Serum microRNA-146a-5p and microRNA-221-3p as potential clinical biomarkers for papillary thyroid carcinoma.

PURPOSE: Papillary thyroid carcinoma (PTC) is the most common malignant thyroid neoplasm, accounting for approximately 85% of all follicular cell-derived thyroid nodules. This study aimed to assess the diagnostic potential of circulating microRNA-146a-5p and microRNA-221-3p as biomarkers for PTC and their usefulness in monitoring disease progression during patient follow-up. METHODS: An observational study was conducted on two cohorts of PTC patients and healthy controls (HCs) using digital PCR. We collected patients' clinical, biochemical, and imaging data during the post-surgery surveillance. We analyzed the levels of circulating miRNAs in serum samples of patients before surgery and during the follow-up, including those with indeterminate/biochemical incomplete response (IndR/BIR) and residual thyroid tissues (Thy Residue). RESULTS: Both miR-146a-5p and miR-221-3p were confirmed as effective biomarkers for PTC diagnosis. They enabled differentiation between pre-surgery PTC patients and HCs with an area under the curve (AUC) of 92% and 87.3%, respectively, using a threshold level of 768,545 copies/uL for miR-146a-5p and 389,331 copies/uL for miR-221-3p. It was found that miRNA fold change levels, rather than absolute levels, can be useful during patient follow-up. In particular, we found that a fold change of 2 for miR-146a-5p and 2.2 for miR-221-3p can identify a progressive disease, regardless of the presence of TgAbs or remnant thyroid. CONCLUSION: MiRNA-146a-5p and miRNA-221-3p, particularly the former, could be valuable diagnostic biomarkers for PTCs. They also seem to be effective in monitoring disease progression during patient follow-up by evaluating their fold change, even when thyroglobulin is uninformative.

A synonymous RET substitution enhances the oncogenic effect of an in-cis missense mutation by increasing constitutive splicing efficiency.

Synonymous mutations continue to be filtered out from most large-scale cancer genome studies, but several lines of evidence suggest they can play driver roles in neoplastic disease. We investigated a case of an aggressive, apparently sporadic medullary thyroid carcinoma (MTC) harboring a somatic RET p.Cys634Arg mutation (a known MTC driver). A germ-line RET substitution (p.Cys630=) had also been found but was considered clinically irrelevant because of its synonymous nature. Next generation sequencing (NGS) of the tumor tissues revealed that the RET mutations were in cis. There was no evidence of gene amplification. Expression analysis found an increase of RET transcript in p.Cys630=;p.Cys634Arg patient compared with that found in 7 MTCs harboring p.Cys634 mutations. Minigene expression assays demonstrated that the presence of the synonymous RET mutation was sufficient to explain the increased RET mRNA level. In silico analyses and RNA immunoprecipitation experiments showed that the p.Cys630 = variant created new exonic splicing enhancer motifs that enhanced SRp55 recruitment to the mutant allele, leading to more efficient maturation of its pre-mRNA and an increased abundance of mature mRNA encoding a constitutively active RET receptor. These findings document a novel mechanism by which synonymous mutations can contribute to cancer progression.

Low-risk papillary thyroid microcarcinoma: Optimal management toward a more conservative approach.

The incidence of papillary thyroid microcarcinoma (microPTC) has dramatically increased in the last decades. Most of these tumors remain small and clinically "silent", only small number progress. Although thyroid surgery used to be the only therapeutic approach, recent guidelines now consider active surveillance for low-risk microPTC. For this reason, more accurate risk stratification of microPTC is needed. The optimal management of low-risk microPTC through accurate risk stratification represents a major clinical issue.

Diagnostic Performance of Neck Ultrasonography in the Preoperative Evaluation for Extrathyroidal Extension of Suspicious Thyroid Nodules.

