Thyroid Nodule Samples Research Articles

Improving the efficiency of thyroid cancer diagnostics in difficult diagnostic conditions using molecular diagnostic methods

Purpose of the study. To develop a diagnostic calculator for assessing the expression of profile microRNAs (miRNAs) that are significant for the oncogenesis of thyroid cancer, to introduce it into clinical practice and to evaluate the effectiveness of the proposed method for early diagnosis of malignant neoplasms of the thyroid gland in uncertain diagnostic situations after fine-needle aspiration biopsy (FNAB).Material and methods. The previously developed «Method for Diagnosing Malignant Neoplasms of the Thyroid Gland» (patent RU2820815 C1) was tested on 284 patients of the dispensary group with nodular formations of the thyroid gland according to the results of ultrasound and an uncertain conclusion after FNAB. The number of patients with papillary thyroid carcinoma was 83 (29,2%), follicular carcinoma 43 (15,1%) and with benign thyroid pathology (follicular adenoma) – 158 (55,6%) people. The expression of miRNA-146b and –574–3p in cytological samples of thyroid nodules after FNAB was assessed using the real-time PCR method.Results. In thyroid cancer patients, the expression of miRNA-146b was higher (p=0,007) compared to patients with benign pathology, while the expression activity of miRNA-574–3p was lower (p=0,013). To assess the index of reciprocal paired dysregulation of miRNA-146b and miRNA-574–3p expression in thyroid cells, it is recommended to determine the ratio of the expression values of the corresponding molecules. If the ratio index of miRNA-146b and miRNA-574–3p expression is higher than 4.5, then a conclusion is made about a malignant neoplasm of the thyroid gland. The diagnostic coefficient calculated by the developed formula using the logistic regression method also allows diagnosing malignant neoplasm of the thyroid gland when compared with the cutoff level. The proposed methods are highly informative.Conclusion. Evaluation of the expression of miRNA-146b and miRNA-574–3p in the cells of thyroid nodules obtained by FNAB allows improving the diagnosis of malignant pathology in mutation-negative thyroid nodules with uncertain FNAB results.

The value of NGS-based multi-gene testing for differentiation of benign from malignant and risk stratification of thyroid nodules.

Fine-needle aspiration (FNA) biopsy is typically used in conjunction with cytopathologic evaluation to differentiate between benign and malignant thyroid nodules. Even so, the cytology results for 20-30% of thyroid nodules are indeterminate. This study sought to evaluate the usefulness of next-generation sequencing (NGS)-based multi-gene panel testing for risk stratification and the differentiation of benign from malignant thyroid nodules. Thyroid nodule samples were obtained from a cohort of 359 patients who underwent FNA. An NGS-based multi-gene panel testing was conducted for these samples, in which single-nucleotide variants (SNVs) and small insertion/deletions (InDels) can be detected in 11 genes and fusion events can be identified in 5 genes. Surgical resection was conducted for 113 patients (113/359), and then histopathology results were obtained. In comparison to cytology alone, the diagnostic sensitivity of NGS combination cytology increased from 0.7245 (95% CI: 0.6289-0.8032) to 0.898 (95% CI: 0.8223-0.9437); the associated AUC was 0.8303 (vs. Cytology AUC: 0.7622, P < 0.001). BRAF V600E was identified in 136 patients, of whom 79 underwent surgery and were diagnosed with papillary thyroid carcinoma (PTC) pathologically. TERT promoter mutations or BRAF/RAS co-mutations with other genes were identified in 5 patients, while 4 patients were diagnosed with malignant thyroid cancer using the pathological method. RAS mutations were identified in 27 patients, while 10 patients underwent surgery, which showed that 3 patients were classified as PTC and 7 cases were benign. In addition, 4 RET fusions, 1 RET activation mutation, and 3 TP53 inactivation mutations were identified in the remaining 8 patients who have not undergone surgery. Negative genetic test results or variants with uncertain significance were identified in 183 patients. Among these patients, 12 malignant thyroid tumors, including 11 PTC and 1 MTC, were diagnosed in 20 patients who received surgery. Thyroid nodules coupled with BRAF V600E, TERT promoter variants, BRAF/RAS co-mutations with other genes, RET fusions, and RET activating mutations were classified as high-risk. Nodules with RAS mutations (NRAS, KRAS, HRAS) and TP53 inactivating mutations were considered to be in the intermediate-risk group, while those with non-pathogenic mutations (negative and variants of uncertain significance) were placed in the low-risk group. When combined with cytopathology, NGS increases the sensitivity of diagnosing benign and malignant thyroid nodules, and the reference is useful for patient risk stratification.

