Blood leukocytes as a non-invasive diagnostic tool for thyroid nodules: a prospective cohort study.

BACKGROUND: Thyroid nodule (TN) patients in China are subject to overdiagnosis and overtreatment. The implementation of existing technologies such as thyroid ultrasonography has indeed contributed to the improved diagnostic accuracy of TNs. However, a significant issue persists, where many patients undergo unnecessary biopsies, and patients with malignant thyroid nodules (MTNs) are advised to undergo surgery therapy. METHODS: This study included a total of 293 patients diagnosed with TNs. Differential methylation haplotype blocks (MHBs) in blood leukocytes between MTNs and benign thyroid nodules (BTNs) were detected using reduced representation bisulfite sequencing (RRBS). Subsequently, an artificial intelligence blood leukocyte DNA methylation (BLDM) model was designed to optimize the management and treatment of patients with TNs for more effective outcomes. RESULTS: The DNA methylation profiles of peripheral blood leukocytes exhibited distinctions between MTNs and BTNs. The BLDM model we developed for diagnosing TNs achieved an area under the curve (AUC) of 0.858 in the validation cohort and 0.863 in the independent test cohort. Its specificity reached 90.91% and 88.68% in the validation and independent test cohorts, respectively, outperforming the specificity of ultrasonography (43.64% in the validation cohort and 47.17% in the independent test cohort), albeit with a slightly lower sensitivity (83.33% in the validation cohort and 82.86% in the independent test cohort) compared to ultrasonography (97.62% in the validation cohort and 100.00% in the independent test cohort). The BLDM model could correctly identify 89.83% patients whose nodules were suspected malignant by ultrasonography but finally histological benign. In micronodules, the model displayed higher specificity (93.33% in the validation cohort and 92.00% in the independent test cohort) and accuracy (88.24% in the validation cohort and 87.50% in the independent test cohort) for diagnosing TNs. This performance surpassed the specificity and accuracy observed with ultrasonography. A TN diagnostic and treatment framework that prioritizes patients is provided, with fine-needle aspiration (FNA) biopsy performed only on patients with indications of MTNs in both BLDM and ultrasonography results, thus avoiding unnecessary biopsies. CONCLUSIONS: This is the first study to demonstrate the potential of non-invasive blood leukocytes in diagnosing TNs, thereby making TN diagnosis and treatment more efficient in China.

[Stratified application of gene expression in diagnosis of thyroid nodules].

Objective: To investigate the value of BRAF V600E and multigene detection and stratified application for the diagnosis of thyroid nodules. Methods: A total of 1 117 patients with thyroid nodules resection at Nanjing Gulou Hospital from December 2020 to July 2022 were enrolled in the study. Fine needle aspiration (FNA) and core biopsy samplings were performed for cytopathologic examination and genetic testings; the findings were combined with BSRTC classification. The diagnostic performance of BRAF V600E and multigene detection were compared. Results: Among the 1, 117 patients who underwent thyroid nodules resection, 285 were male and 832 were female, with a median age of 46 years (range: 24-76 years). Postoperative histopathologic examination confirmed 1 040 cases of thyroid cancer and 77 cases of benign nodules. The sensitivity (87.0% vs. 80.8%, P<0.01) and diagnostic accuracy (87.9% vs. 82.1%, P<0.01) of multigene detection were significantly higher than those of BRAF V600E detection. The result of multigene detection showed that BRAF V600E mutation was the most common finding, followed by CCDC6-RET (E1-E12) fusion, ETV6-NTRK3 fusion, and KRAS mutation. Multigene detection had a higher sensitivity (81.9% vs. 72.8%, P<0.01) and lower cancer risk in wild-type (47.6% vs. 57.7%, P=0.069) than BRAF V600E detection in BSRTCⅠ-Ⅴ lesions. Compared with BRAF V600E detection, multigene had no significant difference of sensitivity in BSRTC Ⅰ lesions, but significantly higher sensitivity (86.3% vs 74.0%, P<0.01) in BSRTC Ⅲ lesions. Conclusions: Genetic detection can be used as an effective tool for the diagnosis of thyroid nodules. A stratified application of molecular markers in the diagnosis of thyroid nodules is proposed. Combined with FNA, single gene or multigene detection both can effectively assist in the diagnosis of thyroid nodules. Moreover, multigene detection is superior to single gene detection. For BSRTC Ⅲ lesion with wild-type BRAF, multigene detection can be considered with a repeated FNA.

RAS-Mutated Cytologically Indeterminate Thyroid Nodules: Prevalence of Malignancy and Behavior Under Active Surveillance.

