Thyroid Fine-needle Aspiration Samples Research Articles

Revisiting the cytomorphological features of poorly differentiated thyroid carcinoma: a comparative analysis with indeterminate thyroid fine-needle aspiration samples.

Poorly differentiated thyroid carcinoma (PDTC) is an uncommon high-grade carcinoma of follicular cell origin that is usually overlooked on preoperative fine-needle aspiration (FNA) due to its rarity and cytomorphological overlap with follicular-patterned neoplasms. Definitive diagnosis of PDTC usually requires histologic examination of the resected thyroid tumor. Herein, we describe the cytological and architectural findings of histologically confirmed PDTC cases. A search for all thyroid FNAs with a corresponding surgical diagnosis of PDTC was performed. Surgical diagnoses were reviewed and confirmed using the Turin criteria. In addition, the control group consisted of indeterminate thyroid nodules (FLUS [follicular lesion of undetermined significance] and FN [follicular neoplasm]) that were either benign or well-differentiated thyroid tumors on resection. The PDTC and control groups were both subjected to cytological assessment using specific cytological and architectural parameters, which included cellularity, growth pattern, mitoses, necrosis, chromatin change, discohesion, and anisonucleosis. A total of 36 thyroid FNAs were included in the study. This consisted of 12 histologically confirmed PDTC FNAs and 24 indeterminate thyroid FNAs (FLUS and FN, 12 each). The most frequent findings among PDTC groups were hypercellularity (75%), trabecular/insular growth pattern (58%), branching capillaries (67%), and cellular discohesion (92%). Necrosis (25%), ≥3 mitoses (50%), and anisonucleaosis (42%) were less frequently observed. A peculiar finding was the presence of adenoid cystic carcinoma-like globules in 50% of PDTC cases. Certain findings such as colloid, necrosis, mitoses, and cellular discohesion were helpful in differentiating the two groups. Thyroid fine-needle aspiration remains an essential diagnostic/triage tool for most thyroid nodules/tumors. PDTC can be diagnosed or at least suspected preoperatively based on the demonstration of certain architectural and cytological alterations. Although mitoses and necroses are not always readily identified, an elevated Ki-67 labeling expression could provide additional clues to the diagnosis in some cases.

Evaluating local thyroid cytopathology practices by molecular quality metrics: A multi-institutional study on 4651 FNAs with a focus on the role of the interventional cytopathologist.

The diagnostic accuracy of thyroid fine-needle aspiration (FNA) can be highly influenced by the technical skills of the operator performing the procedure and by interobserver variability in microscopic interpretation. This is particularly true for the indeterminate categories. Recently, molecular testing has been proposed as an ancillary tool for monitoring the performance of different thyroid cytopathology practices. The objective of this multicenter study was to evaluate the quality of different local cytopathology practices by assessing the impact of interventional cytopathologists on FNA adequacy for molecular testing and the variations in mutation rates across different health care centers operating in the Campania region. The study included 4651 thyroid FNA samples diagnosed in different Southern Italian clinical laboratories belonging to the TIRNET (the Tiroide Network). FNA samples were collected by different proceduralists and were classified by local cytopathologists according to The Bethesda System for Reporting Thyroid Cytopathology. FNAs classified as atypia of undetermined significance, follicular neoplasm, suspicious for malignancy, and malignant were centralized for a real-time polymerase chain reaction-based, seven-gene test at the authors' institution. Centers that employed interventional cytopathologists obtained fewer unsatisfactory FNA samples for molecular testing (11.3%) than centers that employed noncytopathologists (16.7%; p<.05). Furthermore, a significant variation in the mutation rate was observed in FNAs diagnosed by different local cytopathologists; indeterminate categories had the highest percentage of mutation rate variability among centers. Interventional cytopathologists obtained higher yields of diagnostic material for molecular testing. Finally, the current results suggest that the variability in mutation rates among different centers may highlight the low reproducibility of microscopic criteria among cytopathologists, particularly for indeterminate cases.

Screening adequacy of unstained thyroid fine needle aspiration samples using a deep learning-based classifier

Fine needle aspiration (FNA) biopsy of thyroid nodules is a safe, cost-effective, and accurate diagnostic method for detecting thyroid cancer. However, about 10% of initial FNA biopsy samples from patients are non-diagnostic and require repeated FNA, which delays the diagnosis and appropriate care. On-site evaluation of the FNA sample can be performed to filter out non-diagnostic FNA samples. Unfortunately, it involves a time-consuming staining process, and a cytopathologist has to be present at the time of FNA. To bypass the staining process and expert interpretation of FNA specimens at the clinics, we developed a deep learning-based ensemble model termed FNA-Net that allows in situ screening of adequacy of unstained thyroid FNA samples smeared on a glass slide which can decrease the non-diagnostic rate in thyroid FNA. FNA-Net combines two deep learning models, a patch-based whole slide image classifier and Faster R-CNN, to detect follicular clusters with high precision. Then, FNA-Net classifies sample slides to be non-diagnostic if the total number of detected follicular clusters is less than a predetermined threshold. With bootstrapped sampling, FNA-Net achieved a 0.81 F1 score and 0.84 AUC in the precision-recall curve for detecting the non-diagnostic slides whose follicular clusters are less than six. We expect that FNA-Net can dramatically reduce the diagnostic cost associated with FNA biopsy and improve the quality of patient care.

