Assessing RET/PTC in thyroid nodule fine-needle aspirates: the FISH point of view

Abstract

RET/PTC rearrangement and BRAF V600E mutation are the two prevalent molecular alterations associated with papillary thyroid carcinoma (PTC), and their identification is increasingly being used as an adjunct to cytology in diagnosing PTC. However, there are caveats associated with the use of the molecular approach in fine-needle aspiration (FNA), particularly for RET/PTC, that should be taken into consideration. It has been claimed that a clonal or sporadic presence of this abnormality in follicular cells can distinguish between malignant and benign nodules. Nevertheless, the most commonly used PCR-based techniques lack the capacity to quantify the number of abnormal cells. Because fluorescence in situ hybridization (FISH) is the most sensitive method for detecting gene rearrangement in a single cell, we compared results from FISH and conventional RT-PCR obtained in FNA of a large cohort of consecutive patients with suspicious nodules and investigated the feasibility of setting a FISH-FNA threshold capable of distinguishing non-clonal from clonal molecular events. For this purpose, a home brew break-apart probe, able to recognize the physical breakage of RET, was designed. While a R3% FISH signal for broken RET was sufficient to distinguish nodules with abnormal follicular cells, only samples with a R6.8% break-apart FISH signal also exhibited positive RT-PCR results. On histological analysis, all nodules meeting the R6.8% threshold proved to be malignant. These data corroborate the power of FISH when compared with RT-PCR in quantifying the presence of RET/PTC in FNA and validate the RT-PCR efficiency in detecting clonal RET/PTC alterations.

Total thyroidectomy without prophylactic central neck dissection in clinically node-negative papillary thyroid cancer: is it an adequate treatment?

“…A wider use of immunocytochemical and genetic markers could prove useful in better defining the high-risk population. For example, patients with RET/PTC oncogene expression have a higher rate of lymph node metastases [49], and this could constitute a useful factor to consider in the future. The development of techniques for the intraoperative identification of metastatic lymph nodes could also help the surgeon in this difficult choice.…”

Genetic Heterogeneity of HER2 Amplification and Telomere Shortening in Papillary Thyroid Carcinoma

“…An arbitrary cut-off of 3% was employed, as control cells showed no split signal in 200 scored nuclei per sample. For the RET gene, a homebrew probe and a previously described cut-off value were used [19]. Microscopic analysis was performed with an Olympus BX41 epifluorescence microscope and a charge-coupled device camera (Cohu, San Diego, CA, USA) interfaced with the CytoVision system (software version 3.9; Applied Imaging, Pittsburg, PA, USA).…”

Assessment of molecular testing in fine-needle aspiration biopsy samples: An experience in a Chinese population

“…Most of those studies used a highly sensitive technique for analysis, which is capable of detecting a very small proportion of cells carrying a particular genetic event. Recently, a study demonstrated that fluorescence in situ hybridization (FISH) (Caria et al, 2013), the most sensitive method for detecting gene rearrangements in a single cell, could be used to distinguish nodules with abnormal follicular morpholoy with a ≥ 3% FISH signal for broken RET. When samples with a 6.8% break-apart FISH signal could be detected by RT-PCR and were proved to be malignant.…”