Thyroid cancer is the most frequent endocrine malignancy, and its incidence is increasing. A current limitation of cytological evaluation of thyroid nodules is that 20-25% are reported as indeterminate. Therefore, an important challenge for clinicians is to determine whether an indeterminate nodule is malignant, and should undergo surgery, or benign, and should be recommended to follow-up. The emergence of precision medicine has offered a valuable solution for this problem, with four tests currently available for the molecular diagnosis of indeterminate cytologies. However, efforts to critically analyze the quality of the accumulated evidence are scarce. This systematic review and meta-analysis is aimed to contribute to a better knowledge about the four available molecular tests, their technical characteristics, clinical performance, and ultimately to help clinicians to make better decisions to provide the best care options possible. For this purpose, we address three critical topics: (i) the proper theoretical accuracy, considering the intended clinical use of the test (rule-in vs rule-out) and the impact on clinical decisions; (ii) the quality of the evidence reported for each test (iii) and how accurate and effective have the tests proved to be after their clinical use. Together with the upcoming evidence, this work provides significant and useful information for healthcare system decision-makers to consider the use of molecular testing as a public health need, avoiding unnecessary surgical risks and costs.

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Genetic testing for indeterminate thyroid cytology: review and meta-analysis.

The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis. We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers. We identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC. We conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.

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CRABP1, C1QL1 and LCN2 are biomarkers of differentiated thyroid carcinoma, and predict extrathyroidal extension.

Aims Analysis of molecular markers in addition to cytological analysis of fine-needle aspiration (FNA) samples is a promising way to improve the preoperative diagnosis of thyroid nodules. Previously, we have developed an algorithm for the differential diagnosis of thyroid nodules by means of a small set of molecular markers. Here, we aimed to validate this approach using FNA cytology samples of Bethesda categories III and IV, in which preoperative detection of malignancy by cytological analysis is impossible. Methods A total of 122 FNA smears from patients with indeterminate cytology (Bethesda III: 13 patients, Bethesda IV: 109 patients) were analysed by real-time PCR regarding the preselected set of molecular markers (the BRAF V600E mutation, normalised concentrations of HMGA2 mRNA, 3 microRNAs, and the mitochondrial/nuclear DNA ratio). The decision tree–based classifier was used to discriminate between benign and malignant tumours. Results The molecular testing detected malignancy in FNA smears of indeterminate cytology with 89.2% sensitivity, 84.6% positive predictive value, 92.9% specificity and 95.2% negative predictive value; these characteristics are comparable with those of more complicated commercial tests. Residual risk of malignancy for the thyroid nodules that were shown to be benign by this molecular method did not exceed the reported risk of malignancy for Bethesda II histological diagnosis. Analytical-accuracy assessment revealed required nucleic-acid input of ≥5 ng. Conclusions The study shows feasibility of preoperative differential diagnosis of thyroid nodules of indeterminate cytology using a small panel of molecular markers of different types by a simple PCR-based method using stained FNA smears.

https://jcp.bmj.com/content/jclinpath/early/2020/03/25/jclinpath-2020-206445.full.pdf

Preoperative detection of malignancy in fine-needle aspiration cytology (FNAC) smears with indeterminate cytology (Bethesda III, IV) by a combined molecular classifier

Objective: To investigate the clinical value of conventional ultrasound, ultrasound elastography and conventional ultrasound combined with ultrasound elastography in differential diagnosis of benign and malignant thyroid micro-nodules.
Methods: Eighty-six patients who were found with thyroid micro-nodules with the maximum diameter no more than 10 mm in the physical examination in our hospital from June 2015 to December 2017 were selected, and 102 nodules were included. All patients were confirmed with thyroid micro-nodules by puncture or surgical pathology and underwent conventional ultrasound and ultrasound elastography. Taking the surgical pathological results as a control, the value of conventional ultrasound, ultrasound elastography and conventional ultrasound combined with ultrasound elastography in differential diagnosis of benign and malignant thyroid micro-nodules were compared. A receiver operating characteristic (ROC) curve was drawn, and the area under the ROC curve was calculated.
Results: One hundred and two thyroid nodules were detected by surgical pathology, including 75 benign nodules (73.53%) and 27 malignant nodules (26.47%). The sensitivity and diagnostic accordance rate of ultrasound elastography were significantly higher than those of conventional ultrasound in the diagnosis of thyroid microcarcinoma, and the missed diagnosis rate of ultrasound elastography was significantly lower than that of conventional ultrasound; the difference was statistically significant (P 0.05). The areas under the ROC curve in the diagnosis of benign and malignant thyroid nodules by conventional ultrasound and ultrasound elastography were 0.735 and 0.743 respectively.
Conclusion: Conventional ultrasound can be used as a routine examination technique in differential diagnosis of benign and malignant thyroid nodules, while ultrasound elastography can improve the sensitivity and diagnostic rate in the diagnosis of thyroid micro carcinoma. It can help to reduce the clinical missed diagnosis, which also can be be used as an effective supplement for conventional ultrasound.
doi: https://doi.org/10.12669/pjms.35.6.777
How to cite this:Wang J, Wei W, Guo R. Ultrasonic elastography and conventional ultrasound in the diagnosis of thyroid micro-nodules. Pak J Med Sci. 2019;35(6):1526-1531. doi: https://doi.org/10.12669/pjms.35.6.777
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Ultrasonic elastography and conventional ultrasound in the diagnosis of thyroid micro-nodules.