Unraveling the significance of TSHR mutations in indeterminate thyroid cytology specimens

We investigated the clinical significance of thyroid-stimulating hormone receptor (TSHR) mutations detected in thyroid fine needle aspiration (FNA) specimens. The pathology archives at our institution were reviewed between 2018 and 2021 for indeterminate (Bethesda category III and IV) specimens with Thyroseq® analysis showing TSHR mutations. A total of 2184 cases diagnosed as atypia/follicular lesion of undetermined significance (AUS/FLUS), and 2625 diagnosed as follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) were identified. A total of 1735 AUS/FLUS and 2339 SFN/FN underwent Thyroseq® analysis; 69 showed TSHR mutations (1.6%, 59 female, 10 male, average age: 55 years). Ten cases showed oncocytic features. Twelve patients underwent radionuclide scans within 1 year of FNA:11 were hyperfunctioning. Nodule size and TSH levels were weakly correlated. Twenty-two different TSHR mutations were identified (most common: M453T). A second mutation was found in five cases (EZH1 n = 2, and EIF1AX n = 3). The expression of sodium-iodide transporter (NIS) mRNA was in the range of 0.01%-62.43% out of all sequencing reads, and was elevated in 49 (71%) cases. Surgical pathology follow-up was available in five cases (all benign except one). On follow-up available for 38 cases (mean: 24 months; range: 7-47 months), 34 (89.5%) nodules remained stable and 3 (8%) increased in size. TSHR mutations in thyroid FNA samples classified as indeterminate are rare, generally benign, and commonly associated with autonomy on scan if performed.

Diagnostic Value of Preparing Additional Liquid-Based Cytology Slides and Cell Blocks from Residue Material in Thyroid fine Needle Aspiration.

After liquid-based cytology (LBC) technique developed for cervical cytology, it has been used for nongynecological samples and has been very successful. It offers having extra slides of the samples for further examination and ancillary tests. Moreover, cell blocks can be formed from the residue material. The study aimed to evaluate the importance of preparing a second LBC slide or a cell block from the residue material of thyroid fine needle aspiration (FNA) samples to reach a definitive diagnosis in cases diagnosed as nondiagnostic (ND) after the first slide. Seventy five cases diagnosed as ND after the first slide were included in study. For 50 cases, the second LBC slides were prepared (LBC group); for 25 cases, cell block was performed from residue material (CB group). Two groups were compared in terms of reaching a definitive diagnosis. At the end of secondary procedures, a definitive diagnosis was reached in a total of 24 cases (32%). Twenty of 50 cases (40%) in LBC group reached a definitive diagnosis while four cases (16%) in the CB group reached a definitive diagnosis. Achieving a definitive diagnosis was found statistically higher in the LBC group in which the second slide was formed compared to the CB group (P =0.036). Preparing a second slide with LBC method is more purposive than preparing a cell block from the residue material of thyroid FNA samples. Reducing the percentage of ND cases will protect patients from complications and morbidity that may arise from repeated FNA.

Identifying metastatic renal cell carcinoma in thyroid fine-needle aspirates by molecular testing.

Renal cell carcinoma (RCC) is the most common type of cancer found to metastasize to the thyroid gland. These tumors may represent a diagnostic challenge in cytology. However, most RCC tumors carry VHL alterations, which are rare in primary thyroid tumors. The aim of this study was to evaluate the utility of molecular testing in detecting metastatic RCC in thyroid fine-needle aspiration (FNA) samples. From November 2017 until March 2022, thyroid FNA samples with ThyroSeq v3 results showing both VHL alterations and low/absent expression of thyroid cell markers were analyzed. Eighteen samples from 15 patients met the inclusion criteria. On molecular analysis, deleterious VHL mutations were found in nine (50%) nodules, VHL copy number alteration (CNA) in two (11%), and both mutations and CNA in seven (39%). None of the cases showed mutations commonly found in thyroid tumors. The mean age of these patients was 68 (range, 49-89) years with a male to female ratio of 2:1. Eight (53%) patients had multiple thyroid nodules on ultrasound. On cytology, 14 (78%) nodules were diagnosed as Bethesda III, 2 (11%) as Bethesda IV, and 2 (11%) as Bethesda V. At the time of cytology review, the history of RCC, sometimes remote, was available for ten patients. Of the 14 patients with medical history or surgical follow-up available, all had history of RCC or renal mass or revealed metastatic RCC on thyroidectomy. This study demonstrates that molecular testing can reliably identify metastatic RCC in thyroid nodules with indeterminate cytology, which could improve patient management.