Background: Genomic profiling is now available for risk stratification of cytologically indeterminate thyroid nodules (ITNs). Mutations in RAS genes (HRAS, NRAS, KRAS) are found in both benign and malignant thyroid nodules, although isolated RAS mutations are rarely associated with aggressive tumors. Because the long-term behavior of RAS-mutant ITNs is not well understood, most undergo immediate surgery. In this multicenter retrospective cohort study, we characterize tumor growth kinetics of RAS-mutant ITNs followed with active surveillance (AS) using serial ultrasound (US) scans and examine the histopathologic diagnoses of those surgically resected. Methods: US and histopathologic data were analyzed retrospectively from two cohorts: (1) RAS-mutant ITNs managed with AS at three institutions (2010-2023) and (2) RAS-mutant ITNs managed with immediate surgery at two institutions (2016-2020). AS cohort subjects had ≥3 months of follow-up and two or more US scans. Cumulative incidence of nodule growth was determined by the Kaplan-Meier method and growth by ≥72% change in tumor volume. Pathological diagnoses for the immediate surgery cohort were analyzed separately. Results: Sixty-two patients with 63 RAS-mutated ITNs under AS had a median diameter of 1.7 cm (interquartile range [IQR] 1.2-2.6) at time of diagnosis. During a median AS period of 23 months (IQR 9.5-53.5 months), growth was observed in 12 of 63 nodules (19.0%), with a cumulative incidence of 1.9% (1 year), 23.0% (3 years), and 28.0% (5 years). Most nodules (81.0%) demonstrated stability. Surgery was ultimately performed in 6 nodules, of which 1 (16.7%) was malignant. In the cohort of 209 RAS-mutant ITNs triaged to immediate surgery, 33% were malignant (23.9% American Thyroid Association [ATA] low-risk cancers, 7.2% ATA intermediate-risk, and 1.9% ATA high-risk. During a median follow-up of 6.9 (IQR 4.4-7.1) years, there were no disease-specific deaths in these patients. Conclusions: We describe the behavior of RAS-mutant ITNs under AS and find that most demonstrate stability over time. Of the resected RAS-mutant nodules, most were benign; of the cancers, most were ATA low-risk. Immediate surgical resection of all RAS-mutant ITNs appears to be a low-value practice. Further research is needed to help define cases most appropriate for AS or immediate surgery.

Molecular Genetics Augment Cytopathologic Evaluation and Surgical Planning of Pediatric Thyroid Nodules.

PURPOSE: Molecular genetic testing in conjunction with cytopathology may improve prediction of malignancy in thyroid nodules, particularly those with indeterminate cytology (Bethesda III/IV). Though now commonplace in adults, pediatric data are limited. This study examines molecular genetics of pediatric nodules with correlation to cytologic and histologic classification at time of surgery and the distribution of mutations. METHODS: Retrospective chart review of 164 patients <22 years who underwent surgical resection of a thyroid nodule between 2002 and 2020 with molecular testing on fine-needle aspiration biopsy (FNA) or final histopathology. RESULTS: 85 (52 %) of 164 patients undergoing thyroid resection had available molecular genetic testing. BRAF V600E testing was performed on the FNA samples of 73 (86 %) patients and on 15 (18 %) surgical specimens; 31 (37 %) were positive. Of the remaining 54 patients, 21 had additional mutation/fusion testing. In 17 (81 %) cases, an alternate mutation/fusion was identified including 8 gene fusions, 3 DICER1 mutations, 4 NRAS mutations, one BRAF variant, and one unknown variant. BRAF, DICER1 mutations, and gene fusions predicted malignancy. Greater than 95 % of BRAF mutations were in Bethesda V/VI lesions and associated with classic variant PTC whereas fusions and DICER1 mutations clustered in Bethesda IV nodules. Bethesda III nodules harbored BRAF and NRAS mutations. In Bethesda IV nodules, a gene fusion or DICER mutation altered the surgical decision-making (upfront thyroidectomy rather than lobectomy) in 70 % of nodules submitted for genetic testing. CONCLUSION: Expanded molecular genetic testing on FNA of pediatric thyroid nodules, particularly Bethesda III/IV, may improve prediction of malignancy and augment surgical decision-making. LEVEL OF EVIDENCE: III.

Does the Adoption of Molecular Testing Cause Decreased Thyroidectomy Rates in a National Cohort? A Quasiexperimental Study of High- Versus Low-Adoption States.

