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
<ol><li>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).</li>
	<li>
执行案例评审。 注意: 对于这项任务, 至少有两位有乳腺病理学经验的病理学家应该讨论诊断幻灯片并解决 collegially 的分歧。使用传统显微镜或远程工具 (有利于后者在多研究)。如果有, 排除所有不和谐的情况。<ol>
<li>根据世界卫生组织 (WHO) 乳腺肿瘤分类的最新版本, 对所有病例进行组织学重新分类18。</li>
		<li>确保每个病例的正常组织, 如果存在于存档临床标本中, 包括至少一个非肿瘤晚期导管-小叶单位。</li>
		<li>使用明亮场显微镜 (由病理学家和将建造 TMAs 的技术员共同执行), 确定所有显示异质性细胞学、建筑或 IHC 特征的肿瘤区。</li>
	</ol>
</li>
	<li>使用玻璃幻灯片上的标记或在数字幻灯片软件上使用适当的工具 (图 1A) 突出显示离散的地形区域。</li>
	<li>根据所选的幻灯片, 从存档中检索相应的 FFPE 块。 注: 为优化偏微结构, 避免组织损耗, 并保持凸模的结构完整性, 应排除 < 1 毫米厚残余组织的病例。</li>
</ol>2. 基于 Kononen 技术的设计与施工 TMAs
<ol><li>
创建并打开一个新的电子表格文件, 其中包含每个案例的不同区域的记录。在单独的列中包括以下数据, 如表1所示: 案例 id、块 id、区域 id、组织类型 (例如、正常组织、侵入性成分 histotype、原位组件 histotype)、地形 (如、肿瘤核心、侵入前), 形态学 (例如, 核级, 建筑, 分裂, 细胞学特征), 生物标志物状态 (即, 雌激素受体 (ER), 孕激素受体(PR), Ki67, 和 HER2), 以及所有其他 IHC 功能。<ol>
<li>保存文档。</li>
	</ol>
</li>
	<li>
创建一个项目。
	<ol>
<li>安装和打开设计软件。</li>
		<li>定义 "可通过" 菜单文件 > "新建 > 块" 或使用 CTRL + B 访问。</li>
		<li>在第一个字段中单击以填充或按 Tab 键, 并键入石蜡块的尺寸: 宽度 35 (mm), 高度 20 (mm)。键入 "2" 作为 "边距" 值 (即, 从石蜡边缘到 mm 的距离) (图 1B)。通过单击正确的按钮保存数据。新的收件人块模板现已保存, 可用于新的组织阵列模板。</li>
		<li>创建组织阵列项目: 访问菜单文件 > 新建 > 组织阵列或使用 CTRL + T。</li>
		<li>填入组织数组、注释和作者的名称, 然后单击 "下一步"。单击 "导入" 图标, 选择以前创建的收件人块的模板文件, 然后单击 "下一步"。</li>
		<li>单击圆形按钮可选择 1 mm 作为光斑大小。选择500µm 作为点间距, 这是相邻点的两个中心之间的空间。单击计算器图标以计算所创建的斑点总数, 应该是231。单击 "下一步"。</li>
		<li>通过单击它来定义点编号模式。单击 "定义" 按钮以创建网格和子网格。删除中央线 (6 行) 和列8和15。保持3点1号线的方向。最终映射应包含183点的总数, 如图 2所示。</li>
		<li>定义组织类型列表: 通过菜单文件 > 新 > 组织类型列表或使用 CTRL + L 进行访问。</li>
		<li>插入先前定义的组织描述。按向下键或单击 + 按钮添加一行。</li>
		<li>定义项目: 通过菜单文件 > 新 > 助推器项目或使用 CTRL + P 访问。</li>
		<li>填入组织数组、注释和作者的名称, 然后单击 "下一步"。导入电子表格文件、组织类型列表和先前通过单击适当图标创建的组织阵列模型。</li>
