Prospective Validation of ThyroSPEC Molecular Testing of Indeterminate Thyroid Nodule Cytology Following Diagnostic Pathway Optimization.

Background: Molecular testing for cytologically indeterminate thyroid nodules (ITNs) is often reported with incomplete data on clinical assessment and ultrasound malignancy risk (USMR) stratification. This study aimed to clinically validate the diagnostic accuracy of a novel molecular test, assess the incremental preoperative malignancy risk of other clinical factors, and measure the impacts of introducing molecular testing at the population level. Methods: Comprehensive clinical data were collected prospectively for the first 615 consecutive patients with ITNs in a centralized health care system following implementation of a reflexive molecular test. Clinical data include patient history, method of nodule discovery, clinical assessment, USMR, cytology, molecular testing, and surgery or follow-up along with surgeon notes on surgical decision-making. Accuracy of molecular testing and the impact of the introduction of molecular testing were calculated. A multivariable regression model was developed to identify which clinical factors have the most diagnostic significance for ITNs. Results: A locally developed, low-cost molecular test achieved a negative predictive value (NPV) of 76-91% [confidence interval, CI 66-95%] and a positive predictive value (PPV) of 46-65% [CI 37-75%] in ITNs using only residual material from standard liquid cytology fine-needle aspiration (FNA). Sensitivity was highest (80%; [CI 63-92%]) in the American Thyroid Association (ATA) intermediate-suspicion ultrasound category, and lowest (46%; [CI 19-75%]) in the ATA high-suspicion ultrasound category. Following implementation of molecular testing, diagnostic yield increased by 14% (p = 0.2442) and repeat FNAs decreased by 24% (p = 0.05). Mutation was the primary reason for surgery in 76% of resected, mutation-positive patients. High-risk mutations were associated with a 58% (p = 0.0001) shorter wait for surgery. Twenty-six percent of patients with a negative molecular test result underwent surgery. Multivariable regression highlighted molecular testing and USMR as significantly associated with malignancy. Conclusions: Molecular testing improves preoperative risk stratification but requires further stratification for intermediate-risk mutations. Incorporation of clinical factors (especially USMR) with molecular testing may increase the sensitivity for detection of malignancy. Introduction of molecular testing offers some clinical benefits even in a low resection rate setting, and directly influences surgical decision-making. This study illustrates the importance of the local diagnostic pathway in ensuring appropriate integrated use of molecular testing for best outcomes.

Clinical value of molecular markers as diagnostic and prognostic tools to guide treatment of thyroid cancer.

OBJECTIVE: Advances in our understanding of the molecular biology of thyroid tumours is being rapidly translated into their clinical management. This review summarizes the current use of molecular testing in thyroid tumours, focusing on their usefulness as diagnostic and prognostic tools to guide treatment with consideration of present limitations. DESIGN: Considerations about molecular testing applications for the diagnosis and treatment of thyroid tumours are divided into four sections/roles: (1) evaluating cytologically indeterminate thyroid nodules; (2) guiding extent of surgery in indeterminate thyroid nodules; (3) completing histological characterization of thyroid tumours and (4) identifying actionable mutations in advanced progressive thyroid cancers. RESULTS: Genomic testing can improve the presurgical malignancy risk assessment in indeterminate thyroid nodules. However, a prior in-depth analysis of institutional quality and outcomes of sonographical, cytological and histological characterization of thyroid tumours is necessary. Presently, it remains uncertain whether knowing the molecular profile of a cytologically indeterminate thyroid nodule might be advantageous to modify the extent of initial surgery. Molecular characterization of thyroid tumours can be a valuable adjunct to morphological diagnosis in some challenging cases, such as in low-risk follicular cell-derived neoplasms, or rare tumours. Finally, as selective kinase inhibitors are available, molecular testing in locally advanced/metastatic progressive thyroid cancers should also be integrated into the institutional clinical management pathway to improve outcomes and limit toxicity. CONCLUSIONS: Molecular testing needs to be implemented into the local evidence-based clinical management thyroid nodule/cancer pathways to improve its diagnostic and prognostic value and to optimize cost-effectiveness.

DIAGNOSIS OF ENDOCRINE DISEASE: Usefulness of genetic testing of fine-needle aspirations for diagnosis of thyroid cancer.