Background: Over the last decade, the utilization of molecular testing (MT) for the evaluation of thyroid nodules has increased. Rates and patterns of adoption of MT and its effect on thyroidectomy rates nationally are unknown. Varying rates of MT adoption at the state level provide an opportunity to study the effects of MT on thyroidectomy rates using a quasiexperimental study design. Methods: We performed a retrospective analysis of American adult patients in the Merative™ MarketScan® Research Databases who underwent thyroid fine-needle aspiration (FNA) from 2011 to 2021. MT included commercially available DNA and RNA platforms and traditional targeted mutational analysis. Interrupted time series analysis was used to evaluate the inflection of MT adoption and thyroidectomy rates after 2015. Difference-in-differences (DID) analysis was used to causally analyze the effect of MT adoption on thyroidectomy rates in high-adoption (at least a 10% increase in MT utilization) versus low-adoption states (no more than 5% increase in MT utilization) from 2015 to 2021. Results: We identified 471,364 patients who underwent thyroid FNA. The utilization of MT increased over the study period from 0.01% [confidence interval, CI: 0.00% to 0.02%] to 10.1% [CI: 9.7% to 10.5%], in 2021, with an immediate (ß2 = 1.61, p = 0.002) and deeper (ß3 = 0.6, p < 0.001) increase in MT adoption after 2015. Utilization of MT was lower in black patients, the elderly, rural areas, and patients with Medicaid (p < 0.05). Thyroidectomy rates were inversely correlated with MT utilization (r = -0.98, p < 0.0001). From 2015 to 2021, the average MT utilization rate increased from 2.4% to 15.3% in high-adoption states and 1.6% to 5.6% in low-adoption states. In low-adoption states, thyroidectomy rates decreased more but to similar levels (18.5-13.2%) compared with high-adoption states (15.9-13.4%) with an adjusted DID rate of -3.3% [CI -5.6% to -0.8%]. Conclusions: The acceleration in adoption of MT after 2015 likely coincides with the publication of American Thyroid Association guidelines. Black, elderly, and rural patients are less likely to receive MT. Although thyroidectomy rates were inversely correlated with MT utilization, our study suggests that this correlation is not causal. The effect of MT on thyroidectomy rates may be overshadowed by decreasing aggressiveness of thyroid nodule evaluation.

Serum MicroRNA-146b Expression for Malignancy Prediction in Euthyroid Patients with Indeterminate Thyroid Nodules.

Thyroid nodules are frequently found, but the vast majority of them are benign. The difficulty in managing thyroid nodules is correctly diagnosing the minority of those who have malignancy. Thyroid fine-needle aspiration cytology (FNAC) with indeterminate cytology continues to raise doubts about the presence of thyroid cancer, leading to an unnecessary thyroidectomy. Circulating miRNAs may be useful as diagnostic and prognostic markers for a variety of cancers, including thyroid cancer. The goal of the present study was to determine the predictive value of serum miRNA-146b expression level for thyroid cancer by estimating its level in a group of euthyroid patients with thyroid nodules with indeterminate FNAC results. This cross-sectional study included 45 euthyroid patients with indeterminate thyroid nodules who visited the Endocrine Outpatient Clinic and Endocrine Surgical Ward at Ain Shams University Hospitals. For all patient thyroid profiles, ultrasound of the thyroid gland and FNAC of the thyroid nodule were performed. In addition, preoperative assessment of serum microRNA-146b expression by real-time PCR was achieved and the results correlated with post-operative thyroid histopathology. There was no difference in serum miRNA-146b expression between patients with benign thyroid nodules versus patients with malignant nodules (p= 0.789). The risk of malignancy increased with the increase in size of the dominant thyroid nodules, as larger nodules had a higher risk of malignancy (p= 0.027). In conclusion, in euthyroid patients with indeterminate thyroid nodules, serum miRNA-146b is a poor predictor of thyroid malignancy, however, the larger the nodule size, the higher the risk of cancer.

Clinical value of multi-gene testing in distinguishing benign and malignant thyroid nodules.

BACKGROUND: The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC). METHODS: A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET. RESULTS: Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%). CONCLUSIONS: Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.

Thyroid Cancer Polygenic Risk Score Improves Classification of Thyroid Nodules as Benign or Malignant.