BACKGROUND: A preoperative neck ultrasound (US) is recommended for all patients with suspected thyroid cancer, to identify features potentially changing surgical extent. The extrathyroidal extension (ETE) is considered an indication for total thyroidectomy, but there is limited consensus on its US definition, and the interobserver reliability is low. This study aimed to evaluate the predictive value of neck US for ETE before surgery and to estimate the diagnostic performance of different US findings, evaluated during real-time examinations. METHODS: Patients referred to surgery between November 1, 2015, and May 31, 2019, for a suspicious thyroid cancer underwent a preoperative neck US, with systematic assessment for ETE. Three definitions were tested: very restrictive (capsular disruption with suspicious images of surrounding tissues invasion), restrictive (including also capsular abutment with evidence of capsular disruption), and nonrestrictive (capsular abutment is sufficient). Histopathology report of ETE involving at least soft tissues was considered positive. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. RESULTS: The study cohort included 128 patients, with 102 (79.7%) confirmed malignancies, and 44 (43.1%) histological ETE. The nonrestrictive definition had good sensitivity (86.4%) but low specificity (29.8%), with an NPV of 80.6%; the restrictive definition had higher specificity (81%), while the very restrictive had specificity and PPV of 100%. CONCLUSIONS: A more extensive surgical approach should not be based on US suspicion of ETE alone, with the possible exception of gross invasion appearance. The absence of any sign of ETE, on the other hand, has a substantial negative predictive value.

Whole exome sequencing identifies a germline MET mutation in two siblings with hereditary wild-type RET medullary thyroid cancer.

Whole exome sequencing (WES) was used to investigate two Italian siblings with wild-type RET genotype, who developed medullary thyroid cancers (MTCs) and, later, primary prostate and breast cancers, respectively. The proband's MTC harbored a p.Met918Thr RET mutation; his sister's MTC was RET/RAS wild-type. Both siblings had a germline mutation (p.Arg417Gln) in the extracellular Sema domain of the proto-oncogene MET. Experiments involving ectopic expression of MET p.Arg417Gln in MET-negative T47D breast cancer cells documented the mutant receptor's functionality and its ability to enhance cell migration and invasion. Our findings highlight a possible link between MET germline mutations and MTCs and suggest that MET p. Arg417Gln may promote an invasive malignant phenotype. The possibility that MTC can be driven/co-driven by a MET mutation has potential management implications, since the tyrosine-kinase inhibitor cabozantinib-approved for treating advanced MTCs-is a specific MET inhibitor.

The Diagnosis and Management of Thyroid Nodules: A Review.

IMPORTANCE: Thyroid nodules are common, being detected in up to 65% of the general population. This is likely due to the increased use of diagnostic imaging for purposes unrelated to the thyroid. Most thyroid nodules are benign, clinically insignificant, and safely managed with a surveillance program. The main goal of initial and long-term follow-up is identification of the small subgroup of nodules that harbor a clinically significant cancer (≈10%), cause compressive symptoms (≈5%), or progress to functional disease (≈5%). OBSERVATIONS: Thyroid function testing and ultrasonographic characteristics guide the initial management of thyroid nodules. Certain ultrasound features, such as a cystic or spongiform appearance, suggest a benign process that does not require additional testing. Suspicious sonographic patterns including solid composition, hypoechogenicity, irregular margins, and microcalcifications should prompt cytological evaluation. Additional diagnostic procedures, such as molecular testing, are indicated only in selected cases, such as indeterminate cytology (≈20%-30% of all biopsies). The initial risk estimate, derived from ultrasound and, if performed, cytology report, should determine the need for treatment and the type, frequency, and length of subsequent follow-up. Management includes simple observation, local treatments, and surgery and should be based on the estimated risk of malignancy and the presence and severity of compressive symptoms. CONCLUSIONS AND RELEVANCE: Most thyroid nodules are benign. A diagnostic approach that uses ultrasound and, when indicated, fine-needle aspiration biopsy and molecular testing, facilitates a personalized, risk-based protocol that promotes high-quality care and minimizes cost and unnecessary testing.

BRAF V600E and risk stratification of thyroid microcarcinoma: a multicenter pathological and clinical study.

