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Evaluation of Pre-Analytical Conditions and Comparison of the Performance of Several Digital PCR Assays for the Detection of Major EGFR Mutations in Circulating DNA From Non-Small Cell Lung Cancers: The CIRCAN_0 Study

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Evaluation of Pre-Analytical Conditions and Comparison of the Performance of Several Digital PCR Assays for the Detection of Major EGFR Mutations in Circulating DNA From Non-Small Cell Lung Cancers: The CIRCAN_0 Study

Jessica Garcia et al. Oncotarget.

Abstract

Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.

Keywords: EGFR mutation; circulating-free DNA; digital PCR; liquid biopsy; lung cancer.

Conflict of interest statement

CONFLICTS OF INTEREST Sébastien Couraud declares grants, personal fees and non-financial support from Astra Zeneca, Sysmex Innostics, Roche, and Boehringher-Ingelheim in relation with the work under consideration; and grants, personal fees and non-financial support from Pfizer, Chugai, BMS, MSD, Lilly, and Novartis outside the submitted work. Jessica Garcia and Léa Payen declares grants, personal fees and non-financial support from Astra Zeneca, Sysmex Innostics, and BioMérieux in relation with the work under consideration; The other authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Optimization of circulating free DNA (cfDNA) extraction and quantification of cfDNA in the samples
(A) Reproducibility of cfDNA extraction using the QIAamp Circulating Acid Kit (Qiagen, Cat No 55114, Valencia, CA, USA) on two independent cfDNA samples extracted from 1 mL (Ai) and 3 mL (Aii) of plasma from NSCLC patients. After extraction, cfDNA was quantified by Qubit dsDNA HS Assay Kit (Life Technologies, Q32854, Carlsbad, CA, USA) according to the manufacturer's instructions. (B) Correlation between the initial volume of plasma 1 mL versus 3 mL (Bi) or 3 mL versus 5 mL (Bii) and the quantity of cfDNA extracted (in ng/μL). (Ci) Fragment size visualization of cfDNA (in bp) from a concentrated (left) and a less concentrated (right) sample obtained using the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (Cii), and average size distribution (10 bp increments) of cfDNA fragments in 77 plasma samples. (D) Correlation between cfDNA concentration measured using the Qubit method and the number of amplifiable copies in the corresponding plasma samples determined using the Quantifiler Kit.
Figure 2
Figure 2. Impact of pre-analytical blood storage conditions in EDTA tubes on circulating free DNA (cfDNA) integrity
(A) Evaluation of the effect of blood storage time (4 hours or 24 hours) and temperature (room temperature RT or 4°C) in EDTA tubes prior to plasma collection, on the concentration (ng/μL) of cfDNA extracted using the Qubit Quantification Kit (Ai), and the number of amplifiable DNA copies using the Quantifiler technique (Aii). Blood samples from the same patient (n = 7) were processed according to the three storage conditions. (B) Exploration of the number of wild-type (WT) copies of 3 independent regions of EGFR gene with the different ddPCR systems detailed in Figure 3 for WT L858R (Bi), WT delEX19 (Bii) and WT T790M (Biii) when samples are processed within 4 hours and within 24 hours after blood sampling (data not paired).
Figure 3
Figure 3. Overview of three Droplet Digital PCR (ddPCR) detection systems
(Ai-Aiv) Detection of 3 EGFR somatic alterations: L858R and T790M substitutions and delEX19 deletions. Top, 2D flow cytometry plots; bottom, schematic diagrams showing the principles of the corresponding ddPCR detection systems. (Ai-Aii) Dual probe system used to detect L858R (Ai) and T790M mutations (Aii) from liquid biopsies of NSCLC patients. This system can be used for the three detection assays described in Table 2, namely Seki's, Life Technologies' or our in-house assay. It is based on the utilization of reverse and forward primers targeting the hotspot and 2 taqman probes (WT and MT) labeled with 2 distinct fluorophores, VIC and FAM. The first anneals to wild-type (WT) copies whereas the latter binds to mutated (MT) copies. (Aiii) Dual labeling system used to detect delEX19 deletions with Seki's (Seki et al., 2016) or our in-house detection primers/probes. This system revolves around the dual labeling of WT copies (by VIC and FAM) and single labeling of MT copies (by VIC only). (Aiv) A single probe system used to detect delEX19 deletions with Life Technologies' assay (Hs00000228_mu), which is designed to detect only MT copies by blocking the WT sequence with a blocker.
Figure 4
Figure 4. Accuracy of the three Droplet Digital PCR (ddPCR) systems used
