我们感谢医院和病人的所有医务和辅助人员同意参加。我们感谢加布里埃尔白狼, 博士, 从 Edanz 集团 (www.edanzediting.com/ac) 编辑本报告草稿。这项研究得到了来自山梨州 (Y.H. 和作案手法) 的基因组研究项目的资助, 以及 YASUDA 医学基金会 (Y.H.) 的赠款。

1. 利用普通玻片进行快速显微评估的 TIC 准备
<ol><li>在临床病理组织材料可用后, 尽快完成 TIC 的准备工作。如果 TIC 标本不能立即准备好, 保持用盐水湿不育纱布覆盖的组织材料, 并贮存在冰箱中, 以防止组织的干燥。</li>
	<li>准备5毫米3组织材料, 如实体肿瘤 (如, 肝, 肺和乳腺组织), 临床上通过手术或内窥镜获得。<ol>
<li>如果组织表面有大量血液, 用无菌纱布涂上生理盐水并清除血液, 轻轻擦拭组织。</li>
		<li>对于显微标本如活检材料, 保持样品湿润与消毒纱布浸泡在生理盐水。</li>
	</ol>
</li>
	<li>切除和修剪正常组织与修剪刀和暴露表面的肿瘤病变, 如果肿块是不可见的严重。</li>
	<li>用戴手套的手将切除标本的肿瘤表面接触到正常的玻璃滑梯上几次。直观地确认触摸区域是正常玻璃滑块的80% 以上。</li>
	<li>轻轻按普通玻璃滑动对聚乙烯乙二醇 (笔) 膜滑动和摩擦轻轻 2-3 倍戴手套的手。目视确认细胞从正常的玻璃滑块转移到笔膜滑动。</li>
	<li>空气干燥两个玻璃和笔膜在室温下滑动5分钟。</li>
	<li>染色正常的玻璃滑块直接细胞学检查。用固定液蘸5秒的玻璃滑块, 然后用姬姆萨染色溶液染色十五年代。</li>
	<li>用显微镜检查和筛查正常玻璃上的肿瘤含量和细胞构成, 并进行快速评估。根据多种标准对肿瘤细胞进行评价;核扩张, 异常核型, 丰富的染色质, 细胞分布不均, 核组分/细胞质组分的比例, 细胞大小和细胞极性。</li>
</ol>2. 用于基因检测的笔膜滑动膜的制备
<ol><li>如果样本显示肿瘤细胞构成超过60% 通过快速显微评估 (步骤 1.8), 削减了用刀和戴手套的手 DNA 提取的笔膜幻灯片的肿瘤接触膜。用 pincette 和戴手套的手将切割膜转移到不育的离心管上。</li>
	<li>如果肿瘤细胞构成通过快速显微评估 (步骤 1.8) 确定为低 (小于60% 的肿瘤含量), 则使用激光捕获显微切割和获得肿瘤样本。<ol>
<li>执行姬姆萨染色, 以评估肿瘤细胞使用标准协议。</li>
		<li>通过适当的激光捕获显微切割, 切下膜的 PEM 膜滑动。</li>
		<li>用 pincette 和戴手套的手将切割膜转移到不育的离心管上。</li>
		<li>将含膜的离心管存放在4摄氏度, 直到 DNA 提取 (该协议可以在此暂停)。</li>
	</ol>
</li>
</ol>3. DNA 提取
<ol><li>根据制造商的指示进行小的修改, 使用 FFPE dna 提取试剂盒从 TIC 或 FFPE 组织样品中提取 dna。FFPE DNA 提取步骤中有一个等价的套件。</li>
	<li>添加180µL 组织裂解缓冲液 (pH = 8.3) 到含膜离心管与手动200µL 吸管。添加20µL 的蛋白酶 K 与手动20µL 吸管和混合涡流与漩涡搅拌机在最大速度 (约 2500 rpm) 5 s。</li>
	<li>用空气孵化器在一夜之间孵化56摄氏度的样品。</li>
	<li>分别在热块中孵育90摄氏度的 FFPE 和 TIC 样品1小时和10分钟。在室温下用微型离心机将离心管在 1500 x g 处旋转5秒。</li>
	<li>用200µL 的吸管将200µL 的溶解缓冲液添加到样品中, 并在5秒内以最大速度混合涡流。</li>
	<li>添加200µL 乙醇 (96-100%) 与200µL 的吸管, 并彻底混合涡流在最大速度 5 s. 在室温下, 用迷你离心机在 1500 x g 处向下旋转离心管, 其 5 s。</li>
	<li>小心地将整个裂解液转移到旋转柱上, 用1000µL 的吸管和离心机在 6000 x g 处, 1 分钟25摄氏度。</li>
	<li>将旋转柱放在一个干净的2毫升收集管与戴手套的手, 并丢弃的收集管包含的流动进入一个塑料处理箱。</li>
	<li>用1000µL 的吸管和离心机将500µL 的洗涤缓冲器添加到旋转柱上, 在25摄氏度时, 在 6000 x g 处为1分钟。</li>
	<li>将旋转柱放在一个干净的2毫升收集管与戴手套的手, 并丢弃的收集管包含的流动进入一个塑料处理箱。</li>