DICER1 Variants in Pediatric and Young Adult Thyroid Nodules.

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

THADA-IGF2BP3 gene fusions in thyroid fine needle aspiration is involved in the pathway to "noninvasive follicular thyroid neoplasm with papillary-like nuclear features".

The increased usage and adaptation of molecular testing of thyroid fine needle aspirations (FNA) has expanded the variety and number of gene fusions identified. While the identified number of molecular alterations is increasing, the definitive association between preoperative molecular analysis and phenotype has yet to be established. The aim of this study was to examine Thyroid adenoma-associated (THADA)-IGF2BP3 molecular fusions with FNA categorization, surgical pathology diagnosis, and other molecular alterations detected by ThyroSeq Genomic Classifier testing. FNA cytology samples of thyroid nodules from 04/2017 to 01/2023 with the diagnosis of atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS; Bethesda III) or follicular neoplasm suspicious for follicular neoplasm (FN/SFN; Bethesda IV) with associated ThyroSeqV3 testing were reviewed. Parameters including patient demographics, FNA diagnosis, ThyroSeq V3 results, and surgical pathology follow up were examined. 87 out of 249 (35%) FNA specimens of thyroid nodules displayed molecular alterations. 64 cases (74%) had a cytology diagnosis of AUS and 23 (26%) had FN. RAS mutation was observed in 48 cases. On surgical follow-up, 17 (35%) cases showed non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), while 14 (29%) patients had a malignant diagnosis. THADA-IGF2BP3 fusions were seen in 8 cases, all with NIFTP on surgical pathology follow-up (100%). Analysis of THADA-IGF2BP3 fusion, in our institutional series, shows close association with NIFTP cases. THADA-IGF2BP3 fusion, which seems to be a favorable prognostic indicator in general, may serve as a molecular marker for non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP).

Deep learning models for thyroid nodules diagnosis of fine-needle aspiration biopsy: a retrospective, prospective, multicentre study in China

Accurately distinguishing between malignant and benign thyroid nodules through fine-needle aspiration cytopathology is crucial for appropriate therapeutic intervention. However, cytopathologic diagnosis is time consuming and hindered by the shortage of experienced cytopathologists. Reliable assistive tools could improve cytopathologic diagnosis efficiency and accuracy. We aimed to develop and test an artificial intelligence (AI)-assistive system for thyroid cytopathologic diagnosis according to the Thyroid Bethesda Reporting System. 11 254 whole-slide images (WSIs) from 4037 patients were used to train deep learning models. Among the selected WSIs, cell level was manually annotated by cytopathologists according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) guidelines of the second edition (2017 version). A retrospective dataset of 5638 WSIs of 2914 patients from four medical centres was used for validation. 469 patients were recruited for the prospective study of the performance of AI models and their 537 thyroid nodule samples were used. Cohorts for training and validation were enrolled between Jan 1, 2016, and Aug 1, 2022, and the prospective dataset was recruited between Aug 1, 2022, and Jan 1, 2023. The performance of our AI models was estimated as the area under the receiver operating characteristic (AUROC), sensitivity, specificity, accuracy, positive predictive value, and negative predictive value. The primary outcomes were the prediction sensitivity and specificity of the model to assist cyto-diagnosis of thyroid nodules. The AUROC of TBSRTC III+ (which distinguishes benign from TBSRTC classes III, IV, V, and VI) was 0·930 (95% CI 0·921-0·939) for Sun Yat-sen Memorial Hospital of Sun Yat-sen University (SYSMH) internal validation and 0·944 (0·929 - 0·959), 0·939 (0·924-0·955), 0·971 (0·938-1·000) for The First People's Hospital of Foshan (FPHF), Sichuan Cancer Hospital & Institute (SCHI), and The Third Affiliated Hospital of Guangzhou Medical University (TAHGMU) medical centres, respectively. The AUROC of TBSRTC V+ (which distinguishes benign from TBSRTC classes V and VI) was 0·990 (95% CI 0·986-0·995) for SYSMH internal validation and 0·988 (0·980-0·995), 0·965 (0·953-0·977), and 0·991 (0·972-1·000) for FPHF, SCHI, and TAHGMU medical centres, respectively. For the prospective study at SYSMH, the AUROC of TBSRTC III+ and TBSRTC V+ was 0·977 and 0·981, respectively. With the assistance of AI, the specificity of junior cytopathologists was boosted from 0·887 (95% CI 0·8440-0·922) to 0·993 (0·974-0·999) and the accuracy was improved from 0·877 (0·846-0·904) to 0·948 (0·926-0·965). 186 atypia of undetermined significance samples from 186 patients with BRAF mutation information were collected; 43 of them harbour the BRAFV600E mutation. 91% (39/43) of BRAFV600E-positive atypia of undetermined significance samples were identified as malignant by the AI models. In this study, we developed an AI-assisted model named the Thyroid Patch-Oriented WSI Ensemble Recognition (ThyroPower) system, which facilitates rapid and robust cyto-diagnosis of thyroid nodules, potentially enhancing the diagnostic capabilities of cytopathologists. Moreover, it serves as a potential solution to mitigate the scarcity of cytopathologists. Guangdong Science and Technology Department. For the Chinese translation of the abstract see Supplementary Materials section.