Objective: Genetic testing is increasingly used to diagnose or rule out thyroid cancer in indeterminate fine-needle aspirations. This review evaluates the usefulness of these methods with considerations of advantages and limitations. Design: Given the diagnostic problem associated with the increasing incidental detection of indeterminate thyroid nodules in the context of thyroid cancer overtreatment, we consider the conditions and respective necessary settings for the role of genetic testing to improve presurgical malignancy risk stratification. Methods: We review diagnostic pathway requirements and commercially available molecular tests with their respective advantages and disadvantages and discuss the prerequisites required for local application and implementation including quality assurance for local ultrasound and cytopathology practices. Results: Recent improvements in available molecular diagnostic tests have brought high sensitivity and specificity in initial validation studies, but whether these promising results translate to other clinical settings depends on the quality of the local thyroid nodule diagnostic pathway. Conclusions: Genetic testing can meaningfully improve presurgical malignancy risk assessment, but more work is needed to implement and use genetic testing effectively in local settings.

Systematic population-based identification of NTRK and RET fusion-positive thyroid cancers.

Objective: The aim of the study was to identify patients with NTRK fusion-positive or RET fusion/mutation-positive thyroid cancers, who could benefit from neurotrophic tyrosine kinase receptor (NTRK) or receptor tyrosine kinase (RET) inhibitors. Methods: Patients were identified in the Calgary prospective thyroid cancer database (N= 482). Patients were 'pre-screened' with clinically available MassARRAY® BRAF test, Colon Panel, Melanoma Panel, or ThyroSPEC™. Mutation-negative tumors were 'screened' for NTRK fusions and RET fusions/mutations with the Oncomine™ Comprehensive Assay v3 (OCAv3). Results: A total of 86 patients were included in 1 of 2 separate analyses. Analysis A included 42 patients with radioactive iodine (RAI)-resistant distant metastases. After pre-screening, 20 BRAF and RAS mutation-negative patients underwent OCAv3 screening, resulting in the detection of 4 patients with NTRKfusions and 4 patients with RET fusions (8/20, 40% of analyzed patients). Analysis B included 44 patients, 42 with American Thyroid Association (ATA) high and intermediate risk of recurrence and 2 with medullary thyroid carcinoma. During pre-screening, 1 patient with an NTRK fusion, 1 patient with a RET fusion, and 30 patients with BRAF mutations were identified. The remaining 9 patients received OCAv3 screening, resulting in detection of 1 patient with an NTRKfusion and 1 with a RET fusion (4/11, 36% of analyzed patients). Conclusions: Our findings indicate a higher rate of NTRK fusions and RETfusions in patients with thyroid cancer with RAI-resistant distant metastases and ATA high or intermediate risk of recurrence. This highlights the importance of early screening to enable intervention with a NTRK or RET inhibitor.

Histology-based molecular profiling improves mutation detection for advanced thyroid cancer.

Advanced cancers frequently show histologic and molecular intratumoral heterogeneity. Therefore, we comprehensively characterized advanced, metastatic, radioiodine-resistant (RAIR) thyroid carcinomas at the molecular level in the context of histologic heterogeneity with the aim to identify potentially actionable mutations that may guide the use of specific tyrosine kinase inhibitor (TKI) treatment. Whole exome sequencing (WES) was applied to 29 macrodissected tissue samples of histologically heterogeneous and homogeneous areas, lymph node and lung metastases from six clinically and histologically well-characterized metastatic RAIR thyroid cancer patients with structural incomplete response to treatment. WES data were analyzed to identify potential driver mutations in oncogenic pathways, copy number alterations, microsatellite instability, mutant-allele tumor heterogeneity, and the relevance of histologic heterogeneity to molecular profiling. In addition to known driver mutations in BRAF, NRAS, EIF1AX, NCOA4-RET, and TERT, further potentially actionable drivers were identified in AKT1, ATM, E2F1, HTR2A, and MLH3. The analysis of the evolutionary history of the mutations and the reconstruction of the molecular phylogeny of the cancers show a remarkable association between histologic and molecular heterogeneity. A comprehensive molecular analysis of the primary tumor guided by histologic analysis may help to better stratify patients for precision medicine approaches. Given the association between the molecular and the histologic heterogeneity, the selection of tumor samples for molecular analysis should be based on meticulous histologic evaluation of the entire tumor.

