Cell-free DNA in the supernatant of pleural effusion as bio-source for diagnostic biomarker tests 217 6 Results The majority of the 44 paired samples were either both positive (30 out of 44) or both negative (8 out of 44) for the original driver mutation. In five cases the driver mutation was only detected in the supernatant and in one case only in the cell pellet (Table 2). Thus, in 36 of the 44 paired samples a driver mutation was detected using both the supernatant and the cell pellet. Testing only the supernatant would detect 35 of the 36 drivers (97%) and analysis of only the cell pellet would detect 31 of the 36 drivers (86%). These results indicated that the supernatant was an excellent biosource for ddPCR driver detection. Optimal sensitivity was reached when both the cell pellet and supernatant were analyzed. In five paired samples the driver mutation was only detected in the supernatant. Reviewing the cytology reports for these cases showed that in two of these samples no tumor cells were seen, in two cases no cytologically analysis was performed, and in one case the tumor cell percentage was only 1%. In four of these cell pellets no mutant copies were found and in one the amount was below the limit of detection. In one sample only the cell pellet was positive for the primary driver mutation. This cell pellet had a very low estimated tumor cell percentage and a borderline result of only 1 mutant copy/µl in the cell pellet (Table S1). No mutant copies were detected in the supernatant by ddPCR. The cfDNA was isolated from 0.75 ml of supernatant and showed a very low concentration of 0.005 ng/µl measured by Qubit (Invitrogen, Carlsbad, CA, USA). Therefore, the mutation could easily be missed in our analysis. To evaluate whether pleural effusion could be used as a bio-source for resistance analysis after progression on EGFR TKIs we analyzed 20 paired pleural effusions sampled from the 16 patients with EGFR-positive tumors for the presence of EGFR T790M. EGFR T790M was detected in seven out of the 20 paired samples. All seven supernatants and cell pellets were positive (Table 3). Four out of the seven EGFR T790M mutation-positive pleural effusion samples had very low estimated tumor cell percentages and in one case no tumor cells were seen by the pathologist (Table S1). Five of the seven pleural effusions were sampled from patients progressing on firstgeneration EGFR TKIs (erlotinib, gefitinib). Furthermore, two cases with a positive supernatant, but with a cell pellet showing a borderline result of 1 copy/µl, identified two patients with a durable response to osimertinib. The cytology reports of these pleural effusion samples showed that in one sample no tumor cells were seen and in one sample the tumor cell percentage was very low (Table S1). These results indicated that the supernatant is a good bio-source to detect EGFR T790M. In four patients EGFR T790M had already been detected in tumor tissue samples by clinically validated
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