(A) Top, correlation between the theoretical expected number of wild-type (WT) copies and experimentally measured WT copies of commercial genomic DNA from the Quantifiler Human DNA Quantification Kit (Applied Biosystems, PN4344790F, Foster City, CA, USA) by ddPCR for the detection of (Ai) L858R substitutions, (Aii) delEX19 deletions and (Aiii) T790M substitutions, according to the detection assay used, namely Seki's assay, Life Technologies' (LT's) assay, or our in-house assay. Bottom, tables summarizing statistical data presented above. (B) Top, correlation between the number of WT copies for (Bi) L858R substitutions, (Bii) delEX19 deletions and (Biii) substitutions T790M and the concentration of cfDNA (in ng/μL) measured by Qubit (Life Technologies, Q32854, Carlsbad, CA, USA) in cfDNA samples. Bottom, equation used to estimate the concentration of cfDNA required to detect a threshold level of 1,000 mutated copies, for each plot presented above.
Figure 5
Figure 5. Specificity of the three Droplet Digital PCR (ddPCR) systems used
(A) Determination of false-positive cases (mutated MT) detected using the three ddPCR systems described in Figure 3 and a commercial genomic wild-type DNA control provided in the Quantifiler Human DNA Kit (Applied Biosystems, PN4344790F, Foster City, CA, USA). The commercial WT DNA was diluted and tested for L858R substitutions (Ai), various delEX19 deletions (Aii) and T790M substitutions (Aiii) using three detection assays: Seki's assay, an in house's system and LT's system (see Table 2); n indicates the number of independent experiments carried out for each conditions. WT and MT colums indicate the mean of absolute detected copies. The numbers and rates of false-positives (% FP) cases are reported. (B) Background of false-positive copies (%MT) for all of the ddPCR mutation systems used to detect L858R substitutions (Bi), various delEX19 deletions (Bii) and T790M substitutions (Biii) from cfDNA of NSCLC patients with a negative or unknown biopsy status at diagnosis and with negative results in ddPCR. The absolute copy number was based on the maximum number of MT copies observed in tables Ai-Aiii (5 MT copies) over the minimum WT detection threshold (500 WT copies).
Figure 6
Figure 6. Sensitivity of the three Droplet Digital PCR (ddPCR) systems used
Correlation between measured mutated (MT) and wild-type (WT) copies with the theoretical percentage of mutated copies of four reference standards DNA (Horizon Diagnostics) for three somatic EGFR alterations L858R and T790M substitutions, and various delEX19 deletions detected using the ddPCR systems described in Figure 3 and Table 2. The commercial standard DNA was tested for (Ai) L858R mutations with Seki's, Life Technologies' (LT's) and our in-house primers/probes. (Aii) delEX19 deletions were detected using the same three systems, while (Aiii) T790M mutations were detected using Seki's and LT's primers/probes. (B) Representation of the size of the amplicons generated during ddPCR with Seki's primer and our in-house primers for the L858R (Bi) and delEX19 (Bii) gene regions. n indicates the number of independent experiments carried out for each conditions. WT and MT colums indicate the mean of absolute detected copies. (C) Histogram presenting the number of WT copies detected using Seki's and LT's primers/probes for L858R mutations (Ci) and delEX19 deletions (Cii). The ratio represents the difference in the number of MT copies detected between our in-house primers and Seki's primers.
Figure 7
Figure 7
Range of the number of mutated copies detected by Digital Droplet PCR (ddPCR) (A) Representation of the range of WT copies using Seki's primers and our in-house primers for the detection of L858R (Ai) and delEX19 (Aii) among patients with or without a positive mutation status at diagnosis. (B) Comparison between pairs of detections systems used to evaluate the number of mutated copies among patients harboring EGFR alterations for: L858R (Bi), various delEX19 (Bii) and T790M (Biii). μ

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References

    1. Pao W, Girard N. New driver mutations in non-small-cell lung cancer. The Lancet Oncology. 2011;12:175–180. - PubMed
    1. Novello S, Barlesi F, Califano R, Cufer T, Ekman S, Levra MG, Kerr K, Popat S, Reck M, Senan S, Simo GV, Vansteenkiste J, Peters S, et al. Metastatic non-small-cell lung cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2016;27:v1–v27. - PubMed
    1. Yu HA, Arcila ME, Rekhtman N, Sima CS, Zakowski MF, Pao W, Kris MG, Miller VA, Ladanyi M, Riely GJ. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19:2240–2247. - PMC - PubMed
    1. Janne PA, Yang JC, Kim DW, Planchard D, Ohe Y, Ramalingam SS, Ahn MJ, Kim SW, Su WC, Horn L, Haggstrom D, Felip E, Kim JH, et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. The New England journal of medicine. 2015;372:1689–1699. - PubMed
    1. Nowak F, Soria JC, Calvo F. Tumour molecular profiling for deciding therapy-the French initiative. Nature reviews Clinical oncology. 2012;9:479–486. - PubMed
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