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Molecular diagnostic testing in the management of thyroid nodules

M3 India Newsdesk Jul 17, 2017

Thyroid cancer, typically presented as a thyroid nodule, is the most common endocrine malignancy.




The gold standard diagnostic procedure for evaluating thyroid nodules is fine needle aspiration (FNA) followed by cytological analysis of the harvested cells. Although in almost 70-80% of the cases FNA yields a final diagnosis but the remaining 20–30% of samples are characterized as cytologically indeterminate for malignancy. The indeterminate diagnoses include the categories of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS, Bethesda category III) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN, Bethesda category IV), based on the TBSRTC. This remains a challenge for clinicians as some of them may come out to be benign after surgery.

Overcoming limitation of FNA cytology by using molecular diagnostic approaches

Recently there has been extensive research to evaluate the detection of molecular markers on FNA specimens to facilitate accurate stratification of patient's risk of malignancy.3 Molecular FNA diagnostics can not only reduce repeated diagnostic testing and unnecessary surgery but can also better define the need for appropriate surgery in patients of thyroid nodules with indeterminate FNA cytology

  Molecular tests for determining benign or malignant lesion

The most common available molecular tests include the following

  • Afirma Gene Expression Classifier
  • ThyGenX 
  • ThyroSeq


The test is based on microarray technology used to analyze the mRNA expression of 167 different genes included in 2 sets, one with 25 genes (less commonly seen entities in the thyroid) and another with 142 genes (most commonly seen entities in the thyroid cancer).

  • Afirma GEC test is used only in lesions with diagnosis included in the Bethesda III and IV categories.
  • Excellent negative predictive value (NPV) of the Afirma test allows it to be used as a “rule out” test.
  • Generates 2 possible results: benign and suspicious.

Benign result in the indeterminate category suggests regular follow up of the patient with no need for surgery. However, suspicious result remains indeterminate and a surgical consultation is recommended

ThyGenX TEST (modified version of miRInform)

  • ThyGenX uses a next-generation-sequencing (NGS) platform to identify more than 100 genetic alterations across 8 genes associated with thyroid malignancy.
  • It analyzes a panel of DNA mutations and RNA translocation fusion markers to assess the risk of malignancy with good NPV and positive predictive value.
  • It requires only 1 dedicated FNA pass (at least 50 ng of cellular material to be collected into the provided RNA preservative vial RNARetain).

Diagnosed cases as AUS/FLUS or FN/SFN are accepted for ThyGenX analysis.


  • This test is based on the analysis of 10 different microRNAs (miRNAs) that are involved in the cell-cycle progression, differentiation, and proliferation in thyroid pathology.

This test is meant to be used in conjunction with ThyGenX when the ThyGenX result is negative

ThyroSeq TEST2

  • ThyroSeq is a NGS-based gene mutation and fusion panel that target 12 cancer genes with 284 mutational hot spots.
  • It has been reported to have the best NPV and positive predictive value combined, suggesting that it can be used as a “rule in” and “rule out” test.
  • ThyroSeq v2 (enhanced version of ThyroSeq) is a NGS-based gene mutation and fusion panel designed to target DNA alterations (14 genes, including >1000 mutations) and RNA alterations (42 fusions, 16 genes for expression). It may potentially function as both “rule out” and “rule in” test for nodules with indeterminate cytology.

As molecular testing is still under debate and research, it must always be performed and interpreted within the context of the clinical, radiographic, and cytologic findings.

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