Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system

Suzawa,Ken; Yamamoto,Hiromasa; Ohashi,Kadoaki; Hashida,Shinsuke; Tomida,Shuta; Kubo,Toshio; Maki,Yuho; Soh,Junichi; Tsukuda,Kazunori; Kiura,Katsuyuki; Miyoshi,Shinichiro; Toyooka,Shinichi

doi:10.3892/or.2017.5567

Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system

Authors:
- Ken Suzawa
- Hiromasa Yamamoto
- Kadoaki Ohashi
- Shinsuke Hashida
- Shuta Tomida
- Toshio Kubo
- Yuho Maki
- Junichi Soh
- Kazunori Tsukuda
- Katsuyuki Kiura
- Shinichiro Miyoshi
- Shinichi Toyooka
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Affiliations: Department of Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Published online on: April 11, 2017 https://doi.org/10.3892/or.2017.5567
Pages: 3100-3106

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Abstract

Though patients with EGFR mutations are initially responsive to EGFR-tyrosine kinase inhibitors (TKIs), most tumors ultimately acquire resistance to EGFR-TKIs. The most frequently reported mechanism is EGFR T790M mutation. In this study, using a droplet digital PCR (ddPCR) system, we assessed optimal conditions for a mutation detection assay for EGFR T790M obtained from circulating cell-free DNA (cfDNA) in plasma. The advantages of locked nucleic acids (LNA) probe, short amplicon size, and blocking oligo using peptide nucleic acids (PNA) were assessed using control DNAs from cell lines to improve the sensitivity of mutation detection. T790M alleles were then analyzed using ddPCR in 59 plasma samples from 24 NSCLC patients with EGFR mutations, and compared to the T790M status which were determined thorough re-biopsies. The assessment of the optimal assay method revealed that the assay using the short amplicon can efficiently detect more fragmented-DNA. The LNA probe and PNA clamp contributed better separation between positive and negative droplets. This PNA-LNA-ddPCR clamp method can detect mutant alleles in the sample with a mutant allele content of 0.01%. In clinical plasma samples, T790M alleles were detected via ddPCR with a sensitivity of 42.8% and specificity of 97.3%. We established a highly-sensitive detection assay for the T790M allele using the PNA-LNA-ddPCR clamp method. ddPCR is a promising method for detecting non-invasive T790M mutation.

