Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer
by
Martin Metzenmacher 1,2
, Renáta Váraljai 3,4, Balazs Hegedüs 5, Igor Cima 4,6, Jan Forster 4,7, Alexander Schramm 8, Björn Scheffler 4,6, Peter A. Horn 9, Christoph A. Klein 10,11, Tibor Szarvas 12,13, Hennig Reis 14, Nicola Bielefeld 4,15, Alexander Roesch 3,4, Clemens Aigner 5, Volker Kunzmann 16, Marcel Wiesweg 1,2, Jens T. Siveke 4,15, Martin Schuler 1,2,4 and Smiths S. Lueong 4,15,*
Martin Metzenmacher 1,2, Renáta Váraljai 3,4, Balazs Hegedüs 5, Igor Cima 4,6, Jan Forster 4,7, Alexander Schramm 8, Björn Scheffler 4,6, Peter A. Horn 9, Christoph A. Klein 10,11, Tibor Szarvas 12,13, Hennig Reis 14, Nicola Bielefeld 4,15, Alexander Roesch 3,4, Clemens Aigner 5, Volker Kunzmann 16, Marcel Wiesweg 1,2, Jens T. Siveke 4,15, Martin Schuler 1,2,4 and Smiths S. Lueong 4,15,*
1
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
2
Division of Thoracic Oncology, University Medicine Essen-Ruhrlandklinik, University Duisburg-Essen, Tüschener Weg 40, 45239 Essen, Germany
3
Department of Dermatology, University Hospital Essen, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
4
German Cancer Consortium (DKTK), Partner site University Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany
5
Department of Thoracic Surgery, Ruhrlandklinik, University Duisburg-Essen, D-45239 Essen, Germany
6
DKFZ-Division Translational Neurooncology at the WTZ, DKTK partner site, University Hospital Essen, 45122 Essen, Germany
7
Chair for Genome Informatics, Department of Human Genetics, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
8
Laboratory for Molecular Oncology, Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, 45122 Essen, Germany
9
Institute for Transfusion Medicine, University Hospital Essen, 45122 Essen, Germany
10
Experimental Medicine and Therapy Research, University of Regensburg, 93053 Regensburg, Germany
11
Fraunhofer-Institute for Toxicology and Experimental Medicine, Division of Personalized Tumour Therapy, 93053 Regensburg, Germany
12
Department of Urology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany
13
Department of Urology, Semmelweis University, H-1085 Budapest, Hungary
14
Institute of Pathology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany
15
Institute for Developmental Cancer Therapeutics, West German Cancer Center, University Hospital Essen, 45122 Essen, Germany
16
Department of Internal Medicine II, University Hospital Würzburg, 97080 Würzburg, Germany
*
Author to whom correspondence should be addressed.
Cancers 2020, 12(2), 353; https://doi.org/10.3390/cancers12020353
Received: 27 December 2019 / Revised: 29 January 2020 / Accepted: 31 January 2020 / Published: 4 February 2020
(This article belongs to the Collection Cancer Biomarkers)
Early detection of cancer holds high promise for reducing cancer-related mortality. Detection of circulating tumor-specific nucleic acids holds promise, but sensitivity and specificity issues remain with current technology. We studied cell-free RNA (cfRNA) in patients with non-small cell lung cancer (NSCLC; n = 56 stage IV, n = 39 stages I-III), pancreatic cancer (PDAC, n = 20 stage III), malignant melanoma (MM, n = 12 stage III-IV), urothelial bladder cancer (UBC, n = 22 stage II and IV), and 65 healthy controls by means of next generation sequencing (NGS) and real-time droplet digital PCR (RT-ddPCR). We identified 192 overlapping upregulated transcripts in NSCLC and PDAC by NGS, more than 90% of which were noncoding. Previously reported transcripts (e.g., HOTAIRM1) were identified. Plasma cfRNA transcript levels of POU6F2-AS2 discriminated NSCLC from healthy donors (AUC = 0.82 and 0.76 for stages IV and I–III, respectively) and significantly associated (p = 0.017) with the established tumor marker Cyfra 21-1. cfRNA yield and POU6F2-AS transcript abundance discriminated PDAC patients from healthy donors (AUC = 1.0). POU6F2-AS2 transcript was significantly higher in MM (p = 0.044). In summary, our findings support further validation of cfRNA detection by RT-ddPCR as a biomarker for early detection of solid cancers.
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Keywords:
liquid biopsy; cfRNA; cancer; ddPCR; NGS; POU6F2-AS2; early detection
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MDPI and ACS Style
Metzenmacher, M.; Váraljai, R.; Hegedüs, B.; Cima, I.; Forster, J.; Schramm, A.; Scheffler, B.; Horn, P.A.; Klein, C.A.; Szarvas, T.; Reis, H.; Bielefeld, N.; Roesch, A.; Aigner, C.; Kunzmann, V.; Wiesweg, M.; Siveke, J.T.; Schuler, M.; Lueong, S.S. Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer. Cancers 2020, 12, 353.
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