Classification of Benign-Malignant Thyroid Nodules Based on Hyperspectral Technology.

In recent years, the incidence of thyroid cancer has rapidly increased. To address the issue of the inefficient diagnosis of thyroid cancer during surgery, we propose a rapid method for the diagnosis of benign and malignant thyroid nodules based on hyperspectral technology. Firstly, using our self-developed thyroid nodule hyperspectral acquisition system, data for a large number of diverse thyroid nodule samples were obtained, providing a foundation for subsequent diagnosis. Secondly, to better meet clinical practical needs, we address the current situation of medical hyperspectral image classification research being mainly focused on pixel-based region segmentation, by proposing a method for nodule classification as benign or malignant based on thyroid nodule hyperspectral data blocks. Using 3D CNN and VGG16 networks as a basis, we designed a neural network algorithm (V3Dnet) for classification based on three-dimensional hyperspectral data blocks. In the case of a dataset with a block size of 50 × 50 × 196, the classification accuracy for benign and malignant samples reaches 84.63%. We also investigated the impact of data block size on the classification performance and constructed a classification model that includes thyroid nodule sample acquisition, hyperspectral data preprocessing, and an algorithm for thyroid nodule classification as benign and malignant based on hyperspectral data blocks. The proposed model for thyroid nodule classification is expected to be applied in thyroid surgery, thereby improving surgical accuracy and providing strong support for scientific research in related fields.

Near-Infrared Spectroscopy for Distinguishing Malignancy in Thyroid Nodules.

Thyroid nodules are common clinical entities, with a significant proportion being malignant. Early, accurate, and non-invasive tools to differentiate benign and malignant nodules can optimize patient management and reduce unnecessary surgery. This study aimed to evaluate the efficacy and accuracy of near-infrared spectroscopy (NIRS) in distinguishing benign from malignant thyroid nodules. A diffuse reflectance spectrum for a total of 20 thyroid nodule samples (10 samples as colloid goiter and 10 samples as thyroid cancer), were acquired in the wavelength range from 1000 to 2500 nm. Spectral data from NIRS were analyzed by means of principal component analysis (PCA), quadratic discriminant analysis (QDA), and linear discriminant analysis (LDA) to classify and differentiate thyroid nodule samples. The present study found that NIRS effectively distinguished colloid goiter and thyroid cancer using the first two principal components (PCs), explaining 90% and 10% of the variance, respectively. QDA discrimination plot displayed a clear separation between colloid goiter and thyroid cancer with minimal overlap, aligning with reported 95% accuracy. Additionally, applying LDA to seven PCs from PCA achieved a 100% accuracy rate in classifying colloid goiter and thyroid cancer from near-infrared spectral data. In conclusion, NIRS offers a promising, non-invasive complementing diagnostic tool for differentiating benign from malignant thyroid nodules with high accuracy. Future work should integrate these results into predictive model development, emphasizing external validation, alternative performance metrics, and protecting against potential overfitting translation of a machine learning model to a clinical setting.

Prevalence of DICER1 variants in large multinodular goiter: thyroid function, clinical and imaging characteristics.

