Molecular Testing Of Thyroid Nodules Research Articles

The negative outlook: Long-term follow up of ThyroSeq negative and low-risk nodules.

Molecular testing of thyroid nodules is an essential tool to help risk stratify nodules with indeterminate cytology. Although ThyroSeq testing has been around for over a decade, there is a paucity of long-term follow-up data on cytologically indeterminate nodules that are determined to be molecularly negative or low-risk. The objective of this study is to assess the outcomes of nodules with indeterminate cytology (Bethesda III or IV) and negative or low-risk ThyroSeq results. This is a single academic institution retrospective cohort study. Patients with at least one thyroid nodule sampled with fine-needle aspiration who underwent ThyroSeq testing from 2012 to 2018 and had negative or low-risk ThyroSeq results on a cytologically indeterminate sample (n=159 patients, 167 nodules) were included in the study. Outcomes include the false-negative rate and negative predictive value of each test version, as well as follow-up length for each nodule. There were 159 patients with a mean age of 58 years (7-84 years) included in this study; the majority were female (81.8%). The mean follow-up was 4.0 years. Of 167 nodules, three were found to be malignant on resection (1.8%). The negative predictive value for the entire cohort was 98.2% and it was 89.3% for the surgical series. ThyroSeq testing has good negative predictive value and can help risk stratify cytologically indeterminate nodules. Routine follow-up allows for safe monitoring of nodules for features suggestive of malignancy.

Molecular Profiling for Bethesda III to VI Nodules: Results of a Multicenter International Retrospective Study

ObjectiveMolecular testing is a well-established tool that assists in the management of thyroid nodules. We describe our experience using molecular testing of thyroid nodules with Bethesda III to VI cytology. MethodsThis is a retrospective multicenter, multinational study of thyroid nodules that underwent preoperative molecular profiling with ThyGenX/ThyGeNEXT or ThyroSeq V3 between 2015 and 2022. The clinical characteristics and mutational profiles of tumors were compared. Collected data included demographics, cytology results, surgical pathology, and molecular alterations. Molecular alterations were categorized into 3 main phenotypes: BRAF-like, RAS-like, and non-BRAF-non-RAS (NBNR). ResultsOverall, 784 patients who had surgery were included, of which 603 (76.2%) were females. The most common histologic type was papillary thyroid cancer (PTC) with 727 (91.9%) cases. In total, 205 (28.2%) cases showed an aggressive subtype of PTC (eg, tall cell and hobnail). BRAF-like alterations were most likely to be found in Bethesda V and VI nodules and show extrathyroidal extension (ETE), nodal disease, and/or aggressive subtypes of PTC (P < .001 for all). RAS-like alterations were more commonly found in Bethesda III and IV nodules and were less likely to show ETE, nodal disease, and/or aggressive histology (P < .001 for all). NBNR alterations were more commonly found in Bethesda III and IV nodules and were less likely to show ETE, nodal disease, and/or aggressive subtypes of PTC. However, they were rarely but significantly associated with poorly differentiated thyroid cancer (P < .005). ConclusionMolecular testing of thyroid nodules can help determine the likelihood of malignancy and classify nodules into several tumor phenotypes, predicting their behaviors and potentially allowing for a more tailored treatment. NBNR alterations should be managed with caution.

Genetic molecular testing of thyroid nodules

Genetic molecular testing of thyroid nodules

Innovative technologies in the diagnosis and safe surgical treatment of thyroid diseases

