Development of a Low-cost NGS Test for the Evaluation of Thyroid Nodules

Ultrasound-guided fine needle aspiration cytology (FNAC) is the preferred method of identifying malignancy in palpable thyroid nodules using the Bethesda reporting system. However, in around 30-40% of FNACs (Bethesda categories III, IV, and V), the results are indeterminate and surgery is required to confirm malignancy. Out of those who undergo surgery, only 10-40% of patients in these categories are found to have malignancies, thus proving surgery to be unnecessary for some patients or to be incomplete in others. While molecular testing on thyroid FNAC material is part of the American Thyroid Association (ATA) guidelines in evaluating thyroid nodules, it is currently unavailable in India due to cost constraints. In this study, we prospectively collected FNAC samples from sixty-nine patients who presented with palpable thyroid nodules. We designed a cost-effective next-generation sequencing (NGS) test to query multiple variants in the DNA and RNA isolated from the fine needle aspirate. The identification of oncogenic variants was considered to be indicative of malignancy, and confirmed by surgical histopathology. The panel showed an overall sensitivity of 81.25% and a specificity of 100%, while in the case of Bethesda categories III, IV, and V, the sensitivity was higher (87.5%) and the specificity was established at 100%. The panel could thereby serve as a rule-in test for the diagnosis of thyroid cancer and therefore help identify patients who require surgery, especially in the indeterminate Bethesda categories III, IV, and V.