Performance of a Multigene Genomic Classifier in Thyroid Nodules with Suspicious for Malignancy Cytology.

Background: Molecular testing is increasingly used to refine the probability of cancer and assess recurrence risk in thyroid nodules with Bethesda III/IV fine needle aspiration (FNA) cytology. However, limited data exist for Bethesda V (suspicious for malignancy [SFM]) samples. This study evaluated the performance of ThyroSeq v3 (TSv3) in thyroid nodules with SFM cytology. Methods: In this single-institution retrospective cohort study, consecutive thyroid FNA samples diagnosed as SFM with TSv3 testing and known surgical outcome were identified. Clinical, pathology, and molecular findings were reviewed. The TSv3 Cancer Risk Classifier was used to determine molecular risk groups (MRGs). For test-negative cases diagnosed as cancer/noninvasive follicular thyroid neoplasm with papillary-like nuclear features, TSv3 was performed on the resected tumors. Results: Among 128 SFM samples studied, 100 (78.1%) were TSv3 positive, and 28 (21.9%) were negative. The cancer prevalence on surgery was 82.8%. Among test-positive samples, 95% were malignant and 5% benign. Among test-negative samples, 17 (60.7%) were benign and 11 (39.3%) malignant. Overall, TSv3 had a sensitivity of 89.6% (confidence interval; CI 82.4-94.1) and a specificity of 77.3% (CI 56.6-89.9). For a cancer prevalence of 50-75% expected in SFM cytology by the Bethesda system, the negative predictive value was expected to range from 71.2% to 88.1% and the positive predictive value from 79.8% to 92.2%. Among test-positive nodules, 20% were MRG-Low (mostly RAS-like alterations), 66% MRG-Intermediate (mostly BRAF-like alterations), and 14% MRG-High. Among patients with cancer, 65 (61.3%) were American Thyroid Association low risk, 25 (23.6%) intermediate risk, and 6 (5.7%) high risk. During the mean follow-up of 51.2 months (range: <1 to 470 months), 12 (13.0%) patients had disease recurrence, which was more common in MRG-High (54.6%) compared with MRG-Intermediate (9.5%) and MRG-Low (0%) cancers (p < 0.001). Upon reexamining tumors with false-negative results, half of evaluable cases had alterations likely missed due to limiting FNA sampling, and the remainder represented low-risk tumors. Potentially targetable alterations were identified in 10 samples. Conclusions: In this large series of SFM thyroid nodules, TSv3 further improved cancer prediction and detected RAS-like, BRAF-like, high-risk, and potentially targetable alterations, all of which may inform more optimal patient management. MRGs were associated with recurrence-free survival, offering potential preoperative cancer risk stratification.

Detection of BRAF V600E in Fine-Needle Aspiration Samples of Thyroid Nodules by Droplet Digital PCR.

Background BRAF exon 15 p.V600E (BRAF V600E) mutation has been established as an important molecular marker for papillary thyroid carcinoma diagnosis by ultrasound-guided fine-needle aspiration biopsy (FNAB). Sanger sequencing is the gold standard for detecting BRAF V600E mutations but fails to identify low-frequency mutations. However, droplet digital PCR (ddPCR) is a popular new method for detecting low-frequency mutations. Here, we compare the efficiency of droplet digital PCR (ddPCR) and Sanger sequencing for detection of the BRAF V600E mutation in thyroid fine-needle aspiration (FNA) samples. Methods Thyroid fine-needle aspiration samples from 278 patients with 310 thyroid nodules were collected. Sanger sequencing and ddPCR were conducted to detect the BRAF V600E mutation. Results The BRAF V600E mutation was found in 94 nodules (30.32%) by ddPCR and 40 nodules (12.90%) by Sanger sequencing in 310 FNA samples. A total of 119 nodules were confirmed PTC by postsurgical pathology. Among which the BRAF mutation was found in 80 (67.23%) nodules by ddPCR and 31 (26.05%) by Sanger sequencing. All nodules carrying the mutation detected by Sanger sequencing (SS+) were verified by ddPCR (ddPCR+). Also, all nodules with no mutation detected by ddPCR were interpreted as wild-type by Sanger sequencing (SS−). In addition. Almost all SS+/ddPCR + nodules (95.00%; 38/40) and SS−/ddPCR + nodules (100.00%; 54/54) displayed a BRAF mutation rate of >5% and <15%, respectively, indicating easy misdetection by Sanger sequencing when the mutation rate is between 5 and 15%. Conclusion ddPCR has higher sensitivity than Sanger sequencing and we propose ddPCR as a supplement to Sanger sequencing in molecular testing of BRAF using FNAB samples.

Should Liquid Based Cytology (LBC) be Applied to Thyroid Fine Needle Aspiration Cytology Samples?: Comparative Analysis of Conventional and LBC Smears.

