Department of Biomedical Sciences, M. Aresu Surgical Sciences, University of Cagliari, Cittadella Universitaria, Monserrato (Cagliari), Italy.
Endocr Relat Cancer. 2013 Jun 27;20(4):527-36. doi: 10.1530/ERC-13-0157. Print 2013 Aug.
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 ≥3% FISH signal for broken RET was sufficient to distinguish nodules with abnormal follicular cells, only samples with a ≥6.8% break-apart FISH signal also exhibited positive RT-PCR results. On histological analysis, all nodules meeting the ≥6.8% 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.
RET/PTC 重排和 BRAF(V600E) 突变是与甲状腺乳头状癌（PTC）相关的两种常见分子改变，其鉴定越来越多地被用作细胞学诊断 PTC 的辅助手段。然而，在细针抽吸（FNA）中使用分子方法存在一些注意事项，特别是对于 RET/PTC，应该考虑这些注意事项。有人声称，滤泡细胞中这种异常的克隆或散在存在可以区分良恶性结节。然而，最常用的基于 PCR 的技术缺乏定量异常细胞数量的能力。由于荧光原位杂交（FISH）是检测单细胞基因重排的最敏感方法，我们比较了在可疑结节的大量连续患者的 FNA 中获得的 FISH 和常规 RT-PCR 的结果，并研究了设置 FISH-FNA 阈值以区分非克隆和克隆分子事件的可行性。为此，设计了一种能够识别 RET 物理断裂的自制断裂探针。虽然≥3%的 FISH 信号用于断裂 RET 足以区分具有异常滤泡细胞的结节，但只有≥6.8%的断裂 FISH 信号的样本也显示出阳性 RT-PCR 结果。在组织学分析中，所有符合≥6.8%阈值的结节均被证实为恶性。这些数据证实了 FISH 与 RT-PCR 相比在定量 FNA 中 RET/PTC 存在方面的强大功能，并验证了 RT-PCR 在检测克隆 RET/PTC 改变方面的效率。