Studies from single institutions have analyzed BRAF in papillary microcarcinomas, sometimes with contradictory results. Most of them have provided limited integration of histological and clinical data. To obtain a comprehensive picture of BRAF V600E-mutated microcarcinomas and to evaluate the role of BRAF testing in risk stratification we performed a retrospective multicenter analysis integrating microscopical, pathological, and clinical information. Three hundred and sixty-five samples from 300 patients treated at six medical institutions covering different geographical regions of Italy were analyzed with central review of all cases. BRAF V600E statistical analysis was conducted on 298 microcarcinomas from 264 patients after exclusion of those that did not meet the required criteria. BRAF V600E was identified in 145/298 tumors (49%) including the following subtypes: 35/37 (95%, P<0.0001) tall cell and 72/114 (64%, P<0.0001) classic; conversely 94/129 follicular variant papillary microcarcinomas (73%, P<0.0001) were BRAF wild type. BRAF V600E-mutated microcarcinomas were characterized by markedly infiltrative contours (P<0.0001) with elongated strings of neoplastic cells departing from the tumor, and by intraglandular tumor spread (P<0.0001), typically within 5 mm of the tumor border. Multivariate analysis correlated BRAF V600E with specific microscopic features (nuclear grooves, optically clear nuclei, tall cells within the tumor, and tumor fibrosis), aggressive growth pattern (infiltrative tumor border, extension into extrathyroidal tissues, and intraglandular tumor spread), higher American Thyroid Association recurrence risk group, and non-incidental tumor discovery. The following showed the strongest link to BRAF V600E: tall cell subtype, many neoplastic cells with nuclear grooves or with optically clear nuclei, infiltrative growth, intraglandular tumor spread, and a tumor discovery that was non-incidental. BRAF V600E-mutated microcarcinomas represent a distinct biological subtype. The mutation is associated with conventional clinico-pathological features considered to be adverse prognostic factors for papillary microcarcinoma, for which it could be regarded as a surrogate marker. BRAF analysis may be useful to identify tumors (BRAF wild type) that have negligible clinical risk.

The natural history of benign thyroid nodules.

IMPORTANCE: Detection of asymptomatic thyroid nodules has increased. Consensus is lacking regarding the optimal follow-up of cytologically proven benign lesions and sonographically nonsuspicious nodules. Current guidelines recommend serial ultrasound examinations and reassessment of cytology if significant growth is observed. OBJECTIVE: To determine the frequency, magnitude, and factors associated with changes in thyroid nodule size. DESIGN, SETTING, AND PARTICIPANTS: Prospective, multicenter, observational study involving 992 consecutive patients with 1 to 4 asymptomatic, sonographically or cytologically benign thyroid nodules. Patients were recruited from 8 hospital-based thyroid-disease referral centers in Italy between 2006 and 2008. Data collected during the first 5 years of follow-up, through January 2013, were analyzed. MAIN OUTCOMES AND MEASURES: Baseline nodule growth (primary end point) was assessed with yearly thyroid ultrasound examinations. Size changes were considered significant for growth if an increase of 20% or more was recorded in at least 2 nodule diameters, with a minimum increase of 2 mm. Baseline factors associated with growth were identified. Secondary end points were the sonographic detection of new nodules and the diagnosis of thyroid cancer during follow-up. RESULTS: Nodule growth occurred in 153 patients (15.4% [95% CI, 14.3%-16.5%]). One hundred seventy-four of the 1567 original nodules (11.1% [95% CI, 10.3%-11.9%]) increased in size, with a mean 5-year largest diameter increase of 4.9 mm (95% CI, 4.2-5.5 mm), from 13.2 mm (95% CI, 12.1-14.2 mm) to 18.1 mm (95% CI, 16.7-19.4 mm). Nodule growth was associated with presence of multiple nodules (OR, 2.2 [95% CI 1.4-3.4] for 2 nodules; OR, 3.2 [95% CI, 1.8-5.6 for 3 nodules; and OR, 8.9 [95% CI, 4.4-18.0] for 4 nodules), main nodule volumes larger than 0.2 mL (OR, 2.9 [95% CI, 1.7-4.9] for volumes >0.2 to <1 mL and OR, 3.0 [95% CI, 1.8-5.1] for volumes ≥1 mL), and male sex (OR, 1.7 [95% CI, 1.1-2.6]), whereas an age of 60 years or older was associated with a lower risk of growth than age younger than 45 years (OR, 0.5 [95% CI 0.3-0.9]). In 184 individuals (18.5% [95% CI, 16.4%-20.9%]), nodules shrank spontaneously. Thyroid cancer was diagnosed in 5 original nodules (0.3% [95% CI, 0.0%-0.6%]). Only 2 had grown. An incidental cancer was found at thyroidectomy in a nonvisualized nodule. New nodules developed in 93 patients (9.3% [95% CI, 7.5%-11.1%]), with detection of one cancer. CONCLUSIONS AND RELEVANCE: Among patients with asymptomatic, sonographically or cytologically benign thyroid nodules, the majority of nodules exhibited no significant size increase during 5 years of follow-up and thyroid cancer was rare. These findings support consideration of revision of current guideline recommendations for follow-up of asymptomatic thyroid nodules.

