Review Article on Molecular Markers in Thyroid nodules
Molecular Signature of Indeterminate Thyroid Lesions: Current Methods to Improve Fine Needle Aspiration Cytology (FNAC) Diagnosis
1. Thyroid Nodules
.Since only a small proportion of these nodules finally result malignant at histology, this approach would imply a tremendous number of unnecessary surgeries and high financial costs. Thyroid nodules must therefore undergo rigorous selection based on a rational diagnostic protocol.
2. Diagnostic Evaluation of Thyroid Nodules: Fine-Needle Aspiration Cytology (FNAC)
The ultimate objective of the diagnostic protocol is to differentiate between benign and malignant nodules. Nowadays, the problem has largely been solved by fine-needle aspirate cytology. In expert hands, FNAC has an overall accuracy of 95%. The sensitivity is between 43% and 98% and the specificity is between 72% and 100%, with positive and negative predictive values of 89–98% and 94–99%, respectively . False positive and false negative results are between 1–11% and 0–7%, respectively.
A particular issue is represented by category III and IV, representing nearly 20% of FNACs. In this case, the aspirate is represented by a monotonous population of follicular cells arranged on cohesive groups, whose cellular and nuclear features are similar whether the nodule is benign or malignant. The distinction is based on the presence of vascular and capsular invasion, which is detectable only at histology. In a meta-analysis of 25,445 thyroid FNAC  cases reported from eight studies using the Bethesda System, 9.6% of all samples were diagnosed as AUS/FLUS (category III) and 10.1% were diagnosed as follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) (category IV) with an average cancer risk at final histology of 15.9% and 26.1%, respectively. It is evident that, both the AUS/FLUS and FN/SFN have a cancer risk that cannot be ignored. However, at final histology, only about 25% of the lesions result malignant, so the risk of cancer is not high enough to definitely support surgery as treatment of all indeterminate lesions.
4. Use of Molecular Markers in the Differential Diagnosis of Thyroid Nodules
In conclusion, mutation panels intended to identify malignancies in indeterminate lesions must include at least BRAF and RAS point mutations (H, K and NRAS), and RET/PTC, PAX8/PPAR-γ rearrangements. Several “homemade” methods comprising PCR with final Sanger sequencing and some commercial kits are available to screen for these alterations with the limitation that they cannot rule out malignancy with a NPV > 95%.
4.2. Afirma Classifier
4.3. Thyroseq and Other NGS Platform
ThyroSeq v2 actually has shown the best results in terms of sensitivity, specificity, PPV and NPV but further studies including a larger number of cases are required for the Ion AmpliSeq Panel.
5. Role of miRNAs in the Differential Diagnosis of Thyroid Lesions
In 2016 two studies [78,79] were published on clinical validation of the RosettaGX Reveal test, a miRNA-based assay which evaluates a set of 24 miRNAs (by real time PCR) specific for cytologically indeterminate thyroid nodules. The assay can be used directly on FNA smears and it is able to categorize benign or suspicious nodules even when as little as 1% of thyroid cells is present or less than 5 ng RNA are extracted. The overall NPV reported was 99%, with sensitivity of 98% and specificity of 78%.
In summary, the purpose of thyroid molecular testing is to discriminate the nature of thyroid nodules and reduce the diagnostic uncertainty of cytologically indeterminate lesions prior to surgery. Mutation panels intended to identify malignancies must include at least BRAF, and RAS point mutations as well as RET/PTC, NTRK, and PAX8/PPARγ rearrangements. Several “home-made” methods and some commercial kits are available to screen for these alterations with the limitation that they cannot rule out malignancy with an NPV >95%. GEC recognizes benign lesions on the basis of an expression pattern of mRNA extracted from one or two dedicated FNA needle passes. A negative result in the Afirma test has resulted in a major decrease in the number of surgeries performed in samples classified as Bethesda categories III and IV. However, Afirma shows a low PPV. On the other hand, the risk of malignancy calculated by ThyroSeq or other NGS platforms is superior to that of the Afirma, reaching an NPV of 95% or more, with good sensitivity and high PPV. The identification of new biomarkers (i.e., miRNA, proteomic profiles) in the thyroid needs to be corroborated in larger studies with final histology as a gold standard and adequate follow up before use in the clinical routine . Therefore, molecular testing must be always performed in specialized laboratories and results interpreted within the context of the clinical, radiographic, and cytological findings. In addition, clinicians may take into account that the interpretation of molecular testing and its utility are strongly influenced by the prevalence of cancer in each cytological category  which can differ among centers. Due to this aspect, molecular test performance may vary significantly.
Comment: We used needle biopsy slides to get the nucleic acid for molecular marker testing recently on one of my cases.