	<li>用1000µL 的吸管和离心机将500µL 的洗涤缓冲器添加到旋转柱上, 在25摄氏度时, 在 6000 x g 处为1分钟。</li>
	<li>将包含流经的收集管丢弃到塑料处理箱中。将旋转柱放在干净的1.5 毫升离心管中, 用手套的手和离心机在 2万 x g 3 分钟在25°c, 以干燥膜。</li>
	<li>将旋转柱放置在 DNA 低结合管中, 戴手套的手。<ol>
<li>添加 40-50 µL 洗脱缓冲液的中心膜与100µL 吸管。</li>
		<li>在室温下孵育5分钟, 离心机在 2万 x g 处为1分钟, 25 摄氏度。</li>
		<li>将 DNA 样本存储在-20 摄氏度, 直到下一步 (该协议可以在此暂停)。</li>
	</ol>
</li>
</ol>4. 定量实时 PCR 法估计 DNA 质量
<ol><li>用吸管制备无菌离心管中的主混合, 如下所示:10 µL 2x 实时 PCR 总混合, 1 µL 20x RNase P 底漆-探针混合 (扩增子尺寸:87 bp), 8 µL 无菌核酸酶水。</li>
	<li>在一个无菌离心管中准备第二个主组合, 如下所示:10 µL 的2x 实时 PCR 主混合, 1 µL 20x RNase P 底漆-探针混合 (扩增子大小: 268 bp), 8 µL 无菌核酸酶水。</li>
	<li>执行人类控制基因组 DNA 的串行稀释 (在试剂盒中提供) 4 次, 用于五点标准曲线并确定绝对 DNA 浓度13。</li>
	<li>将两个准备好的主混合 (在步骤4.1 和4.2 中准备) 的19µL 添加到具有20µL 吸管的光学96阱反应板的分离井中。</li>
	<li>添加1µL 的 FFPE dna 或 TIC dna, 以分离含有与 2-µL 吸管的反应混合物的水井。将1µL 的无核酸酶水添加到一个单独的井中, 其中包含没有模板控制的反应组合。</li>
	<li>保持光学胶膜的非粘接面, 并从胶片的中心剥离保护背衬。轻轻地将涂抹器拖到胶片上, 并将胶片密封在96井板上。</li>
	<li>在十年代的室温下, 以2000转每分钟的温度, 用96井板式搅拌机轻轻地混合96井板。在室温下, 离心板以 1000 x g 为3分钟。</li>
	<li>对实时 PCR 仪进行电源, 并插入96井板。使用以下协议运行 PCR 反应:95 °c 二十年代, 其次45周期95°c 为 1 s 和60°c 为二十年代. 使用 "标准曲线" 和 "快速模式"。</li>
	<li>用 dna 比值 (相对量化) 评估 dna 碎片;RQ) 获得的长扩增子 (268 bp) 到短扩增子 (87 bp)。RQ 是长扩增子的平均值/短扩增子13的平均值。</li>
</ol>5. 下一代测序库的编制
<ol><li>根据制造商的说明, 为下一代测序准备序列库。</li>
	<li>在无菌离心管中制备多路 pcr 主混合体, 如下所示: 4 µL 的5x 多重 pcr 反应溶液, 4 µL 5x 引物池, ≤6µL TIC 或 FFPE DNA (1-100 ng), 并添加核酸酶免费水多达20µL。<ol>
<li>将多路 pcr 主混合添加到 pcr 管中, 通过轻敲管轻轻混合。</li>
		<li>在室温下用微型离心机将离心管在 1500 x g 处旋转5秒。</li>
	</ol>
</li>
	<li>使用以下协议运行 PCR 反应:99 °c 为2分钟, 其次20个周期99°c 为十五年代和60°c 为4分钟和保持步骤10°c。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。 注: 根据底漆对的数量确定循环次数。</li>
	<li>打开 PCR 管的盖子, 并添加2µL 的限制酶与2µL 的吸管。闭上 pcr 管的盖子, 轻轻地用 pcr 管轻拌。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。</li>
	<li>使用以下协议运行 PCR 反应:50 °c 为10分钟, 55 °c 为10分钟, 60 °c 为20分钟, 并且保持步骤10°c。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。</li>
	<li>将适配器结扎主组合添加到包含已消化 PCR amplicons 的每个井中, 如下所示: 4 µL 适配器结扎液, 0.5 µL 条码, 0.5 µL 适配器, 2 µL 无核酸酶水, 2 µL DNA 连接酶。关闭 PCR 管的盖子, 轻轻地搅拌。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。</li>