Mutations in DICER1 are found in differentiated thyroid carcinoma (DTC) and in multinodular goiter (MNG) at a younger age with other tumors, which characterizes DICER1 syndrome. DICER1 is one driver to DTC; however, it is also found in benign nodules. We speculated that patients with mutations in DICER1 may present long-lasting MNG. Our aim was to investigate the frequency of DICER1 variants in patients with MNG. Patients who submitted to total thyroidectomy due to large MNG with symptoms were evaluated. DICER1 hotspots were sequenced from thyroid nodule samples. To confirm somatic mutation, DNA from peripheral blood was also analyzed. Among 715 patients, 154 were evaluated with 56.2 ± 12.3 years old (28-79) and the thyroid volume was 115.7 ± 108 mL (16.2-730). We found 11% with six DICER1 variations in a homo or heterozygous state. Only rs12018992 was a somatic DICER1 variant. All remaining variants were synonymous and likely benign, according to the ClinVar database. The rs12018992 was previously described in an adolescent with DTC, measuring 13 mm. There were no significant differences according to gender, familial history of goiter, age, thyroid volume, TSH and TI-RADS classification between DICER1 carriers. Free T4 were lower in patients with DICER1 polymorphisms (13.77 ± 1.8 vs. 15.44 ± 2.4 pmol/L, p = 0.008), regardless of TSH levels. We conclude that germline DICER1 variants can be found in 11% of large goiters but no second-hit somatic mutation was found. DICER1 is one driver to thyroid lesion and a second-hit event seems unnecessary in the MNG development.

SRT: Swin-residual transformer for benign and malignant nodules classification in thyroid ultrasound images

With the advancement of deep learning technology, computer-aided diagnosis (CAD) is playing an increasing role in the field of medical diagnosis. In particular, the emergence of Transformer-based models has led to a wider application of computer vision technology in the field of medical image processing. In the diagnosis of thyroid diseases, the diagnosis of benign and malignant thyroid nodules based on the TI-RADS classification is greatly influenced by the subjective judgment of ultrasonographers, and at the same time, it also brings an extremely heavy workload to ultrasonographers. To address this, we propose Swin-Residual Transformer (SRT) in this paper, which incorporates residual blocks and triplet loss into Swin Transformer (SwinT). It improves the sensitivity to global and localized features of thyroid nodules and better distinguishes small feature differences. In our exploratory experiments, SRT model achieves an accuracy of 0.8832 with an AUC of 0.8660, outperforming state-of-the-art convolutional neural network (CNN) and Transformer models. Also, ablation experiments have demonstrated the improved performance in the thyroid nodule classification task after introducing residual blocks and triple loss. These results validate the potential of the proposed SRT model to improve the diagnosis of thyroid nodules' ultrasound images. It also provides a feasible guarantee to avoid excessive puncture sampling of thyroid nodules in future clinical diagnosis.

The diagnostic and prognostic role of miR-146b-5p in differentiated thyroid carcinomas.

Samples classified as indeterminate correspond to 10-20% of cytologies obtained by fine needle biopsy of thyroid nodules, preventing an adequate distinction between benign and malignant lesions and leading to diagnostic thyroidectomies that often prove unnecessary, as most cases are benign. Furthermore, although the vast majority of patients with differentiated thyroid cancer (DTC) have such a good prognosis that active surveillance is permitted as an initial therapeutic option, relapses are not rare, and a non-negligible number of patients experience poor outcomes. MicroRNAs (miR) emerge as potential biomarkers capable of helping to define more precise management of patients in all these situations. Aiming to investigate the clinical utility of miR-146b-5p in the diagnostic of thyroid nodules and evaluating its prognostic potential in a realworld setting, we studied 89 thyroid nodule samples, correlating miR-146b-5p expression with clinical tools such as the 8th edition from the American Joint Committee on Cancer (AJCC/UICC) and the American Thyroid Association Guideline Stratification Systems for the rate of recurrence (RR). miR-146b-5p expression levels distinguished benign from malignant thyroid FNA samples (p< 0.0001). For indeterminate nodules, overexpression of miR-146b-5p with a cut-off of 0.497 was able to diagnose malignancy with a 90% accuracy; specificity=87.5%; sensitivity=100%. An increased expression of miR-146b-5p was associated with greater RR (p=0.015). A cut-off of 2.21 identified cases with more vascular involvement (p=0.013) and a cut-off of 2.420 was associated with a more advanced TNM stage (p-value=0.047). We demonstrated that miR-146b5p expression in FNA samples is able to differentiate benign from malignant indeterminate nodules and is associated with an increased risk of recurrence and mortality, suggesting that this single miRNA may be a useful diagnostic and prognostic marker in the personalized management of DTC patients.

One nodule-one punction-one slide: Optimizing thyroid fine-needle aspiration for a digital workflow.

