Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts
Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts
复制标题
多重数字 PCR 检测可通过最小样本量检测与胰腺癌发生相关的多种 KRAS 和 GNAS 突变
DOI：
10.1016/j.jmoldx.2023.02.007
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Liss Andre
中科院分区：
文献类型：
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作者：
Maeda Chiho;Ono Yusuke;Hayashi Akihiro;Takahashi Kenji;Taniue Kenzui;Kakisaka Rika;Mori Miyuki;Ishii Takahiro;Sato Hiroki;Okada Tetsuhiro;Kawabata Hidemasa;Goto Takuma;Tamamura Nobue;Omori Yuko;Takahashi Kuniyuki;Katanuma Akio;Karasaki Hidenori;Liss Andre
数字PCR（dPCR）允许对低频突变进行高灵敏度定量，并有助于癌症的早期检测。然而，当使用多个探针时，dPCR中单个热点的低通量靶向阻碍了变体特异性。我们开发了一种dPCR方法来同时鉴定与胰腺癌发生相关的主要变异。使用在两种荧光探针池的优化浓度下的液滴荧光的二维图，确定不同KRA和GNAS变体的绝对定量。在来自19名患者的24份手术切除的肿瘤样本和来自胰腺导管腺癌患者的22份细针穿刺样本中验证了多个驱动突变的成功检测。通过使用浓度低至1至10 ng的模板DNA，优化了变异等位基因频率的精确定量。此外，使用高保真聚合酶成功富集了靶向多个热点的扩增子，具有较少的假阳性结果，允许在少量细胞/组织标本中以高概率检测各种KRA和GNAS突变。使用这种靶点富集，在小的残留组织中，如细针抽吸针冲洗和切除标本中的显微病变，突变率为90%，被成功地确定。所提出的方法允许低成本、准确地检测驱动突变以诊断癌症，即使收集的组织最少。
Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low-throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. We developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a two-dimensional plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, the absolute quantification of differentKRASandGNASvariants was determined. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 fine-needle aspiration samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized by using template DNA at a concentration as low as 1 to 10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false-positive findings using high-fidelity polymerase, allowing for the detection of variousKRASandGNASmutations with high probability in small amount of cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the fine-needle aspiration needle flush and microscopic lesions in resected specimens, were successfully identified. The proposed method allows for low-cost, accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.