This study clarified the results of using innovative technologies in the diagnosis and surgical treatment of patients with thyroid diseases by evaluating the role of modern diagnostic methods and effectiveness of minimally invasive thyroid surgery. The results of a comprehensive examination and treatment of 332 patients with thyroid diseases, who were examined using modern diagnostic methods and underwent conventional and various minimally invasive approaches, were analyzed. The sensitivity, specificity, accuracy, and positive and negative predictive values of multiparametric neck ultrasonography, 99mTc-MIBI thyroid scintigraphy, fine-needle cytology, and molecular testing of thyroid nodules were compared. The minimally invasive procedure was performed in 70.4% of the patients. Postoperative complications were found in 4.9% of the patients who underwent surgery, functional dysphonia in 0.9%, and transitory hypoparathyroidism in 2.4%. The use of the main criteria that determine the choice of a rational minimally invasive surgery, such as the nodule size and thyroid volume, hyperfunctioning thyroid, clinical thyroiditis, substernal extension, extrathyroid extension, and necessity of implementation of central and lateral neck dissection due to lymph node metastases, avoids the conversion of access to the traditional approach, prevents damage to the recurrent laryngeal nerve with the development of transitory or permanent recurrent laryngeal nerve palsy, and minimizes injury to the parathyroid glands with the development of isolated cases of transient hypoparathyroidism. Therefore, results of the multiparametric analysis of the examination and surgical treatment outcomes of patients with thyroid diseases show the feasibility of using innovative technologies in their diagnosis and surgical treatment. Modern examination methods allow us to determine with great accuracy the morphological form of thyroid disease and choose a rational volume and surgical intervention. In this study, minimally invasive procedures are optimal and safe in the surgical treatment of this category of patients.

Molecular Testing of Atypical Thyroid Nodules with Corresponding Surgical Correlation: Five-Year Retrospective Review in Veterans Population.

ObjectiveWe report the results of a retrospective five-year study within a veteran population aimed at correlating abnormal thyroid fine-needle aspiration (FNA) diagnosis with associated molecular testing to the histology of the surgical resection.MethodsA retrospective analysis of abnormal thyroid FNAs with associated molecular testing and surgical outcome was conducted from January 1, 2015 to December 31, 2020. Aspirates were classified using the Bethesda system for reporting thyroid cytopathology, including atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), suspicious for malignancy (SM), and malignant. Pertinent data, including patient demographics, imaging, and ancillary testing were reviewed. A thyroid cancer mutation panel assessing the most common mutations and rearrangements associated with neoplasia was utilized. The results of molecular testing were directly compared and correlated with final cytological and histological diagnosis.ResultsA total of 1850 thyroid aspirates were performed, 200 of which were given an abnormal cytologic diagnosis. Thirty-six samples were submitted for molecular testing and subsequent surgical follow-up. Four were called malignant on cytology. 32 were placed in an indeterminate category (89%). Within indeterminate cases: 53% exhibited positive molecular mutations (n=17), 34% no mutation detected (n=11), and 13% insufficient quantity for testing (n=4). Upon surgical resection in the mutation-positive group: 18% had no malignancy (n=3), and the remaining 82% were positive for malignancy (n=14). Mutations in the histologically malignant group included: 57% BRAF (n=8), 21% NRAS (n=3), 7% HRAS (n=1), 7% KRAS (n=1), and 7% PAX8/PPAR gamma (n=1). In indeterminate cases with no mutation detected, 10 cases were found to be benign, and one case of malignancy was diagnosed. The probability of indeterminate diagnosis in combination with no mutation yielded a 91% chance of benign entity and 9% chance of malignancy. We demonstrated 93% sensitivity and 91% negative predictive value (NPV) for the risk of malignancy in indeterminate cytology specimens with ancillary molecular testing. There was 77% specificity and 82% positive predictive value (PPV) for our data set.ConclusionsIn indeterminate samples, the detection of a mutation was highly predictive of malignancy and a strong indicating factor for surgery with a high sensitivity and NPV. Molecular testing refined or established the diagnosis in 89% of the cases. Our results indicate that molecular testing of thyroid nodules enhances the accuracy of FNA cytology and the subsequent surgical outcome.

Preoperative Molecular Testing of Thyroid Nodules

Robust molecular testing is commercially available for adjuvant assessment of cytologically indeterminate thyroid nodules. Testing has been developed and optimized for fine needle aspiration biopsy collections of thyroid nodules typically under ultrasound evaluation. These assays use a combination of gene expression and/or DNA and RNA assessments for molecular alterations to stratify indeterminate thyroid nodules as benign with risk level similar to benign cytologic read or suspicious with increased risk of malignancy. Guidelines for when to consider adjuvant molecular testing will be discussed.

