原位杂交的色原性杂交是HPV相关头颈部癌症诊断的工具
Summary
人皮疹病毒(HPV)RNA原位染色体杂交被认为是肿瘤内活性人皮疹病毒感染检测的黄金标准之一。它允许通过定位和半定量评估其信号来可视化HPV E6-E7 mRNA表达。
Abstract
人皮球瘤病毒 (HPV) 感染是食管鳞状细胞癌 (OPSCC) 亚型的主要危险因素,该病毒往往与与酒精和烟草相关的 OPSCC 更好的结果相关。HPV病毒RNA的原位杂交(CISH)可实现对致癌蛋白E6和E7病毒转录本的半定量评价,并具有良好的空间分辨率的原位可视化。此技术允许通过肿瘤HPV感染细胞中的HPV转录可视化来诊断活动感染。这种技术的一个优点是避免来自肿瘤附近的非肿瘤HPV感染细胞的污染。总体而言,其良好的诊断性能被认为是主动HPV感染鉴定的黄金标准。由于E6和E7病毒蛋白与细胞蛋白pRb和p3的相互作用是强制性的细胞转化,HPV RNA CISH在功能上相关,并敏锐地反映活性致源性HPV感染。这种技术在临床上也相关,因为”低”或”高”HPV转录水平有助于识别HPV相关p16阳性头颈部癌症患者中的两个预后组。在这里,我们介绍在正式固定石蜡嵌入 (FFPE) 幻灯片上执行的手动 HPV RNA CISH 的协议,该幻灯片使用从制造商处获得的工具包。RNA原位杂交也可以用荧光导光(RNA FISH)进行,而不是染色体性揭示。它也可以与传统的免疫染色相结合。
Introduction
HPV RNA CISH 是检测活动 HPV 感染的有力工具,在诸如口腔或子宫颈等不同部位的良性或恶性病变中可能证明至关重要。活动HPV感染的检测可能支持HPV引起的病变的诊断,从而影响其治疗和预后。
HPV是最常见的性传播感染,超过100种病毒基因型已被描述为1。从原理上讲,低风险基因型(如基因型6和11)已知可诱发生殖器疣、复发性呼吸道性手皮瘤病和其他良性病变,而高风险基因型(如基因型16和18)是大多数宫颈癌的罪魁祸首和肛门癌,并在HNSCC的肿瘤发生发挥作用,比例变化,根据区域流行病学数据2。
有几种工具可用于检测 HPV 感染。由于高危HPV感染导致病毒致癌蛋白E6和E73的表达,E6和E7转录本的检测被广泛认为是主动HPV感染鉴定4的黄金标准。HPV RNA CISH 可在 FFPE 样本上执行,这些样本很容易从患有各种 HPV 相关疾病的患者身上获得。其性能在子宫颈、肛门和阴道的鳞状宫内肿瘤中,以及宫颈、肛门和上呼吸道的侵入性鳞状细胞癌中进行了评估:其灵敏度达到98%以上在HPVDNA聚合酶链反应(PCR)阳性病例中。这略好于p16免疫染色(93%)和HPVDNA原位杂交(DNA ISH:97%),这是更常用的。在另一组57例鳞状细胞癌(SCC)中,与HPV DNA ISH相比,HPV RNA CISH具有更好的敏感性(100%对88%)。和特异性(87%对74%)6 。
P16免疫染色是一种间接标记,反映细胞周期中断,这可能是由HPV感染4,7引起(但不完全) 引起的。这种具有成本效益的测试具有良好的灵敏度和负预测值,被美国病理学家学院(CAP)和国际联盟推荐为奥述林克斯癌症(OPC)高危HPV感染的代用品标志物。癌症控制(UICC)8.
虽然本文只关注HNSCC中HPV的检测,但HPV RNA CISH在涉及HPV感染的各种其他条件下与临床相关。例如,这种技术可以提高诊断的低级鳞状颅内病变的宫颈(LSIL,以前称为宫颈内皮瘤,1级[CIN1])的形态不明确的病例9的诊断的准确性。关于口管SCC,HPV RNA CISH允许识别HPV相关SCC,在最近第八版《头颈癌TNM分类》(国际癌症联盟)中,该SCC与HPV无关的口管SCC有别于HPV。控制 [UICC])10.由于HPV相关SCC表现出更好的预后,更长的生存期和增强的放疗和化疗敏感性比HPV无关SCC11,12,13,HPV感染的检测可能会影响病人管理14,15.此外,HPV RNA CISH可用于诊断HPV相关多表皮癌,其信号高于HPVDNACISH16。几个多变量分析表明,E6 和 E7 转录的检测与奥法林格 SCC 总体7、15、17、18 和p16阳性性食管SCC19、20的子组。
在这里,我们介绍在FFPE幻灯片上执行的手动HPV RNA CISH的协议,使用从制造商获得的工具包。
Protocol
Representative Results
Discussion
HPV RNA CISH 使用购买的试剂盒执行,是检测病毒记录组的强大工具,它表明 HPV 感染活跃。手动执行,协议的步骤整体上易于遵循,并且购买的工具包很方便。该技术允许一次染色19个组织学样本和一个控制幻灯片,测定持续约8小时。除非另有说明,否则不要让样品在步骤之间干燥,这一点至关重要。预处理条件可能需要根据操纵的组织进行调整。
HPV E6-E7 mRNA 表达信号以精确的空间分辨?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢欧洲霍皮塔兰·乔治·蓬皮杜和内克尔病理学系(劳里安·尚博勒、埃洛迪·米歇尔和吉塞勒·勒格尔);PARCC组织学平台,欧洲霍皮塔·乔治·蓬皮杜(科林娜·莱萨弗雷);弗吉尼亚克拉克语言编辑;亚历山德拉·埃尔巴基扬为她的贡献。
Materials
| Hematoxylin solution, Gill No. 1 | Merck | GHS132 | |
| HybEZ Oven (110v) | Advanced Cell Diagnostics Inc. | 321710 | |
| HybEZ slide rack | Advanced Cell Diagnostics Inc. | 300104 | |
| ImmEdge Hydrophobic Barrier Pen | Advanced Cell Diagnostics Inc. | 310018 | |
| RNAscope 2.5 HD Detection Reagents-BROWN | Advanced Cell Diagnostics Inc. | 322310 | This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B |
| RNAscope 3-Plex Negative Control Probe | Advanced Cell Diagnostics Inc. | 320871 | DAPB |
| RNAscope 3-Plex Positive Control Probe | Advanced Cell Diagnostics Inc. | 320861 | PPIB |
| RNAscope H202 & Protease Plus Reagent | Advanced Cell Diagnostics Inc. | 322330 | Hydrogen Peroxyde x2 and Protease Plus x 1 |
| RNAscope Probe- HPV16/18 | Advanced Cell Diagnostics Inc. | 311121 | |
| RNAscope Target Retrieval Reagents | Advanced Cell Diagnostics Inc. | 322000 | |
| RNAscope Wash Buffer Reagents | Advanced Cell Diagnostics Inc. | 310091 | Wash Buffer 50X x4 |
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