Context:Liquid-based cytology (LBC) is a cyto-preparatory technique that may be applied to fine-needle aspiration (FNA) samples. However, its efficacy over conventional smears (CS) in thyroid is controversial.Aims:The aim of this study was to compare CS versus LBC (SurePath, BD) preparations in thyroid FNA samples for diagnostic efficacy.Settings and Design:Prospective case-control study using split sample analysis in 200 non-consecutive cases of thyroid FNA reported as per the Bethesda system.Methods and Material:Detailed cytomorphological features were evaluated in CS and LBC preparations. Cellularity was scored as 0–3. Based on diagnostic efficacy, they were categorized into three groups:(i) CS and LBC equivalent for diagnosis, (ii) CS better than LBC, and (iii) LBC better than CS for providing the diagnosis.Statistical Analysis Used:Paired t test for cellularity scores and descriptive for diagnostic efficacy.Results:There were 7 unsatisfactory, 118 benign, 10 atypia or follicular lesion of undetermined significance, 25 follicular neoplasms, 6 suspicious for malignancy, and 34 malignant thyroid FNA cases based on routine conventional smears. Cellularity of conventional smears was significantly higher than paired LBC smears (paired t test, P < 0.005). Comparison of overall diagnostic efficacy showed that LBC and CS were equivalent in 59% of cases; CS was superior to LBC in 37% cases and LBC was better than CS in 4% cases only. LBC smears showed higher unsatisfactory rate as compared to CS (18% vs. 3.5%). In category 2, the two techniques were equivalent in 87% of cases. In categories 4 and 6, CS were superior to LBC.Conclusions:In thyroid FNA cases, conventional smears are superior to LBC preparation in terms of cellularity and diagnostic efficacy and hence, LBC preparations should not replace conventional smears for making a routine cytomorphological diagnosis.

BRAF V600E as an accurate marker to complement fine needle aspiration (FNA) cytology in the guidance of thyroid surgery in the Chinese population: evidence from over 1000 consecutive FNAs with follow-up.

Currently, several commercial molecular tests have been developed for reclassifying thyroid nodules with indeterminate fine needle aspiration cytology. These tests are quite expensive and not available in China. Previous studies demonstrated a very high prevalence of the BRAF V600E mutation in Asian people. A high incidence may result in a robust sensitivity. We conducted this study to determine the prevalence of BRAF V600E mutation and its ability to reclassify cytologically indeterminate thyroid nodules in the Chinese population. Between January 2016 and October 2018, consecutive patients who underwent a fine needle aspiration procedure and agreed to provide materials for molecular analysis in our hospital were recruited in this study. All were followed up until they had a thyroidectomy and a final pathological diagnosis or until January 2019 (those did not have surgery). A total of 1960 patients were included in this study. Until January 2019, 1240 patients underwent surgery. Using histopathological diagnosis as a gold standard, the overall sensitivity and specificity of the BRAF V600E mutational analysis for the discrimination of benign nodules from cancer in thyroid fine needle aspiration samples were 83.3% (81.0-85.3%) and 96.0% (77.7-99.8%), respectively, with an area under the ROC curve of 0.90 (95% CI 0.85-0.95, P<0.001). Among cases with indeterminate cytology, BRAF-positive cases were showing malignancy in the final pathology, and BRAF-negative cases were showing safer to be followed up. The BRAF V600E mutation is highly prevalent in the Chinese population and can accurately complement cytopathology in the guidance of thyroid surgery.Mini-abstract: The BRAF V600E mutation has both high specificity and sensitivity to predict thyroid malignancy in the Chinese population. It can accurately complement cytopathology in the guidance of thyroid surgery.