Clinical Utility of BRAF, NRAS, and TERT Promoter Mutation in Preoperative Thyroid Fine-Needle Aspiration Biopsy: A Diagnostic Study From Dharmais Cancer Hospital.

Objective:Molecular testing of thyroid nodules becomes important for improving the accuracy of fine-needle aspiration biopsy (FNAB). This study aimed to investigate the diagnostic utility of BRAF, NRAS, and TERT promoter mutation in thyroid nodules at Dharmais Cancer Hospital. Methods:We performed a prospective diagnostic study involving 50 patients with thyroid nodules who needed surgery between September 2013 and August 2014. Mutational hotspots in BRAF exon 15, NRAS exon 3, and TERT promoter region were analyzed by Sanger sequencing from FNAB specimens. Cytology and molecular data were compared to histopathology results. Results:Of the 50 cases included in the analysis, 39 cases (78%) were thyroid malignancies. Mutations of BRAF, NRAS, and TERT promoter were detected in 31% (12/39), 18% (7/39), and 13% (5/39) cases, respectively. BRAF and NRAS mutations were found mutually exclusive, while all of TERT promoter mutation was found coexistent either with BRAF (40%) or NRAS (60%). The combination of FNAB cytology and molecular testing resulted in 69% sensitivity, 100% specificity, 100% positive predictive value, 48% negative predictive value, and 76% accuracy. Conclusion:Molecular testing of BRAF, NRAS, and TERT mutations improve the sensitivity of thyroid FNAB and is beneficial for more definitive treatment in selective cases. However, the NPV is relatively low to avoid the need for diagnostic surgery. Therefore, further studies to identify more sensitive methods and more comprehensive molecular markers in the diagnosis of thyroid nodules are needed.

Thyroid cytology smear slides: An untapped resource for ThyroSeq testing.

Molecular testing of thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology is commonly used to guide patient management and is typically performed on freshly collected FNA samples. In this study, the authors evaluated the performance of the ThyroSeq test in cytology smear slides. Air-dried Diff-Quik (DQ)-stained and alcohol-fixed Papanicolaou (Pap)-stained smears were used to determine required cellularity and sensitivity of mutation detection and to compare ThyroSeq v3 Genomic Classifier (GC) results obtained in cytology smears and fresh FNA samples from the same nodules. ThyroSeq testing of 31 cytology smears revealed that 25 smears (81%) were adequate for ThyroSeq analysis, including 14 Pap-stained smears (100%) and 11 DQ-stained smears (65%), whereas 6 DQ-stained smears (35%) failed RNA sequencing. The overall accuracy for detecting molecular alterations was 98%, with 100% concordance for mutations and gene expression alterations, 96% concordance for fusions, and 94% concordance for copy number alterations. Cytology smears were adequate for ThyroSeq analysis when at least 200 to 300 cells were present in 1 to 3 slides. ThyroSeq detected all studied mutations down to 5% allele frequency and BRAF mutations down to 1% allele frequency. Testing of smears yielded a positive ThyroSeq GC result in all nodules originally classified as positive. Thyroid FNA cytology smear slides with adequate cellularity can be successfully used for ThyroSeq GC testing in approximately 80% of cases, with an even higher success rate in Pap-stained smears. Compared with FNA samples collected into preservative solution, 94% to 100% of different genetic alterations could be accurately detected in smears, validating cytology smears as an alternative for ThyroSeq testing in patients with indeterminate thyroid cytology.

