Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer

Metzenmacher, Martin GND; Váraljai, Renáta GND; Hegedüs, Balazs GND; Cima, Igor; Forster, Jan; Schramm, Alexander GND; Scheffler, Björn GND; Horn, Peter A. GND; Klein, Christoph GND; Szarvas, Tibor GND; Reis, Henning GND; Bielefeld, Nicola GND; Rösch, Alexander GND; Aigner, Clemens GND; Kunzmann, Volker; Wiesweg, Marcel GND; Siveke, Jens T. GND; Schuler, Martin GND; Lueong, Smiths S. GND

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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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., 2020. Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer. https://doi.org/10.3390/cancers12020353
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