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Clinical ThyroidologyVol. 32, No. 5 Letters to the EditorFree AccessHandling of Thyroid FNA Samples During the COVID-19 PandemicLiron PantanowitzLiron PantanowitzProfessor of Pathology, Director of Cytopathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A. Search for more papers by this authorEmail the corresponding author at pantanowitzl@upmc.eduPublished Online:6 May 2020https://doi.org/10.1089/ct.2020;32.239-241AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Dear Editor,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly described pathogen that causes coronavirus disease-2019 (COVID-19). After its initial outbreak in China in late 2019, widespread transmission of this virus quickly resulted in the current pandemic. Countries such as Italy were severely stricken and soon reported COVID-19 positive cases and related deaths that surpassed those in China. Not surprisingly, this raised concern among practicing pathologists in Italy who were receiving samples from infected patients. As a result, pathology laboratories in Italy sought to implement biosafety measures to minimize spread and protect pathologists, as well as laboratory staff, who were asked to handle infectious samples.In a recent publication (1), a group of pathologists from Rome, Italy advised several cytopathology measures which are helpful to highlight. They advocate that cytological evaluations only be performed on patients for essential cases, especially when procuring pulmonary samples, that cytology specimens be handled in a laboratory with a controlled ventilation system, and processing be performed only by trained technologists wearing appropriate personal protective equipment (PPE). These rigorous measures were recommended because most liquid-based cytology specimens contain low alcohol concentrations. As an additional precautionary measure, this group has modified their cytology specimen processing protocol; their “off-label” method uses fixative solutions containing a minimum of 70% ethyl alcohol and, when needed, 99% ethanol is also added to their fixation solutions. To further minimize risk, they have stopped preparing cell blocks for cytology specimens. Despite the potential for such modifications to alter the diagnostic quality of cytologic samples, the authors felt strongly that adoption of appropriate laboratory biosafety during this pandemic was also important (1).There is a paucity of literature on COVID-19 and the thyroid gland. However, based on a small autopsy series (2), we know that there is both thyroid follicular and parafollicular cell injury in patients with severe acute respiratory syndrome (SARS). From these data (2), SARS-CoV appears to cause distortion of follicular architecture and damages follicular epithelium, which was believed to be the reason why some infected patients have low serum T3 and T4 levels. Whilst it is unclear if SARS-CoV-2 may be detected in extrapulmonary specimens, such as those from a thyroid fine needle aspiration (FNA), adopting proactive measures that limit aerosolization and viral transmission, in addition to universal precautions, is reasonable. SARS-CoV-2 has been classified as a risk group 3 pathogen, which is similar to the SARS-CoV and Middle East respiratory syndrome (MERS) coronaviruses (3). The U.S. Centers for Disease Control and Prevention (CDC) recommend that handling of these potentially infectious samples be performed only in a standard biosafety level (BSL)-2 laboratory (4).If performing a thyroid FNA is deemed necessary during this COVID-19 pandemic, it is important to consider: (i) how best to handle and evaluate the aspirated sample on-site, (ii) how to transport this specimen to the cytology laboratory, and (iii) how this FNA sample should be processed. During a thyroid FNA procedure, all specimens collected for cytologic evaluation should be considered potentially infectious. Hence, universal precautions which includes the use of PPE should be mandatory. While rapid on-site evaluation (ROSE) by a cytologist is well-known to ensure specimen adequacy, the proceduralist performing the thyroid FNA will need to use clinical judgment on a case-by-case basis to determine if this is absolutely necessary. The pathology group from Rome discourages the use of ROSE procedures at this time (1). Limiting the number of people present in the biopsy room is also desirable. Therefore, once a slide is made by a cytotechnologist on-site, it could be remotely evaluated by a cytopathologist using telecytology, if such technology is available and has been validated for this purpose. If on-site evaluation is not needed, the aspirated material can be directly placed into a vial with appropriate fixative. Leaving the aspirate in the needle syringe until a slide can be safely made in the cytology lab should be avoided, because samples tend to clot and thus may become very difficult to later expel. If a slide must be prepared on-site for immediate evaluation the following steps should be undertaken. The specimen must not be squirted onto the glass slide, but rather gently dropped on the slide which will reduce aerosolization (5). Air drying the slide and using methods to try speed up drying (e.g. waving the slide around, hand-held fan, heat drying) should be avoided. During this pandemic, several cytology laboratories have actually converted from air drying slides on-site to rather using alcohol-based fixatives (e.g. 95% alcohol) with rapid staining procedures (e.g. with Diff Quik, Toluidine blue, or rapid H&E stains). Also, all slides should be immediately coverslipped. Since we now know that SARS-CoV-2 can survive on several contaminated surfaces and fomites (6), all work area surfaces and equipment (e.g. microscope) that are used should be regularly cleaned and disinfected.After the thyroid FNA procedure is complete, all material (i.e. specimen and slides) should be delivered to the pathology laboratory in properly-labeled, leak-proof packaging. This should be delivered by hand, and not using a pneumatic tube system that may result in spills (7). The personnel transporting the specimen should be trained to safely handle such samples, as well as potential spill decontamination. Also, the cytology laboratory needs to be notified in a timely manner to expect this sample. In the laboratory, specimen processing should ideally be carried out under a Class II Biological Safety Cabinet (BSC) or similar containment device. Cytopreparatory steps that may generate aerosol droplets (e.g. centrifugation) should be circumvented if possible. Fixatives such as formalin have been reported to inactivate SARS-CoV and SARS-CoV-2 (8). However, most cytology fixatives utilize variants of alcohol (ethanol or methanol). Currently, it is unknown if proprietary commercial cytology fixatives (e.g. PreservCyt, CytoLyt, SurePath) that contain low concentrations of alcohol adequately inactivate the new coronavirus (9). Utilizing solutions with at least >70% alcohol is recommended because they are biocidal to SARS-CoV-2 (5–6).In summary, when handling a thyroid FNA and/or biopsy it is important to follow universal precautions and biosafety guidelines recommended by organizations such as the U.S. CDC and WHO (10–11). However, until we learn more about SARS-CoV-2, undertaking additional safety practices specific to cytopathology in order to mitigate the risk of transmission is appropriate (1).The author has no disclosures.Liron Pantanowitz, MDProfessor of PathologyDirector of CytopathologyUniversity of Pittsburgh Medical CenterPittsburgh, Pennsylvania, U.S.A.pantanowitzl@upmc.edu

HMGA2 Gene Expression in Fine-needle Aspiration Samples of Thyroid Nodules as a Marker for Preoperative Diagnosis of Thyroid Cancer.