MON-498 Thyroseq V3 GC for Bethesda III and IV: An Institutional Experience

Background:Molecular testing of thyroid nodules classified as indeterminate on fine-needle aspiration (FNA) is used for patient management. ThyroSeq® v3 genomic classifier is a commercially available test that examines a wide spectrum of genomic alterations in a thyroid FNA sample based on algorithmic analysis and reports test results as either negative (including currently negative) or positive. This study reviews our institutional experience with Thyroseq® V3 to distinguish between benign disease versus cancer in thyroid nodules diagnosed as Bethesda III or IV on cytology.Methods:Thyroid nodules with Bethesda III or IV cytology diagnoses and available Thyroseq® V3 results from 12/17 to 8/19 were retrieved from the pathology database. Cytopathology diagnoses were correlated with molecular testing and histopathology.Results:416 cases (Bethesda III n=252, Bethesda IV n=164) were retrieved: 295 (71%) were reported as Thyroseq® V3 negative and 121 (29%) as positive. The 82.1% (207/252) benign call rate (BCR) of Thyroseq® v3 for Bethesda III was significantly higher (p<0.001) than that for Bethesda IV, BCR 54% (88 /164). Histopathologic follow-up was available for 128 cases (96 ThyroSeq® v3 positive, 32 ThyroSeq® v3 negative): 57 benign and 71 malignant (including NIFTP). For Bethesda III and IV diagnoses respectively, the test demonstrated 91.7 % (95% Cl 73%-99%) and 91.5% (95% Cl 80%-98%) sensitivity, 94% (95% Cl 90%-97%) and 82.4% (95% Cl 74%-89%) specificity, 99% (95% Cl 96%-99%) and 95% (95% Cl 89%-98%) negative predictive value and 63% (95% Cl 50%-74%) and 70.5% (95% Cl 61%-79%) positive predictive value, given malignancy rates 10% Bethesda III; 32% Bethesda IV. 45 unique combinations of genetic alterations were detected in the 96 operated ThyroSeq® v3 positive cases—34 combinations (76%) were present in <2 cases and only 3 combinations occurred 7 or more cases. Forty-six (48%) nodules had RAS mutations, either combined with other mutations 31% (23/30, 77% malignant) or alone, 17% (10/16, 63% malignant), followed byTHADA/IGF2BP3 gene fusion changes,11.5% (10 /11 malignant). Three cases with BRAF mutations (1 V600E, 1 K601E, 1 K601N) were malignant. Copy numbers alterations alone were present in 10 (5/ 10 malignant) and the gene expression profile alone was positive in 5 cases (all malignant). Mutations that were associated with benign pathology were PTEN, DICER1, E1F1AX and TP53. There were 6 false negative cases, 5 low risk cancers by American Thyroid Association criteria and 1 NIFTP.Conclusion:The high BCR of Thyroseq® v3 for Bethesda III category avoids surgery for majority of patients. A more comprehensive mutational and fusion panel reveals the complexity of the genetic signature of indeterminate nodules. Future larger and likely multicenter studies will be required to define the associated cancer risk and potential prognosis associated with adjunct molecular testing.

Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features Accounts for More Than Half of "Carcinomas" Harboring RAS Mutations.

Molecular testing of thyroid nodules is increasingly being utilized to guide clinical management decisions. RAS mutations are the most frequent mutations detected in the context of an indeterminate fine-needle aspiration (FNA) diagnosis. The term "noninvasive follicular thyroid neoplasm with papillary-like nuclear features" (NIFTP) was recently introduced to promote conservative management of tumors previously classified as noninvasive follicular variant of papillary thyroid carcinoma (FVPTC). This change in terminology was based on the indolent clinical behavior of these tumors and their molecular profile, which includes frequent RAS mutations. The aim of this study was to determine the percentage of RAS-mutant "carcinomas" that would now be classified as NIFTPs. A search was performed for cases with known activating RAS mutations in a database of 199 thyroid carcinomas that underwent molecular characterization as part of Profile:Oncopanel between July 2013 and July 2015. Cases of FVPTC were re-reviewed to identify tumors that now would be categorized as NIFTP. Preceding FNA diagnoses were recorded, and cases with an indeterminate FNA result (defined as a diagnosis of atypia/follicular lesion of undetermined significance, suspicious for follicular neoplasm, or suspicious for malignancy) were identified. A total of 27 RAS-mutant thyroid tumors were identified. Fifteen (56%) cases had an NRAS mutation, nine (33%) had an HRAS mutation, and three (11%) had a KRAS mutation. Twenty-four (89%) cases had a preceding FNA, 19 (79%) of which had an indeterminate FNA diagnosis. The surgical resection specimen demonstrated FVPTC in 20 (74%) cases, classical type PTC in two (7%), solid variant of PTC in one (4%), and follicular thyroid carcinoma in four (15%). Of the 20 FVPTCs, 16 (80%) would now be classified as NIFTP. NIFTPs accounted for 59% of RAS-mutant carcinomas overall and 63% of RAS-mutant carcinomas with a prior indeterminate FNA diagnosis. NIFTPs accounted for more than half of RAS-mutant "carcinomas" in this cohort. In cases where clinical and sonographic data support a low-risk phenotype, these results suggest that a lobectomy should be considered as the initial surgical approach for a nodule with an indeterminate FNA diagnosis and a RAS mutation.