CONTEXT: Thyroid nodule ultrasound-based risk stratification schemas rely on the presence of high-risk sonographic features. However, some malignant thyroid nodules have benign appearance on thyroid ultrasound. New methods for thyroid nodule risk assessment are needed. OBJECTIVE: We investigated polygenic risk score (PRS) accounting for inherited thyroid cancer risk combined with ultrasound-based analysis for improved thyroid nodule risk assessment. METHODS: The convolutional neural network classifier was trained on thyroid ultrasound still images and cine clips from 621 thyroid nodules. Phenome-wide association study (PheWAS) and PRS PheWAS were used to optimize PRS for distinguishing benign and malignant nodules. PRS was evaluated in 73 346 participants in the Colorado Center for Personalized Medicine Biobank. RESULTS: When the deep learning model output was combined with thyroid cancer PRS and genetic ancestry estimates, the area under the receiver operating characteristic curve (AUROC) of the benign vs malignant thyroid nodule classifier increased from 0.83 to 0.89 (DeLong, P value = .007). The combined deep learning and genetic classifier achieved a clinically relevant sensitivity of 0.95, 95% CI [0.88-0.99], specificity of 0.63 [0.55-0.70], and positive and negative predictive values of 0.47 [0.41-0.58] and 0.97 [0.92-0.99], respectively. AUROC improvement was consistent in European ancestry-stratified analysis (0.83 and 0.87 for deep learning and deep learning combined with PRS classifiers, respectively). Elevated PRS was associated with a greater risk of thyroid cancer structural disease recurrence (ordinal logistic regression, P value = .002). CONCLUSION: Augmenting ultrasound-based risk assessment with PRS improves diagnostic accuracy.

[Possibilities of the personalized approach to solitary thyroid nodules based on molecular profiling]. / Möglichkeiten des personalisierten Vorgehens bei solitären Schilddrüsenknoten basierend auf molekularem Profiling.

BACKGROUND: Molecular profiling is currently not yet widespread in Germany and its potential for personalized treatment of solitary thyroid nodules is not exhausted. METHOD: Narrative assessment of the available options and their evidence based on the current international literature as well as discussion of the options in the German context from the perspective of a thyroid surgeon and a thyroid pathologist. RESULTS: The commercial tests available, especially in the USA, for molecular profiling of thyroid nodules offer good rule-in and somewhat poorer rule-out options, as many differentiated thyroid carcinomas do not display genetic alterations. The costs of these tests and molecular pathology for nodule assessment are still high and the individual mutations have not yet been clearly characterized. CONCLUSION: It can be assumed that molecular profiling will become more important in the coming years and find its way into the clinical routine. At this point in time, however, there is not yet sufficient evidence of the clinical significance of most mutations in thyroid carcinomas to derive therapeutic consequences from them.

[Molecular and other ancillary tests proposed by The Bethesda system for reporting thyroid cytopathology 2023]. / Les tests moléculaires et autres techniques ancillaires en cytologie thyroïdienne selon Bethesda 2023.

For the first time the 2023 version of The Bethesda System for Reporting Thyroid Cytology dedicates a whole chapter (chapter 14) to ancillary studies almost exclusively represented by molecular testing. The latest data reported bring some evidence that molecular testing could help to optimize the diagnostic performance of « indeterminate ¼ categories (AUS and NF). Other studies suggest a promising role to guide the management of suspicious of malignancy and malignant categories. Indeed, the recognition of prognostic and predictive biomarkers analyzed on cytological samples, regardless of how it is collected, has progressed thanks to advances in our knowledge of molecular abnormalities of thyroid tumors. The chapter 14 is presented here highlighting the current and emerging roles of « in-house ¼ and commercialized molecular testing as presented by TSBRTC.

Molecular Diagnostics and [18F]FDG-PET/CT in Indeterminate Thyroid Nodules: Complementing Techniques or Waste of Valuable Resources?

