Break–apart interphase fluorescence in situ hybridization assay in papillary thyroid carcinoma: on the road to optimizing the cut-off level for RET/PTC rearrangements

Abstract

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 WT 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 WT PTCs were analyzed by FISH for RET rearrangements. As controls, six BRAFV600E mutated PTCs, 13 follicular adenomas (FA), and ten normal thyroid parenchyma were also analyzed. Methods: We performed FISH analysis on formalin-fixed, paraffin-embedded 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 one spontaneous FA (1/13, 7.7%). Overall, the data obtained by FISH matched well with qRT-PCR results. Challenging findings were observed in five 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.

Differentiated Thyroid Carcinoma in Pediatric Age: Genetic and Clinical Scenario

“…To identify RET/PTC rearrangements (either 10q11.2 inversions or translocations), FISH was performed using the REPEAT-FREE POSEIDON RET (10q11) break-apart probe (Kreatech Diagnostics, Amsterdam, Netherlands) on FFPE samples. The FISH procedure was completed according to the Kreatech protocol with some modifications, as previously described (9). Slides were examined using an Olympus BIX-61 microscope (Olympus, Hamburg, Germany) with appropriate fluorescence excitation/emission filters.…”

AGK‐BRAF is associated with distant metastasis and younger age in pediatric papillary thyroid carcinoma

“…The mean value () and the SD of cells with split signal in control tissues were calculated. The cut‐off value was calculated considering the mean value + 3SD ( ± 3xSD) …”

Iodine regulates G2/M progression induced by CCL21/CCR7 interaction in primary cultures of papillary thyroid cancer cells with RET/PTC expression

“…RET serves a regulatory role in cell survival, growth, differentiation and migration (9). In PTC, RET fuses with different ubiquitous genes on the same or alternate chromosomes to yield various RET/PTC fusion rearrangements, leading to the abnormal expression of a chimeric RET protein that is constitutively activated in thyroid follicular cells (10). Among the 13 fusion patterns of RET with 12 different genes reported at present (11), RET/PTC1 and RET/PTC3 are the major variants, while the others are rare and hypothesized to be of little clinical significance.…”