Interventional pathologists have expanded their expertise by acquiring proficiency in ultrasound-guided thyroid fine-needle aspiration biopsy (FNAB) and are now required to optimize punction procedures due to low resources and digital workflows. The aim of this study is to compare FNAB sample adequacy in two series with one versus two slides available for cytopathological analysis and its influence on diagnosis categorization, time taken to reach a final diagnosis, scanning time and size of the digital files produced. Patients were retrospectively selected based on the sampling of thyroid nodules using either two glass slides (two-slide group) or one slide only (one-slide group) and cytological diagnosis was performed using the second edition of the Bethesda system. For each group, the initial 15 cases were sorted to be scanned. From a total of 713 procedures, 328 were sampled into two slides and 385 on one slide only. No significant differences were found regarding nodule size, location or EU-TIRADS classification between the two groups. The one-slide group did not exhibit a higher prevalence of non-diagnostic or atypia of undetermined significance (AUS) categories. As expected, the mean time taken to finalize diagnoses in cases where only one slide was prepared was 1.2 days faster. Scanning time and total file size were also significantly smaller in the one-slide group. Adopting the 'one nodule-one puncture-one slide' strategy for thyroid FNAB optimization enhances procedural efficiency in digital workflows, leading to cost savings without compromising diagnostic accuracy.

A Matched-Pair Analysis of Nuclear Morphologic Features Between Core Needle Biopsy and Surgical Specimen in Thyroid Tumors Using a Deep Learning Model.

Core needle biopsy (CNB) is a method that can be used as an alternative to fine-needle aspiration for the sampling of thyroid nodules to provide a preoperative diagnosis. Nuclear atypia is of paramount importance in diagnosing thyroid tumors. We aimed to identify the differences in nuclear morphologic features between CNB and surgical specimens. Quantitative image analysis was performed on whole slide images (WSI) of paired CNB and surgical specimens from 50 follicular cell-derived tumors including adenoma, non-invasive follicular thyroid neoplasm with papillary-like nuclear features, and carcinoma. The nuclear features of tumor cells were extracted from WSI using a pre-trained deep-learning model. In matched-pair analysis, the ratios (surgical specimens-to-CNB) of the mean nuclear diameters and the areas of the tumor cells were 1:0.75-0.85 and 1:0.54-0.73 according to the tumor types, respectively. The ratio of the nuclear optical density of tumor cells was 1.68-2.44 times higher in CNB specimens than it was in their corresponding surgical specimens. The significant nuclear size reduction and optical density increase in CNB were most prominent in papillary carcinoma cells. Normal follicular cells showed nuclear size reduction in CNB but no change in nuclear optical density between CNB and surgical specimens. Nuclear vacuolar artifacts mimicking nuclear pseudoinclusions were frequently seen in CNB regardless of the tumor types. In conclusion, tumor cells in CNB showed marked nuclear shrinkage, darker nuclear staining, and nuclear vacuolar changes. Awareness of the morphological differences according to the sampling types can facilitate correct diagnosis in thyroid histopathology.

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.

Application of biomarkers in the diagnosis of uncertain samples of core needle biopsy of thyroid nodules

Core needle biopsy (CNB) is now more frequently used for the preoperative diagnosis of thyroid nodules. Based on morphology alone, 5–20% of CNB samples cannot be determined as malignant or benign. Compared to fine-needle biopsy (FNB), samples collected by CNB are more accessible for various tests. Therefore, studying biomarkers’ application in distinguishing uncertain CNB samples of thyroid nodules is a practical need. Patients of thyroid nodules with both CNB and matched resected specimens were reviewed. Cases classified as indeterminate lesions, follicular neoplasms, and suspicious for malignancy were retrieved. All CNB samples were stained by immunohistochemistry (IHC) using antibodies against CK19, galectin-3, HBME-1, and CD56 and detected by next-generation sequencing (NGS) using an OncoAim® thyroid cancer multigene assay kit (Singlera Genomics) that detected 26 genes. Taking the resected specimens’ classification as the gold standard, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy of a single biomarker, and various combinations for discriminating malignancy from benignity were calculated. The sensitivity, specificity, PPV, NPV, and accuracy for preoperative malignancy evaluation were as follows. In the cohort of non-follicular-neoplasm-lesions (non-FN-lesion), they were 95.16%, 53.85%, 90.77%, 70.00%, and 88.00% for CK19; 95.16%, 38.46%, 88.06%, 62.50%, and 85.33% for galectin-3; 77.42%, 76.92%, 94.12%, 41.67%, and 58.00% for HBME-1; 66.13%, 100.00%, 100.00%, 38.24%, and 72.00% for CD56; 90.32%, 92.31%, 98.25%, 66.67%, and 90.67% for NGS; and 88.71%, 92.30%, 98.21%, 63.16%, and 89.33% for integrated IHC. In the cohort of follicular neoplasms (FN), they were 30.43%, 77.77%, 77.77%, 30.43%, and 43.75% for CK19; 73.91%, 66.67%, 85.00%, 50.00%, and 71.88% for galectin-3; 26.09%, 88.89%, 85.71%, 32.00%, and 43.75% for HBME-1; 26.09%, 100.00%, 100.00%, 34.62%, and 46.88% for CD56; 52.17%, 88.89%, 92.31%, 42.11%, and 62.50% for NGS; 82.61%, 66.67%, 86.36%, 60.00%, and 78.13% for integrated IHC; and 100%, 66.67%, 88.46%, 100%, and 90.63% for integrated IHC-NGS. The application of biomarkers in distinguishing uncertain CNB samples of thyroid nodules is available and capable. CD56 negative or NGS positive suggests malignancy strongly for both FN and non-FN-lesion, which may be used as a “rule in” tool. The negative predictive value of the integrated IHC and the integrated IHC-NGS implies a high possibility to be benign for non-FN-lesion and FN separately, which can work as a “rule out” tool. Considering the balance of specificity and sensitivity, NGS is the best for non-FN-lesion and the integrated IHC-NGS is the best for FN.