Molecular Testing of Thyroid Nodules: A Review of Current Available Tests for Fine-Needle Aspiration Specimens.

- Fine-needle aspiration of thyroid nodules is a reliable diagnostic method to determine the nature of thyroid nodules. Nonetheless, indeterminate cytology diagnoses remain a diagnostic challenge. The development of multiplex molecular techniques and the identification of genetic alterations associated with different follicular cell-derived cancers in the thyroid have led to the introduction of several commercially available tests. - To summarize the most common commercially available molecular testing in thyroid cancer, focusing on the technical features and test performance validation. - Peer-reviewed original articles, review articles, and published conference abstracts were reviewed to analyze the advantages and limitations of the most common tests used in the evaluation of thyroid needle aspirations. - The most common tests available include the Afirma Gene Expression Classifier, ThyGenX, and ThyroSeq. The excellent negative predictive value (NPV) of the Afirma test allows it to be used as a "rule out" test. ThyGenX analyzes a panel of DNA mutations and RNA translocation fusion markers to assess the risk of malignancy with good NPV and positive predictive value. ThyroSeq is a next-generation sequencing-based gene mutation and fusion test that has been reported to have the best NPV and positive predictive value combined, suggesting that it can be used as a "rule in" and "rule out" test. Molecular testing of cytology specimens from thyroid nodules has the potential to play a major role in the evaluation of indeterminate thyroid lesions.

A 2015 Survey of Clinical Practice Patterns in the Management of Thyroid Nodules.

The management of thyroid nodules has changed dramatically over the past two decades. In the interim, technological advances including high-resolution ultrasound and molecular testing of thyroid nodules have been introduced. We sought to document current practices in the management thyroid nodules and assess the extent to which technological advances have been incorporated into current practice. We further sought to compare current practice to recommendations made in a recently updated American Thyroid Association (ATA) clinical practice guideline (CPG) and examine differences in thyroid nodule management among international members of U.S.-based endocrine societies. Members of The Endocrine Society, ATA, and American Association of Clinical Endocrinologists were invited to participate in a Web-based survey dealing with testing, treatment preference, and modulating factors in patients with thyroid nodules. A total of 897 respondents participated in the survey, including 661 members of The Endocrine Society, 454 American Association of Clinical Endocrinologists members, and 365 ATA members. Thyroid fine-needle aspiration (FNA) in 2015 is generally performed by endocrinologists (56.6%) and radiologists (31.9%), most frequently using ultrasound guidance (83.3%). Respondents in general have a lower threshold for FNA of thyroid nodules than that recommended in the updated ATA CPG. Management depends on the FNA result, with follicular lesion of undetermined significance/atypia of undetermined significance resulting in molecular testing (38.8% of respondents), repeat FNA cytology (31.5%), or immediate referral for thyroid surgery (24.4%). Nodules showing follicular neoplasm by FNA are referred for thyroid surgery by 61.2% of respondents (46.6 % lobectomy, 14.6 % total thyroidectomy) or molecular testing (29.0 %). Nodules found suspicious but not conclusive for malignancy (Bethesda category V), are referred for thyroid surgery (86.0%) and rarely undergo molecular testing (9.5%). During pregnancy, only 47.6% of respondents would perform FNA in the absence of nodular growth, with most respondents deferring FNA until after pregnancy. Endocrinologists are 64.2% less likely to perform FNA in an octogenarian than a younger patient with a comparable thyroid nodule. Striking international differences were identified in the routine measurement of calcitonin and in the use of molecular testing of thyroid nodules. In summary, our survey of clinical endocrinologists on the management of thyroid nodules documents current practice patterns and demonstrates both concordance and focal discordance with recently updated CPGs. Both international differences and a change in practice patterns during the past two decades are demonstrated.