There is a great interest in molecular markers that would help in the preoperative diagnosis of malignant thyroid nodules in cases of indeterminate fine-needle aspiration cytology. The aim of this study was to determine the diagnostic accuracy of HMGA2 gene expression in discriminating benign from malignant thyroid nodules. In this study, 237 preoperative thyroid fine-needle aspiration samples were analyzed prospectively for the expression of the HMGA2 gene by real-time reverse transcription polymerase chain reaction. The results were evaluated against the postoperative histopathologic diagnosis or definitive cytologic diagnosis in cases of nodular goiter and Hashimoto thyroiditis. Among 237 samples from patients with thyroid nodules that were analyzed, 231 were adequate for real-time reverse transcription polymerase chain reaction analysis. With a cutoff value of 8.71 for relative gene expression, HMGA2 was positive in 19 (16.4%) of 116 nodular goiter, 1 (2.6%) of 39 Hashimoto thyroiditis, 9 (28.1%) of 32 follicular adenoma, 0 (0%) of 5 Hurthle cell adenoma, 32 (88.9%) of 36 papillary carcinoma, and 3 (100%) of 3 follicular carcinoma samples. In discriminating between malignant and benign thyroid nodules, HMGA2 has shown specificity of 84.5%, sensitivity of 91.9%, positive predictive value of 53.1%, and negative predictive value of 98.2%. High sensitivity and negative predictive value of HMGA2 for preoperative detection of malignant thyroid nodules shown in this study indicate that it may have a role as an ancillary marker in cytology in the management of patients with thyroid nodules.

Diagnosing atypia of undetermined significance in thyroid fine needle aspiration samples using nuclear scoring

BackgroundAtypia of undetermined significance (AUS) is an indeterminate category in the Bethesda system for reporting thyroid cytopathology. Cytological features described as atypia are not always observed in every case, and it is difficult to determine how the small population of cells with enlarged nuclei, a few grooves, and rare elongated nuclei should be classified. Therefore, there is inter-intra observer variability considering these cell types, even though the cytological criteria are well defined. Therefore, this study aimed to establish a nuclear scoring system to help in the differential diagnosis of AUS. MethodsFine needle aspiration (FNA) samples that showed AUS and had surgical follow-up were included in this study. The aspirate was scored for the presence of intanuclear cytoplasmic inclusions, nuclear grooves, overlapping, enlargement, and elongation individually. The total nuclear score for each case was calculated. Statistical analysis of the association between each nuclear feature and the presence of papillary thyroid cancer (PTC) in the surgical specimens was performed. Cut-off points from the total score of these nuclear features were also calculated. ResultsNuclear grooves and overlapping were more common in malignant cases (p < 0.001 and p = 0.048, respectively). A cut-off point of ≥5.5 for the total score was sensitive and specific for defining malignancy. ConclusionThe risk of PTC was higher in nodules with more prominent nuclear overlapping or nuclear groove in their FNA samples. In order to achieve a more confident AUS diagnosis, our scoring system can be helpful for thyroid FNA samples.

GLIS Rearrangement is a Genomic Hallmark of Hyalinizing Trabecular Tumor of the Thyroid Gland.

Hyalinizing trabecular tumor (HTT) is a rare thyroid neoplasm with a characteristic trabecular growth pattern and hyalinization. This lesion has been the subject of long-term controversy surrounding its genetic mechanisms, relationship to papillary thyroid carcinoma (PTC), and malignant potential. Due to the presence of nuclear features shared with PTC, HTT frequently contributes to a false-positive cytology, which hampers patient management. The goal of this study was to apply genome-wide sequencing analyses to elucidate the genetic mechanisms of HTT and its relationship to PTC. Whole-exome, RNA-Seq, and targeted next-generation sequencing analyses were performed to discover and characterize driver mutations in HTT. RNA-Seq results were used for pathway analysis. Tissue expression of GLIS3 and other proteins was detected by immunohistochemistry. The prevalence of GLIS fusions was studied in 17 tumors initially diagnosed as HTT, 220 PTC, and 10,165 thyroid fine-needle aspiration samples. Using whole-exome and RNA-Seq analyses of the initial three HTT, no known thyroid tumor mutations were identified, while in-frame gene fusion between PAX8 exon 2 and GLIS3 exon 3 was detected in all tumors. Further analysis identified PAX8-GLIS3 in 13/14 (93%) and PAX8-GLIS1 in 1/14 (7%) of HTT confirmed after blind pathology review. The fusions were validated by Sanger sequencing and FISH. The fusions resulted in overexpression of the 3'-portion of GLIS3 and GLIS1 mRNA containing intact DNA-binding domains of these transcription factors and upregulation of extracellular matrix genes including collagen IV. Immunohistochemistry confirmed upregulation and deposition of collagen IV and pan-collagen in HTT. The analysis of 220 PTC revealed no PAX8-GLIS3 and one PAX8-GLIS1 fusion. PAX8-GLIS3 was prospectively identified in 8/10,165 (0.1%) indeterminate cytology fine-needle aspiration samples; 5/5 resected fusion-positive nodules were HTT on surgical pathology. This study demonstrates that GLIS rearrangements, particularly PAX8-GLIS3, are highly prevalent in HTT but not in PTC. The fusions lead to overexpression of GLIS, upregulation of extracellular matrix genes, and deposition of collagens, which is a characteristic histopathologic feature of HTT. Due to unique genetic mechanisms and an indolent behavior, it is proposed to rename this tumor as "GLIS-rearranged hyalinizing trabecular adenoma."

