S7 Table -

S7A Table. EGFR phenotypes obtained for cohort I as detected by the TheraScreen® (QIAGEN), the SensiScreen® EGFR Liquid assay (PentaBase) and the Ion Torrent® (Thermo Fisher Scientific) platforms. Abbreviations: del, deletion; EGFR, Epidermal growth factor receptor; IOT, Ion Torrent methodology; WT, wild type. aFor one of the three samples (Sample 11) with this combinatorial phenotype, using 9 ng of cfDNA as input for the ctEGFR Mutation Detection Kit (EntroGen) did only detect the EGFR exon 19 deletion variant, not the p.T790M variant. Triplicate measurements using 18 ng of cfDNA as input per PCR reaction and absolute quantification resulted in detection of the p.T790M variant in addition to the exon 19 deletion variant. From the standard curve, it was estimated that the copy number of the p.T790M variant was at the level 1–2 copies in 18 ng of cfDNA input. The SensiScreen® EGFR Liquid assay (PentaBase ApS) simplex did detect the exon 19 deletion using 9 ng of cfDNA per PCR reaction. bFor two of the three samples (Sample 21 & 22) with this combinatorial phenotype, the DNA input amount available was too low for detection of the EGFR p.T790M variant by the SensiScreen® EGFR Liquid assay (PentaBase ApS) as previously evaluated from the low copy number of p.T790M detected at higher DNA input amounts by the ctEGFR Mutation Detection Kit (EntroGen). Hence, these two samples should be considered as agreement samples, both detecting the exon 19 deletion. cWhile the ctEGFR Mutation Detection Kit (EntroGen) detected an EGFR exon 19 deletion, the p.T790M variant but not the exon 19 deletion was detected by the SensiScreen® EGFR Liquid assay (PentaBase) (Sample 31). Duplicate measurements using the ctEGFR Mutation Detection Kit (EntroGen) showed two amplification curves for the p.T790M variant however below the threshold. S7B Table. Overview of the EGFR gene status for each plasma sample in cohort II as detected by the Entrogen ctEGFR Mutation Detection Kit (EntroGen) and the SensiScreen® EGFR Liquid assay (PentaBase ApS). Right column: EGFR gene status detected by previous analysis of tissue months to years prior to blood sampling for cfDNA EGFR analysis. aInitially for sample 11, triplicate measurements using 18 ng of cfDNA as input per PCR reaction resulted in detection of both the EGFR p.T790M and the exon 19 deletion variants. From standard curve analysis, it was estimated that the copy number of the p.T790M variant was 1–2 copies in 18 ng of cfDNA. Using 9 ng of cfDNA as input per PCR reaction for both the ctEGFR Mutation Detection Kit (EntroGen) (Triplicate measurements) and the SensiScreen® EGFR Liquid assay (PentaBase ApS) (single replicate) did only result in detection of the exon 19 deletion variant. Hence, it is likely that in 9 ng of cfDNA, no DNA copies of the c.2369C>T (p.T790M) variant was present and therefore could not be detected by either the SensiScreen® EGFR Liquid assay (PentaBase ApS) or the ctEGFR Mutation Detection Kit (EntroGen). bThe cfDNA amount available for test of the SensiScreen® EGFR Liquid assay (PentaBase ApS) was too low to detect the EGFR p.T790M variant as only few mutant copies were detected initially by the Entrogen ctEGFR Mutation Detection Kit (EntroGen). For sample 21, at 25 ng cfDNA input per PCR reaction used for the ctEGFR Mutation Detection Kit (EntroGen) the EGFR exon 19 deletion was detected, and the inclusion of copy number standards revealed that the p.T790M variant was estimated to be present in around 1–3 copies. Hence as evaluated from the copy number estimation using the ctEGFR Mutation Detection Kit (EntroGen) and with the lowest value detectable of the SensiScreen® EGFR Liquid assay (PentaBase ApS) in mind, it was expected that only the exon 19 deletion could be detected by the SensiScreen® EGFR Liquid assay at the 5 ng of cfDNA available for test. Using the SensiScreen® EGFR Liquid assay (PentaBase ApS), a low/late PCR amplification curve for the EGFR p.T790M variant was observed, however below the threshold. For sample 22, at 23 ng cfDNA as input per PCR reaction used for the ctEGFR Mutation Detection Kit (EntroGen) the EGFR exon 19 deletion was detected, and the inclusion of copy number standards revealed that the p.T790M variant was estimated to be present in around 1–2 copies. Only 10 ng of cfDNA was available for the test of the SensiScreen® EGFR Liquid assay (PentaBase ApS). Hence as evaluated from the copy number estimation using the ctEGFR Mutation Detection Kit (EntroGen) and with the lowest value detectable with the SensiScreen® EGFR Liquid assay (PentaBase ApS) in mind it was expected that only the exon 19 deletion may be detected by the SensiScreen® EGFR Liquid assay (PentaBase ApS). Using the SensiScreen® EGFR Liquid assay (PentaBase ApS), a low/late PCR amplification curve for the EGFR p.T790M variant was observed, however below the threshold. cFor sample 31, while the ctEGFR Mutation Detection Kit (EntroGen) detected an EGFR exon 19 deletion, the p.T790M variant but not the exon 19 deletion was detected by the SensiScreen® EGFR Liquid assay (PentaBase ApS). * For sample 13, TKI treatment may be based on inclusion in a clinical trial based on mutations in other genes than EGFR. Abbreviations: ctEGFR, circulating tumor Epidermal growth factor receptor; ctDNA, circulating tumor DNA; del, deletion; EGFR, Epidermal growth factor receptor; WT, wild type.
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