建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性
1Division of Pathology,Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, 2School of Medicine,University of Milan, 3School of Biology,University of Pavia, 4Division of Breast Surgery,Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, 5Division of Medical Oncology,Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, 6Department of Biomedical, Surgical, and Dental Sciences,University of Milan
Summary
HER2-positive 细胞的异质分布可以在乳腺癌的一个子集中观察到, 并产生临床难题。在这里, 我们介绍了一个可靠的和成本-有效的协议, 以定义, 量化, 并比较 HER2 内肿瘤遗传异质性的大系列异处理乳腺癌。
Abstract
针对人类表皮生长因子受体 2 (HER2) 的靶向治疗已经从根本上改变了 HER2-positive 乳腺癌患者的预后。然而, 少数病例显示 HER2-positive 细胞的异质分布, 从而产生重大的临床挑战。到目前为止, 没有可靠和标准化的协议, 以表征和量化的HER2异构基因扩增在大的队列中提出。在这里, 我们提出了一个高通量的方法, 以同时评估 HER2 的地位, 跨不同地形地区的多个乳腺癌。特别是, 我们说明了实验室的程序, 以构建增强的组织芯片 (TMAs) 纳入了肿瘤的目标定位。对福尔马林固定石蜡包埋 (FFPE) 乳腺组织中的银原位杂交 (SISH), 所有的偏置参数都进行了具体的优化。免疫组化分析的预后和预测生物标记 (即, ER, PR, Ki67, 和 HER2) 应使用自动程序执行。一个定制的 SISH 协议已被实施, 以允许在多个组织的高品质的分子分析, 接受不同的固定, 处理, 和存储程序。在这项研究中, 我们提供了一个证据的原则, 即特定的 DNA 序列可以同时在不同的地形地区的多个和异加工乳腺癌使用一个有效的和成本效益的方法。
Introduction
HER2是一种原癌基因, 在所有浸润性乳腺癌的 15-30% 中被表达和放大,1,2。HER2 过度表达是由 > 10% 细胞的存在, 强膜免疫组织化学 (IHC) 染色 (3 +), 而基因放大可以评估时, 无论是 HER2/centromere 比率是≥2或基因复制数是≥6, 在计数至少20细胞由原位杂交 (一)3。
在乳腺癌中, 肿瘤的遗传异质性已经被广泛的描述, 这对生物标志物评估和治疗反应的潜在不利因素4。根据美国病理学家学院 (CAP), HER2 的异质性存在, 如果HER2放大 > 5% 和 < 50% 浸润肿瘤细胞5。令人遗憾的是, 乳腺癌 HER2 空间异质性的实际发病率在病理学家中仍然是一个争议的话题, 一些作者认为这是一个极其罕见的事件, 而另一些人则认为, 多达40% 的病例是HER2-heterogeneous1,5,6,7,8,9,10。尽管支持这一条件的生物学机制尚未完全阐明, 但肿瘤 HER2 异质性的预后和临床影响对于乳腺癌患者是至关重要的11。
最近, 亮场分子技术, 如显像 (CISH) 和银 (SISH), 已成为可靠的方法, 以检测 HER2 异质性 FFPE 组织, 有一些优势相比, 荧光 (鱼)12。遗憾的是, 对单个病例的大量分析在大规模研究中仍然不切实际。一些团体认为, 组织化学、IHC 和与偏价技术相结合可以在癌症生物学研究中代表一个有价值的策略13,14,15,16。通过这种广泛采用的方法, 可以同时分析不同患者的组织样本, 最大限度地减少所使用的组织和试剂, 从而促进对一大系列病例的统一分析14。但是, 在试剂、固定时间和使用的保存方法等不同处理的多组织样品的同时高通量分子表征方面, 没有任何协议可供使用, 如存档块.
