Search

RAS p.Q61R is the most prevalent hot-spot mutation in RAS and RAS-like mutated thyroid nodules. A few studies evaluated RAS p.Q61R by immunohistochemistry (RASQ61R-IHC). We performed a retrospective study of an institutional cohort of 150 patients with 217 thyroid lesions tested for RASQ61R-IHC, including clinical, cytologic and molecular data. RASQ61R-IHC was performed on 217 nodules (18% positive, 80% negative, and 2% equivocal). RAS p.Q61R was identified in 76% (n = 42), followed by RAS p.Q61K (15%; n = 8), and RAS p.G13R (5%; n = 3). NRAS p.Q61R isoform was the most common (44%; n = 15), followed by NRAS p.Q61K (17%; n = 6), KRAS p.Q61R (12%; n = 4), HRAS p.Q61R (12%; n = 4), HRAS p.Q61K (6%; n = 2), HRAS p.G13R (6%; n = 2), and NRAS p.G13R (3%; n = 1). RASQ61R-IHC was positive in 47% of noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP; 17/36), 22% of follicular thyroid carcinomas (FTC; 5/23), 10% of follicular thyroid adenomas (FTA; 4/40), and 8% of papillary thyroid carcinomas (PTC; 9/112). Of PTC studied (n = 112), invasive encapsulated follicular variant (IEFVPTC; n = 16) was the only subtype with positive RASQ61R-IHC (56%; 9/16). Overall, 31% of RAS-mutated nodules were carcinomas (17/54); and of the carcinomas, 94% (16/17) were low-risk per American Thyroid Associated (ATA) criteria, with only a single case (6%; 1/17) considered ATA high-risk. No RAS-mutated tumors recurred, and none showed local or distant metastasis (with a follow-up of 0-10 months). We found that most RAS-mutated tumors are low-grade neoplasms. RASQ61R-IHC is a quick, cost-effective, and reliable way to detect RAS p.Q61R in follicular-patterned thyroid neoplasia and, when malignant, guide surveillance., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Reliable preoperative diagnosis of thyroid nodules remained challenging because of the inconclusiveness of fine-needle aspiration (FNA) cytology. In the present study, 583 formalin-fixed paraffin embedded (FFPE) thyroid nodule tissues and 161 FNA specimens were enrolled retrospectively. Then BRAF V600E, EZH1 Q571R, SPOP P94R, and ZNF148 mutations among these samples were identified using Sanger sequencing. Based on this four-gene genomic classifier, we proposed a two-step modality to diagnose thyroid nodules to differentiate benign and malignant thyroid nodules. In the FFPE group, thyroid cancers were effectively diagnosed in 37.7% (220/583) of neoplasms by the primary BRAF V600E testing, and 15.7% (57/363) of thyroid nodules could be further determined as benign by subsequent EZH1 Q571R, SPOP P94R, and ZNF148 (we called them "ESZ") mutation testing. In the FNA group, 161 BRAF wild-type specimens were classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). A total of 7 mutated samples fell within Bethesda categories III-IV, and the mutation rate of "ESZ" in Bethesda III-IV categories was 8.4%. The two-step genomic classifier could further improve thyroid nodule diagnosis, which may inform more optimal patient management., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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