Centrifuged supernatants from FNA provide a liquid biopsy option for clinical next-generation sequencing of thyroid nodules.

Molecular testing is recommended as an adjunct to improve the preoperative diagnosis of fine-needle aspiration (FNA) of thyroid nodules. Centrifuged supernatants from FNA samples, which are typically discarded, have recently emerged as a novel liquid-based biopsy for molecular testing. This study evaluates the use of thyroid FNA supernatants for detecting clinically relevant mutations. Supernatants from thyroid FNA samples (n=156) were evaluated. A 50-gene next-generation sequencing (NGS) assay was used, and mutation analysis results from a subset of samples were further compared with those of paired FNA smears and/or cell blocks. All 156 samples yielded adequate DNA (median, 135 ng; range, 11-3180 ng), and 129 of these samples (83%) were successfully sequenced by NGS. The most frequently detected somatic mutations included BRAF and RAS mutations, which were followed by RET, TP53, PTEN, CDKN2A, and PIK3CA mutations. Eleven of 31 cases with an indeterminate cytologic diagnosis and 9 of 12 cases that were suspicious for malignancy had somatic mutations, including the BRAF V600E mutation, which is highly definitive for papillary thyroid carcinoma (PTC). Seven of the 9 indeterminate and suspicious cases with the BRAF V600E mutation had surgical follow-up, and they were all confirmed to be PTC. A comparison of the mutation profiles derived from supernatants with those of paired smears and/or cell blocks in a small subset of cases (n=8) showed 100% concordance. This study provides evidence that FNA supernatants can be used as a surrogate for thyroid molecular testing to improve diagnostic accuracy in indeterminate nodules, provide prognostic/predictive information, and improve overall patient management.

Cell block is a valuable adjunct to conventional smear for thyroid fine needle aspiration: 2395 cases with histological correlation.

Conventional smears (CS) of samples obtained by fine needle aspiration (FNA) have proven useful in thyroid nodules evaluation, but the additional contribution of cell block (CB) has only been investigated in a limited fashion. In this study, we aimed to evaluate whether the auxiliary application of CB adds to the diagnostic accuracy of the CS by a College of American Pathologists-accredited hospital laboratory. All thyroid FNA samples processed with CS only or combined CB and CS in West China Hospital from January 2011 to December 2015 were retrospectively collected. All specimens were classified according to Bethesda System, and the distribution of Bethesda (2009) categories was compared between CS and combined CS and CB. Further, we compared the diagnostic performance between these two groups for nodules with follow-up histopathology. A total of 11011 thyroid nodules from 10206 patients were included. Of these, 2395 nodules from 2211 patients underwent surgical resection. The unsatisfactory rate decreased significantly from 18.1% to 9.8% in the total group and from 1.7% to 0.8% in the group with surgery after combined use of CS and CB. The proportion of atypia/follicular lesion of undetermined significance also declined slightly. Furthermore, all of the sensitivities, specificities, accuracies and positive predictive values increased significantly after the combined use of CS and CB for different calculation methods. Combined use of CS and CB can significantly decrease the unsatisfactory rate of thyroid FNAs, improve the diagnostic efficacy, and thus should be routinely applied in thyroid nodule evaluation if available.

Diagnosis of intrathyroidal ectopic thymus in thyroid fine needle aspiration samples

AimsIntrathyroidal ectopic thymus (ITET) is a rare cause of paediatric thyroid nodules. Although ultrasonography of ITET demonstrates a characteristic appearance similar to that of normal thymus, accurate differentiation from other...

An Automatable Method for Determining Adequacy of Thyroid Fine-Needle Aspiration Samples.

