Multiplex 1 cfdna reference standard

The ddPCR assays were performed using the PrimePCR™ ddPCR™ Mutation Detection Assay kit, and the PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories, Hercules, CA, USA) [11 (link)]. Tumor-derived nucleic acids (cfDNA and exoTNA) were extracted from 1 to 2 mL of plasma or pleural fluid. The ddPCR assay for detecting EGFR mutations was validated using the Multiplex I cfDNA Reference Standards (Horizon Discovery, Cambridge, UK) and healthy control samples from a previous study [11 (link)]. Briefly, the amplifications were performed in a reaction volume of 20 μL on a QX100 Droplet Digital PCR System (Bio-Rad). The 20 μL of the PCR mixture was composed of 10 μL Bio-Rad Super mix TaqMan, 1–2 μL of each amplification primer/probe mix, and 8–9 μL of nucleic acids (NAs). Thermal cycling comprised an initial denaturing and polymerase hot-start activating step of 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s and 55 °C for 60 s. The results were analyzed with QuantaSoft v.1.7.2 software (Bio-Rad) and reported as copies per milliliter of plasma. The ddPCR assay was validated for detecting EGFR mutations and determining the limit of detection (LOD) in a previous study [11 (link)]. The assays were considered “positive” if the measured event rate was ≥ 2 events/assay and “negative” if the event rate within a gated region was < 2 events/assay.

To prepare spiked materials with known mutant allele frequencies and mutant DNA copies, Multiplex I cfDNA Reference Standards (Horizon Discovery, Cambridge, UK) were purchased. This set is composed of wild-type cfDNA with mutant allele frequencies of 5%, 1%, 0.1%, and 0%. For each reference standard, copies per μL of wild-type and mutant DNA were measured using digital droplet PCR and compared with the values provided by the manufacturer (Supplementary Table S1).
Four levels (levels 1 to 4) of spiked NHP (2 mL per sample) were prepared to determine the LOD of the detection assays. Intended mutant allele frequencies were 5%, 2.5%, 1%, and 0.1%, with 4 to 760 mutant copies in a background of about 10,000~16,000 wild-type copies in a spiked NPH samples, depending on the mutation. cfDNA was extracted from 2 mL spiked NHP using MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher Scientific). Concentration and fragment size distribution of cfDNA were assessed using a 2200 TapeStation Instrument (Agilent Technologies). Average fragment size was about 190 bp, and the range of cfDNA was 60.42 ng to 80.18 ng. Details are provided in Supplementary Table S1.

For the cobas EGFR assay, 75 μL DNA from each sample was loaded into three reaction wells (25 μL DNA per well). Amplification and detection were performed using the cobas z 480 analyzer (Roche Molecular Systems, Inc.). Data were interpreted by the cobas z 480 software if positive and negative controls showed valid results. When a mutation was detected, semiquantitative index (SQI) values for each mutation are reported automatically by the software using the observed threshold cycle for the target mutation. The SQI was developed to measure trends in the amount of mutant cfDNA in a patient [13 (link)].
The analytical performance of the cobas EGFR assay was additionally evaluated using five NHP samples spiked with different mutant allele frequencies that were made using Multiplex I cfDNA Reference Standards (Horizon Discovery, Cambridge, UK). These test samples had expected mutant allele frequencies of 3.85~5.19%, 1.94~2.65%, 0.72~1.10%, 0.34~0.51%, and 0.05~0.12% (Supplementary Table and Figure 1).

A total of 93 EGFR exon 19 deletions identified by fragment analysis in routine practice in 2015 were subjected to PNA clamping with droplet digital PCR (ddPCR) and Sanger sequencing to confirm exon 19 deletion. PNA clamping with ddPCR was carried out using a Bio-Rad QX200 droplet system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. The primers and probes were identical to those previously reported [25 ], but a modified EX19_PNA probe was used. The sequence of the EX19_PNA probe was: 5’-AGAGAAGCAACATCT-3’, which targets the common exon 19 deletion region and could cover more subtypes of exon 19 deletions than the primer previously used, according to our clinical data.
Briefly, the PCR amplification system was as follows: 10 μL of 2× digital PCR supermix for probes (Bio-Rad Laboratories), 1.8 μL of 10 μM exon19 forward and reverse primers mix, 1.8 μL of 10 μM exon 2 forward and reverse primers mix, 1.8 μL of 10 μM EX19_PNA probe, 1 μL of 10 μM exon 19 probe, 1 μL of 10 μM exon 2 probe, 1 μL of 20 ng/μL DNA, and water added to 20 μL. Multiplex I cfDNA Reference Standard (HD780, Horizon Discovery, Cambridge, UK) was used to assay the sensitivity for detecting EGFR exon 19 deletions at 5%, 1%, 0.1%, and 0.1% allelic frequencies.

Reference samples used to evaluate the ctDNA detection threshold and performance were purchased from Horizon Discovery (Cambridge, UK), including Quantitative Multiplex Formalin Compromised (Mild) Reference Standard (cat# HD798), 1% Multiplex I cfDNA Reference Standard (cat# HD778), and 100% Wild-type (Tru-Q 0) (cat# HD752). The references were mixed and serially diluted to generate standards with allelic frequencies of 15%, 10%, 6%, 3%, 1%, 0.5%, 0.25%, and 0%.

ddPCR assays were performed with the PrimePCR™ ddPCR™ Mutation Detection Assay kit and PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories, Hercules, CA, USA) (Additional file 1: Table S1). We selected three hotspot mutations of Ex19del, L858R, and T790M. L858R and Ex19del are the most common forms of EGFR sensitizing mutations (85%) that are responsive for EGFR TKI treatment [23 (link)]. In case of progression on 1st generation TKI treatment, T790M mutation testing is recommended as acquired T790M mutation is the most common resistance mechanism (> 50%) that is responsive for 3rd generation TKI treatment [23 (link), 24 (link)]. The limitation of detection (LOD) was determined as the lowest mutant concentration above the 95% confidence interval (CI) of the wild-type (WT) control, which was determined using a Poisson model (Additional file 1: Table S2). Validation ddPCR was performed using Multiplex I cfDNA Reference Standard (Horizon Discovery, Cambridge, UK) (Additional file 1: Table S3). The ability to detect EGFR mutation based on type of input nucleic acid (short-length exoTNA and a size range of exoDNA) was evaluated (Additional file 1: Figures S1 and S2). Analytic performance of isolated cfDNA and short-length exoTNA was evaluated using ddPCR. We assessed the influence on cfDNA levels and short-length exoTNA according to storage period.