Imkamp Florian, von Wasielewski Reinhard, Musholt Thomas J, Musholt Petra B
Department of Urology, Hannover University Medical School, Hannover, Germany.
J Surg Res. 2007 Dec;143(2):350-63. doi: 10.1016/j.jss.2006.10.033. Epub 2007 Jul 26.
In few papillary thyroid carcinomas (PTC) and oxyphilic thyroid carcinoma, the clinical impact of the 15 known RET hybrid oncogene variants (RET/PTC 1 to 12, 1L, 3r2, 3r3) is subject to controversial discussions. Large patient cohorts and exploitation of pathological thyroid tissue archives are essential to study the prognostic significance of RET/PTC chimeras.
Formalin-fixed and paraffin-embedded thyroid neoplasms were subjected to manual punching macrodissection and subsequent extraction of total RNA. Following reverse transcriptase polymerase chain reaction (RT-PCR)-based screening for RET rearrangements, hybrid-specific expression analyses were carried out for samples indicative of chimeric transcripts. Due to lack of tissue specimen harboring the rare RET chimeras, artificially constructed hybrid sequences of all known RET/PTC variants served as PCR controls.
Manual punching dissection successfully diminished RET wild-type contamination originating from C-cells dispersed throughout normal thyroid tissues. The average amount of 27.4 mug RNA extracted allowed for repeated molecular analyses (>60 PCRs). Hybrid-specific expression analysis identified 10 of 15 RET rearrangements (8x RET/PTC 1, 2x RET/PTC 3, 5x RET/PTC x) to be found in 54 oxyphilic thyroid tumors examined. Successful amplification of each artificial hybrid sequence ensured the absence of rare chimeric transcripts. Therefore, RET/PTC x represent either common chimeras not amplifiable due to archival RNA degradation or truly novel hybrid oncoproducts.
The fast and simple techniques described here were used to examine oxyphilic carcinomas and adenomas. These microdissection and RT-PCR procedures can easily be put into practice in any molecular biology research laboratory to enable screening of large numbers of archival thyroid tumors for known as well as yet unknown RET rearrangements.
在少数乳头状甲状腺癌（PTC）和嗜酸细胞性甲状腺癌中，15种已知的RET融合致癌基因变体（RET/PTC 1至12、1L、3r2、3r3）的临床影响存在争议。大型患者队列以及对甲状腺病理组织档案的利用对于研究RET/PTC嵌合体的预后意义至关重要。
对福尔马林固定石蜡包埋的甲状腺肿瘤进行手工打孔宏观解剖，随后提取总RNA。基于逆转录聚合酶链反应（RT-PCR）筛选RET重排后，对指示嵌合转录本的样本进行杂交特异性表达分析。由于缺乏含有罕见RET嵌合体的组织标本，所有已知RET/PTC变体的人工构建杂交序列用作PCR对照。
手工打孔解剖成功减少了源自散布于整个正常甲状腺组织中的C细胞的RET野生型污染。提取的平均27.4微克RNA量允许进行重复的分子分析（>60次PCR）。杂交特异性表达分析在54例嗜酸细胞性甲状腺肿瘤中鉴定出15种RET重排中的10种（8例RET/PTC 1、2例RET/PTC 3、5例RET/PTC x）。每种人工杂交序列的成功扩增确保了不存在罕见的嵌合转录本。因此，RET/PTC x要么代表由于存档RNA降解而无法扩增的常见嵌合体，要么代表真正的新型杂交致癌产物。
本文所述的快速简单技术用于检查嗜酸细胞性癌和腺瘤。这些显微解剖和RT-PCR程序可在任何分子生物学研究实验室轻松实施，以便对大量存档甲状腺肿瘤进行已知和未知RET重排的筛查。
