Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR

Abstract 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 detect...

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Autores principales:	Yusuke Ono, Akihiro Hayashi, Chiho Maeda, Mayumi Suzuki, Reona Wada, Hiroki Sato, Hidemasa Kawabata, Tetsuhiro Okada, Takuma Goto, Hidenori Karasaki, Yusuke Mizukami, Toshikatsu Okumura
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/44da39f9b2d04b38a658977559d6f81c
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spelling	oai:doaj.org-article:44da39f9b2d04b38a658977559d6f81c2021-12-02T16:26:36ZTime-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR10.1038/s41598-020-69221-62045-2322https://doaj.org/article/44da39f9b2d04b38a658977559d6f81c2020-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-69221-6https://doaj.org/toc/2045-2322Abstract 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 detect KRAS mutations 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 mutant KRAS in 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 oncogenic KRAS pancreatic tumor samples, the "water-burst" method resulted in a positive mutation call. We describe a dPCR-based, super-sensitive screening protocol for determining KRAS mutation 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.Yusuke OnoAkihiro HayashiChiho MaedaMayumi SuzukiReona WadaHiroki SatoHidemasa KawabataTetsuhiro OkadaTakuma GotoHidenori KarasakiYusuke MizukamiToshikatsu OkumuraNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-10 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yusuke Ono Akihiro Hayashi Chiho Maeda Mayumi Suzuki Reona Wada Hiroki Sato Hidemasa Kawabata Tetsuhiro Okada Takuma Goto Hidenori Karasaki Yusuke Mizukami Toshikatsu Okumura Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
description	Abstract 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 detect KRAS mutations 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 mutant KRAS in 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 oncogenic KRAS pancreatic tumor samples, the "water-burst" method resulted in a positive mutation call. We describe a dPCR-based, super-sensitive screening protocol for determining KRAS mutation 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.
format	article
author	Yusuke Ono Akihiro Hayashi Chiho Maeda Mayumi Suzuki Reona Wada Hiroki Sato Hidemasa Kawabata Tetsuhiro Okada Takuma Goto Hidenori Karasaki Yusuke Mizukami Toshikatsu Okumura
author_facet	Yusuke Ono Akihiro Hayashi Chiho Maeda Mayumi Suzuki Reona Wada Hiroki Sato Hidemasa Kawabata Tetsuhiro Okada Takuma Goto Hidenori Karasaki Yusuke Mizukami Toshikatsu Okumura
author_sort	Yusuke Ono
title	Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
title_short	Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
title_full	Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
title_fullStr	Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
title_full_unstemmed	Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR
title_sort	time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital pcr
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/44da39f9b2d04b38a658977559d6f81c
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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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