Cell-free DNA in the supernatant of pleural effusion as bio-source for diagnostic biomarker tests 219 6 Discussion This study investigated whether cfDNA from the supernatant of pleural effusions could be used for detecting driver mutations and EGFR resistance mutations. Our results clearly show that the cell-free fraction of pleural effusions is an excellent source for cfDNA and that it can be used to detect driver as well as resistance mutations effectively with ddPCR. We showed a high concordance rate between supernatant and cell pellet, which is in line with a previous study6, but a slightly lower rate has been reported elsewhere7. Furthermore, our results suggest that by using cfDNA in pleural effusion as a bio-source, the molecular testing has gained in sensitivity and that optimal sensitivity can be reached by analyzing both the supernatant and the cell pellet. In addition to analysis of cfDNA in pleural effusion from NSCLC patients, we could also detect KRAS mutations in the supernatant samples from patients with colon carcinoma, appendiceal carcinoma and adenocarcinoma of unknown primary, which has not been reported before. In five pleural effusion samples driver mutations were detected only in the supernatant even though no or very few tumor cells were seen in the cytopathological results. In our study, mutations were only called when the amount of copies found by ddPCR was above the limit of detection, determined according to the CLSI EP17 protocol11. The limit of detection was set with a confidence level of 99% to prevent false-positive results. Tumor DNA in the five corresponding cell pellets was not present or too low to detect the driver mutation. Importantly, since the detection of a driver mutation in the supernatant provides no proof of actual presence of tumor cells it can at present not be used for staging purposes. The origin of cfDNA in the supernatant has not been well studied, although several theories have been described5,8,9. Most likely this cfDNA is released from necrotic cells in the pleural cavity, but may also have been leaked from the circulation as a transudate. Alternatively, the cfDNA could have been derived from tumor cells damaged in the pre-analytical phase, e.g. by centrifugation5, or from exosomes secreted by the tumor8,9. Therefore, a high detection rate in the supernatant is expected and also observed in our study. In one pleural effusion sample only the cell pellet was positive for the driver mutation. Most likely too little tumor DNA was present in the supernatant. Resistance mutations are subclonal events in the tumor that often occur during TKI treatment. The EGFR T790M mutation, for example, is common in tumors of patients progressing on first-generation EGFR TKI (erlotinib, gefitinib)2. Osimertinib is frequently given as alternative treatment after this mutation is detected in a biopsy of a progressive lesion2. As obtaining tumor tissue is challenging and EGFR
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