		<li>单击 "全部选择", 并定义每个组织类型的斑点数;单击 "匹配" 按钮以应用设置。将会出现一个表, 列出将被取样并转移到此项目中的收件人块的核心总数。保存项目并关闭程序。</li>
	</ol>
</li>
	<li>
准备受体块 (图 1B)。
	<ol>
<li>在65° c 时用弹力石蜡填充清洗过的金属模具。</li>
		<li>将石蜡块留在室温下, 在金属模具内过夜降温。</li>
		<li>从金属模具中提取石蜡块。如果出现裂纹, 请丢弃该块并重复步骤 2.3. 1-2. 3.3。</li>
	</ol>
</li>
	<li>
使用自动或半自动的机器人19构造该方法。
	<ol>
<li>使用注释检索所有选定的 FFPE 块和相应的 H & E 节。</li>
		<li>打开机器人并在机器人传送带上插入受体块。</li>
		<li>打开机器人控制站软件, 单击 "新组织阵列", 然后加载以前创建的项目 (图 1C)。</li>
		<li>单击 "继续", 然后从菜单中选择施主块的位置。</li>
		<li>将起始位置定义在每个收件人块上: 使用箭头键移动激光光源, 并将其与接收器块的石蜡上的左上边缘对齐。</li>
		<li>单击 "下一步", 并对垂直对齐重复。</li>
		<li>单击 "下一步", 在收件人 (受体) 块的先前定义的坐标中自动创建一个孔。</li>
		<li>把拳打空</li>
		<li>放置供方块进行取样, 并从先前标注的感兴趣区域取出组织芯。</li>
		<li>在接收方 (受体) 块的孔中插入供体组织芯。</li>
		<li>从机器人中取出施主块。</li>
		<li>重复步骤 2.4. 5-2. 4.11, 直到完成。</li>
	</ol>
</li>
	<li>将新生成的偏压块的组织侧放在一个无菌的空白幻灯片上, 并将其放置在45° c 5 分钟的实验室烤箱中。</li>
	<li>轻轻地按下在幻灯片上的方块 5 s, 以铺平组织核心。重复一次 (总共2次)。</li>
	<li>从烤箱中取出与滑块一起, 将其翻转, 然后轻轻地从幻灯片中分离出该块。</li>
	<li>在65° c 的情况下, 用一层薄薄的弹力石蜡覆盖该组织表面。</li>
	<li>在室温下保持2小时。</li>
	<li>在4° c 的24小时转移。 注: 该区块可贮存在4° c, 直至耗尽。</li>
</ol>3. 组织化学和免疫组织化学分析
<ol><li>
削减的部分。
	<ol>
<li>放置在-10 ° c 的表面上的蜡侧的偏位受体块 (s) 10 分钟的寒意石蜡。</li>
		<li>用超纯水填充浮选槽, 将热量设置为38° c。</li>
		<li>在旋转切片上放置一个新的刀片, 并将该块插入切片卡盘, 使蜡块面向刀片, 并在垂直平面上对齐。</li>
		<li>小心地与刀片块, 并削减一些薄薄的部分, 以确保定位是正确的;必要时进行调整。</li>
		<li>切割3µm 厚的部分。</li>
		<li>用镊子拿起这些部分, 并将它们漂浮在水浴表面。</li>
		<li>在普通的和 IHC 的玻璃滑梯上挑出水浴的部分。</li>
		<li>将幻灯片放在机架上, 在45° c 过夜将它们存放在烤箱中。 注: 一旦准备好, 应在24小时内染色。</li>
	</ol>
</li>
	<li>
使用 autostainer 进行组织化学染色 (H & E)。
	<ol>
<li>在 autostainer 机架上插入幻灯片, 并将其放置在60° c 为10分钟。</li>
		<li>打开 autostainer 的装载抽屉, 并在一个带有标准 H & E 剪辑的负载站中向外插入机架。</li>
