Cell-free DNA in the supernatant of pleural effusion as bio-source for diagnostic biomarker tests 215 6 Methods Supernatant and corresponding cell pellets from 39 stage IV patients, presented at the Netherlands Cancer Institute (Amsterdam, The Netherlands) between 2009 and 2016, with EGFR (n=16) or KRAS (n=23) mutation-positive tumors were obtained from the Netherlands Cancer Institute biobank. In total, 44 paired (supernatant and cell pellet) samples were available from 39 patients diagnosed with NSCLC (n=32), colon carcinoma (n=4), appendiceal carcinoma (n=2), and adenocarcinoma of unknown primary (n=1) (Table 1). The reason for pleurocentesis was either diagnostic (n=16) or therapeutic (n=28). These samples were leftover material that had been stored as routine laboratory assessment after diagnostics. This study was approved by the Institutional Research Board of the Netherlands Cancer Institute (CFMPB497). All driver gene and EGFR T790M mutations were detected in tumor tissue or cytology samples with clinically validated assays using high resolution melting, fragment analysis, Sanger sequence analysis, MassARRAY technology or next-generation sequencing (NGS) (data not shown). These analyses were done in multiple hospitals in the Netherlands. The supernatant was separated from the cell pellet after centrifugation (1700xg for 10 min). The pellet was resuspended in 0.5 ml of 0.9% NaCl. Both samples were stored at -30°C. In total, 400µl from the cell pellet was isolated using the QIAsymphony DSP DNA Midi Kit (Qiagen, Hilden, Germany). At least 10% from the sample was analyzed, from a median eluted volume of 200µl. CfDNA was isolated from a median (range) of 1 (0.75-4) ml pleural effusion using the QIAsymphony Circulating DNA kit (Qiagen). At least 20% of the sample was analyzed, from a median eluted volume of 90µl. The Bio-Rad (Hercules, CA, USA) QX200 droplet digital PCR (ddPCR) was used for mutation detection using Bio-Rad PrimePCR ddPCR mutation assays for KRAS (KRAS Screening Multiplex 186-3506), EGFR T790M (dHsaCP2000019 and dHsaCP2000020), EGFR exon 19 deletion screening assay10, EGFR L858R (dHsaCP2000021 and dHsaCP2000022), EGFR G719X (validated laboratory-developed method using IDT (Coralville, IA, USA), EGFR wild-type for G719 (HEX), EGFR G719S and EGFR G719A (both FAM). The limit of blank and the limit of detection were determined for each individual assay using a Clinical and Laboratory Standards Institute (CLSI) EP17 protocol11. Results were analyzed using Quantasoft software version 1.6.6 (www.quantasoft.com). Supernatant results were normalized to the amount of cfDNA in 1 ml of fluid.
RkJQdWJsaXNoZXIy MTk4NDMw