Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

Abstract The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient’s tumour status, chances of survival, and response to therapy from serum or plasma samp...

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Autores principales:	T. Ehlert, S. Tug, A. Brahmer, V. Neef, F. Heid, C. Werner, B. Jansen-Winkeln, W. Kneist, H. Lang, I. Gockel, P. Simon
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/deef6877bf8c477fbd96d8ff951df021
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spelling	oai:doaj.org-article:deef6877bf8c477fbd96d8ff951df0212021-12-02T12:32:54ZEstablishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection10.1038/s41598-017-09137-w2045-2322https://doaj.org/article/deef6877bf8c477fbd96d8ff951df0212017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-09137-whttps://doaj.org/toc/2045-2322Abstract The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient’s tumour status, chances of survival, and response to therapy from serum or plasma samples. We established PNB-qPCR (Pooled, Nested, WT-Blocking qPCR), a highly specific nested qPCR with various modifications to detect and quantify minute amounts of circulating tumour DNA (ctDNA) from very limited blood plasma samples. PNB-qPCR is a nested qPCR technique combining ARMS primers, blocking primers, LNA probes, and pooling of multiple first round products for sensitive quantification of the seven most frequent point mutations in KRAS exon 2. Using this approach, we were able to characterize ctDNA and total cell-free DNA (cfDNA) kinetics by selective amplification of KRAS mutated DNA fragments in the blood plasma over the course of tumour resection and the surrounding days. Whereas total cfDNA concentrations increased over the surgical and regenerative process, ctDNA levels showed a different scheme, rising only directly after tumour resection and about three days after the surgery. For the first time, we present insights into the impact of surgery on the release of ctDNA and total cfDNA.T. EhlertS. TugA. BrahmerV. NeefF. HeidC. WernerB. Jansen-WinkelnW. KneistH. LangI. GockelP. SimonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q T. Ehlert S. Tug A. Brahmer V. Neef F. Heid C. Werner B. Jansen-Winkeln W. Kneist H. Lang I. Gockel P. Simon Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection
description	Abstract The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient’s tumour status, chances of survival, and response to therapy from serum or plasma samples. We established PNB-qPCR (Pooled, Nested, WT-Blocking qPCR), a highly specific nested qPCR with various modifications to detect and quantify minute amounts of circulating tumour DNA (ctDNA) from very limited blood plasma samples. PNB-qPCR is a nested qPCR technique combining ARMS primers, blocking primers, LNA probes, and pooling of multiple first round products for sensitive quantification of the seven most frequent point mutations in KRAS exon 2. Using this approach, we were able to characterize ctDNA and total cell-free DNA (cfDNA) kinetics by selective amplification of KRAS mutated DNA fragments in the blood plasma over the course of tumour resection and the surrounding days. Whereas total cfDNA concentrations increased over the surgical and regenerative process, ctDNA levels showed a different scheme, rising only directly after tumour resection and about three days after the surgery. For the first time, we present insights into the impact of surgery on the release of ctDNA and total cfDNA.
format	article
author	T. Ehlert S. Tug A. Brahmer V. Neef F. Heid C. Werner B. Jansen-Winkeln W. Kneist H. Lang I. Gockel P. Simon
author_facet	T. Ehlert S. Tug A. Brahmer V. Neef F. Heid C. Werner B. Jansen-Winkeln W. Kneist H. Lang I. Gockel P. Simon
author_sort	T. Ehlert
title	Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection
title_short	Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection
title_full	Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection
title_fullStr	Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection
title_full_unstemmed	Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection
title_sort	establishing pnb-qpcr for quantifying minimal ctdna concentrations during tumour resection
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/deef6877bf8c477fbd96d8ff951df021
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Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

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