		<li>关闭抽屉后, 系统将自动识别该剪辑, 并且以下协议将开始: 烘箱站在37° c (6 分钟), 二甲苯 (2 分钟), 二甲苯 (2 分钟), 乙醇 100% (2 min), 乙醇 100% (2 分钟), 乙醇 95% (2 分钟), 乙醇 95% (2 分钟), 蒸馏水 (4 分钟), Carazzi 的苏木精 (9 分钟), 低压自来水 (2 分钟), 曙红 1% (1 分钟), 低压自来水 (2 分钟), 乙醇 95% (二十年代), 乙醇 95% (二十年代), 乙醇 95% (二十年代), 乙醇 95% (二十年代), 乙醇 100% (十五年代), 乙醇 100% (十五年代), 二甲苯 (三十年代), 二甲苯 (三十年代)。</li>
		<li>从抽屉中卸下机架, 然后用盖板装入幻灯片。<ol>
<li>收集一滴装载与吸管, 并把它放在一个外边缘的封面幻灯片。</li>
			<li>将滑块的湿侧放在滑梯上, 让安装干30分钟。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
使用自动 immunostainer 进行 ER、PR、Ki67 和 HER2 的 IHC 染色。 注: 在开始染色运行前, 启动系统, 并检查消耗品试剂瓶 (材料表) 和废容器。<ol>
<li>放置在60° c 的偏析幻灯片分析10分钟。</li>
		<li>启动 immunostainer 仪器和软件。</li>
		<li>在 "主页" 视图中, 单击 "协议" 按钮, 然后单击 "创建/编辑协议"。</li>
		<li>选择标准的民建联 (3, 3 '-diaminobenzidine) IHC 程序修改基本模板。</li>
		<li>将 "Deparaffinization" 标记在方框列表中, 并将介质温度设置为72° c。</li>
		<li>标记 cc1 揭露溶液, 将培养基温度设在95° c, 孵育时间在36分钟。</li>
		<li>标记主抗体盒, 插入 ER 内嵌分配器的 ID (仪器软件将在试剂传送带上识别分配器), 并将潜伏期设置为16分钟。</li>
		<li>标记 Counterstaining 框, 选择苏木素 I, 并设置潜伏期在12分钟。</li>
		<li>单击 "另存为" 按钮并命名协议。</li>
		<li>重复步骤 3.3. 3-3. 3.9 为剩余的抗体使用以下主要抗体潜伏期: PR 16 min、Ki67 16 min 和 HER2 12 min. 使用预稀释的现成使用 (RTU) 抗体。</li>
		<li>在 "主页" 视图中, 单击 "创建标签" 按钮。</li>
		<li>单击 "协议" 按钮, 并添加 ER、PR、Ki67 和 HER2 协议, 以便对每个要分析的。</li>
		<li>单击 "关闭/打印" 按钮。</li>
		<li>当标签模板出现时, 输入标签的 "对中" id。</li>
		<li>单击 "打印" 按钮以打印标签, 然后将其应用于 "偏" 幻灯片。</li>
		<li>单击仪器图标, 并将仪器启动模式设置为 "就绪"。</li>
		<li>打开覆盖试剂传送带的引擎盖, 放置检测系统和主抗体, 然后关闭引擎盖。</li>
		<li>单击幻灯片抽屉中的前按钮以打开它, 加载偏压幻灯片, 然后通过单击同一按钮关闭抽屉。</li>
		<li>将仪表启动模式设置为 "正在运行" 并按照说明进行操作。</li>
		<li>在运行结束时, 通过按下打开的所有抽屉, 在仪表滑块上按下 "已完成的幻灯片" 按钮, 并将其用温水肥皂水冲洗, 以除去剩余的试剂。</li>
		<li>在低压冷水运行水中冲洗滑块, 脱水片, 并在每张幻灯片上应用玻璃。</li>
	</ol>
</li>
	<li>