	<li>使用以下协议运行 PCR 反应:22 °c 为30分钟, 68 °c 为5分钟, 72 °c 为5分钟, 并且保持步骤10°c。</li>
	<li>根据制造商的指示, 用磁性珠子净化测序库。</li>
	<li>将适配器结扎的库解决方案转移到1.5 毫升的 DNA 低结合管中。在 1st纯化的 DNA 低结合管中添加45µL 磁性珠子。轻轻地搅拌, 在室温下孵化5分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 然后在室温下孵化2分钟, 直到溶液清晰为止。在不干扰磁性珠子的情况下, 用200µL 吸管小心地丢弃上清液。</li>
	<li>添加150µL 新鲜准备的70% 乙醇与一个200µL 的吸管, 然后移动管侧的磁铁, 以洗涤珠。小心丢弃上清, 不干扰磁性珠子。</li>
	<li>重复步骤5.11 以进行第二次清洗。</li>
	<li>在室温下, 用迷你离心机在 1500 x g 处向下旋转管, 5 秒。将 DNA 低结合管放置在磁性机架中, 用10µL 吸管小心地丢弃乙醇液滴。</li>
	<li>将50µL 的低 TE 加入到含有磁性珠子颗粒的 DNA 低结合管中, 以驱散珠子。室温下孵育2分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 在室温下孵育2分钟, 直到溶液清晰为止。</li>
	<li>将50µL 的上清液转移到新的 DNA 低结合管中, 并在 2nd纯化中添加75µL 的磁性珠子和100µL 吸管。轻轻地搅拌, 在室温下孵化5分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 然后在室温下孵育2分钟, 直到溶液清晰为止。在不干扰磁性珠子的情况下, 用200µL 吸管小心地丢弃上清液。</li>
	<li>添加150µL 新鲜准备的70% 乙醇与一个200µL 的吸管, 然后移动管侧的磁铁, 以洗涤珠。小心丢弃上清, 不干扰磁性珠子。</li>
	<li>重复步骤5.18 以进行第二次清洗。</li>
	<li>在室温下, 用微型离心机在 1500 x g 处以5秒的温度旋转管子。将 DNA 低结合管放置在磁性机架中, 用10µL 吸管小心地丢弃乙醇液滴。</li>
	<li>将50µL 的低 TE 加入到含有磁性珠子颗粒的 DNA 低结合管中, 以驱散珠子。室温下孵育2分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 在室温下孵育2分钟, 直到溶液清晰为止。</li>
	<li>将含有纯化库的45µL 的清液转移到新的 DNA 低结合管中, 用100µL 的吸管。</li>
</ol>6. 定量实时 PCR 量化图书馆集中度
<ol><li>根据制造商的说明13确定每个库的集中程度。<ol>
<li>准备一个20倍稀释溶液如下: 混合2µL 纯化的图书馆和38µL 核酸酶无水的 DNA 低结合管与2µL 和100µL 吸管。</li>
		<li>将未稀释的库存储在-20 °c, 直到步骤7.3。</li>
	</ol>
</li>
	<li>准备一个200倍稀释溶液如下: 混合5µL 的20倍稀释纯化库 (准备在步骤 6.1) 和45µL 核酸酶无水的 DNA 低结合管。</li>
	<li>准备一个2000倍稀释溶液如下: 混合5µL 的200倍稀释纯化库 (准备在步骤 6.2) 和45µL 核酸酶无水的 DNA 低结合管。</li>
	<li>准备反应大师组合如下: 混合10µL 2x 主混合溶液和1µL 20x 底漆探针检测溶液在无菌离心管与吸管, 然后混合通过攻丝管。将反应主混合的11µL 加入光学96阱反应的井中。</li>
	<li>添加9µL 2000 倍稀释库, 每标准控制9µL, 或9µL 的核酸酶水, 每井与10µL 吸管。</li>
	<li>保持光学胶膜的不粘接面, 并从胶片的中心剥离保护背衬。<ol>
<li>轻轻地将涂抹器拖到胶片上, 并将胶片密封在96井板上。</li>
		<li>在室温下, 在十年代用96井板式搅拌机轻轻混合96井板。</li>
		<li>在室温下, 离心板以 1000 x g 为3分钟。</li>
	</ol>
</li>