Sodium iodide symporter (NIS) in extrathyroidal malignancies: focus on breast and urological cancer.

BACKGROUND: Expression and function of sodium iodide symporter (NIS) is requisite for efficient iodide transport in thyrocytes, and its presence in cancer cells allows the use of radioiodine as a diagnostic and therapeutic tool in thyroid neoplasia. Discovery of NIS expression in extrathyroidal tissues, including transformed cells, has opened a novel field of research regarding NIS-expressing extrathyroidal neoplasia. Indeed, expression of NIS may be used as a biomarker for diagnostic, prognostic, and therapeutic purposes. Moreover, stimulation of endogenous NIS expression may permit the radioiodine treatment of extrathyroidal lesions by concentrating this radioisotope. RESULTS: This review describes recent findings in NIS research in extrathyroidal malignancies, focusing on breast and urological cancer, emphasizing the most relevant developments that may have clinical impact. CONCLUSIONS: Given the recent progress in the study of NIS regulation as molecular basis for new therapeutic approaches in extrathyroidal cancers, particular attention is given to studies regarding the relationship between NIS and clinical-pathological aspects of the tumors and the regulation of NIS expression in the experimental models.

Histone post-translational modifications induced by histone deacetylase inhibition in transcriptional control units of NIS gene.

Histone post-translational modifications (HPTMs) play a major role in control of gene transcription. Among them, histone acetylation and methylation have been extensively investigated. Histone acetylation at different residues is generally associated to active gene transcription. In contrast, histone methylation can be associated either to transcriptional activation or repression, depending primarily on the histone residue that is subjected to the modification. Herein, effects of the histone deacetylase inhibitor SAHA on the sodium-iodide symporter (NIS) gene expression were investigated in breast cancer cells (MDA157 and MDA468). SAHA treatment induces high increase of NIS mRNA levels in MDA468 cells (300-fold), but moderate increase in MDA157 cells (fivefold). Histone H3 HPTMs (acetylation and methylations) on transcriptional units of NIS gene were investigated in these cell lines upon SAHA treatment. Our data indicate that HPTMs, particularly the H3 lysine 27 trimethylation, may operate in contrast to current models that relate epigenetic modifications with transcriptional activity.

Thyroid nodules: diagnosis and management.

Thyroid nodules, with a prevalence of almost 25% in the general population, are a common occurrence. Their prevalence varies considerably depending on demographics such as age and sex as well as the presence of risk factors. This article provides a comprehensive overview of the prevalence, risk stratification and current management strategies for thyroid nodules, with a particular focus on changes in diagnostic and therapeutic protocols that have occurred over the past 10 years. Several sonography-based stratification systems (such as Thyroid Imaging Reporting and Data Systems (TIRADS)) might help to predict the malignancy risk of nodules, potentially eliminating the need for biopsy in many instances. However, large or suspicious nodules necessitate cytological evaluation following fine-needle aspiration biopsy for accurate classification. In the case of cytology yielding indeterminate results, additional tools, such as molecular testing, can assist in guiding the management plan. Surgery is no longer the only treatment for symptomatic or malignant nodules: active surveillance or local ablative treatments might be beneficial for appropriately selected patients. To enhance clinician-patient interactions and discussions about diagnostic options, shared decision-making tools have been developed. A personalized, risk-based protocol promotes high-quality care while minimizing costs and unnecessary testing.