根据美国临床肿瘤协会 (ASCO)/CAP 指南和 Ki67 国际 Ki67 在乳腺癌工作组中的建议, 对 ER、PR 和 HER2 进行 IHC 分析 (表 2)。 注: 此分析应分别在分离的组织斑点的病理学家和经验的乳房病理。<ol>
<li>如果≥1% 的肿瘤细胞核是免疫反应性的, 则评估 ER 表达式为阳性20,21,22,23。</li>
		<li>评估 PR 表达为阳性, 如果≥1% 的肿瘤细胞核为免疫反应性20,21,22,23。</li>
		<li>评估 HER2 表达式为: a) 评分 3 + 如果周膜染色是完整和激烈的, b) 评分 2 + 如果周膜染色不完全和/或弱/中 > 10% 的侵袭性肿瘤细胞或完整和周膜染色在浸润性肿瘤细胞的≤10% 内是剧烈的, c) 评分 1 + 如果不完整的膜染色微弱/勉强察觉, 并在 > 10% 的浸润性肿瘤细胞, d) 阴性, 如果没有染色是存在或膜染色是不完整和微弱/勉强察觉和在≤10% 浸润性肿瘤细胞3,24。</li>
		<li>在至少1000个肿瘤细胞中随机选择超过10高功率场 (放大倍数, 400X)25的 Ki67 指数作为细胞核染色的细胞百分比。</li>
	</ol>
</li>
</ol>4. 对HER2的 SISH 分析
<ol><li>切口部分 (重复步骤 3.1)。</li>
	<li>
使用自动 immunostainer 对 HER2 执行 SISH。
	<ol>
<li>放置在60° c 的偏析幻灯片分析10分钟。</li>
		<li>启动 immunostainer 仪器和软件。</li>
		<li>在 "主页" 视图中, 单击 "协议" 按钮, 然后单击 "创建/编辑协议"。</li>
		<li>选择 "U 双电路" 程序修改基本模板。</li>
		<li>标志 "烘干" 在箱子名单和设置温度在63° c 为20分钟。</li>
		<li>标记 "细胞调理" 在盒子里的列表, 然后 "细胞调理 CC2", 并设定温度在86° c 和孵育时间在4分钟。</li>
		<li>旗 CC2 温和 (周期 1), 标准 (周期 2), 并延长 (周期 3) 为8分钟, 12 分钟, 和8分钟分别。</li>
		<li>在盒子列表中标记 "蛋白酶 3", 并将潜伏期设定在16分钟。</li>
		<li>选择探头 HER2DNP 和 CHR17DIG, 并设置潜伏期在6小时。</li>
		<li>设置洗涤在76° c 为8分钟。</li>
		<li>设置 SISH multimer (32 分钟) 孵育时间。</li>
		<li>设置银原 (CHRC) 孵育时间在4分钟。</li>
		<li>设置红 multimer (红点挖 AP) 孵育时间在24分钟。</li>
		<li>设置红色原 (红色的) 孵育时间在8分钟。</li>
		<li>在方框列表中标记 "Counterstaining", 选择 "苏木素 II", 并将潜伏期设置为8分钟。</li>
		<li>在方框列表中标记 "后 Counterstaining", 选择 "发蓝试剂", 并将潜伏期设置为4分钟。</li>
		<li>重复步骤 3.3. 11-3. 3.15 (选择修改 U 双电路协议)。</li>
		<li>打开覆盖试剂传送带的引擎盖, 放置洗涤和检测系统, 然后关闭引擎盖。</li>
		<li>重复步骤从 3.3. 18-3. 3.21。</li>
	</ol>
</li>
	<li>对HER2执行 SISH 分析: 如果HER2/CEP17 比率是≥2.0, 并且每个单元格的平均HER2复制数≥4.0 信号, 并且HER2/CEP17 比率为 < 2.0, 则将HER2基因放大率评估为正数。使用平均HER2复制号 < 4.0 信号/单元格3,24。 注: 类似于 IHC, 这种分析应分别在离散组织斑点的病理学家和经验的乳腺病理。</li>
</ol>

