Tetra-color break-apart probe to simultaneously locate RET and PPARg gene breakage in single thyroid cancer cells.

The “marriage of cytology and cytogenetics” has recently been celebrated, as reported by Dal Cin et al. (Cancer Cytopathol, 2013). To make it a long-lasting marriage, however, it is important to set new DNA FISH probes, targeting specific DNA regions involved in changes (gene loss, amplification, gene fusion) in various tumors. Differentiated thyroid cancer accounts for more than 80 % of all thyroid cancers, and is represented by papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) histotype variants. Expanding knowledge on the molecular pathways implicated in their pathobiology has revealed that several gene changes play a cogent role in the pathogenesis of these carcinoma: BRAFV600E mutation, rearrangements of the tyrosine kinase receptor genes RET and TRK, and RAS mutations are mutually exclusive in PTC (found in more than 70 %), whereas RAS mutations or PPARg fusion mutations (PAX8- PPARg and CREB3L2-PPARg), also mutually exclusive, are identified in up to 75 % of FTC. On these bases, the use of some of these abnormalities as tumor biomarkers is becoming part of clinical practice, and their preoperative examination is increasingly being used as an adjunct to cytology. Eco-guided fine needle aspiration is a precious source of pre-operative material, in which the presence of RET-PTC variants and PPARg fusion mutations might be tested using FISH. To optimize the use of this material, simultaneously studying RET and PPARg genes integrity - in a single FISH experiment - is mandatory. To pursue this aim, we designed a tetra-color break-apart cocktail probe, containing 5′ and 3′ flanking sequences of each gene, using four different fluorophores: SpectrumGreen and SpectrumGold for RET and SpectrumAqua and SpectrumRed for PPARg. The probe was able to correctly identify RET and PPARg breakage in samples positive for RET/PTC and PAX8/PPARg rearrangements assessed by RT-PCR. Supported by the Banco Sardegna Foundation.