Data-Driven Thyroglobulin Cutoffs for Low- and Intermediate-Risk Thyroid Cancer Follow-Up: ITCO Real-World Analysis.

CONTEXT: The utility of thyroglobulin (Tg) in the follow-up of differentiated thyroid cancer (DTC) patients has been well-documented. Although third-generation immunoassays have improved accuracy, limitations persist (interfering anti-Tg antibodies and measurement variability). Evolving treatment strategies require a reevaluation of Tg thresholds for optimal patient management. OBJECTIVE: To assess the performance of serum Tg testing in two populations: patients receiving total thyroidectomy and radioiodine remnant ablation (RRA), or treated with thyroidectomy alone. DESIGN: Prospective observational study. Setting. Centers contributing to the Italian Thyroid Cancer Observatory (ITCO) database. PATIENTS: We included 540 patients with 5 years of follow-up and negative anti-Tg antibodies. INTERVENTIONS: Serum Tg levels assessed at 1-year follow-up visit. MAIN OUTCOME MEASURE: Detection of structural disease within 5 years of follow-up. RESULTS: After excluding 26 patients with structural disease detected at any time point, the median Tg did not differ between patients treated with or without radioiodine. Data-driven Tg thresholds were established based on the 97th percentile of Tg levels in disease-free individuals: 1.97 ng/mL for patients undergoing thyroidectomy alone (lower than proposed by the MSKCC protocol and ESMO Guidelines, yet demonstrating good predictive ability, with a negative predictive value (NPV) of 98%) and 0.84 ng/mL for patients receiving post-surgical RRA. High sensitivity and NPV supported the potential of these thresholds in excluding structural disease. CONCLUSIONS: This real-world study provides evidence for the continued reliability of 1-year serum Tg levels. The data-driven Tg thresholds proposed offer valuable insights for clinical decision-making in patients undergoing total thyroidectomy with or without RRA.

A new germline VHL gene mutation in three patients with apparently sporadic pheochromocytoma.

CONTEXT: Germline mutations in four genes (RET, VHL, SDHB and SDHD) are detected in about 17% of patients with apparently sporadic pheochromocytoma. Thus, genetic screening of all patients with this disease is suggested for a rational diagnostic approach and management. OBJECTIVE: To report the clinical, biochemical and genetic analysis of three unrelated patients affected by pheochromocytoma. DESIGN AND PATIENTS: All the coding regions and exon-intron boundaries of RET, VHL, SDHB and SDHD genes were sequenced in three unrelated patients with intra-adrenal pheochromocytoma: a 17-year-old girl, a 15-year-old boy and a 73-year-old man. The family history of all three cases was negative for von Hippel-Lindau lesions or other types of endocrine tumours. Structural modelling of the VHL protein was then performed. RESULTS: We identified a novel germline VHL gene point mutation, a G to A nucleotide substitution in exon 3, leading to an aspartate to asparagine amino acid change in codon 197 (D197N). No mutations were found in RET, SDHB and SDHD genes. Structural modelling of the VHL protein suggests that the D197N mutation could have a functional role. CONCLUSIONS: Our study expands the number of VHL gene known mutations and indicates the usefulness of performing the genetic analysis in all patients with apparently sporadic pheochromocytoma.

A Prospective Multicenter Study Examining the Relationship Between Thyroid Cancer Treatment Outcomes and the Presence of Autoimmune Thyroiditis.