<ol>
	<li>Allison, K. H., Dintzis, S. M., Schmidt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+HER2+heterogeneity+by+fluorescence+in+situ+hybridization+according+to+CAP+expert+panel+recommendations:+time+for+a+new+look+at+how+to+report+heterogeneity.">Frequency of HER2 heterogeneity by fluorescence in situ hybridization according to CAP expert panel recommendations: time for a new look at how to report heterogeneity.</a> Am J Clin Pathol. 136 (6), 864-871 (2011).</li><li>Montemurro, F., Scaltriti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+drugs+targeting+HER-family+signalling+in+cancer.">Biomarkers of drugs targeting HER-family signalling in cancer.</a> J Pathol. 232 (2), 219-229 (2014).</li><li>Wolff, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+human+epidermal+growth+factor+receptor+2+testing+in+breast+cancer:+American+Society+of+Clinical+Oncology/College+of+American+Pathologists+clinical+practice+guideline+update.">Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.</a> J Clin Oncol. 31 (31), 3997-4013 (2013).</li><li>Ng, C. K., Pemberton, H. N., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+intratumor+genetic+heterogeneity:+causes+and+implications.">Breast cancer intratumor genetic heterogeneity: causes and implications.</a> Expert Rev Anticancer Ther. 12 (8), 1021-1032 (2012).</li><li>Vance, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+heterogeneity+in+HER2+testing+in+breast+cancer:+panel+summary+and+guidelines.">Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines.</a> Arch Pathol Lab Med. 133 (4), 611-612 (2009).</li><li>Murthy, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+HER2/Neu+status+by+fluorescence+in+situ+hybridization+in+immunohistochemistry-equivocal+cases+of+invasive+ductal+carcinoma+and+aberrant+signal+patterns:+a+study+at+a+tertiary+cancer+center.">Assessment of HER2/Neu status by fluorescence in situ hybridization in immunohistochemistry-equivocal cases of invasive ductal carcinoma and aberrant signal patterns: a study at a tertiary cancer center.</a> Indian J Pathol Microbiol. 54 (3), 532-538 (2011).</li><li>Ohlschlegel, C., Zahel, K., Kradolfer, D., Hell, M., Jochum, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+genetic+heterogeneity+in+breast+carcinoma.">HER2 genetic heterogeneity in breast carcinoma.</a> J Clin Pathol. 64 (12), 1112-1116 (2011).</li><li>Chang, M. C., Malowany, J. I., Mazurkiewicz, J., Wood, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Genetic+heterogeneity'+in+HER2/neu+testing+by+fluorescence+in+situ+hybridization:+a+study+of+2,522+cases.">'Genetic heterogeneity' in HER2/neu testing by fluorescence in situ hybridization: a study of 2,522 cases.</a> Mod Pathol. 25 (5), 683-688 (2012).</li><li>Bartlett, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+HER2+gene+amplification:+impact+on+patient+outcome+and+a+clinically+relevant+definition.">Heterogeneous HER2 gene amplification: impact on patient outcome and a clinically relevant definition.</a> Am J Clin Pathol. 136 (2), 266-274 (2011).</li><li>Seol, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+heterogeneity+of+HER2+gene+amplification+in+breast+cancer:+its+clinicopathological+significance.">Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance.</a> Mod Pathol. 25 (7), 938-948 (2012).</li><li>Hanna, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+in+situ+hybridization+in+breast+cancer:+clinical+implications+of+polysomy+17+and+genetic+heterogeneity.">HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity.</a> Mod Pathol. 27 (1), 4-18 (2014).</li><li>Sanguedolce, F., Bufo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+assessment+by+silver+in+situ+hybridization:+where+are+we+now?.">HER2 assessment by silver in situ hybridization: where are we now?.</a> Expert Rev Mol Diagn. 15 (3), 385-398 (2015).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Contrasting+Role+of+p16Ink4A+Patterns+of+Expression+in+Neuroendocrine+and+Non-Neuroendocrine+Lung+Tumors:+A+Comprehensive+Analysis+with+Clinicopathologic+and+Molecular+Correlations.">The Contrasting Role of p16Ink4A Patterns of Expression in Neuroendocrine and Non-Neuroendocrine Lung Tumors: A Comprehensive Analysis with Clinicopathologic and Molecular Correlations.