Background: An accurate preoperative workup of cytologically indeterminate thyroid nodules (ITN) may rule out malignancy and avoid diagnostic surgery for benign nodules. This study assessed the performance of molecular diagnostics (MD) and 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG)-positron emission tomography/computed tomography (PET/CT) in ITN, including their combined use, and explored whether molecular alterations drive the differences in [18F]FDG uptake among benign nodules. Methods: Adult, euthyroid patients with a Bethesda III or IV thyroid nodule were prospectively included in this multicenter study. They all underwent MD and an [18F]FDG-PET/CT scan of the neck. MD was performed using custom next-generation sequencing panels for somatic mutations, gene fusions, and copy number alterations and loss of heterozygosity. Sensitivity, specificity, negative and positive predictive value (NPV, PPV), and benign call rate (BCR) were assessed for MD and [18F]FDG-PET/CT separately and for a combined approach using both techniques. Results: In 115 of the 132 (87%) included patients, MD yielded a diagnostic result on cytology. Sensitivity, specificity, NPV, PPV, and BCR were 80%, 69%, 91%, 48%, and 57% for MD, and 93%, 41%, 95%, 36%, and 32% for [18F]FDG-PET/CT, respectively. When combined, sensitivity and specificity were 95% and 44% for a double-negative test (i.e., negative MD plus negative [18F]FDG-PET/CT) and 68% and 86% for a double-positive test, respectively. Concordance was 63% (82/130) between MD and [18F]FDG-PET/CT. There were more MD-positive nodules among the [18F]FDG-positive benign nodules (25/59, 42%, including 11 (44%) isolated RAS mutations) than among the [18F]FDG-negative benign nodules (7/30, 19%, p = 0.02). In oncocytic ITN, the BCR of [18F]FDG-PET/CT was mere 3% and MD was the superior technique. Conclusions: MD and [18F]FDG-PET/CT are both accurate rule-out tests when unresected nodules that remain unchanged on ultrasound follow-up are considered benign. It may vary worldwide which test is considered most suitable, depending on local availability of diagnostics, expertise, and cost-effectiveness considerations. Although complementary, the benefits of their combined use may be confined when therapeutic consequences are considered, and should therefore not routinely be recommended. In nononcocytic ITN, sequential testing may be considered in case of a first-step MD negative test to confirm that withholding diagnostic surgery is oncologically safe. In oncocytic ITN, after further validation studies, MD might be considered. Clinical Trial Registration: This trial is registered with ClinicalTrials.gov: NCT02208544 (August 5, 2014), https://clinicaltrials.gov/ct2/show/NCT02208544.

A retrospective analysis of molecular testing in cytologically indeterminate thyroid nodules with histologic correlation: Experience at a heterogenous multihospital system.

INTRODUCTION: Thyroid malignancy is one of the most common types of cancer in developed nations. Currently, fine-needle aspiration cytology (FNAC) is the most practical screening test for thyroid nodules. However, cytologically indeterminate samples comprise approximately 15%-30% of cases. These include cases classified as atypia of undetermined significance (AUS), follicular neoplasm (FN), and suspicious for malignancy (SFM). Indeterminate cases can be sent for molecular testing for more definitive classification to help guide management and prevent overtreatment of benign thyroid nodules. We conducted a retrospective review on molecular testing of indeterminate thyroid FNAC and reviewed subsequent histologic diagnoses in resection specimens to assess how molecular testing supported a diagnosis and its effect on clinical management of patients at our institution. METHODS: A retrospective chart review was performed on all thyroid FNAC specimens, corresponding molecular testing, and subsequent surgical resection specimens over a 6-year period. RESULTS: A total of 10,253 thyroid FNAC were performed in our hospital system during our study period, of which 10% (n = 1102/10,253) had indeterminate FNAC results. Molecular testing was performed in 16% (n = 178/1102) of indeterminate cytology cases. Genetic alterations were identified in 39% (n = 69/178) of the cases sent for molecular testing. The majority of cytologically indeterminate cases sent for molecular testing were follicular-patterned lesions and their corresponding resection specimens revealed mostly low grade follicular derived neoplasms (i.e., follicular adenoma, non-invasive follicular thyroid neoplasm with papillary-like nuclear features, and follicular variant of papillary thyroid carcinoma). Of the cases with identified genetic alterations, 75% (n = 52/69) were treated surgically. In cases with no genetic alterations identified, only 18% (n = 20/109) were treated surgically. DISCUSSION/CONCLUSION: Molecular testing on cytologically indeterminate thyroid nodules can help provide a more accurate risk of malignancy assessment in patients with lesions that are difficult to diagnosis based solely on FNAC morphology. The types of genetic alterations identified in the resected thyroid lesions were consistent with what has been previously described in the literature. Additionally, we found that in the patients with indeterminate thyroid FNAC with adjunct molecular testing, more than half did not undergo surgical resection. This finding emphasizes the value of adding molecular testing in patients, particularly when attempting to reduce unnecessary surgical intervention.

US of thyroid nodules: can AI-assisted diagnostic system compete with fine needle aspiration?

