Objective: Chromosomal rearrangements of the RET proto-oncogene is one of the most common molecular events in papillary thyroid carcinoma (PTC). However, their pathogenic role and clinical significance are still debated. This study aimed to investigate the prevalence of RET/PTC rearrangement in a cohort of BRAF-wild type PTCs by fluorescence in situ hybridization (FISH) and to search a reliable cut-off level in order to distinguish clonal or non-clonal RET changes. Design: Forty BRAF wild-type PTCs were analyzed by FISH for RET rearrangements. As controls, 6 BRAFV600E mutated PTCs, 13 follicular adenomas (FA) and 10 normal thyroid parenchyma (NTP) were also analyzed. Methods: We performed FISH analysis on formalin-fixed paraffin-embedded (FFPE) tissue using a commercially available RET Break-apart probe. A cut-off level equivalent to 10.2% of aberrant cells was accepted as significant. To validate FISH results, we analyzed the study cohort by qRT-PCR. Results: Split RET signals above the cut-off level were observed in 25% (10/40) of PTCs, harboring a percentage of positive cells ranging from 12% to 50%, and in 1 spontaneous FA (1/13, 7.7%). Overall, the data obtained by FISH matched well with qRT-PCR results. Challenging findings were observed in 5 cases showing a frequency of rearrangement very close to the cut-off. Conclusions: FISH approach represents a powerful tool to estimate the ratio between broken and non-broken RET tumor cells. Establishing a precise FISH cut-off may be useful in the interpretation of the presence of RET rearrangement, primarily when this strategy is used for cytological evaluation or for targeted therapy.
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