</a> PLoS One. 10 (12), e0144923 (2015).</li><li>Albanghali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+tissue+microarrays+from+core+needle+biopsies+-+a+systematic+literature+review.">Construction of tissue microarrays from core needle biopsies - a systematic literature review.</a> Histopathology. 68 (3), 323-332 (2016).</li><li>Navani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+evaluation+of+tissue+microarrays+in+a+high-throughput+research+project:+The+contribution+of+Indian+surgical+pathology+to+the+Human+Protein+Atlas+(HPA)+project.">Manual evaluation of tissue microarrays in a high-throughput research project: The contribution of Indian surgical pathology to the Human Protein Atlas (HPA) project.</a> Proteomics. 16 (8), 1266-1270 (2016).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+in+gastric+cancer:+a+digital+image+analysis+in+pre-neoplastic,+primary+and+metastatic+lesions.">HER2 in gastric cancer: a digital image analysis in pre-neoplastic, primary and metastatic lesions.</a> Mod Pathol. 26 (6), 816-824 (2013).</li><li>Amin, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AJCC Cancer Staging Manual. , (2017).</li><li>Lakhani, S. R., Ellis, I. O., Schnitt, S. J., Tan, P. H., van de Vijver, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WHO Classification of Tumours of the Breast. , (2012).</li><li>Kononen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays+for+high-throughput+molecular+profiling+of+tumor+specimens.">Tissue microarrays for high-throughput molecular profiling of tumor specimens.</a> Nat Med. 4 (7), 844-847 (1998).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+quandaries:+basaloid+adenoid+cystic+carcinoma+or+breast+cylindroma?+The+role+of+massively+parallel+sequencing.">Resolving quandaries: basaloid adenoid cystic carcinoma or breast cylindroma? The role of massively parallel sequencing.</a> Histopathology. 68 (2), 262-271 (2016).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+events+in+the+progression+of+adenoid+cystic+carcinoma+of+the+breast+to+high-grade+triple-negative+breast+cancer.">Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer.</a> Mod Pathol. 29 (11), 1292-1305 (2016).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+NAB2-STAT6+gene+fusions+and+oestrogen+receptor-&#945;+expression+in+pulmonary+adenofibromas.">Recurrent NAB2-STAT6 gene fusions and oestrogen receptor-&#945; expression in pulmonary adenofibromas.</a> Histopathology. 70 (6), 906-917 (2017).</li><li>Hammond, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Society+of+Clinical+Oncology/College+Of+American+Pathologists+guideline+recommendations+for+immunohistochemical+testing+of+estrogen+and+progesterone+receptors+in+breast+cancer.">American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer.</a> J Clin Oncol. 28 (16), 2784-2795 (2010).</li><li>Fusco, N., Bosari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+aberrations+and+heterogeneity+in+cancers+of+the+digestive+system:+Implications+for+pathologists+and+gastroenterologists.">HER2 aberrations and heterogeneity in cancers of the digestive system: Implications for pathologists and gastroenterologists.</a> World J Gastroenterol. 22 (35), 7926-7937 (2016).</li><li>Dowsett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Ki67+in+breast+cancer:+recommendations+from+the+International+Ki67+in+Breast+Cancer+working+group.">Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group.</a> J Natl Cancer Inst. 103 (22), 1656-1664 (2011).</li><li>Dekker, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+sensitivity+and+specificity+of+HER2+testing+in+breast+cancer+using+a+tissue+micro-array+approach.">Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach.</a> Breast Cancer Res. 14 (3), R93 (2012).</li><li>De Sio, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+MALDI-Mass+Spectrometry+Imaging+method+applicable+to+different+formalin-fixed+paraffin-embedded+human+tissues.">A MALDI-Mass Spectrometry Imaging method applicable to different formalin-fixed paraffin-embedded human tissues.</a> Mol Biosyst. 11 (6), 1507-1514 (2015).</li></ol>