Follicular-patterned lesions often have indeterminate results (diagnostic category III or IV) by core needle biopsy (CNB) and fine needle aspiration (FNA). However, CNB diagnoses follicular neoplasm (category IV) more frequently than FNA. Therefore, we aimed to develop a risk stratification system for CNB samples with category III/IV using immunohistochemistry (IHC). The specificity of the RAS Q61R antibody was validated on 58 thyroid nodules with six different types of RAS genetic variants and 40 cases of RAS wild-type. We then applied IHC analysis of RAS Q61R to 207 CNB samples with category III/IV in which all patients underwent surgical resection. RAS Q61R IHC had 98% sensitivity and 98% specificity for detecting the RAS p.Q16R variant. In an independent dataset, the positive rate of RAS Q61R was significantly higher in NIFTP (48%) and malignancies (45%) than in benign tumors (19%). The risk of NIFTP/malignancy was highest in the group with nuclear atypia and RAS Q61R expression (86%) and lowest in the group without both parameters (32%). The high-risk group with either nuclear atypia or RAS Q61R had 67.3% sensitivity, 73.4% specificity, 75.2% positive predictive value, and 65.1% negative predictive value for identifying NIFTP/malignancy. We conclude that RAS Q61R IHC can be a rule-in diagnostic test for NIFTP/malignancy in CNB category III/IV results. Combining of the histologic parameter (nuclear atypia) with RAS Q61R IHC results can further stratify CNB category III/IV into a high-risk group, which is sufficient for a surgical referral, and a low-risk group sufficient for observation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In cytologically indeterminate thyroid nodules undergoing molecular testing, estimated risk of malignancy is variable. Identification of a non-cancer-specific mutation (RAS-like) confirms a neoplastic process but does not differentiate between benign, malignant, and low-risk neoplasms. This study aims to retrospectively evaluate institutional experience of Interpace (ThyGeNEXT ® and ThyraMIR ® ; Pittsburgh, PA) testing and to determine the rate of malignancy in resected nodules, stratified by mutational analysis and microRNA profile. Of 1917 fine need aspirations, 140 (7.3%) underwent Interpace testing: 47 (33.6%) were molecular-not-benign (harbored mutation, fusion, and/or positive miRNA) and 93 (66.4%) were molecular-benign (no mutations or fusions and negative microRNA). Surgery was spared in 79.6% of molecular-benign and 61.4% of all tested patients. Fifty-four (38.6%) underwent resection. Seventeen (89.5%) of the resected molecular-benign were benign and 2 were malignant. Thirteen (37.1%) of the resected molecular-not-benign were benign, 7 (20%) were noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), and 15 (42.9%) were malignant (p < 0.05, negative predictive value (NPV) 89.4-95.6%, positive predictive value (PPV) 22.3-42.8%). Most molecular-not-benign (72.3%) had RAS-like mutation. Twenty-three were resected: 3 were malignant and 7 were NIFTP. Nodules with non-RAS-like mutations (BRAF V600E-like, others) were more likely to be malignant than RAS-like (H/N/KRAS, BRAF K601E) (p < 0.05, NPV 86.9-96.5%, PPV 100%). Most nodules had RAS-like mutations and most were benign or low-risk neoplasms (NIFTP). This study supports the role of histologic examination in the distinction of malignancy in RAS-like thyroid neoplasms and underscores the role of molecular testing in risk stratification, patient counseling, and operative management., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Despite several reports on the association between molecular profiling, aggressive histology, and clinical outcomes, the association between mutation expression and pre-operative cytology is yet to be demonstrated. Therefore, we performed a retrospective, single-center study, including all patients who underwent molecular profiling of thyroid nodules in Bethesda System for Reporting Thyroid Cytopathology (BSRTC) categories III to VI, between 2018 and 2019. Medical records were reviewed to collect demographics, cytology results according to BSRTC, final pathology (presence of malignancy and its type, as well as presence of aggressive features, including extrathyroidal extension, positive neck lymph nodes, and multifocality), and the identified genetic variants stratified by risk levels, according to the 2015 ATA guidelines. We supplemented this analysis with a systematic review to identify the variant distributions across the literature. We included data on 55 nodules from 48 patients for the final analysis. A significant positive correlation was found between BSRTC categories and the mutation risk level, shown by an increase in the intermediate to high-risk mutation rate in the higher BSRTC categories (Rs = 0.660, p ≤ 0.001). A significant positive correlation was also found between mutation risk levels and the presence of malignancy and aggressive tumor features (Rs = 0.637, p < 0.001 and Rs = 0.459, p = 0.006, respectively). This novel positive and significant correlation between BSRTC categories and the mutation risk level provides additional insight to aid clinicians in the interpretation of BSRTC results and may contribute to the discussion of appropriate management of thyroid nodule with patients., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The ThyroSeq next-generation sequencing test refines the risk of malignancy in cytologically indeterminate thyroid nodules. Specific genetic alterations have distinct cancer probabilities and clinical phenotypes. There is limited data on the association between specific genetic alterations and histopathologic features. The aim of this study was to evaluate specific ThyroSeq alterations in prognosticating high-risk histopathologic characteristics. We performed a retrospective single-institution study of all patients diagnosed with indeterminate thyroid nodules (May 2016-December 2019) who had a mutation identified with ThyroSeq v2 or v3 and underwent surgical resection. Specific genetic alterations were correlated with surgical histopathology. The main outcomes were risk of malignancy and structural recurrence risk based on histopathologic features and the 2015 American Thyroid Association (ATA) risk stratification. Of the 78 nodules, 50 (64%) were thyroid cancer or noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) on surgical histopathology. Nodules with high-risk TERT or TP53 combination mutations (TERT/TP53) and those with BRAF-like mutations were associated with a 100% probability of cancer and higher rates of extrathyroidal extension and regional nodal involvement than nodules with RAS-like mutations. Among nodules with RAS-like mutations, there was an even distribution between benign, NIFTP, and malignant results, the latter of which were all ATA low risk for structural disease recurrence. Overall, TERT/TP53 and BRAF-like ThyroSeq mutations are associated with an increased cancer probability and risk of recurrence defined by histopathologic features, while RAS-like mutations are associated with lower cancer probability and indolent disease. Individualized management, including extent of surgery, should be considered based on specific genetic alterations found in cytologically indeterminate thyroid nodules.