OBJECTIVES: Artificial intelligence (AI) systems can diagnose thyroid nodules with similar or better performance than radiologists. Little is known about how this performance compares with that achieved through fine needle aspiration (FNA). This study aims to compare the diagnostic yields of FNA cytopathology alone and combined with BRAFV600E mutation analysis and an AI diagnostic system. METHODS: The ultrasound images of 637 thyroid nodules were collected in three hospitals. The diagnostic efficacies of an AI diagnostic system, FNA-based cytopathology, and BRAFV600E mutation analysis were evaluated in terms of sensitivity, specificity, accuracy, and the κ coefficient with respect to the gold standard, defined by postsurgical pathology and consistent benign outcomes from two combined FNA and mutation analysis examinations performed with a half-year interval. RESULTS: The malignancy threshold for the AI system was selected according to the Youden index from a retrospective cohort of 346 nodules and then applied to a prospective cohort of 291 nodules. The combination of FNA cytopathology according to the Bethesda criteria and BRAFV600E mutation analysis showed no significant difference from the AI system in terms of accuracy for either cohort in our multicenter study. In addition, for 45 included indeterminate Bethesda category III and IV nodules, the accuracy, sensitivity, and specificity of the AI system were 84.44%, 95.45%, and 73.91%, respectively. CONCLUSIONS: The AI diagnostic system showed similar diagnostic performance to FNA cytopathology combined with BRAFV600E mutation analysis. Given its advantages in terms of operability, time efficiency, non-invasiveness, and the wide availability of ultrasonography, it provides a new alternative for thyroid nodule diagnosis. CLINICAL RELEVANCE STATEMENT: Thyroid ultrasonic artificial intelligence shows statistically equivalent performance for thyroid nodule diagnosis to FNA cytopathology combined with BRAFV600E mutation analysis. It can be widely applied in hospitals and clinics to assist radiologists in thyroid nodule screening and is expected to reduce the need for relatively invasive FNA biopsies. KEY POINTS: • In a retrospective cohort of 346 nodules, the evaluated artificial intelligence (AI) system did not significantly differ from fine needle aspiration (FNA) cytopathology alone and combined with gene mutation analysis in accuracy. • In a prospective multicenter cohort of 291 nodules, the accuracy of the AI diagnostic system was not significantly different from that of FNA cytopathology either alone or combined with gene mutation analysis. • For 45 indeterminate Bethesda category III and IV nodules, the AI system did not perform significantly differently from BRAFV600E mutation analysis.

Clinical utility of a microRNA classifier in cytologically indeterminate thyroid nodules with RAS mutations: A multi-institutional study.

BACKGROUND: Molecular testing guides the management of cytologically indeterminate thyroid nodules. We evaluated the real-world clinical benefit of a commercially available thyroid mutation panel plus microRNA risk classifier in classifying RAS-mutated nodules. METHODS: We performed a subgroup analysis of the results of molecular testing of Bethesda III/IV nodules using the ThyGenX/ThyGeNEXT-ThyraMIR platform at 3 tertiary-care centers between 2017 and 2021, defining a positive result as 10% or greater risk of malignancy. RESULTS: We identified 387 nodules from 375 patients (70.7% female, median age 59.3 years) who underwent testing. Positive nodules (32.3%) were associated with increased surgical intervention (74.4% vs 14.9%, P < .0001) and carcinoma on surgical pathology (46.4% vs 3.4%, P < .0001) compared to negative modules. RAS mutations were the most common mutations, identified in 71 of 380 (18.7%) nodules, and were classified as ThyraMIR- (28 of 71; 39.4%) or ThyraMIR+ (43 of 71; 60.6%). Among RAS-mutated nodules, there was no significant difference in operative rate (P = .2212) or carcinoma diagnosis (P = .6277) between the ThyraMIR+ and ThyraMIR- groups, and the sensitivity, specificity, negative predictive value, and positive predictive value of ThyraMIR were 64.7%, 34.8%, 40.0%, and 59.5%, respectively. CONCLUSION: Although testing positive is associated with malignancy in surgical pathology, the ThyraMIR classifier failed to differentiate between benign and malignant RAS-mutated nodules. Diagnostic lobectomy should be considered for RAS-mutated nodules, regardless of microRNA expression status.

Diagnostic performance of molecular testing in indeterminate (Bethesda III and IV) thyroid nodules with Hürthle cell cytology.

BACKGROUND: Indeterminate thyroid nodules with Hürthle cell cytology remain a diagnostic challenge. The low benign call rate and positive predictive value of first-generation molecular tests precluded their use to rule out malignancy. We examined the diagnostic performance of current tests. METHOD: This subset analysis of our prospective randomized trial compared the benign call rate and positive predictive value of Afirma Gene Sequencing Classifier and Thyroseq v3 in Bethesda III and IV nodules with Hürthle cell cytology. Molecular test samples were obtained at initial fine-needle aspiration (8/2017-7/2022) and reflexively sent for processing. RESULTS: Molecular testing was performed on 140 Hürthle cell nodules. Of 79 nodules tested with the Afirma Gene Sequencing Classifier, the benign call rate was 84% (66/79). Nine of 66 nodules with benign results were resected, with no malignancies. Twelve of 13 nodules with suspicious results were resected, revealing 3 malignancies-2 papillary thyroid carcinomas and one Hürthle cell carcinoma (positive predictive value 25%). Of 61 nodules tested with Thyroseq v3, the benign call rate was 56% (34/61; (P < .01 versus Afirma Gene Sequencing Classifier). Five of 34 nodules with negative results were resected, with no malignancies. Nineteen of 27 nodules with positive results were resected, revealing 3 malignancies-2 papillary thyroid carcinomas and 1 Hürthle cell carcinoma (positive predictive value 16%). CONCLUSION: The high benign call rate of current molecular tests in Hürthle cell nodules strengthens their value in enabling patients to avoid surgery.

