Next Generation Sequencing Identifies a MicroRNA Panel that Accurately Discriminates between Malignant and Benign Thyroid Nodules Deemed Indeterminate by Cytopathology of Fine Needle Aspiration Biopsies

Background: Fine needle aspiration biopsy (FNAB) is the gold-standard procedure for evaluation of thyroid nodules suspicious for cancer. Indeterminate cytology is reported in 10-40% of cases and molecular testing may guide management in this setting. Current commercial options are costly and have suboptimal sensitivity or specificity. The aim of this study was to utilize next generation sequencing (NGS) to identify informative diversities in the microRNA (miRNA) expression profiles of malignant versus benign thyroid nodules and test their utility in classifying indeterminate nodules.

Methods: FNAB samples were obtained ex-vivo from thyroid specimens of patients that underwent thyroidectomy at a referral center. miRNA levels were determined using multiplexed NGS. Statistical analyses identified differential expression between normal and malignant samples and miRNA expression profiles that associate with malignancy were established. The accuracy of the miRNA signature in detecting histological malignancy was validated using specimens from patients with indeterminate cytology results.

Results: 274 samples were obtained from 102 patients undergoing thyroidectomy. Of these samples, 71% were benign and 29% were malignant. Nineteen miRNAs were identified as statistically different between benign and malignant samples and were used to classify 35 additional nodules with indeterminate cytology (validation). The sensitivity, specificity, negative and positive predictive values and overall accuracy of the miRNA panel quantification were 91%, 100%, 87%, 100% and 94%, respectively.

Conclusion: Using deep sequencing of small RNA we identified a panel of 19 miRNAs that may be utilized to distinguish benign from malignant thyroid nodules with indeterminate cytology. Our panel may classify indeterminate thyroid nodules at higher accuracy than commercially available molecular tests.