作者没有利益冲突。

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?...

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。

<table><tbody><tr><td>Surgipath Paraplast</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>39601006</td><td>Tissue embedding medium, 56 &amp;deg;C melting point</td></tr><tr><td>Eosin Y&amp;nbsp; 1% water solution</td><td>Bio Optica, Milan, Italy, EU</td><td>510002</td><td>Eosin yellowish, water-soluble</td></tr><tr><td>Carazzi&amp;rsquo;s hematoxylin</td><td>Bio Optica, Milan, Italy, EU</td><td>506012</td><td>Alum hematoxylin ripened using potassium iodate</td></tr><tr><td>Diamond quality</td><td>Laboindustria, Arzergrande, Italy, EU</td><td>33533</td><td>26x76 mm microscope slides</td></tr><tr><td>Leica CV Mount</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>14046430011</td><td>Mounting medium, with no monomers, based on polymers of butylmethacrylate in xylene</td></tr><tr><td>FLEX IHC microscope slides</td><td>Agilent Technologies (Dako),&amp;nbsp; Santa Clara, CA, USA</td><td>K8020</td><td>Coated microscope slides for adhesion of FFPE for use in IHC</td></tr><tr><td>BenchMark ULTRA</td><td>Ventana medical system, Tucson, AZ, USA</td><td>N750-BMKU-FS</td><td>Slide staining system</td></tr><tr><td>CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-4324</td><td>Primary antibody, ready-to-use</td></tr><tr><td>CONFIRM anti-Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-2223</td><td>Primary antibody, ready-to-use</td></tr><tr><td>CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-4286</td><td>Primary antibody, ready-to-use</td></tr><tr><td>PATHWAY HER2 (4B5) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-4493</td><td>Primary antibody, ready-to-use</td></tr><tr><td>ultraView Universal DAB Detection Kit</td><td>Ventana medical system, Tucson, AZ, USA</td><td>760-500</td><td>Indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies</td></tr><tr><td>INFORM HER2 Dual ISH DNA Probe Cocktail</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-4422</td><td>INFORM HER2 Dual ISH assay - Dual color in situ hybridization FDA approved automated assay for determining HER2 gene status in breast cancer patients&amp;nbsp;</td></tr><tr><td>ultraView Silver ISH DNP Detection Kit</td><td>Ventana medical system, Tucson, AZ, USA</td><td>800-098</td><td></td></tr><tr><td>ultraView Red ISH DIG Detection Kit</td><td>Ventana medical system, Tucson, AZ, USA</td><td>800-505</td><td></td></tr><tr><td>ISH Protease 3</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-4149</td><td>Used in the ISH process to remove protein that surrounds the target DNA sequences of interest</td></tr><tr><td>Hematoxylin</td><td>Ventana medical system, Tucson, AZ, USA</td><td>760-2021</td><td>Modified Gill&#039;s hematoxylin counterstain reagent</td></tr><tr><td>Hematoxylin II Counterstaining</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-2208</td><td>Modified Meyer&#039;s hematoxylin counterstain reagent</td></tr><tr><td>Bluing reagent</td><td>Ventana medical system, Tucson, AZ, USA</td><td>760-2037</td><td>Aqueous solution of buffered lithium carbonate for bluing hematoxylin stained sections on glass slides</td></tr><tr><td>HybReady</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-4409</td><td>Formamide-based buffer for ISH assays</td></tr><tr><td>EZ Prep (10x)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-102</td><td>Concentrate solution for paraffin removal from tissue samples during IHC and ISH reactions, and to dilute 1:10.</td></tr><tr><td>SSC Buffer (10X)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-110</td><td>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.</td></tr><tr><td>ULTRA LCS</td><td>Ventana medical system, Tucson, AZ, USA</td><td>650-210</td><td>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</td></tr><tr><td>Reaction Buffer (10x)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-300</td><td>Tris based buffer solution (pH 7.6 &amp;plusmn; 0.2) to rinse slides between staining steps during IHC and ISH. Dilute 1:10.</td></tr><tr><td>ULTRA Cell Conditioning (ULTRA CC2)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-223</td><td>Pretreatment steps in the processing of tissue samples during IHC and ISH. Ready to use.</td></tr><tr><td>ULTRA Cell Conditioning (ULTRA CC1)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-224</td><td></td></tr><tr><td>ultraView Silver Wash II</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-003</td><td>Ready-to-use solution to rinse slides between IHC and ISH staining steps</td></tr><tr><td>Microtome</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>RM 2255</td><td>Automated rotary microtome</td></tr><tr><td>Multistainer</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>ST 5020</td><td>Workstation for automated staining and coverslipping</td></tr><tr><td>Minicore 1</td><td>Alphelys, Plaisir, France, EU</td><td>00-MICO-1</td><td>Semi-automatic arrayer for TMA contruction with TMA Designer 2 embedded software</td></tr><tr><td>Aperio ScanScope CS2</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>K080254</td><td>Image capture device - digital pathology scanner</td></tr><tr><td>Tissue-Tek III Uni-Cassette</td><td>Sakura Finetek Europe B.V</td><td>4135</td><td>Cassette</td></tr><tr><td>Tissue-Tek Paraform Standard Base Mold</td><td>Sakura Finetek Europe B.V</td><td>7055</td><td>Stainless-Steel base metal mold</td></tr></tbody></table>