DNA Methylation Status of HYAL1 in Malignant and Benign Thyroid Nodules.

Differentiation between benign and malignant thyroid nodules has been a challenge in clinical practice. Exploring a novel biomarker to determine the malignancy of thyroid nodules has important implications. We semi-quantitatively determined the DNA methylation levels of four CpG sites located at the gene body of HYAL1 in formalin-fixed paraffin-embedded (FFPE) tissue samples from 190 early-stage papillary thyroid cancer (PTC) cases and 190 age- and gender-matched subjects with benign thyroid nodule (BTN). HYAL1 expression was evaluated by immunohistochemical (IHC) staining in another cohort of 55 PTC and 55 matched BTN cases. Covariates-adjusted odds ratios (ORs) for 10% increased methylation were calculated by binary logistic regression. A 165 bp amplicon covering four CpG sites at the second exon of HYAL1 gene was designed. After adjusted for all covariates, higher methylation level of HYAL1_CpG_3,4 in the FFPE tissue was associated with PTC (OR per 10% increased methylation=1.53, p=0.025), even with stage І PTC (OR per 10% increased methylation=1.58, p=0.021). Hypermethylation of HYAL1_CpG_3,4 had a significant association with early-stage PTC in the females (OR per 10% increased methylation=1.60, p=0.028) rather than in the males. Besides, we found the higher expression of HYAL1 protein in PTC than that in BTN patients (IHC score: 2.3 vs. 0.5, p=1.00E-06). Our study suggested altered methylation and expression of HYAL1 could be a novel and potential biomarker in distinguishing malignant and benign thyroid nodules.

The Diagnostic Value of BRAF V600E Gene Detection Combined with DNA Ploidy Analysis in Thyroid Cancer.

BACKGROUND: The thyroid cancer incidence has been experimenting an accelerated growth all over the world. The serine/threonine-protein kinase (BRAF) V600E gene detection or the DNA ploidy analysis has been employed in the identification of thyroid cancer type. This study aimed to evaluate the diagnostic value of the BRAF V600E gene integrated with DNA ploidy analysis in thyroid cancer. METHODS: From August 2022 to May 2023, 400 individuals from the thyroid surgery outpatient department of our hospital were enrolled in this study. The participants were divided into low-risk groups (Ⅰ+Ⅱ+Ⅲ group; n = 200) and high-risk groups (Ⅳ+Ⅴ group; n = 200) based on the Thyroid Imaging Reporting and Data System (TI-RADS). A total of the patients were subjected to the DNA ploidy analysis, the BRAF V600E gene detection, or the combination of both techniques. We evaluated the diagnostic value of the above techniques and considered the postoperative pathology results as gold standard for cancer diagnosis. The negative predictive value (NPV), accuracy, specificity, sensitivity, and positive predictive value (PPV) of TI-RADS, BRAF V600E gene detection, DNA ploidy analysis, and BRAF V600E gene detection joined with DNA ploidy analysis were calculated. RESULTS: Among 400 subjects, 238 presented thyroid cancer and 162 had benign lesions, according to the postoperative pathology results. The obtained sensitivity, specificity, accuracy, PPV, and NPV values of TI-RADS were 55.88%, 58.64%, 57.00%, 66.50%, 47.50%, respectively; of BRAF V600E gene detection were 81.93%, 69.75%, 77.00%, 79.92%, 72.44%, respectively; of DNA ploidy analysis were 83.19%, 72.84%, 79.00%, 81.82%, 74.68%, respectively; of BRAF V600E gene combined with DNA ploidy analysis were 90.34%, 76.54%, 84.75%, 84.98%, 84.35%, respectively. Compared with TI-RADS, the sensitivity, specificity, accuracy, PPV, and NPV values of DNA ploidy analysis, BRAF V600E gene detection, and the conjunction of these last two methods were increased (p < 0.05). The combination of DNA ploidy analysis and BRAF V600E gene detection had the highest values among them all. CONCLUSIONS: BRAF V600E gene detection in conjunction with DNA ploidy analysis showed a better diagnostic value than both methods separately or TI-RADS.

