Chapter 6 220 T790M is a subclonal event, sensitive techniques are needed for molecular testing. Plasma genotyping is an alternative approach, but the sensitivity of detecting EGFR T790M in plasma is only 70%11. Therefore, other bio-sources for molecular testing could be useful. In our study, seven paired samples were positive for EGFR T790M, of which all were concordant between the supernatant and the cell pellet. Based on these results, both the supernatant and the cell pellet performed equally well as bio-sources for detecting EGFR T790M. The pleural effusion samples from four of the five patients progressing on first-generation EGFR TKIs showed a higher amount of T790M copies in both the supernatant and the cell pellet compared with the two patients showing a durable response to osimertinib. In prospective studies it will be interesting to see if the amount of EGFR T790M copies, measured by ddPCR in the supernatant and the cell pellet of pleural effusions, correlates to outcome in patients with EGFR-positive tumors treated with osimertinib. This study is limited by the small sample size. The supernatant performed better as a bio-source than the cell pellet for detecting EGFR or KRAS primary driver mutations, but not for detecting EGFR T790M. Studies with a larger sample size are needed to confirm our results. Besides this, we could only compare a subset of the paired samples with results obtained in molecular diagnostics with NGS/TSACP version 1.0 using the cell pellet. A direct comparison with a larger set of paired samples could confirm the additive value of using cfDNA in the supernatant as a bio-source for molecular profiling in a clinical setting. In conclusion, we showed that analysis of cfDNA in pleural effusion can robustly detect EGFR and KRAS driver mutations and EGFR resistance mutations. Therefore, cfDNA is a valuable bio-source for molecular testing, even when tumor cell purity is low. Acknowledgements We would like to acknowledge the Netherlands Cancer Institute Core Facility Molecular Pathology and Biobanking (CFMPB; Amsterdam, The Netherlands) for supplying NKI-AVL biobank material and laboratory support.
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