In patients with thyroid fine-needle aspiration (FNA) report of suspicious for malignancy (SFM), both lobectomy and thyroidectomy might be considered. BRAF mutation analysis could guide towards accurate surgical therapy. The primary outcome was the reliability of BRAF (V600E) in detecting malignancy in nodules with FNA reading of SFM. The secondary outcome was to analyze its positive predictive value (PPV) and negative predictive value (NPV) considering the surgical histology as gold standard. A literature search of online databases was performed in June 2019. BRAF prevalence among thyroid nodules with FNA read as SFM according to the most popular classification systems (i.e., Bethesda V, Thy4, TIR4 category) was searched. The random-effects model was used. Three hundred sixty original articles were identified and 34 were finally included in the study. There were 1428 thyroid nodules with FNA read as SFM and 1287 (90.1%) lesions underwent surgery with a cancer rate 89.6%. The pooled prevalence of BRAF (V600E) mutation among all nodules with SFM cytology was 47% (95% CI = 40 to 54, I2 = 85.5%). Pooled PPV and NPV of BRAF testing were 99% (95% CI, 97-99) and 24% (95% CI, 16-32), respectively. BRAF (V600E) mutation was found in about one in two nodules with thyroid FNA read as SFM, its PPV to detect cancers was excellent, and its NPV was very poor. The routine BRAF testing in FNA read as SFM cannot be recommended. BRAF (V600E) test may be useful to extend surgical approach in selected cases with further suspicious clinical/ultrasound features.

A percentage of 15 to 30% of thyroid fine-needle aspiration (FNA) is indeterminate, i.e., TIR3A and TIR3B according to the Italian consensus for thyroid cytology. Afirma, a molecular analysis of thyroid FNA specimens, has recently gained popularity as an adjunct to microscopic cytological evaluation, in order to avoid diagnostic surgery in patients with indeterminate thyroid cytology. We described the first Italian experience with Afirma tests in a single Italian institution and assessed the performance of the Afirma test in TIR3B. Moreover, this is a preliminary study to assess the patient response to the offer of Afirma testing. The Afirma test was proposed to 67 patients with thyroid nodules that had yielded TIR3B cytology. Fifty-one patients (76.1%) chose the Afirma test, 25/51 underwent the test but the remaining 26 did not because of cost. A total of 41/67 (61.2%) patients underwent surgery, and 22/41 (53.7%) nodules were carcinomas. Of the 25 tested by Afirma, 9 (36%) were classified as Afirma-suspicious (Afirma-S); seven of them underwent surgery, and in 6/7 (85.7%), a cancer was proven at histology. Afirma is the procedure that many Italian patients with TIR3B lesions would choose. However, its routine clinical application in Italy is currently limited by high costs for the patient. When Afirma is performed in this setting of patients, the cancer risk of suspicious result is higher than that expected in all the series of TIR3B. Therefore, testing for Afirma in these nodules may be useful for managing patients and tailoring their surgical approach.

Immunohistochemistry (IHC) in evaluating thyroid surgical specimens may facilitate diagnostic and prognostic evaluation, with potential therapeutic implications. We performed a systematic review and meta-analysis examining the analytic validity of IHC in detecting BRAFV600E mutations in thyroid cancer (primary or metastatic). We screened citations from three electronic databases (until December 20, 2018), supplemented by a hand search of authors' files and cross-references of reviews. Citations and full-text papers were independently reviewed in duplicate, and consensus was achieved on inclusion of papers. Two reviewers independently critically appraised and abstracted data from included papers. Random-effect meta-analyses were conducted for sensitivity and specificity estimates. We reviewed 1499 unique citations and 93 full-text articles. We included 1 systematic review and 30 original articles. The published review (from 2015) needed to be updated as there were multiple subsequent original studies. The pooled sensitivity of IHC in detecting a BRAFV600E mutation was 96.8% (95% confidence interval [CI] at 94.1%, 98.3%) (29 studies, including 2659 BRAFV600E mutant tumors). The IHC pooled specificity was 86.3% (95% CI 80.7%, 90.4%) (28 studies, including 1107 BRAFV600E wild-type specimens). These meta-analyses were subject to statistically significant heterogeneity, partly explained by antibody type (sensitivity and specificity) and tissue/tumor type (specificity). In conclusion, BRAF IHC is highly sensitive and reasonably specific in detecting the BRAFV600E mutation; however, there is some variability in analytic performance.