building up a high-throughput screening platform to assess the heterogeneity of her2 gene amplification in breast cancers

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。

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...

Watch this Scientific Journal Video about Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers at JoVE.com

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

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。

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

building-up-high-throughput-screening-platform-to-assess

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.4K 次观看.  Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico. 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。

视频: Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

医学

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating investigators to analyze and interpret genes, samples and clinical data across different types of cancer. Herein, we describe a practical operation procedure, taking ID1 (Inhibitor of DNA binding proteins 1) as an example, to characterize the expression patterns of biomarker and survival predictors of breast cancer based on pooled clinical datasets derived from online accessible databases, including ONCOMINE, bcGenExMiner v4.0 (Breast cancer gene-expression miner v4.0), GOBO (Gene expression-based Outcome for Breast cancer Online), HPA (The human protein atlas), and Kaplan-Meier plotter. The analysis began with querying the expression pattern of the gene of interest (e.g., ID1) in cancerous samples vs. normal samples. Then, the correlation analysis between ID1 and clinicopathological characteristics in breast cancer was performed. Next, the expression profiles of ID1 was stratified according to different subgroups. Finally, the association between ID1 expression and survival outcome was analyzed. The operation procedure simplifies the concept to integrate multidimensional data types at the gene level from different databases and test hypotheses regarding recurrence and genomic context of gene alteration events in breast cancer. This method can improve the credibility and representativeness of the conclusions, thereby, present informative perspective on a gene of interest.

Here, we present a protocol to explore the biomarker and survival predictor of breast cancer based on the comprehensive analysis of pooled clinical datasets derived from a variety of publicly accessible databases, using the strategy of expression, correlation and survival analysis step by step.

利用多个可公开访问的数据库对乳腺癌生物标志物进行数据挖掘和综合分析

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating ...

癌症研究

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

使用快速和定量的细胞内胆碱膜模型发现转移调节器

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

增强癌症组织样本中致癌驱动因素的 FISH 分析

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

作者聚焦：FISH 作为癌症标本中精确基因扩增评估的工具

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

在使用定量免疫肿瘤的异质性蛋白表达的映射

利用多个可公开访问的数据库对乳腺癌生物标志物进行数据挖掘和综合分析

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

使用快速和定量的细胞内胆碱膜模型发现转移调节器

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

摘要

探索更多视频

此视频中的章节

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

在使用定量免疫肿瘤的异质性蛋白表达的映射

利用多个可公开访问的数据库对乳腺癌生物标志物进行数据挖掘和综合分析

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

使用快速和定量的细胞内胆碱膜模型发现转移调节器

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心