Next-Generation Sequencing Enhances the Diagnosis Efficiency in Thyroid Nodules.

BackgroundThough fine-needle aspiration (FNA) improved the diagnostic methods of thyroid nodules, there are still parts of nodules that cannot be determined according to cytology. In the Bethesda system for reporting thyroid cytopathology, there are two uncertain cytology results. Thanks to the development of next-generation sequencing technology, it is possible to gain the genetic background of pathological tissue efficiently. Therefore, a combination of the cytology and genetic background may enhance the accuracy of diagnosis in thyroid nodules.MethodsDNA from 73 FNA samples of thyroid nodules belonging to different cytology types was extracted and exome sequencing was performed by the ThyroLead panel. Test for BRAF mutation was also performed by ARMS-qPCR. Information including age, sex, preoperative cytology, BRAF mutation status tested by ARMS-qPCR, and surgical pathology was collected in electronic medical record system.ResultsA total of 71 single nucleotide variants, three fusion gene, and two microsatellite instability-high status were detected in 73 FNA samples. BRAF V600E mutation is the most common mutation in these malignant thyroid nodules. After combining the cytology and genetic background detected by next-generation sequencing, the diagnosis sensitivity was increased from 0.582 (95% CI: 0.441–0.711) to 0.855 (95% CI: 0.728–0.930) (P < 0.001) in our group, while the specificity, 1,000 (95% CI: 0.732–1.000) compared to 0.857 (95% CI: 0.562–0.975) (P = 0.25), did not get affected.ConclusionsNext-generation sequencing in thyroid nodules can enhance the preoperative diagnosis sensitivity by fine-needle aspiration alone. It can also provide genetic background for direction of medication. It is possible for clinicians to combine cytology with genetic alterations for a more precise diagnosis strategy of thyroid nodules.

Differences of US-FNA BSRTC class, postoperative pathology, and mutation landscape of thyroid nodules between China and other countries.