	<li>对实时 PCR 仪进行电源, 并插入96井板。使用以下协议运行 PCR 反应:50 °c 为2分钟, 95 °c 为二十年代, 跟随40个周期95°c 为 1 s 和60°c 为二十年代. 使用 "标准曲线" 和 "快速模式"。</li>
	<li>通过将 qPCR 确定的浓度乘以 2000, 计算未稀释的库浓度。</li>
</ol>7. 下一代测序
<ol><li>计划运行条件并在软件中设置运行参数。<ol>
<li>单击 [计划选项卡] 和 [模板], 然后选择适当的运行方法。</li>
		<li>选择应用程序和技术类型, 然后单击 [下一步]。</li>
		<li>选择仪器、样品准备套件 (可选)、库套件类型、模板套件、排序工具包、基本校准模式、芯片类型、控制序列 (可选) 和条形码集, 然后单击 [下一步]。</li>
		<li>选择插件, 然后单击 [下一步]。</li>
		<li>选择项目并单击 [下一步]。</li>
		<li>选择目标区域的默认参考和床文件。</li>
		<li>键入示例名称, 选择条形码, 然后单击 [计划运行]。</li>
	</ol>
</li>
	<li>根据制造商的指示, 在自动仪器中执行模板准备和芯片加载。使用前将试剂盒在室温下解冻45分钟。</li>
	<li>根据步骤6.8 中计算的库集中度和晚上20点的图书馆, 稀释未稀释的图书馆与无核酸酶的水。<ol>
<li>准备一个汇集库, 用于排序和储存在冰上。</li>
		<li>增加25µL 的汇集图书馆与100µL 吸管在样品管的底部。在48小时内使用池库。</li>
	</ol>
</li>
	<li>开启并打开自动仪器的盖子。<ol>
<li>将测序芯片、芯片适配器、浓缩盒、尖端墨盒、pcr 板、pcr 框架密封、回收管、溶液盒和试剂盒放到自动仪器的适当位置。</li>
		<li>在屏幕上触摸 [设置运行] 和 [逐步]。</li>
		<li>在屏幕上关闭盖子并触摸 [开始检查]。</li>
		<li>在甲板扫描过程之后, 触摸屏幕上的 [下一页]。</li>
		<li>检查显示内容 (套件类型、芯片类型、芯片 id、样本 id、计划)、设置时间和触摸屏幕上的 [确定]。</li>
	</ol>
</li>
	<li>完成芯片加载后:<ol>
<li>触摸 [下一步] 在屏幕上打开盖子。</li>
		<li>从芯片适配器卸下带手套的手的测序芯片。</li>
		<li>将芯片放入芯片容器, 说唱与 parafilm 和存储在4°c, 直到排序反应。</li>
		<li>从自动仪器的适当位置取出浓缩盒、pcr 板、pcr 框架密封、回收管、溶液盒和试剂盒, 戴手套的手。</li>
		<li>将空尖端墨盒转移到自动仪器的废料尖端位置, 戴手套的手。</li>
		<li>触摸 [下一步] 并关闭盖子。</li>
		<li>触摸 [开始] 和清洁自动仪器的紫外线4分钟。</li>
	</ol>
</li>
	<li>用0.22 µm 滤池过滤器将亚氯酸钠片溶解在1000毫升的超纯水和过滤液中。<ol>
<li>在测序仪上通电。</li>
		<li>在测序仪的屏幕上触摸 [清洁] 和 [下]。</li>
		<li>用250毫升过滤灭菌的亚氯酸钠溶液和随后250毫升的超纯水清洗测序仪。</li>
	</ol>
</li>
	<li>触摸 [初始化] 并在屏幕上选择适当的排序工具包。<ol>
<li>安装一个灰色托运人在适当的位置戴手套的手。</li>
		<li>用洗涤液 (套件中提供)、ph 调节溶液 (含350µL 100 毫米氢氧化钠) 和 ph 标准溶液 (在试剂盒中提供) 初始化测序仪。</li>
	</ol>
</li>
	<li>在50毫升的试管中添加20µL 的 dATP、dGTP、dCTP 和 dTTP 核苷酸 (在套件中提供), 用 100-µL 吸管。<ol>
<li>安装一个灰色托运人在适当的位置戴手套的手。</li>
		<li>将50毫升管和螺钉装入测序仪。</li>
		<li>触摸屏幕上的 [下一页] 开始初始化步骤, 大约需要25分钟。</li>
	</ol>
</li>
	<li>完成初始化步骤后:<ol>
<li>在屏幕上触摸 [运行] 并选择适当的库准备工具。</li>
		<li>扫描芯片的二维条码。</li>
		<li>在适当的位置插入测序芯片。</li>
		<li>关闭芯片钳和仪表门。</li>
		<li>触摸 [芯片检查], [下一页] 和 [OK] 在屏幕上开始排序运行。</li>
	</ol>
</li>
	<li>在排序反应之后, 在排序服务器13、17上传输数据并执行数据分析管道。</li>
</ol>