The objective of this meta-analysis was to evaluate the performance of the Gene Expression Classifier (GEC) and ThyroSeq v2 (ThyroSeq) in the preoperative diagnosis of thyroid nodules with indeterminate fine-needle aspiration biopsy results. We searched literature databases from January 2001 to April 2018. The bivariate model analysis was performed to estimate pooled sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR-), positive predictive value (PPV), and negative predictive value (NPV). Pooled data from 1086 nodules with histopathologic confirmation from 16 GEC studies enabled calculation of diagnostic parameters (95% confidence interval): sensitivity 98% (96-99%), specificity 12% (8-20%), PPV 45% (37-53%), and NPV 91% (85-96%). Pooled data from five ThyroSeq studies assessing 459 nodules showed sensitivity of 84% (74-91%), specificity 78% (50-92%), PPV 58% (31-81%), and NPV 93% (89-97%). When both tools were compared, GEC had a significantly higher sensitivity (p = 0.003), while ThyroSeq had a significantly higher specificity (p < 0.001) and accuracy (p = 0.015). Pooled LR+ was higher for ThyroSeq: 3.79 (1.40-10.27) vs. 1.12 (1.05-1.20). Pooled LR- was higher for GEC, 0.20 (0.10-0.39) vs. 0.13 (0.05-0.31). The bivariate summary estimates of sensitivity and specificity for GEC and ThyroSeq and their pooled accuracy showed a superiority of the ThyroSeq test. The GEC with a high sensitivity and NPV may be helpful in ruling out malignancy in cases of indeterminate thyroid nodule cytology. ThyroSeq has a significantly higher specificity and accuracy with an acceptable sensitivity so that it has the potential for use as an all-round test of malignancy of thyroid nodules.

Thyroid fine-needle aspiration (FNA) cytology is the primary tool for the diagnostic evaluation of thyroid nodules. BRAF mutation analysis is employed as an ancillary tool in indeterminate cases, as recommended by the American Thyroid Association management guidelines. Hereby, we report the case of a 73-year-old woman who presented an 8-mm-size, ill-defined, left thyroid nodule. FNA resulted "suspicious for papillary thyroid carcinoma". BRAF mutation status was analyzed, and somatic BRAF (V600E) mutation identified. The patient underwent a total thyroidectomy. At histological examination, the nodule was composed of Langerhans cells, admixed with many eosinophils. A final diagnosis of Langerhans cell histiocytosis of the thyroid was made. Our case emphasizes the critical diagnostic pitfalls due to the use of BRAF (V600E) mutation analysis in thyroid FNA. Notably, BRAF (V600E) mutation is common in melanoma, colorectal carcinoma, lung carcinoma, ovarian carcinoma, brain tumors, hairy cell leukemia, multiple myeloma, and histiocytoses. Therefore, in cases of indeterminate FNA with unclassifiable atypical cells BRAF (V600E) mutated, the possibility of a localization of hystiocytosis or a secondary thyroid malignancy should be taken into account.

