Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.

<h4>Background</h4>Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q...

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Autores principales:	Vassiliki Kotoula, Elpida Charalambous, Bart Biesmans, Andigoni Malousi, Eleni Vrettou, George Fountzilas, George Karkavelas
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e241b17456aa4daf9e42a247a3ac19c2
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spelling	oai:doaj.org-article:e241b17456aa4daf9e42a247a3ac19c22021-11-25T06:28:19ZTargeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.1932-620310.1371/journal.pone.0007746https://doaj.org/article/e241b17456aa4daf9e42a247a3ac19c22009-11-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19888477/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.<h4>Methodology/principal findings</h4>Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect<or=1% mutant cells, provided that samples yielded cycle thresholds (Ct)<29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).<h4>Conclusions/significance</h4>Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.Vassiliki KotoulaElpida CharalambousBart BiesmansAndigoni MalousiEleni VrettouGeorge FountzilasGeorge KarkavelasPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 11, p e7746 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Vassiliki Kotoula Elpida Charalambous Bart Biesmans Andigoni Malousi Eleni Vrettou George Fountzilas George Karkavelas Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
description	<h4>Background</h4>Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.<h4>Methodology/principal findings</h4>Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect<or=1% mutant cells, provided that samples yielded cycle thresholds (Ct)<29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).<h4>Conclusions/significance</h4>Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.
format	article
author	Vassiliki Kotoula Elpida Charalambous Bart Biesmans Andigoni Malousi Eleni Vrettou George Fountzilas George Karkavelas
author_facet	Vassiliki Kotoula Elpida Charalambous Bart Biesmans Andigoni Malousi Eleni Vrettou George Fountzilas George Karkavelas
author_sort	Vassiliki Kotoula
title	Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
title_short	Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
title_full	Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
title_fullStr	Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
title_full_unstemmed	Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
title_sort	targeted kras mutation assessment on patient tumor histologic material in real time diagnostics.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/e241b17456aa4daf9e42a247a3ac19c2
work_keys_str_mv	AT vassilikikotoula targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics AT elpidacharalambous targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics AT bartbiesmans targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics AT andigonimalousi targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics AT elenivrettou targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics AT georgefountzilas targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics AT georgekarkavelas targetedkrasmutationassessmentonpatienttumorhistologicmaterialinrealtimediagnostics
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Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.

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