RUNX1 methylation as a cancer biomarker in differentiating papillary thyroid cancer from benign thyroid nodules.

Aim: It remains a challenge to accurately identify malignancy of thyroid nodules when biopsy is indeterminate. The authors aimed to investigate the abnormal DNA methylation signatures in papillary thyroid cancer (PTC) compared with benign thyroid nodules (BTNs). Methods: The authors performed genome profiling by 850K array and RNA sequencing in early-stage PTC and BTN tissue samples. The identified gene was validated in two independent case-control studies using mass spectrometry. Results: Hypomethylation of RUNX1 in PTC was identified and verified (all odds ratios: ≥1.50). RUNX1 methylation achieved good accuracy in differentiating early-stage PTC from BTNs, especially for younger women. Conclusion: The authors disclosed a significant association between RUNX1 hypomethylation and PTC, suggesting RUNX1 methylation as a potential biomarker for companion diagnosis of malignant thyroid nodules.

Fine-needle biopsy of thyroid nodules and the contribution of molecular analysis of BRAF and RAS mutations.

BACKGROUND: Thyroid cancer is the most common endocrine malignancy. There is a significant overdiagnosis of thyroid carcinomas that would never clinically manifest, with consequent unnecessary surgical treatment. The fine-needle biopsy and subsequent cytologic examination is of crucial importance in the differential diagnosis of thyroid nodules. On the other hand, a significant portion of the results are indeterminate. OBJECTIVE: To assess the relationship of BRAF/RAS mutations in biopsy specimens to histological characteristics of thyroid nodules in individuals who undergone fine-needle biopsy and surgery. METHODS: This cross-sectional study involves 170 subjects with indeterminate cytology analyzed for BRAF/RAS mutations. RESULTS: Of all 170 patients with indeterminate cytological finding, 103 were indicated for surgery. Of these, 31 were BRAF and 25 RAS positive. Thyroid cancer was diagnosed in 59 patients, while 44 patients had non-malignant thyroid lesions. The BRAF V600E mutation was detected in 30 patients, and the RAS (K-RAS, N-RAS, and H-RAS) mutation in 13 patients with thyroid cancer. In all BRAF-positive nodules, thyroid cancer was histologically confirmed. This means a 100 % positive predictive value of BRAF testing in our study. CONCLUSION: Stratification of thyroid lesions with uncertain results of fine-needle cytology using genetic markers can help to deliver more tailored medical treatment (Tab. 6, Ref. 19).

Significance of LINC02082 and LOC105369812 in differentiating papillary thyroid cancer from benign nodules.

OBJECTIVE: To explore the significance of LINC02082 and LOC105369812 in the differential diagnosis of papillary thyroid carcinoma (PTC) and benign nodules. METHODS: Cancer tissues and benign nodules from 8 patients were sequenced and constructed using high-throughput sequencing. Differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) with significant differences were screened. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the mRNAs co-expressed by DEmRNAs and DElncRNAs. LncRNAs with significant differences, good consistency, and enrichment in the PI3K-AKt signaling pathway were selected as candidate lncRNAs, and the target lncRNAs were screened by correlation analysis. Target lncRNAs and co-expressed mRNAs enriched in the PI3K-AKt signaling pathway and microRNAs (miRNAs) interacting with each other were used to construct a competing endogenous RNA (ceRNA) network. Finally, the PTC-related gene set (GSE33630) was downloaded from the GEO database and the expression of the genes obtained by sequencing was compared. Differential expression was verified using quantitative real-time PCR (qRT-PCR). Finally, the receiver operating characteristic (ROC) curve was used to evaluate the value of the target lncRNAs in diagnosis, when used alone or in combination. RESULTS: A total of 1113 differential RNAs (DE RNAs) were identified, of which 338 were DElncRNAs and 775 were DEmRNAs. Three lncRNAs enriched in the PI3K-AKt signaling pathway, LINC02082, LOC105369812, and LOC105375170, were used as candidate lncRNAs. After correlation analysis with known biomarkers, LINC02082 and LOC105369812 were selected as the target lncRNAs. The qRT-PCR results showed that the target lncRNAs were significantly different among the 3 tissues. The ROC curve showed that LOC105369812 could be used to differentiate PTC from benign thyroid nodules, whereas LINC02082 and its combination had lower predictive value. CONCLUSIONS: LOC105369812 is valuable for differentiating benign from malignant thyroid nodules, whereas LINC02082 has lower diagnostic value.