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Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients

A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the flu...

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Autores principales: Tanaka, Junko, Nakagawa, Tatsuo, Harada, Kunio, Morizane, Chigusa, Tanaka, Hidenori, Shiba, Satoshi, Ohba, Akihiro, Hijioka, Susumu, Takai, Erina, Yachida, Shinichi, Kamura, Yoshio, Ishida, Takeshi, Yokoi, Takahide, Uematsu, Chihiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9944920/
https://www.ncbi.nlm.nih.gov/pubmed/36810451
http://dx.doi.org/10.1038/s41598-023-30131-y
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author Tanaka, Junko
Nakagawa, Tatsuo
Harada, Kunio
Morizane, Chigusa
Tanaka, Hidenori
Shiba, Satoshi
Ohba, Akihiro
Hijioka, Susumu
Takai, Erina
Yachida, Shinichi
Kamura, Yoshio
Ishida, Takeshi
Yokoi, Takahide
Uematsu, Chihiro
author_facet Tanaka, Junko
Nakagawa, Tatsuo
Harada, Kunio
Morizane, Chigusa
Tanaka, Hidenori
Shiba, Satoshi
Ohba, Akihiro
Hijioka, Susumu
Takai, Erina
Yachida, Shinichi
Kamura, Yoshio
Ishida, Takeshi
Yokoi, Takahide
Uematsu, Chihiro
author_sort Tanaka, Junko
collection PubMed
description A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity.
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spelling pubmed-99449202023-02-23 Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients Tanaka, Junko Nakagawa, Tatsuo Harada, Kunio Morizane, Chigusa Tanaka, Hidenori Shiba, Satoshi Ohba, Akihiro Hijioka, Susumu Takai, Erina Yachida, Shinichi Kamura, Yoshio Ishida, Takeshi Yokoi, Takahide Uematsu, Chihiro Sci Rep Article A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity. Nature Publishing Group UK 2023-02-21 /pmc/articles/PMC9944920/ /pubmed/36810451 http://dx.doi.org/10.1038/s41598-023-30131-y Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Tanaka, Junko
Nakagawa, Tatsuo
Harada, Kunio
Morizane, Chigusa
Tanaka, Hidenori
Shiba, Satoshi
Ohba, Akihiro
Hijioka, Susumu
Takai, Erina
Yachida, Shinichi
Kamura, Yoshio
Ishida, Takeshi
Yokoi, Takahide
Uematsu, Chihiro
Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients
title Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients
title_full Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients
title_fullStr Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients
title_full_unstemmed Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients
title_short Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients
title_sort efficient and accurate kras genotyping using digital pcr combined with melting curve analysis for ctdna from pancreatic cancer patients
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9944920/
https://www.ncbi.nlm.nih.gov/pubmed/36810451
http://dx.doi.org/10.1038/s41598-023-30131-y
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