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Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples
The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor’s temporal and spatial het...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10185647/ https://www.ncbi.nlm.nih.gov/pubmed/37097304 http://dx.doi.org/10.1007/s00216-023-04696-6 |
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author | Oliveira, Beatriz B. Costa, Beatriz Morão, Barbara Faias, Sandra Veigas, Bruno Pereira, Lucília Pebre Albuquerque, Cristina Maio, Rui Cravo, Marília Fernandes, Alexandra R. Baptista, Pedro Viana |
author_facet | Oliveira, Beatriz B. Costa, Beatriz Morão, Barbara Faias, Sandra Veigas, Bruno Pereira, Lucília Pebre Albuquerque, Cristina Maio, Rui Cravo, Marília Fernandes, Alexandra R. Baptista, Pedro Viana |
author_sort | Oliveira, Beatriz B. |
collection | PubMed |
description | The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor’s temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-023-04696-6. |
format | Online Article Text |
id | pubmed-10185647 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-101856472023-05-17 Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples Oliveira, Beatriz B. Costa, Beatriz Morão, Barbara Faias, Sandra Veigas, Bruno Pereira, Lucília Pebre Albuquerque, Cristina Maio, Rui Cravo, Marília Fernandes, Alexandra R. Baptista, Pedro Viana Anal Bioanal Chem Research Paper The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor’s temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-023-04696-6. Springer Berlin Heidelberg 2023-04-25 2023 /pmc/articles/PMC10185647/ /pubmed/37097304 http://dx.doi.org/10.1007/s00216-023-04696-6 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 | Research Paper Oliveira, Beatriz B. Costa, Beatriz Morão, Barbara Faias, Sandra Veigas, Bruno Pereira, Lucília Pebre Albuquerque, Cristina Maio, Rui Cravo, Marília Fernandes, Alexandra R. Baptista, Pedro Viana Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples |
title | Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples |
title_full | Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples |
title_fullStr | Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples |
title_full_unstemmed | Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples |
title_short | Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples |
title_sort | combining the amplification refractory mutation system and high-resolution melting analysis for kras mutation detection in clinical samples |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10185647/ https://www.ncbi.nlm.nih.gov/pubmed/37097304 http://dx.doi.org/10.1007/s00216-023-04696-6 |
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