3055 Background: Ultrasound and ultrasound-guided fine needle aspiration (US-FNA) are the first choice for judging benign and malignant thyroid nodules. This study will report on the differences of US-FNA BSRTC class, postoperative pathology and mutation landscape of thyroid nodules between China and other countries. Methods: We conducted a prospective study containing 383 FNA samples of thyroid nodules. For most of these FNA samples, genomic DNA and RNA were extracted and sequenced with FSZ-Thyroid NGS Panel V1, and postoperative pathology were followed up. Moreover, we also compared results of this study with those of West China Hospital in China, Yamashita Thyroid Hospital in Japan, and Cleveland Clinic in the United States. Results: Among the 383 FNA samples, the proportions of BSRTC class I to VI were 10.7%, 6.3%, 18.8%, 3.7%, 12.3%, and 48.3% respectively. Compared with study in other countries, the proportion of class II was significantly lower than that in Japan and the United States. Meanwhile, the proportion of class V and VI were significantly higher than the above two countries. Subsequently, 232 thyroid nodules were surgically removed. Postoperative pathology showed that the proportion of malignant tumors (85.3%) was also significantly higher than reported in Japan and the United States. But compared with other studies in China, there was no significant difference. Most of the malignant tumors were papillary thyroid cancer (PTC, 96%), accompanied with 2 follicular thyroid cancer (FTC), 3 medullary cancer (MTC) and 3 anaplastic thyroid cancer (ATC). Compared with study in the United States, the proportion of PTC and FTC were elevated (96% vs. 85.3%) and reduced (1% vs. 9.3%) respectively. At last, we also analysis the mutation landscape of 180 malignant tumors. Compared with TCGA study, the frequency of BRAF V600E in PTC in our study was significantly higher than that of TCGA (73.3% vs. 58%), and the frequency of RAS mutation was significantly lower (1.2% vs. 12.6%). And compared with an institutional experience of ThyroSeq v3 for Bethesda III and IV at the University of Pittsburgh Medical Center, the frequency of BRAF V600E and RAS mutation in Bethesda III-IV malignant tumors was also significantly higher (45.8% vs. 1.4%) and lower (8.3% vs. 47.1%). Conclusions: There were significant differences in BSRTC class and postoperative pathology between China and other countries, such as Japan and the United States. The possible reasons included that the indications for FNA in China were different. For example, most of patients who underwent FNA in this study had suspicious clinical/ultrasound features. So the proportion of BSRTC class V and VI as well as the malignant rate were elevated. On the other hand, more BRAF V600E and less RAS mutations were detected in malignant tumors in this study which might result from racial differentiation and discrepancy in proportion of PTC and FTC.

Significance of molecular detection in diagnosis of benign and malignant thyroid nodules in China.

e15028 Background: Ultrasound and ultrasound-guided fine needle aspiration (US-FNA) are a preferred method for judging benign and malignant thyroid nodules. For thyroid nodules that can’t be determined by FNA, molecular markers of thyroid cancer can be detected as assistant method. This study aim to verify the significance of molecular detection in diagnosis of benign and malignant thyroid nodules in China. Methods: We carried out a prospective study of 383 FNA samples of thyroid nodules, and performed targeted multi-gene NGS on most samples with FSZ-Thyroid NGS Panel V1, the result of which would be incorporated into the reference index for clinicians to decide whether a patient should undergo surgery. And postoperative pathology were followed up. The NGS targets were divided into three categories: high risk (BRAF V600E, TP53, PIK3CA, AKT1, CTNNB1, RET, KRAS with VAF≥30%, HRAS with VAF≥30%, NRAS with VAF≥30%, RET fusion, NTRK1 fusion, NTRK3 fusion, ALK fusion, BRAF fusion), low risk (BRAF non-V600E, EIF1AX, GNAS, TSHR, TERT, KRAS with VAF &lt; 30%, HRAS with VAF &lt; 30%, HRAS with VAF &lt; 30%, PPARG fusion, THADA fusion), benign like (EZH1, SPOP, ZNF148 or no mutations). Results: Among the 383 FNA samples, 333 of them were sequenced with FSZ-Thyroid NGS Panel V1, and 211 thyroid nodules were resected. Finally, postoperative pathology showed that 180 nodules was malignant and 31 was benign. Among the 180 malignant tumors, BRAF V600E (71.1%), RET fusion (7.8%), and TERT mutation (2.8%) were the top three most mutated genes; and there were also significant differences in frequency of some mutations between malignant tumors in different BSRTC class, including more BRAF V600E (83.6%) in class VI malignant tumors, more gene fusions (26.1%) in class V malignant tumors which was significantly higher than that in class VI (7%). And, the frequency of RAS mutations (8.3%) in class III and IV malignant tumors was higher than that of class V and VI. Here a test was considered as positive if a genetic alteration was annotated with High-Risk, and negative if Low-Risk or Benign-Like, the sensitivity and specificity for detecting malignant tumors in BSRTC class III-IV were 66.7% and 100%, respectively, and the total sensitivity and specificity for BSRTC class I-VI malignant tumors were 85.6% and 100%. In addition, 21 of 383 patients underwent Re-FNA. Among these 21 patients, 11 patients detected high risk mutations and 90.9% (10/11) underwent upgrade of BSRTC class on Re-FNA. As a comparation, among the remaining 10 patients who detected low risk or benign like sites, only 30% (3/10) underwent upgrade of BSRTC class on Re-FNA which suggested that the detection of high risk sites might predict the upgrade of BSRTC class on Re-FNA. Conclusions: Molecular testing can distinguish between benign and malignant thyroid nodules and predict the upgrade of BSRTC class on Re-FNA which could provide the reference for treatment decision-making of thyroid nodules in China.

