Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
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使用数字 PCR 从细针抽吸物中直接扩增和捕获最少量胰腺肿瘤衍生突变的省时方法
DOI：
10.1038/s41598-020-69221-6
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发表时间：
2020
影响因子：
4.6
通讯作者：
Toshikatsu Okumura
中科院分区：
文献类型：
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作者：
Yusuke Ono;Akihiro Hayashi;Chiho Maeda;Mayumi Suzuki;Reona Wada;Hiroki Sato;Hidemasa Kawabata;Tetsuhiro Okada;Takuma Goto;Hidenori Karasaki;Yusuke Mizukami;Toshikatsu Okumura
利用微量的肿瘤液体和组织碎片进行细胞病理学诊断是具有挑战性的。因此，我们开发了一种快速、准确、低成本的方法，用于从有限数量的肿瘤样本和低肿瘤细胞度的样本中检测肿瘤细胞来源的DNA，使用数字PCR （dPCR）检测胰腺导管癌（PDA）中的kras突变。核心发明是基于将肿瘤样品悬浮在纯水中，从而引起渗透爆裂；粗悬液可直接在平台上进行乳状PCR。我们使用手术切除的胰腺肿瘤标本（n = 12）的针抽吸器来检验该方法的可行性。我们成功地扩增并检测了12个携带突变的肿瘤样本中的突变体krasin 11；阳性突变频率低至0.8%。我们使用细针抽吸/活检和针冲洗过程的残余标本（n = 10）进行方法验证。在10个致癌基因的胰腺肿瘤样本中，有9个“水爆裂”方法产生了阳性突变。我们描述了一种基于dpcr的超灵敏筛选方案，用于确定krasmutation的可用性，该方案使用简单步骤处理的pda的微小针抽吸器。这种方法可能使病理学家能够使用微小的组织碎片获得更准确的微创诊断。
It is challenging to secure a cytopathologic diagnosis using minute amounts of tumor fluids and tissue fragments. Hence, we developed a rapid, accurate, low-cost method for detecting tumor cell-derived DNA from limited amounts of specimens and samples with a low tumor cellularity, to detectKRASmutations in pancreatic ductal carcinomas (PDA) using digital PCR (dPCR). The core invention is based on the suspension of tumor samples in pure water, which causes an osmotic burst; the crude suspension could be directly subjected to emulsion PCR in the platform. We examined the feasibility of this process using needle aspirates from surgically resected pancreatic tumor specimens (n = 12). We successfully amplified and detected mutantKRASin 11 of 12 tumor samples harboring the mutation; the positive mutation frequency was as low as 0.8%. We used residual specimens from fine-needle aspiration/biopsy and needle flush processes (n = 10) for method validation. In 9 of 10 oncogenicKRASpancreatic tumor samples, the "water-burst" method resulted in a positive mutation call. We describe a dPCR-based, super-sensitive screening protocol for determiningKRASmutation availability using tiny needle aspirates from PDAs processed using simple steps. This method might enable pathologists to secure a more accurate, minimally invasive diagnosis using minute tissue fragments.