Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR.
Sci Rep
; 10(1): 12332, 2020 07 23.
Article
en En
| MEDLINE
| ID: mdl-32704002
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.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Neoplasias Pancreáticas
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Reacción en Cadena de la Polimerasa
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Proteínas Proto-Oncogénicas p21(ras)
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Mutación
Tipo de estudio:
Guideline
Idioma:
En
Revista:
Sci Rep
Año:
2020
Tipo del documento:
Article