The correlation between ultrasonographic features, bFGF, and the local invasiveness of thyroid papillary carcinoma.

The present study aimed to investigate the correlation between ultrasonographic features, basic fibroblast growth factor (bFGF), and the local invasiveness of papillary thyroid carcinoma (PTC).A total of 350 samples of thyroid nodules were collected. Routine ultrasonography was performed before the operation and routine pathological diagnosis and bFGF detection were performed after the operation.’These 350 samples of thyroid nodules included 90 samples of nodular goiter, 36 samples of focal thyroiditis, and 224 samples of PTC. A total of 326 thyroid nodules were examined for bFGF. The results revealed that the difference in the expression of bFGF between the benign and malignant groups was statistically significant (P < .05) and the difference in the positive expression of bFGF between the invasive and non-invasive PTC groups was statistically significant (P < .05).Whether the shape of PTC is regular or not and whether there is micro-calcification in PTC and other ultrasonographic features, the size and location of the lesions and the age of the patient help make a preliminary prediction of local invasiveness before the operation. Postoperative detection of bFGF is helpful for further risk assessments of PTC.

Effect of BRAF V600E mutation detection of fine-needle aspiration biopsy on diagnosis and treatment guidance of papillary thyroid carcinoma

ObjectiveThe aim of this study was to evaluate the diagnostic value of detection of BRAF V600E mutation in the fine-needle aspiration cytology (FNAC) specimens of thyroid nodules and the relationship between BRAF V600E mutation and the clinicopathological characteristics of papillary thyroid carcinoma (PTC). MethodsA total of 252 patients who underwent initial thyroid surgery were retrospectively analysed. All the patients underwent a preoperative FNAC at our institution, and the thyroid puncture cell fluid was used for both the cytological diagnosis and BRAF V600E mutational analysis using quantitative polymerase chain reaction. The Cochran-Mantel-Haenszel test was used to evaluate the diagnostic value of BRAF V600E mutation in FNAC fluid in diagnosing PTC. The association between BRAF V600E mutation and the clinicopathological parameters of PTC was analysed using the χ2 test. ResultsThrough FNAC, 21 (8%), 60 (24%), and 171 (68%) cases were cytologically diagnosed as benign, indeterminate, and malignant, respectively. Postoperatively, 242 cases were histopathologically diagnosed as PTCs and 10 as goitre nodules. In the FNAC samples, 12 (57 %) of the 21 benign, 48 (80 %) of the 60 indeterminate, and 152 (88.9 %) of the 171 malignant cases showed BRAF V600E mutation. The histopathological diagnosis was used as the gold standard. The sensitivity and specificity of BRAF V600E mutational analysis in the FNAC samples for the diagnosis of PTC were 91.7 % and 100 % in benign, 82.8 % and 100 % in the indeterminate, and 89.4 % and 100 % in the malignant cases, respectively. ConclusionBRAF V600E mutational analysis in FNAC samples of thyroid nodules can be used an effective supplementary diagnostic method at our institution. However, BRAF V600E mutation was not associated with aggressive characteristics in PTC.

PIWI-interacting RNAs piR-13643 and piR-21238 are promising diagnostic biomarkers of papillary thyroid carcinoma.

Emerging studies demonstrate that PIWI-interacting RNAs (piRNAs) participate in the development of cancers. 75 pairs of papillary thyroid carcinoma (PTC) samples and 31 benign thyroid nodule samples were included in this three-phase biomarker identifying study. First, piRNA expression profiles of five pairs of PTC samples were acquired piRNA sequencing. The expression of all upregulated piRNAs were further validated by RT-qPCR. Paired t and nonparametric test were used to evaluate the association between all upregulated piRNAs and clinic stage. The expression levels of key piRNAs were corrected by demographic data to construct a multivariate model to distinguish malignant nodules from benign. Additionally, the intersection between target genes of key piRNAs and differentially expressed genes in The Cancer Genome Atlas (TCGA) PTC samples were used to perform enrichment analysis. Only piR-13643 and piR-21238 were significantly upregulated in PTC and associated with clinic stage. Moreover, both piR-13643 (Area Under Curve (AUC): 0.821) and piR-21238 (AUC: 0.823) showed better performance in distinguishing malignant nodules from benign than currently used biomarkers HBME1 (AUC: 0.590). Based on our findings, piR-13643 and piR-21238 were observed to be significantly upregulated in human PTC. PIWI-interacting RNAs could serve as promising novel biomarkers for accurate detection of PTC.