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Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples

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dc.contributor.authorOliveira, Beatriz B.
dc.contributor.authorCosta, Beatriz
dc.contributor.authorMorão, Barbara
dc.contributor.authorFaias, Sandra
dc.contributor.authorVeigas, Bruno
dc.contributor.authorPereira, Lucília Pebre
dc.contributor.authorAlbuquerque, Cristina
dc.contributor.authorMaio, Rui
dc.contributor.authorCravo, Marília
dc.contributor.authorFernandes, Alexandra R.
dc.contributor.authorBaptista, Pedro Viana
dc.date.accessioned2023-04-27T14:18:43Z
dc.date.available2023-04-27T14:18:43Z
dc.date.issued2023
dc.description© The Author(s) 2023. 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/.pt_PT
dc.description.abstractThe 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.pt_PT
dc.description.sponsorshipThis work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. FCT-MCTES is also acknowledged for 2020.07660.BD for BBO. Open access funding provided by FCT|FCCN (b-on).pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationAnal Bioanal Chem. 2023 Apr 25pt_PT
dc.identifier.doi10.1007/s00216-023-04696-6pt_PT
dc.identifier.eissn1618-2650
dc.identifier.issn1618-2642
dc.identifier.urihttp://hdl.handle.net/10451/57280
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherSpringer Naturept_PT
dc.relationApplied Molecular Biosciences Unit
dc.relationApplied Molecular Biosciences Unit
dc.relationSimplified chip prototyping for improved screening of gene-silencing therapeutics using 3D cell models.
dc.relation.publisherversionhttps://www.springer.com/journal/216pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectARMS-HRMApt_PT
dc.subjectCirculating tumor DNApt_PT
dc.subjectMutation detectionpt_PT
dc.subjectPancreatic cancerpt_PT
dc.titleCombining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samplespt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleApplied Molecular Biosciences Unit
oaire.awardTitleApplied Molecular Biosciences Unit
oaire.awardTitleSimplified chip prototyping for improved screening of gene-silencing therapeutics using 3D cell models.
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F04378%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04378%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/OE/2020.07660.BD/PT
oaire.citation.titleAnalytical and Bioanalytical Chemistrypt_PT
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStreamOE
person.familyNameCravo
person.givenNameMarília
person.identifier.orcid0000-0001-8309-4599
person.identifier.scopus-author-id35551480200
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublication01f30332-6fe1-4d53-9f07-905ec4697d78
relation.isAuthorOfPublication.latestForDiscovery01f30332-6fe1-4d53-9f07-905ec4697d78
relation.isProjectOfPublicationae38cc55-a200-4d0c-8c45-ec3fcb87b03e
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relation.isProjectOfPublication295bb210-9a0a-4141-9843-bedd00d6945f
relation.isProjectOfPublication.latestForDiscovery5d9bae41-3108-4dac-b589-fbd8b25249a2

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