鉴于乳腺癌 HER2 空间异质性的预后和临床意义, 我们开发了一个综合的分子平台, 以评估它在大系列的异处理的案件。在这里, 我们描绘了实验室的策略, 产生和分析的肿瘤内的非均质性的HER2扩增在乳腺癌的高产 TMAs 的手段, SISH。下面的协议已经开发的肿瘤测量 > 5 毫米 (> pT1b 根据 TNM 2017)17。对于较小的病变, 我们建议对全脸串行切片进行分析。我们的程序允许同时 IHC 和 SISH 分析多达30乳腺癌, 包括平均6个不同的领域 (范围 4-8) 为每个案件。一共, 180 组织核心的直径1毫米, 与500µm 之间的核心, 和2毫米之间的网格和边缘将生成每一个偏的区块。
Protocol
这项研究是由 IRCCS Ca 的格基金会, Policlinico 医院, 意大利米兰的机构审查委员会批准的。 1. 病人和组织标本的选择 检索所有要分析的案例的存档幻灯片, 包括所有可用的苏木精和曙红 (H & E) 和 IHC 幻灯片的原始诊断, 如果目前, 一个 H & E 的匹配非肿瘤性乳腺组织 (例如, 手术保证金). 执行案例评审。注意: 对于这项任务, 至少有两位有乳腺病…
Representative Results
总的来说, 444 浸润性乳腺癌被纳入 15 TMAs 专门针对分析的优化。在抽样的2664点中, 2651 (99.5%) 代表了先前选定的区域, 因此被认为适于随后的分析。肿瘤的异质性是通过 IHC 和 SIH 的方法来确定的, 特别着重于肿瘤不同地形区 HER2-positive 无性系的异质分布。表 3描述了所分析案例的生物学特性, 重点介绍了不同 histotypes 中的 ER、PR、Ki67 和 HER2 状态。特别是, 18% 的病…
Discussion
在这里, 我们详细的实验室策略, 以执行 SISH 分析的HER2基因及其相应的丝在高产 TMAs 异处理乳腺癌。这一方法具有成本效益, 可以在大多数实验室进行, 以研究HER2基因放大异质性的大群乳腺癌检索形式的病理档案。
由于 HER2 检测在乳腺癌中的临床重要性以及它的异质表达所带来的挑战, 我们开发了一个高通量测试协议来评估肿瘤的内部和间的HER2遗传异质性?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
没有.
Materials
| Surgipath Paraplast | Leica Biosystems, Wetzlar, Germany, EU | 39601006 | Tissue embedding medium, 56 °C melting point |
| Eosin Y 1% water solution | Bio Optica, Milan, Italy, EU | 510002 | Eosin yellowish, water-soluble |
| Carazzi’s hematoxylin | Bio Optica, Milan, Italy, EU | 506012 | Alum hematoxylin ripened using potassium iodate |
| Diamond quality | Laboindustria, Arzergrande, Italy, EU | 33533 | 26×76 mm microscope slides |
| Leica CV Mount | Leica Biosystems, Wetzlar, Germany, EU | 14046430011 | Mounting medium, with no monomers, based on polymers of butylmethacrylate in xylene |
| FLEX IHC microscope slides | Agilent Technologies (Dako), Santa Clara, CA, USA | K8020 | Coated microscope slides for adhesion of FFPE for use in IHC |
| BenchMark ULTRA | Ventana medical system, Tucson, AZ, USA | N750-BMKU-FS | Slide staining system |
| CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody | Ventana medical system, Tucson, AZ, USA | 790-4324 | Primary antibody, ready-to-use |
| CONFIRM anti-Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody | Ventana medical system, Tucson, AZ, USA | 790-2223 | Primary antibody, ready-to-use |
| CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody | Ventana medical system, Tucson, AZ, USA | 790-4286 | Primary antibody, ready-to-use |
| PATHWAY HER2 (4B5) Rabbit Monoclonal Primary Antibody | Ventana medical system, Tucson, AZ, USA | 790-4493 | Primary antibody, ready-to-use |
| ultraView Universal DAB Detection Kit | Ventana medical system, Tucson, AZ, USA | 760-500 | Indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies |
| INFORM HER2 Dual ISH DNA Probe Cocktail | Ventana medical system, Tucson, AZ, USA | 780-4422 | INFORM HER2 Dual ISH assay – Dual color in situ hybridization FDA approved automated assay for determining HER2 gene status in breast cancer patients |
| ultraView Silver ISH DNP Detection Kit | Ventana medical system, Tucson, AZ, USA | 800-098 | |
| ultraView Red ISH DIG Detection Kit | Ventana medical system, Tucson, AZ, USA | 800-505 | |