Background: The Afirma gene expression classifier (GEC) has been used to aid in the diagnosis and management of thyroid nodules having Bethesda category III fine-needle aspiration cytologic diagnosis (B3 nodules). The American Thyroid Association sonographic risk stratification system for thyroid nodules (ATA-US) may stratify B3 nodules and aid in the decision to order a molecular test. The aim of this study was to assess the association between ATA-US and GEC as well as to determine their individual and combined diagnostic performances when applied to B3 nodules. Methods: A retrospective single-center study included B3 nodules that had undergone evaluation by GEC. Each ultrasound was reviewed by three radiologists, and nodules were classified using the 2015 ATA sonographic risk categories. Nodules were determined to be benign or malignant based on surgical pathology or minimum 11 months of follow-up. Positive predictive values (PPV) and negative predictive values (NPV) were calculated for GEC, ATA-US, and GEC across all ATA-US categories. Results: One hundred twenty-six B3 nodules with GEC results were included and deemed benign or malignant based on final pathology or follow-up. Prevalence of malignancy was 32%. The rate of malignancy was similar in the ATA-US high suspicion (HS) and intermediate suspicion (IS) categories at 42% and 38%, respectively; and lower in nodules with low suspicion sonography (LS) and very low suspicion sonography (VLS) at 23% and 11%, respectively. The PPV and NPV of ATA-US was calculated by designating HS or IS sonography as a "positive" test and the lower risk categories as "negative." ATA-US had a PPV of 40% and NPV of 79%. The GEC PPV was 40% and NPV was 83%. The PPV of GEC was 50% in nodules with HS or IS ATA-US and lower at 28% and 20%, respectively, in LS and VLS nodules. The NPV of GEC was 80% in HS, 77% in IS, 84% in LS, and 100% in VLS sonography categories. Conclusions: In B3 nodules, ATA-US and GEC have similar diagnostic performance. The PPV of GEC varies across ATA-US categories, while the NPV remains similar. These data support the need for future prospective studies.

American Thyroid Association Sonographic Risk and Afirma Gene Expression Classifier Alone and in Combination for the Diagnosis of Thyroid Nodules with Bethesda Category III Cytology.

In recent years there has been an increased awareness of the genetic alterations underlying both benign and malignant neoplasms of the thyroid. Next-generation sequencing (NGS) is an emerging technology that allows for rapid detection of a large number of genetic mutations in thyroid fine-needle aspiration (FNA) specimens. NGS for targeted mutational analysis in thyroid tumors has been proposed as a tool to assist in the diagnosis of thyroid nodules with indeterminate FNA cytology. Results of genomic testing of thyroid nodules and thyroid cancers could also have prognostic implications and play a role in determining optimal treatment strategies including targeted therapies. We provide a critical review of existing studies assessing the performance of the ThyroSeq NGS test for the diagnosis and management of patients with thyroid nodules with indeterminate cytopathology and discuss the applicability of findings from these studies to clinical practice. While there are early indications to suggest a possible utility of data obtained from NGS to aid in prognostication and therapeutic decision-making in thyroid cancer, we recommend judicious use and cautious interpretation of such molecular testing until results of ongoing clinical trials become available. Lastly, we discuss recommendations provided from clinical practice guidelines regarding the use of mutation detection via NGS in the diagnostic evaluation of thyroid nodules.

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Marcela Perez Bustamante

Papers

Genomic Profiling of Thyroid Nodules: Current Role for ThyroSeq Next-Generation Sequencing on Clinical Decision-Making.