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1	Collisson EA, Campbell JD, Brooks AN, Berger AH, Lee W, Chmielecki J, Beer DG, Cope L, Creighton CJ, Danilova L, et al: Cancer Genome Atlas Research Network: Comprehensive molecular profiling of lung adenocarcinoma. Nature. 511:543–550. 2014. View Article : Google Scholar : PubMed/NCBI
2	Kris MG, Johnson BE, Berry LD, Kwiatkowski DJ, Iafrate AJ, Wistuba II, Varella-Garcia M, Franklin WA, Aronson SL, Su PF, et al: Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 311:1998–2006. 2014. View Article : Google Scholar : PubMed/NCBI
3	Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, et al: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 361:947–957. 2009. View Article : Google Scholar : PubMed/NCBI
4	Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, Turke AB, Fidias P, Bergethon K, Shaw AT, Gettinger S, Cosper AK, et al: Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 3:75ra262011. View Article : Google Scholar : PubMed/NCBI
5	Soucheray M, Capelletti M, Pulido I, Kuang Y, Paweletz CP, Becker JH, Kikuchi E, Xu C, Patel TB, Al-Shahrour F, et al: Intratumoral heterogeneity in EGFR-Mutant NSCLC results in divergent resistance mechanisms in response to EGFR tyrosine kinase inhibition. Cancer Res. 75:4372–4383. 2015. View Article : Google Scholar : PubMed/NCBI
6	Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, Johnson BE, Eck MJ, Tenen DG and Halmos B: EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 352:786–792. 2005. View Article : Google Scholar : PubMed/NCBI
7	Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ, Orme JP, Finlay MR, Ward RA, Mellor MJ, et al: AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 4:1046–1061. 2014. View Article : Google Scholar : PubMed/NCBI
8	Crowley E, Di Nicolantonio F, Loupakis F and Bardelli A: Liquid biopsy: Monitoring cancer-genetics in the blood. Nat Rev Clin Oncol. 10:472–484. 2013. View Article : Google Scholar : PubMed/NCBI
9	Oxnard GR, Paweletz CP, Kuang Y, Mach SL, O'Connell A, Messineo MM, Luke JJ, Butaney M, Kirschmeier P, Jackman DM, et al: Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res. 20:1698–1705. 2014. View Article : Google Scholar : PubMed/NCBI
10	Diaz LA Jr and Bardelli A: Liquid biopsies: Genotyping circulating tumor DNA. J Clin Oncol. 32:579–586. 2014. View Article : Google Scholar : PubMed/NCBI
11	Nagai Y, Miyazawa H, Huqun Tanaka T, Udagawa K, Kato M, Fukuyama S, Yokote A, Kobayashi K, Kanazawa M, et al: Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res. 65:7276–7282. 2005. View Article : Google Scholar : PubMed/NCBI
12	Tanaka T, Nagai Y, Miyazawa H, Koyama N, Matsuoka S, Sutani A, Huqun Udagawa K, Murayama Y, Nagata M, et al: Reliability of the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based test for epidermal growth factor receptor mutations integrated into the clinical practice for non-small cell lung cancers. Cancer Sci. 98:246–252. 2007. View Article : Google Scholar : PubMed/NCBI
13	Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J, Peyton M, Zou Y, Kurie JM, Dimaio JM, et al: Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64:9027–9034. 2004. View Article : Google Scholar : PubMed/NCBI
14	Gandhi J, Zhang J, Xie Y, Soh J, Shigematsu H, Zhang W, Yamamoto H, Peyton M, Girard L, Lockwood WW, et al: Alterations in genes of the EGFR signaling pathway and their relationship to EGFR tyrosine kinase inhibitor sensitivity in lung cancer cell lines. PLoS One. 4:e45762009. View Article : Google Scholar : PubMed/NCBI
15	Schwarzenbach H, Hoon DS and Pantel K: Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 11:426–437. 2011. View Article : Google Scholar : PubMed/NCBI
16	Giacona MB, Ruben GC, Iczkowski KA, Roos TB, Porter DM and Sorenson GD: Cell-free DNA in human blood plasma: Length measurements in patients with pancreatic cancer and healthy controls. Pancreas. 17:89–97. 1998. View Article : Google Scholar : PubMed/NCBI
17	Heitzer E, Ulz P and Geigl JB: Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem. 61:112–123. 2015. View Article : Google Scholar : PubMed/NCBI
18	Sikora A, Zimmermann BG, Rusterholz C, Birri D, Kolla V, Lapaire O, Hoesli I, Kiefer V, Jackson L and Hahn S: Detection of increased amounts of cell-free fetal DNA with short PCR amplicons. Clin Chem. 56:136–138. 2010. View Article : Google Scholar : PubMed/NCBI
19	Weisenberger DJ, Trinh BN, Campan M, Sharma S, Long TI, Ananthnarayan S, Liang G, Esteva FJ, Hortobagyi GN, McCormick F, et al: DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight. Nucleic Acids Res. 36:4689–4698. 2008. View Article : Google Scholar : PubMed/NCBI
20	Hyman DM, Diamond EL, Vibat CR, Hassaine L, Poole JC, Patel M, Holley VR, Cabrilo G, Lu TT, Arcila ME, et al: Prospective blinded study of BRAFV600E mutation detection in cell-free DNA of patients with systemic histiocytic disorders. Cancer Discov. 5:64–71. 2015. View Article : Google Scholar : PubMed/NCBI
21	Janku F, Vibat CR, Kosco K, Holley VR, Cabrilo G, Meric-Bernstam F, Stepanek VM, Lin PP, Leppin L, Hassaine L, et al: BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease. Oncotarget. 5:3607–3610. 2014. View Article : Google Scholar : PubMed/NCBI