Peitz, Constantin, Spruessel, Annika, Linke, Rasmus B., Astrahantseff, Kathy, Grimaldi, Maddalena, Schmelz, Karin, Toedling, Joern, Schulte, Johannes H., Fischer, Matthias, Messerschmidt, Clemens ORCID: 0000-0001-8632-656X, Beule, Dieter, Keilholz, Ulrich, Eggert, Angelika ORCID: 0000-0003-3476-8184, Deubzer, Hedwig E. and Lodrini, Marco (2020). Multiplexed Quantification of Four Neuroblastoma DNA Targets in a Single Droplet Digital PCR Reaction. J. Mol. Diagn., 22 (11). S. 1309 - 1324. NEW YORK: ELSEVIER SCIENCE INC. ISSN 1943-7811

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Abstract

The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALK(F1174L) (exon 23 position 3522, C>A) and ALK(R1275Q) (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Peitz, ConstantinUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Spruessel, AnnikaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Linke, Rasmus B.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Astrahantseff, KathyUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Grimaldi, MaddalenaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Schmelz, KarinUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Toedling, JoernUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Schulte, Johannes H.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Fischer, MatthiasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Messerschmidt, ClemensUNSPECIFIEDorcid.org/0000-0001-8632-656XUNSPECIFIED
Beule, DieterUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Keilholz, UlrichUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Eggert, AngelikaUNSPECIFIEDorcid.org/0000-0003-3476-8184UNSPECIFIED
Deubzer, Hedwig E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lodrini, MarcoUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-313799
DOI: 10.1016/j.jmoldx.2020.07.006
Journal or Publication Title: J. Mol. Diagn.
Volume: 22
Number: 11
Page Range: S. 1309 - 1324
Date: 2020
Publisher: ELSEVIER SCIENCE INC
Place of Publication: NEW YORK
ISSN: 1943-7811
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
ANAPLASTIC LYMPHOMA KINASE; CIRCULATING TUMOR DNA; IN-SITU HYBRIDIZATION; LARGE-CELL LYMPHOMA; MYCN AMPLIFICATION; N-MYC; ACTIVATING MUTATIONS; ALK KINASE; COPY; HETEROGENEITYMultiple languages
PathologyMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/31379

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