<ol>
	<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Path+to+Cancer+-+Three+Strikes+and+You're+Out.">The Path to Cancer - Three Strikes and You're Out.</a> N Engl J Med. 373 (20), 1895-1898 (2015).</li><li>Weinstein, J. 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+cancer+genome+atlas+pan-cancer+analysis+project.">The cancer genome atlas pan-cancer analysis project.</a> Nat. Genet. 45 (10), 1113-1120 (2013).</li><li>Nagasaki, 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=Rare+variant+discovery+by+deep+whole-genome+sequencing+of+1,070+Japanese+individuals.">Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals.</a> Nat Commun. 6 (8018), (2015).</li><li>Vogelstein, 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=Cancer+genome+landscapes.">Cancer genome landscapes.</a> Science. 339 (6127), 1546-1558 (2013).</li><li>Zehir, A., 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=Mutational+landscape+of+metastatic+cancer+revealed+from+prospective+clinical+sequencing+of+10,000+patients.">Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.</a> Nat Med. 23 (6), 703-713 (2017).</li><li>Garraway, L. A., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+the+cancer+genome.">Lessons from the cancer genome.</a> Cell. 53 (1), 17-37 (2013).</li><li>Chalkley, R., Hunter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone-histone+propinquity+by+aldehyde+fixation+of+chromatin.">Histone-histone propinquity by aldehyde fixation of chromatin.</a> Proc Natl Acad Sci U S A. 72 (4), 1304-1308 (1975).</li><li>Ben-Ezra, J., Johnson, D. A., Rossi, J., Cook, N., Wu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixation+on+the+amplification+of+nucleic+acids+from+paraffin-embedded+material+by+the+polymerase+chain+reaction.">Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.</a> J Histochem Cytochem. 39 (3), 351-354 (1991).</li><li>Srinivasan, M., Sedmak, D., Jewell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixatives+and+tissue+processing+on+the+content+and+integrity+of+nucleic+acids.">Effect of fixatives and tissue processing on the content and integrity of nucleic acids.</a> Am.J.Pathol. 161 (6), 1961-1971 (2002).</li><li>Adhya, A. K., Mohanty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+touch+imprint+cytology+in+the+preoperative+diagnosis+of+malignancy+in+low+resource+setting.">Utility of touch imprint cytology in the preoperative diagnosis of malignancy in low resource setting.</a> Diagn Cytopathol. 45 (6), 507-512 (2017).</li><li>Lumachi, F., Marino, F., Zanella, S., Chiara, G. B., Basso, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Touch+Imprint+Cytology+and+Frozen-section+Analysis+for+Intraoperative+Evaluation+of+Sentinel+Nodes+in+Early+Breast+Cancer.">Touch Imprint Cytology and Frozen-section Analysis for Intraoperative Evaluation of Sentinel Nodes in Early Breast Cancer.</a> Anticancer Research. 32 (8), 3523-3526 (2012).</li><li>Sumiyoshi, K., 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=Usefulness+of+intraoperative+touch+smear+cytology+in+breast-conserving+surgery.">Usefulness of intraoperative touch smear cytology in breast-conserving surgery.</a> Exp Ther Med. 1 (4), 641-645 (2012).</li><li>Amemiya, K., 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=Touch+imprint+cytology+with+massively+parallel+sequencing+(TIC-seq):+a+simple+and+rapid+method+to+snapshot+genetic+alterations+in+tumors.">Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.</a> Cancer Med. 5 (12), 3426-3436 (2016).</li><li>Goto, T., 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=Mutational+analysis+of+multiple+lung+cancers:+Discrimination+between+primary+and+metastatic+lung+cancers+by+genomic+profile.">Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile.</a> Oncotarget. 8 (19), 31133-31143 (2017).</li><li>Goto, T., 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=Detection+of+tumor-derived+DNA+dispersed+in+the+airway+improves+the+diagnostic+accuracy+of+bronchoscopy+for+lung+cancer.">Detection of tumor-derived DNA dispersed in the airway improves the diagnostic accuracy of bronchoscopy for lung cancer.</a> Oncotarget. 8 (45), 79404-79413 (2017).</li><li>Hirotsu, Y., 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=Targeted+and+exome+sequencing+identified+somatic+mutations+in+hepatocellular+carcinoma.">Targeted and exome sequencing identified somatic mutations in hepatocellular carcinoma.</a> Hepatol Res. 46 (11), 1145-1151 (2016).</li><li>Hirotsu, Y., 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=Comparison+between+two+amplicon-based+sequencing+panels+of+different+scales+in+the+detection+of+somatic+mutations+associated+with+gastric+cancer.">Comparison between two amplicon-based sequencing panels of different scales in the detection of somatic mutations associated with gastric cancer.</a> BMC Genomics. 17 (1), 833 (2016).</li><li>Hirotsu, Y., 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=Intrinsic+HER2+V777L+mutation+mediates+resistance+to+trastuzumab+in+a+breast+cancer+patient.">Intrinsic HER2 V777L mutation mediates resistance to trastuzumab in a breast cancer patient.</a> Med Oncol. 34 (1), 3 (2017).</li><li>Hirotsu, Y., 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=Detection+of+BRCA1+and+BRCA2+germline+mutations+in+Japanese+population+using+next-generation+sequencing.">Detection of BRCA1 and BRCA2 germline mutations in Japanese population using next-generation sequencing.</a> Mol Genet Genomic Med. 3 (2), 121-129 (2015).</li><li>Hirotsu, Y., 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=Multigene+panel+analysis+identified+germline+mutations+of+DNA+repair+genes+in+breast+and+ovarian+cancer.">Multigene panel analysis identified germline mutations of DNA repair genes in breast and ovarian cancer.</a> Mol Genet Genomic Med. 3 (5), 459-466 (2015).</li><li>Li&#232;vre, A., 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=KRAS+mutation+status+is+predictive+of+response+to+cetuximab+therapy+in+colorectal+cancer.">KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer.</a> Cancer Res. 66 (8), 3992-3995 (2006).</li><li>Mok, T. 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=Osimertinib+or+Platinum-Pemetrexed+in+EGFR+T790M-Positive+Lung+Cancer.">Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer.</a> N Engl J Med. 376 (7), 629-640 (2017).</li><li>Wong, S. Q., 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=Targeted-capture+massively-parallel+sequencing+enables+robust+detection+of+clinically+informative+mutations+from+formalin-fixed+tumours.">Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours.</a> Sci rep. 13 (3), 3493 (2013).</li><li>Wong, S. Q., 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=Sequence+artefacts+in+a+prospective+series+of+formalin-fixed+tumours+tested+for+mutations+in+hotspot+regions+by+massively+parallel+sequencing.">Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing.</a> BMC Med Genomics. 13 (7), 23 (2014).</li></ol>