Malignancy is in the eye of the beholder: Pathologic diagnosis of challenging follicular neoplasms in the era of noninvasive follicular thyroid neoplasms with papillary-like nuclear features and immunohistochemical and molecular adjuncts.

BACKGROUND: Classification of thyroid follicular neoplasms can be challenging for pathologists. Introduction of noninvasive follicular thyroid neoplasms with papillary-like nuclear features, the utilization of immunohistochemistry, and molecular analysis are all thought to be valuable diagnostic adjuncts. Our aim was to determine whether interobserver variability for follicular neoplasms has improved since the application of these adjuncts. METHODS: One representative section from a cohort of follicular neoplasms previously proven difficult for pathologists were examined independently by 7 pathologists and assigned to 1 of 3 diagnostic categories (benign, neoplasms with papillary-like nuclear features, or malignant). This process was carried out separately 3 times: (1) after viewing hematoxylin and eosin stain slides, (2) hematoxylin and eosin stain in conjunction with immunohistochemistry, and (3) hematoxylin and eosin stain/immunohistochemistry in conjunction with molecular analysis. The interobserver variability and overall agreement were then calculated using the free-marginal kappa coefficient. RESULTS: Agreement on hematoxylin and eosin stain was 57%, with a kappa coefficient of 0.36 (minimal agreement). The agreement improved slightly with the application of immunohistochemistry (kappa coefficient = 0.49 [weak agreement] and a percentage agreement 67%). The level of agreement decreased slightly after the addition of molecular analysis (kappa coefficient = 0.43 [weak agreement] and percentage agreement 62%). CONCLUSION: Despite attempts to standardize the diagnostic criteria for neoplasms with papillary-like nuclear features and the utilization immunohistochemistry and molecular analysis, attaining pathologic consensus for difficult follicular neoplasms of the thyroid remains a challenge.

Sensitive Sequencing Analysis Suggests Thyrotropin Receptor and Guanine Nucleotide-Binding Protein G Subunit Alpha as Sole Driver Mutations in Hot Thyroid Nodules.

Background: Constitutively activating mutations in the thyrotropin receptor (TSHR) and the guanine nucleotide-binding protein G subunit alpha (GNAS) are the primary cause of hot thyroid nodules (HTNs). The reported prevalence of TSHR and GNAS mutations in HTNs varies. Previous studies show TSHR mutations in 8-82% of HTNs and GNAS mutations in 8-75% of HTNs. With sensitive and comprehensive targeted next-generation sequencing (tNGS), we re-evaluated the prevalence of TSHR and GNAS mutations in HTNs. Methods: Samples from three previous studies found to be TSHR and GNAS mutation negative were selected and re-evaluated using high-resolution melting (HRM) PCR. Remaining mutation negative samples were further reanalyzed by tNGS with a sequencing depth between 3000 × and 10,000 × . Our tNGS panel covered the entire TSHR coding sequence along with mutation hot spots in GNAS. Sequencing reads were aligned to reference and variants were called using Torrent Suite software v5.8. Results: In total, 154 of 182 previously mutation negative HTNs were positive for TSHR or GNAS mutations, resulting in an 85% prevalence of TSHR and GNAS mutations in HTNs, 79% and 6%, respectively. In a subset of 25 HTNs with multiple samples per nodule, and analyzed by tNGS at high sequencing depth, TSHR mutations were detected in 23 (92%) HTNs and 1 GNAS mutation was detected in 1 (4%) HTN, 96% mutation positive HTNs in this subset. Conclusions: Owing to the higher sensitivity of tNGS as compared with denaturing gradient gel electrophoresis and HRM-PCR, TSHR or GNAS mutations could be detected in 85% of HTNs. The detection of TSHR and GNAS mutations occurred in 96% of HTNs in a sample set with multiple samples per nodule analyzed by tNGS. Taken together with the fact that no other driver mutations could be identified by whole exome sequencing, our study strongly supports the hypothesis that TSHR and GNAS mutations are the main somatic mutations leading to HTNs.