Background: There is some controversy on the potential relationship between autoimmune processes and clinicopathologic features as well as prognosis of differentiated thyroid cancer (DTC), and the evidence is limited by its largely retrospective nature. We examined the relationship between the presence of autoimmune thyroiditis (AT) and 1-year thyroid cancer treatment outcomes in a large multicenter study using prospectively collected data. Methods: We included data from consecutive DTC patients enrolled in the Italian Thyroid Cancer Observatory (ITCO) database (NCT04031339). We divided the groups according to the presence (AT) or absence (no autoimmune thyroiditis [noAT]) of associated AT. We used propensity score matching to compare the clinical features and outcomes between the two groups at 1-year follow-up. Results: We included data from 4233 DTC patients, including 3172 (75%) females. The American Thyroid Association (ATA) risk levels were as follows: 51% (2160/4233) low risk, 41.3% (1750/4233) intermediate risk, and 7.6% (323/4233) high risk. There were 1552 patients (36.7%) who had AT. Before propensity score matching, AT patients were significantly younger and had a smaller and bilateral tumor (p < 0.0001). Patients with AT more frequently fell into the low- and intermediate-risk categories, while the ATA high risk was more frequent among noAT patients (p = 0.004). After propensity score matching, patients with AT more frequently showed evidence of disease (structural/biochemical incomplete response) versus excellent/indeterminate response, compared with patients without AT (7.3% vs. 4.5%, p = 0.001), with an odds ratio of 1.86 ([confidence interval: 1.3-2.6], p = 0.0001). However, when considering only structural persistence as the outcome, no statistically significant differences were observed between patients with or without AT (3.4% vs. 2.7%, p = 0.35). The elevated risk associated with the ATA intermediate and high risk at diagnosis remained consistently statistically significant. Conclusions: In this large prospective series, biochemical persistence was more frequent, at 1-year follow-up, in AT patients. However, there was no significant association between the presence of AT and structural persistence of disease. These findings may be explained by the presence of a residual thyroid tissue.

A Data-Driven Approach to Refine Predictions of Differentiated Thyroid Cancer Outcomes: A Prospective Multicenter Study.

CONTEXT: The risk stratification of patients with differentiated thyroid cancer (DTC) is crucial in clinical decision making. The most widely accepted method to assess risk of recurrent/persistent disease is described in the 2015 American Thyroid Association (ATA) guidelines. However, recent research has focused on the inclusion of novel features or questioned the relevance of currently included features. OBJECTIVE: To develop a comprehensive data-driven model to predict persistent/recurrent disease that can capture all available features and determine the weight of predictors. METHODS: In a prospective cohort study, using the Italian Thyroid Cancer Observatory (ITCO) database (NCT04031339), we selected consecutive cases with DTC and at least early follow-up data (n = 4773; median follow-up 26 months; interquartile range, 12-46 months) at 40 Italian clinical centers. A decision tree was built to assign a risk index to each patient. The model allowed us to investigate the impact of different variables in risk prediction. RESULTS: By ATA risk estimation, 2492 patients (52.2%) were classified as low, 1873 (39.2%) as intermediate, and 408 as high risk. The decision tree model outperformed the ATA risk stratification system: the sensitivity of high-risk classification for structural disease increased from 37% to 49%, and the negative predictive value for low-risk patients increased by 3%. Feature importance was estimated. Several variables not included in the ATA system significantly impacted the prediction of disease persistence/recurrence: age, body mass index, tumor size, sex, family history of thyroid cancer, surgical approach, presurgical cytology, and circumstances of the diagnosis. CONCLUSION: Current risk stratification systems may be complemented by the inclusion of other variables in order to improve the prediction of treatment response. A complete dataset allows for more precise patient clustering.

Precision oncology for RET-related tumors.

Aberrant activation of the RET proto-oncogene is implicated in a plethora of cancers. RET gain-of-function point mutations are driver events in multiple endocrine neoplasia 2 (MEN2) syndrome and in sporadic medullary thyroid cancer, while RET rearrangements are driver events in several non-medullary thyroid cancers. Drugs able to inhibit RET have been used to treat RET-mutated cancers. Multikinase inhibitors were initially used, though they showed modest efficacy and significant toxicity. However, new RET selective inhibitors, such as selpercatinib and pralsetinib, have recently been tested and have shown good efficacy and tolerability, even if no direct comparison is yet available between multikinase and selective inhibitors. The advent of high-throughput technology has identified cancers with rare RET alterations beyond point mutations and fusions, including RET deletions, raising questions about whether these alterations have a functional effect and can be targeted by RET inhibitors. In this mini review, we focus on tumors with RET deletions, including deletions/insertions (indels), and their response to RET inhibitors.

Prevalence of Thyroid Nodules and Thyroid Cancer in Individuals with a First-Degree Family History of Non-Medullary Thyroid Cancer: A Cross-Sectional Study Based on Sonographic Screening.

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.