作者没有什么可透露的。

在本研究中, 我们提出了一种从临床病理标本中获取肿瘤 DNA 的替代方法。TIC 准备是非常简单的, 需要较少的时间与 FFPE 方法相比, 没有特殊仪器的要求10。从 TIC 准备到 DNA 提取的所有程序都可以在两天内完成 (图 1)。因此, 这种方法缩短了进行基因检测的周转时间。值得注意的是, 这为缩短分子分析所需的天数提供了重要的优势。这一短暂的周转时间使我们?...

下一代测序技术为研究人员在基因变异、孟德尔病、遗传性倾向和癌症方面的基因组信息分析提供了重要的进展1,2,3.癌症基因图谱 (TCGA) 和国际癌症基因组联合会 (ICGC) 一直在寻找几种常见癌症的基因改变的鉴定4。数以百计的基本癌症驱动基因已被成功地确定, 其中一些分子被瞄准药物开发1,5,6。
在临床环境中, FFPE 标本通常用于各种疾病, 包括癌症的病理诊断和分子检测。然而, 在福尔马林固定过程中, dna 蛋白或 dna dna 交联发生, 导致 dna 分裂。因此, FFPE dna 样本并不总是适合遗传分析, 因为质量和数量的 dna7,8,9。此外, 准备 FFPE 标本需要数天的时间, 技术技能是准确准备剖面的必要条件。因此, 建立一种简便、快速的获取高质量完整 DNA 的方法是可取的。
细胞学是病理诊断的一种替代方法。与 FFPE 准备10相比, 细胞学样品制备方法更简单、成本更低、更快速。采用 TIC 技术对乳腺癌患者的前哨淋巴结和边缘组织进行了一些年的术中快速诊断, 其方法为11,12。然而, 很少有报告研究了高质量的基因组 DNA 是否可以从 TIC 标本中提取, 并用于后续的遗传分析。细胞学标本通常染色阴道 (Pap) 或姬姆萨染色, 我们以前报告说, 从 TIC 标本 (特别是姬姆萨染色样品) 提取的 DNA 的数量和质量优于从 FFPE 获得的样本组织13。与 Pap 染色相比, 姬姆萨染色在要求较少染色的过程中具有优势。在 Pap 染色, 样品被固定和染色后, 必须安装在安装培养基 (例如, Malinol), 以区分样本内容, 如肿瘤细胞, 正常细胞和炎症细胞在显微镜下。如果在没有安装步骤的情况下制备 Pap 标本, 在显微镜下观察细胞几乎是不可能的, 因为试样是干的。相比之下, 姬姆萨染色可以观察到干燥状态, 因此, 安装步骤是不必要的快速细胞评估。对于显微切割, 姬姆萨染色更适合, 因为它需要干燥标本。
在本报告中, 我们介绍了一个简单快速的方法来制备 tic 标本与姬姆萨染色, 并表明 TIC 是一个更好的来源的 DNA 比较 FFPE 标本。

<table border="0" cellpadding="0" cellspacing="0" width="796">
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			<td height="41" style="height:41px;width:283px;">FINE FROST white20 micro slide glass</td>
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			<td height="41" style="height:41px;">Arcturus PEN Membrane Glass Slides&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>LCM0522</td>
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			<td height="41" style="height:41px;">Cyto Quick A solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20571</td>
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			<td height="41" style="height:41px;">Cyto Quick B solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20581</td>
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			<td height="41" style="height:41px;">May-Grunwald Solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15053</td>
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			<td height="41" style="height:41px;">Giemsa solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15002</td>
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			<td height="41" style="height:41px;">QIAamp DNA FFPE tissue kit</td>
			<td>Qiagen</td>
			<td>56404</td>
			<td></td>
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			<td height="41" style="height:41px;">TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444557</td>
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			<td height="41" style="height:41px;">TaqMan RNase P Detection Reagents Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316831</td>
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			<td height="41" style="height:41px;">TaqMan Assay from FFPE DNA QC Assay v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324034</td>
			<td></td>
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			<td height="41" style="height:41px;">MicroAmp Fast Optical 96-Well Reaction Plate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4346907</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp optical Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4311971</td>
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			<td height="41" style="height:41px;width:283px;">MicroMixer E36</td>
			<td style="width:191px;">TITEC</td>
			<td style="width:143px;">0027765-000</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:283px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">VIIA7-03</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Himac CF16RXII</td>
			<td style="width:191px;">Hitachi-koki</td>
			<td style="width:143px;">CF16RII</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion Library TaqMan Quantitation Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4468802</td>
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			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Cancer Hotspot Panel v2</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4475346</td>
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			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Library Kit 2.0</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4480442</td>
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			<td height="41" style="height:41px;width:283px;">Ion Xpress Barcode Adapters 1-16 Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4471250</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Hi-Q View Sequencing Kit (200 base)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">A30044</td>