| ISH Protease 3 | Ventana medical system, Tucson, AZ, USA | 780-4149 | Used in the ISH process to remove protein that surrounds the target DNA sequences of interest |
| Hematoxylin | Ventana medical system, Tucson, AZ, USA | 760-2021 | Modified Gill's hematoxylin counterstain reagent |
| Hematoxylin II Counterstaining | Ventana medical system, Tucson, AZ, USA | 790-2208 | Modified Meyer's hematoxylin counterstain reagent |
| Bluing reagent | Ventana medical system, Tucson, AZ, USA | 760-2037 | Aqueous solution of buffered lithium carbonate for bluing hematoxylin stained sections on glass slides |
| HybReady | Ventana medical system, Tucson, AZ, USA | 780-4409 | Formamide-based buffer for ISH assays |
| EZ Prep (10x) | Ventana medical system, Tucson, AZ, USA | 950-102 | Concentrate solution for paraffin removal from tissue samples during IHC and ISH reactions, and to dilute 1:10. |
| SSC Buffer (10X) | Ventana medical system, Tucson, AZ, USA | 950-110 | Sodium Chloride Sodium Citrate buffer solution is used for stringency washes and to rinse slides between staining steps and provide a stable aqueous environment for the in situ hybridization reactions. Dilute 1:5. |
| ULTRA LCS | Ventana medical system, Tucson, AZ, USA | 650-210 | Prediluted (ready-to-use) coverslip solution used as a barrier between the aqueous reagents and the air to prevent evaporation in the IHC and ISH reactions |
| Reaction Buffer (10x) | Ventana medical system, Tucson, AZ, USA | 950-300 | Tris based buffer solution (pH 7.6 ± 0.2) to rinse slides between staining steps during IHC and ISH. Dilute 1:10. |
| ULTRA Cell Conditioning (ULTRA CC2) | Ventana medical system, Tucson, AZ, USA | 950-223 | Pretreatment steps in the processing of tissue samples during IHC and ISH. Ready to use. |
| ULTRA Cell Conditioning (ULTRA CC1) | Ventana medical system, Tucson, AZ, USA | 950-224 | |
| ultraView Silver Wash II | Ventana medical system, Tucson, AZ, USA | 780-003 | Ready-to-use solution to rinse slides between IHC and ISH staining steps |
| Microtome | Leica Biosystems, Wetzlar, Germany, EU | RM 2255 | Automated rotary microtome |
| Multistainer | Leica Biosystems, Wetzlar, Germany, EU | ST 5020 | Workstation for automated staining and coverslipping |
| Minicore 1 | Alphelys, Plaisir, France, EU | 00-MICO-1 | Semi-automatic arrayer for TMA contruction with TMA Designer 2 embedded software |
| Aperio ScanScope CS2 | Leica Biosystems, Wetzlar, Germany, EU | K080254 | Image capture device – digital pathology scanner |
| Tissue-Tek III Uni-Cassette | Sakura Finetek Europe B.V | 4135 | Cassette |
| Tissue-Tek Paraform Standard Base Mold | Sakura Finetek Europe B.V | 7055 | Stainless-Steel base metal mold |
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Citer Cet Article
Ercoli, G., Lopez, G., Ciapponi, C., Corti, C., Despini, L., Gambini, D., Runza, L., Blundo, C., Sciarra, A., Fusco, N. Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers. J. Vis. Exp. (130), e56686, doi:10.3791/56686 (2017).