Some thyroid nodules such as follicular adenomas (FAs), follicular variant of papillary thyroid carcinomas (FVPTCs), and follicular thyroid carcinomas (FTCs) exhibit similar clinical presentations and gross morphologic appearances. The differential diagnosis of these lesions is sometimes difficult based on morphologic, cytologic, or clinical features alone. miR-146b-5p and miR-21 deregulation has been associated with progression and metastasis of thyroid cancers. However, the utility of in situ hybridization (ISH) to determine the cellular localization, diagnostic, and prognostic significance of miR-146b-5p and miR-21 expression in thyroid tumors has not been extensively analyzed. In order to examine the expression of miR-146b-5p and miR-21 in benign and malignant thyroid tissues and to determine if these microRNAs could be assigned to distinct histomorphological types of thyroid nodules, we analyzed miR-146b-5p and miR-21 expression in thyroid nodules on tissue microarrays (TMAs) with 193 thyroid specimens by ISH. miR-146b-5p and miR-21 expression in thyroid tissues was also analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). miR-146b-5p was highly expressed (89%) in papillary thyroid carcinomas (PTCs) and 41% of FVPTC. The expression of miR-146b-5p was not expressed in most FTCs, anaplastic thyroid carcinomas (ATCs), poorly differentiated thyroid carcinomas (PDTCs), or FAs (7, 8, 0, and 0%, respectively). MiR-21 was overexpressed in 83% of ATCs, 79 % of PTCs, 34% of FVPTCs, and 19% of PDTCs. The expression of miR-21 was not expressed in most FAs (9%) or FTCs (4%). Normal thyroid tissues and most benign goiters were negative for miR-146b-5p and miR-21. qRT-PCR analysis supported the ISH findings. PTC cases with positive expression of miR-146b-5p and miR-21 had significantly poorer disease-free survival rates. Immunohistochemical staining for HBME-1 showed positive staining in PTCs (100 %) and FVPTCs (92 %) with a subset of FTC (40%) staining positive, while all FAs were negative. Since miR-146b-5p was mainly expressed in PTC including FVPTC and was not expressed in most FTC, PDTC, or ATC, it may serve as a useful diagnostic marker for PTC. ISH is a useful method to analyze microRNA expression in formalin-fixed paraffin-embedded thyroid tissues.

Fine-needle aspiration biopsy (FNA) is currently the best initial diagnostic test for evaluation of a thyroid nodule. FNA cytology cannot discriminate between benign and malignant thyroid nodules in up to 30% of thyroid nodules. Therefore, an adjunct to FNA is needed to clarify these lesions as benign or malignant. Using differential display-polymerase chain reaction method, the gene expression differences between follicular and classic variants of papillary thyroid carcinoma (PTC) and benign thyroid nodules were evaluated in a group of 42 patients. Computational gene function analyses via Cytoscape, FuncBASE, and GeneMANIA led us to a functional network of 17 genes in which a core sub-network of five genes coexists. Although the exact mechanisms underlying in thyroid cancer biogenesis are not currently known, our data suggest that the pattern of transformation from healthy cells to cancer cells of PTC is different in follicular variant than in classic variant.

Activating mutations of GNAq protein in a hotspot at codon 209 have been recently described in uveal melanomas. Since these neoplasms share with thyroid carcinomas a high frequency of MAP kinase pathway-activating mutations, we hypothesized whether GNAq mutations could also play a role in the development of thyroid carcinomas. Additionally, activating mutations of another subtype of G protein (GNAS1) are frequently found in hyperfunctioning thyroid adenomas, making it plausible that GNAq-activating mutations could also be found in some of these nodules. To investigate thyroid papillary carcinomas and thyroid hyperfunctioning nodules for GNAq mutations in exon 5, codon 209, a total of 32 RET/PTC, BRAF, and RAS negative thyroid papillary carcinomas and 13 hyperfunctioning thyroid nodules were evaluated. No mutations were identified. Although plausible, GNAq mutations seem not to play an important role in the development of thyroid follicular neoplasms, either benign hyperfunctioning nodules or malignant papillary carcinomas. Our results are in accordance with the literature, in which no GNAq hotspot mutations were found in thyroid papillary carcinomas, as well as in an extensive panel of other tumors. The molecular basis for MAP-kinase pathway activation in RET-PTC/BRAF/RAS negative thyroid carcinomas remains to be determined.

Our understanding of oncocytic change in thyroid nodules is evaluated in light of the recent progress in understanding the mitochondrial DNA, its mutations, and somatic mutations that affect mitochondrial function. These changes are largely unrelated to the genetic events that result in proliferation and neoplastic transformation of thyroid follicular epithelial cells. The criteria for diagnosing lesions that are composed predominantly of oncocytic cells are the same as those applied to follicular lesions that do not contain oncocytic cells, including follicular variant papillary carcinomas, based on nuclear morphology, immunohistochemical profiles, and molecular markers.

BRAF gene mutations are identified in about 45% of papillary thyroid carcinomas (PTC) and represent the most common genetic event in this tumor. Here, we report a case of PTC, solid variant, with a complex BRAF mutation that involves one nucleotide substitution, C1796T, and a CTT triplet insertion, 1798&#95;1799insCTT, located on the same allele. This mutation leads to the replacement of a threonine with an isoleucine, T599I, and replacement of a valine with an alanine and a leucine, V600delinsAL. This mutation was identified both in the preoperative fine needle aspirate sample and in the surgical specimen after total thyroidectomy. Other rare BRAF mutations in PTC are reviewed.