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			<td height="41" style="height:41px;width:283px;">Ion Chef System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4484177</td>
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			<td height="41" style="height:41px;width:283px;">Veriti 96-well Thermal Cycler</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>Veriti200</td>
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			<td height="41" style="height:41px;width:283px;">Ion 318 Chip Kit v2 BC</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4488150</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>PGM11-001</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Wash 2 Bottle kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>A25591</td>
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			<td style="width:143px;">A63881</td>
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			<td height="41" style="height:41px;width:283px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM12450</td>
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			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM9938</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Optical 96-well Reaction Plates</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306737</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Clear Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306311</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:143px;">A63881</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ethanol(99.5)</td>
			<td style="width:191px;">Nacalai Tesque</td>
			<td style="width:143px;">08948-25</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Sodium hydroxide (10M)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:143px;">72068</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">DTU-Neo</td>
			<td style="width:191px;">TAITEC</td>
			<td style="width:143px;">0063286-000</td>
			<td></td>
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		<tr height="41">
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		<tr height="41">
			<td height="41" style="height:41px;width:283px;">ECLIPSE Ci-L</td>
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			<td style="width:143px;">704354</td>
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		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-2XLS+</td>
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			<td>17014393</td>
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		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-10XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014388</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014392</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-100XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014384</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014391</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014382</td>
			<td style="width:180px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;">petit-change</td>
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			<td>MODEL8864</td>
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		<tr height="41">
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		<tr height="41">
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			<td style="width:191px;">Osaki</td>
			<td style="width:143px;">11138</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Refrigerator MediCool</td>
			<td style="width:191px;">SANYO</td>
			<td style="width:143px;">MPR-312DCN-PJ</td>
			<td style="width:180px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.130</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.260</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER&#160; S</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">FA-10</td>
			<td style="width:180px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Vortex Genius 3</td>
			<td style="width:191px;">IKA</td>
			<td>41-0458&#160;</td>
			<td style="width:180px;">Vortex mixer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Pincette</td>
			<td style="width:191px;">NATSUME</td>
			<td style="width:143px;">A-5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">1.5 mL microtube</td>
			<td style="width:191px;">BIOBIK</td>
			<td style="width:143px;">RC-0150</td>
			<td style="width:180px;"></td>
		</tr>
	</tbody>
</table>

simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology

在癌症患者的特定分子靶向药物的治疗前, 确定肿瘤的突变状态是至关重要的。在临床环境中, 福尔马林固定石蜡 (FFPE) 组织广泛用于基因检测。然而, FFPE DNA 在福尔马林固定过程中通常会受到损伤和分裂。因此, 由于 dna 质量和数量的降低, FFPE dna 有时不足以进行基因检测。在这里, 我们提出了触摸印记细胞学 (TIC) 的方法, 以获得肿瘤细胞的基因组 DNA, 可以在显微镜下观察到。细胞形态学和癌细胞数量可以用 TIC 标本来评估。此外, 从 TIC 样品中提取基因组 DNA 可以在两天内完成。利用该方法获得的 TIC dna 的总量和质量均高于 FFPE dna。这种快速简便的方法使研究人员能够获得高质量的基因检测 DNA (例如、下一代测序分析、数字 pcr 和定量实时 pcr), 并缩短报告结果的周转时间。

图 1显示了从准备 TIC 标本到 DNA 提取的整个过程。值得注意的是, 该手术仅需两天时间才能从 TIC 样品中获得基因组 DNA。我们评估了在滑动处理前肿瘤贮存的任何影响。我们发现, 当组织标本立即触及到幻灯片上时, 肿瘤细胞附着在玻璃滑梯上, 当组织被保存在生理盐水湿润的无菌纱布中时, 1 小时 (图 2)。然而, 当组织保持在室温...

Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

从肿瘤组织获得高质量的基因组 DNA 是利用下一代测序分析基因改变的重要第一步。本文提出一种简便、快速的方法来丰富肿瘤细胞, 从接触印记细胞学标本中获得完整的 DNA。

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

simple-rapid-method-to-obtain-high-quality-tumor-dna-from-clinical

Research

JoVE Journal

Cancer Research

10.8K 次观看.  Yamanashi Central Hospital. 从肿瘤组织获得高质量的基因组 DNA 是利用下一代测序分析基因改变的重要第一步。本文提出一种简便、快速的方法来丰富肿瘤细胞, 从接触印记细胞学标本中获得完整的 DNA。

视频: Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

捕获和血液中的活循环肿瘤细胞释放

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

22C3 Anti-PD-L1 抗体精矿在非小细胞肺癌活检及细胞学标本中的应用

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

用于肿瘤球体半高通量分析的快速光学清除

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

摘要

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此视频中的章节

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

捕获和血液中的活循环肿瘤细胞释放

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

22C3 Anti-PD-L1 抗体精矿在非小细胞肺癌活检及细胞学标本中的应用

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

患者生物流体中肿瘤相关循环DNA的检测与监测

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

用于肿瘤球体半高通量分析的快速光学清除

来自切除人肿瘤标本的离体器官型腹膜假粘液瘤肿瘤切片的培养和成像

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

摘要

探索更多视频

此视频中的章节

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

捕获和血液中的活循环肿瘤细胞释放

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

22C3 Anti-PD-L1 抗体精矿在非小细胞肺癌活检及细胞学标本中的应用

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

患者生物流体中肿瘤相关循环DNA的检测与监测

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

用于肿瘤球体半高通量分析的快速光学清除

来自切除人肿瘤标本的 离体 器官型腹膜假粘液瘤肿瘤切片的培养和成像

来自切除人肿瘤标本的离体器官型腹膜假粘液瘤肿瘤切片的培养和成像