• miRNA
• molecular markers
• mutational status
• Nanostring technology
• NIFTP
• thyroid cancer

Background: The encapsulated noninvasive follicular variant of papillary thyroid carcinoma has an indolent behavior. Recently, it has been reclassified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). Although since the reclassification of NIFTP several studies attest the low malignant potential of this lesion, patients with regional lymph nodes and distant metastases have been reported. More than one research group has shown the presence of BRAF V600E mutation and coexistence of RAS and TERT promoter mutations in NIFTP.
On the one hand the NIFTP seems to have benign characteristics but on the other hand it seems to show malignant aspects.
However, the genetic landscape of NIFTP has yet to be fully investigated. A more accurate molecular characterization of NIFTPs is necessary to better classify their clinical and biological behavior.
Aim of the study: On this basis, the present study investigated miRNA expression profile and mutational status in NIFTPs. The molecular features of NIFTPs have been compared to those of both benign lesions - follicular adenomas (FAs) - and malignant lesions - invasive/infiltrative follicular variant of papillary thyroid carcinomas (IFVPTCs).
Materials and methods: The expression of a panel of 798 miRNAs using Nanostring technology, mutational status in BRAF gene, RAS family genes, TERT promoter gene and PAX8/PPARγ gene fusions were tested in 53 patients with follicular-patterned thyroid tumors: 18 FAs, 19 NIFTPs and 16 IFVPTCs. First we correlated miRNA expression profiles with mutational status, then we compared miRNA expression profiles of NIFTPs to those of FAs and IFVPTCs using clustering analysis.
To clarify the biological role of deregulated miRNAs, we used DIANA-miRPath v.3.0 software.
The expression data of miRNA and mRNA of more than 400 patients reported in The Cancer Genome Atlas (TCGA) database were used to confirm our results and our hypothesis.
Results: Twelve miRNAs have been found differentially expressed between BRAF and RAS mutated NIFTPs and wild-type ones. Four of these (miR-221-5p, miR-221-3p, miR-222-3p and miR-146b-5p) were upregulated whereas 8 (miR-181a-3p, miR-28-5p, miR-363-3p, miR-342-3p, miR-1285-5p, miR-152-3p, miR-25-3p, miR-30e-3p) were downregulated in the mutated lesions.
Comparing miRNA expression profiles of NIFTPs to those of FAs and invasive/infiltrative FVPTCs, we found that wild-type NIFTPs showed a miRNA expression profile comparable to that of FAs, whereas mutated NIFTPs had a miRNA expression profile comparable to that of invasive/infiltrative FVPTCs.
The analyses conducted on TCGA data confirmed that the upregulation of miR-222-3p and the downregulation of miR-152-3p, miR-30e-3p, miR-363-3p could be likely involved in potential invasive processes of mutated NIFTPs.
By using DIANA miRPath software, we identified 12 enriched pathways and 282 target genes potentially deregulated by miRNA differentially expressed between wild-type and mutated NIFTPs. The analysis conducted on 483 patients of TCGA database identified 137 differentially expressed genes between patients with extrathyroidal extension and patients without extrathyroidal extension.
Conclusion: Our results suggest that NIFTPs represent a heterogeneous group of lesions. Their miRNA expression profile differs according to mutational status. The deregulation of 12 miRNAs distinguishes wild-type from BRAF- and RAS- mutated NIFTPs. Wild-type NIFTPs have a miRNA expression profile similar to that of FAs, while NIFTPs harboring mutations in BRAF or RAS genes have a miRNA expression profile similar to that of invasive/infiltrative FVPTCs. TCGA investigations confirmed that miR-222-3p, miR-363-3p, miR-30e-3p and found that a panel of 137 target genes may promote an invasive phenotype of mutated NIFTPs. Additional studies are warranted to confirm the miRNA and gene expression effects on the NIFTP phenotype but we could speculate that only mutated NIFTPs represent a precursor of invasive/infiltrative FVPTCs.