Stepwise Molecular Testing of Thyroid Nodules with Atypia of Undetermined Significance Is Cost-Effective in the United States but Not in Canada

Clinical ThyroidologyVol. 26, No. 10 SummariesStepwise Molecular Testing of Thyroid Nodules with Atypia of Undetermined Significance Is Cost-Effective in the United States but Not in CanadaJerome M. HershmanJerome M. HershmanSearch for more papers by this authorPublished Online:15 Oct 2014https://doi.org/10.1089/ct.2014;26.271-272AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Stepwise Molecular Testing of Thyroid Nodules with Atypia of Undetermined Significance Is Cost-Effective in the United States but Not in Canada." Clinical Thyroidology, 26(10), pp. 271–272FiguresReferencesRelatedDetails Volume 26Issue 10Oct 2014 InformationCopyright 2014 American Thyroid Association, Inc.To cite this article:Jerome M. Hershman.Stepwise Molecular Testing of Thyroid Nodules with Atypia of Undetermined Significance Is Cost-Effective in the United States but Not in Canada.Clinical Thyroidology.Oct 2014.271-272.http://doi.org/10.1089/ct.2014;26.271-272Published in Volume: 26 Issue 10: October 15, 2014PDF download

Molecular characterization of 54 cases of false-negative fine-needle aspiration among 1347 papillary thyroid carcinomas.

Fine-needle aspiration (FNA) has been widely accepted as the most crucial step in the preoperative assessment of thyroid nodules, but the false-negative rates are generally reported to be between 3.6% and 10.2%. To lower the overall incidence of this false-negative testing, new reporting systems encourage the molecular testing of thyroid nodules. However, to the authors' knowledge, the role of molecular testing in false-negative FNA has not yet been evaluated. In total, 1347 consecutive papillary thyroid carcinomas (PTCs) with both cytological and histological diagnoses were collected from the same center. A blinded revision of the false-negative cases was performed. An analysis of the BRAF and Ras genes in the false-negative cases was then performed. The false-negative rate at the time of primary FNA diagnosis was 4.8% (65 of 1347 cases). False-negative cases were 15 follicular variant PTCs, 2 classical variant, and 1 solid variant that lacked peculiar PTC cytomorphological features. Adequate cellular material for molecular analysis was available only in 54 of the 65 false-negative cases. Mutations were found in 6 cases (11%), and Ras alterations were present in 16 cases (29.6%). The addition of molecular analysis decreased the false-negative rate to 0.4% (5 of 1347 cases). The results of the current study confirm the feasibility of BRAF and Ras analysis in routine FNA. However, when the false-negative FNA rate is low, the cost-benefit analysis of the detection of BRAF and Ras mutations should be carefully evaluated. Consequently, the authors suggest that preoperative molecular assessment could be helpful for benign nodules, but only in the presence of clinical suspicion of malignancy.

Molecular Testing of Thyroid Nodules: Distinguishing Misuse from Appropriate Use

Molecular Testing of Thyroid Nodules: Distinguishing Misuse from Appropriate Use

Molecular diagnosis on tissues and cells: how it affects training and will affect practice in the future

