Sensitive cell-free tumor DNA analysis in supernatant pleural effusions supports therapy selection and disease monitoring of lung cancer patients.

Supernatant pleural effusions (PE) have shown to be a valuable source for the detection of driver mutations in circulating tumor DNA (ctDNA). In this prospective study, the clinical value of ctDNA analysis in supernatant PE to support therapy selection and disease monitoring in lung cancer patients is assessed. Paired PE and plasma samples were collected from lung cancer patients before initiation of therapy (N = 2) and from EGFR positive patients during therapy (N = 3). Supernatant PE and plasma were tested for mutations in EGFR, KRAS and BRAF by droplet digital PCR. In PE of two patients with suspected lung cancer, a KRAS mutation was detected with a 5- and 8-fold higher fractional abundance (FA) compared to plasma. For three patients with progressive disease during therapy, both the EGFR L858R and T790M mutations were detected in PE. However, in plasma only for two of these patients the L858R mutation was detected with a 46- and 14- fold lower FA, and only for one patient the T790M mutation was detected with a 8-fold lower FA. For one patient, longitudinal ctDNA analysis in PE revealed the T790M and L858R mutations already two months prior to detection of progressive disease by CT-scan. In this study, a higher ctDNA concentration and FA was obtained from PE compared to the corresponding blood samples, which enables more sensitive mutation analysis. Thus, PE is a valuable liquid biopsy, complementing plasma, for ctDNA analysis to support therapy selection and disease monitoring in lung cancer patients.

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR.

The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.