of unnecessary diagnostic surgeries.

Background: PAX8/PPARγ rearrangement is a common genetic alteration in follicular thyroid carcinoma (FTC) and has been reported with variable frequency in papillary thyroid carcinoma (PTC). The diagnostic and phenotypic features of thyroid nodules positive for PAX8/PPARγ on preoperative examination are not well understood., Methods: The prevalence of PAX8/PPARγ rearrangement was analyzed in a series of 2015 consecutive thyroid nodules that underwent molecular analysis on cytology specimens and in 446 surgically removed PTCs. For all PAX8/PPARγ positive cases, cytology and surgical pathology slides were examined and the available clinical records were reviewed., Results: Twenty-two PAX8/PPARγ rearrangements were identified, including 16 detected preoperatively and 6 postoperatively. The incidence of PAX8/PPARγ in PTC was 1.1%. Cytologically, most of these nodules were diagnosed as a follicular neoplasm (73%), followed by the diagnosis of atypia of undetermined significance (19%), and none of the cases was diagnosed as cytologically malignant. All nodules with PAX8/PPARγ detected preoperatively and surgical follow-up available were found to be malignant, among which the most common diagnosis was the encapsulated follicular variant of PTC. Overall, among 20 PAX8/PPARγ-positive tumors that were surgically excised, 17 (85%) were PTC and 3 (15%) were FTC. On follow-up available for 17 patients (mean, 22.4 months), 16 PAX8/PPARγ-positive cancers showed no evidence of biochemical or structural recurrence, whereas 1 patient with FTC developed bone metastasis., Conclusions: In this series, PAX8/PPARγ rearrangement found in thyroid nodules had a 100% predictive value for differentiated thyroid cancer, and was more predictive of PTC than FTC. However, almost all PTC carrying PAX8/PPARγ were encapsulated follicular-pattern tumors, distinguished from FTC only by nuclear features. Although most tumors carrying this mutation appear to be clinically indolent, at least on short-term follow-up, distant metastasis can develop from FTC positive for PAX8/PPARγ.

Background: Autonomously functioning, "hot", thyroid nodules are not common in children and adolescents. Such nodules are not considered alarming because they are assumed to be benign adenomas. Herein, we present a 15-year-old girl with a papillary thyroid carcinoma of 3.5 cm in diameter, which was functionally autonomous and scintigraphically hot., Patient Findings: The patient, initially referred to our Endocrine Unit because of a thyroid nodule, returned 6 months later for symptoms of hyperthyroidism. Hyperthyroidism was confirmed biochemically. Radioactive iodine ((131)I) thyroid scintigraphy was consistent with an autonomous thyroid nodule. As per guidelines, the patient underwent surgery and a pathological examination revealed papillary carcinoma, follicular variant. The excised nodule was examined for activating mutations of the thyrotropin receptor (TSHR), Gsα (GNAS1), H-RAS, N-RAS, K-RAS, and BRAF genes by direct sequencing. No mutations were found. Nevertheless, two combined nonfunctioning mutations were detected: a single-nucleotide polymorphism (SNP) of the TSHR gene, in exon 7, at codon 187 (AAT→AAC, both encoding asparagine), and a SNP within exon 8 of the Gsα gene at codon 185 (ATC→ATT, both encoding isoleucine). Both SNPs were also identified in the germline DNA of the patient. The same SNPs were sought in the parents and brother of our patient. Her father was heterozygous for the TSHR SNP, her mother heterozygous for the Gsα SNP, and her brother was wild type., Conclusions: This case demonstrates that the presence of hyperfunctioning thyroid nodule(s) does not rule out cancer and warrants careful evaluation, especially in childhood and adolescence to overlook malignancy.

Background: The cribriform-morular variant of papillary thyroid carcinoma (cmvPTC) is rare. There are few if any studies of the ultrasonographic (US) features of cmvPTC. The aim of this study was to determine the characteristic US and clinical features of the cmvPTC., Methods: A retrospective review of the US and clinical features was performed on 18 surgically confirmed cmvPTCs in five patients who were seen at our institution between January 2000 and December 2010., Results: All patients were female with a mean age of 28 years (range, 19-46 years). Two patients presented with palpable lesions, and the other patients were incidentally detected during screening US. On US, the majority of nodules had well-defined, oval to round shapes, and were hypoechoic and solid without calcifications. However, 6 (33.3%) of 18 nodules did have a cystic change. The size of the lesions varied from 0.3 to 3.0 cm (mean, 1.11 cm). None of the nodules were diagnosed as malignant based on the US criteria, but all except one patient had a cytology of their thyroid nodules that was read as malignant, without revealing the subtype of their PTC. Two of the five patients had familial adenomatous polyposis (FAP), and they had bilateral multiple nodules. No metastatic lymph nodes or extrathyroidal extension were identified. To date, none of the patients has had recurrence or metastasis during their mean follow-up of 25 months after thyroidectomy., Conclusion: It appears that most cases of cmvPTC do not have features of malignancy on US and that they are indolent tumors as far as their clinical and histological features are concerned.