On 25th November 2011, a symposium organized by the Royal College of Pathologists, entitled 'Molecular diagnosis on tissues and cells', took place in London. As trainees in histopathology and cytopathology, we were stimulated to consider the role that molecular biology is likely to play in future practice and how this is addressed by our own training. The symposium provided a basis for this article. Routine samples requiring molecular analysis are equally relevant to histopathologists and cytopathologists, and molecular biology laboratories are now using cytological as well as histological material for diagnostic testing, allowing different specimen types to be used as and when they are most appropriate. The most widely used types of molecular analysis in routine cellular pathology are EGFR testing in lung cancer, molecular testing of thyroid nodules, fluorescence in situ hybridization testing of urine samples, clonality analysis in lymphoma testing, HER2 testing in breast and gastric cancer, KRAS testing in colorectal cancer, intraoperative assessment of breast cancer sentinel nodes, molecular testing of gastrointestinal stromal tumours and mismatch repair protein analysis. Of these, the majority in the UK are carried out on histopathology samples, although many are applicable to cytological samples if adequate material is obtained. We are particularly encouraged by the potential of molecular diagnostic cytology in traditionally difficult areas, such as intraoperative assessment. We believe that increasing reliance on molecular diagnostic techniques will also herald changes in training.

Prevalence of RET/PTC rearrangement in benign and malignant thyroid nodules and its clinical application

Fine-needle aspiration cytology (FNAC) is the primary means to distinguish benign thyroid nodules from malignant ones. About 20% of FNAC yields indeterminate results leading to unnecessary or delayed surgery. Many studies of tissue samples, the majority of which are retrospective advocate testing for RET rearrangements as a diagnostic adjunctive tool in thyroid nodules with indeterminate cytological findings. Because of the uncertain prevalence of RET rearrangements, its utility as a tumor marker is still controversial. The goal of this study was to establish the prevalence and the utility of testing for RET rearrangements in FNAC suspicious of cancer in a clinical setting. In this prospective study, we analysed a large series of thyroid aspirates by RT-PCR only and Southern blot on RT-PCR products for type 1 and 3 RET rearrangements. Results were compared with clinical findings, cytological diagnosis and final histopathology. By the higher sensitive Southern-blot on RT-PCR method, RET rearrangements were present in 36% of papillary thyroid carcinomas (RET/PTC-1, 12%; RET/PTC-3, 20%; both, 4%) and of 13.3% of benign nodules. By means of RT-PCR only, RET rearrangements were disclosed only in 14.3% of PTC and in 3.6% of benign nodules. No significant correlation was found between RET rearrangements and clinicopathological features of patients. These results indicate that molecular testing of thyroid nodules for RET/PTC must take into account of its high prevalence in benign nodules, inducing false positive diagnoses when the highly sensitive assay Southern-blot on RT-PCR is used. Its searching by means of RT-PCR only, has a specificity superior of conventional cytology and can be used to refine inconclusive FNAC.

Molecular Testing for Mutations in Improving the Fine-Needle Aspiration Diagnosis of Thyroid Nodules

Thyroid nodules are common in adults, but only a small fraction of them are malignant. Fine-needle aspiration (FNA) with cytological evaluation is the most reliable tool for cancer diagnosis in thyroid nodules. However, 10-40% of nodules are diagnosed as indeterminate by cytology, making it difficult to optimally manage these patients. The aim of this study was to establish the feasibility and role of testing for tumor-specific mutations in improving the FNA diagnosis of thyroid nodules. The prospective study included 470 FNA samples of thyroid nodules from 328 patients. At the time of aspiration, a small portion of the material was collected and tested for BRAF, RAS, RET/PTC, and PAX8/PPARgamma mutations. The mutational status was correlated with cytology and either surgical pathology diagnosis or follow-up (mean, 34 months). A sufficient amount of nucleic acids were isolated in 98% of samples. Thirty-two mutations were found, including 18 BRAF, eight RAS, five RET/PTC, and one PAX8/PPARgamma. The presence of any mutation was a strong indicator of cancer because 31 (97%) of mutation-positive nodules had a malignant diagnosis after surgery. A combination of cytology and molecular testing showed significant improvement in the diagnostic accuracy and allowed better prediction of malignancy in the nodules with indeterminate cytology. These results indicate that molecular testing of thyroid nodules for a panel of mutations can be effectively performed in a clinical setting. It enhances the accuracy of FNA cytology and is of particular value for thyroid nodules with indeterminate cytology.