• nanostring
• indeterminate cytological thyroid nodules
• miRNA

Thyroid cancer (TC) is the most common malignancy of the endocrine system, and from 20% to 30% of fine-needle aspiration cytology samples yield indeterminate cytologic diagnoses leading many patients undergo diagnostic surgery to ascertain the effective nature of the nodule at the histologic diagnosis. To date, molecular testing is used to support the differential diagnosis between benign and malignant lesion, analyzing mainly gene mutations (such as BRAF and RAS point mutations) and gene fusions but in the recent years, some studies have evaluated the microRNA (miRNA) profile of thyroid tumours, highlighting that the miRNA expression analysis could be useful to clarify the gray area of indeterminate thyroid FNA.
The aim of the study was to analyse miRNA expression profiles of samples with indeterminate cytology (TIR 3A, TIR 3B) and wild-type for the most common mutations in order to identify miRNAs able to differentiate them into benign and malignant nodules.
Sixty-nine tissue specimens (27 follicular adenoma, FA, 8 noninvasive follicular neoplasm with papillary-like nuclear features, NIFTP and 33 follicular variant of papillary thyroid carcinoma, FVPTC) and the respective cytologic smears were selected. DNA was extracted from paraffin tissue specimens and then the mutational status for BRAF (exon 15) and NRAS (exon 3) was tested with high resolution melt analysis and Sanger sequencing. RNA was extracted from mutation negative cytologic specimens and then miRNA expression profiles were analyzed by a hybridization-based digital counting system (nCounter miRNA Expression Assay, NanoString Technologies). Statistical analyses were performed on normalized data in R environment and differences in miRNA expression between histological classes were investigated by a moderated t-statistics with Benjamini-Hochberg correction.
The mutational status analysis shows that out of 69 specimens, 55 (82%) were wild type for BRAF and NRAS and therefore suitable for miRNA analysis. To date, 24 samples (11 FA, 13 FVPTC) were examined with the nCounter analysis: hierarchical clustering analysis showed that the samples do not clearly separate according to their histological classes. Statistical analysis revealed that one miRNA, the hsa-miR-7-5p, had a significantly different expression between FA and FVPTC (p-value 0,0004; adjusted p-value 0,038).
Although samples do not show peculiar miRNA expression profiles by hierarchical clustering, likely due to the small number of analyzed samples, one miRNA was significantly down-regulated in FVPTC vs FA, consistently with literature data; in fact, miR-7 has been described as a tumor suppressor miRNA. This result is noteworthy since it was obtained on real clinical samples with cytological indeterminated diagnosis that were negative for the main gene mutations. Therefore, miR-7 has the potential to be used as a marker in the differential diagnosis between benign and malignant thyroid nodules, and its expression will be further investigated in larger series of thyroid nodules.
Irene Sofia Burzi, Pisa, 08/01/2021