Background: Seventy-five percent of thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology are found to be benign postoperatively. A novel genomic test, the Afirma gene expression classifier (AGEC), has been available for clinical use in the United States, since late 2010. In 2010, two modest-sized validation studies showed that the AGEC could identify a benign gene expression signature in indeterminate cytology thyroid FNA samples with a negative predictive value >95%. The objective of this study was to evaluate how the AGEC impacted the joint decision of the endocrinologist and patient to operate when FNA cytology was indeterminate, but the AGEC reading of the nodule was benign., Methods: In this cross-sectional cohort study, data were contributed retrospectively by 51 endocrinologists at 21 practice sites that had previously obtained ≥3 benign AGEC readings in ≥1 cm nodules with indeterminate FNA cytology readings. Information regarding demographic data, nodule size and location, decision to operate, surgery type (hemithyroidectomy [HT] or total thyroidectomy [TT]), and reason for recommending surgery was retrospectively collected., Results: Compared to a 74% previous historical rate of surgery for cytologically indeterminate nodules, the operative rate fell to 7.6% during the period that AGEC were obtained in the clinical practices, a highly significant reduction in the decision to operate (p<0.001). The rate of surgery on cytologically indeterminate nodules that were benign by the AGEC reading did not differ from the historically reported rate of operation on cytologically benign nodules (p=0.41). The four primary reasons reported by the physicians for operating on nodules with a benign AGEC reading, in descending order: large nodule size (46.4%), symptomatic nodules (25.0%), rapidly growing nodules (10.7%), or a second suspicious or malignant nodule in the same patient (10.7%). These reasons are concordant with those typically given for operation on cytologically benign nodules., Conclusions: In a substantial group of medical practices, obtaining an AGEC test in patients with cytologically indeterminate nodules was associated with a striking reduction in the rate of diagnostic thyroidectomy. Approximately, one surgery was avoided for every two AGEC tests run on thyroid FNAs with indeterminate cytology.

Background: Papillary carcinomas with diameters that are less than or equal to 1 cm (thyroid papillary microcarcinoma [mPTC]) are quite common but can carry more risk than previously thought. The proper treatment and management of these lesions is still being debated. Even though fine needle aspiration cytology (FNAC) is considered the best method for the diagnosis of thyroid nodules, its efficacy is still questioned for mPTC. We investigated the role of BRAF gene status in preoperative cytological samples, using manual macrodissection as an additional tool to improve the diagnostic accuracy of mPTC., Methods: DNA was extracted directly from stained FNAC smears of 95 patients including 85 with histological diagnoses of papillary thyroid carcinoma (PTC) ≤1 cm and 10 with goiters. The cytological diagnoses of the 95 cases included the following: 42 samples were suspicious for papillary carcinoma, 38 were PTCs, and 15 were indeterminate lesions. DNA was then extracted from the FNAC slides after performing a "manual macrodissection" procedure. The BRAF(V600E) mutational status was determined by sequence analysis in all the patients., Results: In this study, we showed that the BRAF(V600E) mutation was present with a high frequency in patients with mPTC (74%). The presence of the mutation was independent of the size of the tumor. In our study, the combination of the cytological diagnosis and the molecular analysis was able to identify 82% of all cases of mPTC, with an increase of 37% compared with a morphological diagnosis alone. The morpho-molecular analysis was able to reduce the number of suspicious cases by >70%. All of the goiters had a wild-type BRAF status., Conclusions: The analysis of BRAF mutational status in FNAC obtained from papillary microcarcinomas demonstrates that molecular pathology, combined with morphology and molecular biology is a powerful tool for cytological diagnosis of mPTC. Our results also confirm the data supporting the biological relevance of PTCs with diameters that are ≤1 cm and the importance of "manual macrodissection" in the molecular analysis of cytological material.