Thyroid nodules are a common clinical problem. Cytologic evaluation via fine-needle aspiration is often employed in the diagnostic workup, and rapid on-site assessment of adequacy can help ensure an adequate sample is obtained. However, rapid on-site assessment of adequacy only examines part of the sample, a part that may not then be available for ancillary testing. Moreover, the procedure is time-consuming and poorly reimbursed. To develop an automatable fluorescence-based image analysis system for assessing the adequacy of thyroid fine-needle aspirations that uses the entire aspirated sample. There were 12 previously diagnosed cases that served as a training set, and 11 cases were used for validation of an image analysis algorithm. The samples were fluorescently stained and imaged using a fluorescent microscope. The images were assessed for adequacy by an image analysis algorithm. Following image analysis, a ThinPrep slide was prepared and blindly scored by a cytopathologist. The standard and computer-derived results were then compared. The algorithm was optimized using the 12 cases in the training set and then applied to the 11 test cases. A total of 8 of 8 adequate samples in the test group were correctly scored as adequate, and 2 of 3 cases that were inadequate were correctly scored as inadequate by the algorithm. One case was erroneously designated as not adequate by the algorithm. Our results demonstrate the feasibility of automating thyroid adequacy assessment using a fluorescent labeling technique followed by computer image analysis.

Cancer risk and clinicopathological characteristics of thyroid nodules harboring thyroid-stimulating hormone receptor gene mutations.

Thyroid-stimulating hormone receptor (TSHR) gene mutations play a critical role in thyroid cell proliferation and function. They are found in 20%-82% of hyperfunctioning nodules, hyperfunctioning follicular thyroid cancers (FTC), and papillary thyroid cancers (PTC). The diagnostic importance of TSHR mutation testing in fine needle aspiration (FNA) specimens remains unstudied. To examine the association of TSHR mutations with the functional status and surgical outcomes of thyroid nodules, we evaluated 703 consecutive thyroid FNA samples with indeterminate cytology for TSHR mutations using next-generation sequencing. Testing for EZH1 mutations was performed in selected cases. The molecular diagnostic testing was done as part of standard of care treatment, and did not require informed consent. TSHR mutations were detected in 31 (4.4%) nodules and were located in exons 281-640, with codon 486 being the most common. Allelic frequency ranged from 3% to 45%. Of 16 cases (12 benign, 3 FTC, 1 PTC) with surgical correlation, 15 had solitary TSHR mutations and 1 PTC had comutation with BRAF V600E. Hyperthyroidism was confirmed in all 3 FTC (2 overt, 1 subclinical). Of 5 nodules with solitary TSHR mutations detected at high allelic frequency, 3 (60%) were FTC. Those at low allelic frequency (3%-22%) were benign. EZH1 mutations were detected in 2 of 4 TSHR-mutant malignant nodules and neither of 2 benign nodules. We report that TSHR mutations occur in ∼5% thyroid nodules in a large consecutive series with indeterminate cytology. TSHR mutations may be associated with an increased cancer risk when present at high allelic frequency, even when the nodule is hyperfunctioning. Benign nodules were however most strongly correlated with TSHR mutations at low allelic frequency.

Different qualifiers of AUS/FLUS thyroid FNA have distinct BRAF, RAS, RET/PTC, and PAX8/PPARg alterations.

The Bethesda System for Reporting Thyroid Cytopathology category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) includes fine-needle aspiration (FNA) specimens that cannot straightforwardly be classified as benign or malignant. To determine whether morphological subcategorization based on atypia qualifiers and molecular testing could improve malignancy risk stratification of AUS/FLUS patients, this study assessed the correlation between these qualifiers and the molecular alterations commonly harbored by thyroid neoplasms. A total of 162 AUS/FLUS cases were subcategorized by atypia qualifiers (Hürthle cell changes, architectural atypia, and cytologic atypia [CyA]) and were tested for BRAF, N-H-KRAS, RET/PTC, and paired box 8 (PAX8)/peroxisome proliferator activated receptor γ (PPARg) mutations. CyA was observed more frequently in mutation-positive AUS/FLUS (14 of 37 [37.84%]) than mutation-negative AUS/FLUS (20 of 125 [16.00%]; P < .0084), and it specifically harbored the BRAFV600E point mutation. Malignancy was confirmed in the available follow-up. Conversely, although RAS was the most frequent mutation identified in AUS/FLUS FNA specimens (26 of 37 cases [70.27%]; P < .0001), it was distributed across various AUS/FLUS subcategories and was not significantly associated with a specific atypia qualifier or malignant outcome according to the available follow-up. Rearrangements of both RET/PTC (n = 1) and PAX8/PPARg (n = 3) were rarely retrieved in the FNA samples. BRAF and RAS mutations are associated with different AUS/FLUS qualifiers and hence have different risks of malignancy. Consequently, a hybrid molecular and morphological subcategorization system could improve the malignancy risk stratification of thyroid FNA samples diagnosed as AUS/FLUS. Cancer Cytopathol 2018;126:317-25. © 2018 American Cancer Society.