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Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer

2015; Elsevier BV; Volume: 17; Issue: 3 Linguagem: Inglês

10.1016/j.jmoldx.2015.01.004

ISSN

1943-7811

Autores

Guanshan Zhu, Xin Ye, Zhengwei Dong, Ya Lu, Yun Sun, Yi Liu, Rose McCormack, Yi Gu, Xiaoqing Liu,

Tópico(s)

Chronic Lymphocytic Leukemia Research

Resumo

Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%–94.81%) and 98.44% (95% CI, 91.60%–99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704–0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%–95.67%) and 95.77% (95% CI, 88.14%–99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571–0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing. Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%–94.81%) and 98.44% (95% CI, 91.60%–99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704–0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%–95.67%) and 95.77% (95% CI, 88.14%–99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571–0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib, erlotinib, and afatinib, were found to be effective treatments for the subpopulation of patients with advanced non-small cell lung cancer (aNSCLC) whose tumors harbor EGFR-activating mutations.1Rosell R. Carcereny E. Gervais R. Vergnenegre A. Massuti B. Felip E. et al.Spanish Lung Cancer Group in collaboration with Groupe Francais de Pneumo-Cancerologie and Associazione Italiana Oncologia ToracicaErlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.Lancet Oncol. 2012; 13: 239-246Abstract Full Text Full Text PDF PubMed Scopus (4436) Google Scholar, 2Mok T.S. Wu Y.L. Thongprasert S. Yang C.H. Chu D.T. Saijo N. Sunpaweravong P. Han B. Margono B. Ichinose Y. Nishiwaki Y. Ohe Y. Yang J.J. Chewaskulyong B. Jiang H. Duffield E.L. Watkins C.L. Armour A.A. Fukuoka M. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.N Engl J Med. 2009; 361: 947-957Crossref PubMed Scopus (7031) Google Scholar, 3Zhou C. Wu Y.L. Chen G. Feng J. Liu X.Q. Wang C. Zhang S. Wang J. Zhou S. Ren S. Lu S. Zhang L. Hu C. Hu C. Luo Y. Chen L. Ye M. Huang J. Zhi X. Zhang Y. Xiu Q. Ma J. Zhang L. You C. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study.Lancet Oncol. 2011; 12: 735-742Abstract Full Text Full Text PDF PubMed Scopus (3402) Google Scholar, 4Sequist L.V. Yang J.C. Yamamoto N. O'Byrne K. Hirsh V. Mok T. Geater S.L. Orlov S. Tsai C.M. Boyer M. Su W.C. Bennouna J. Kato T. Gorbunova V. Lee K.H. Shah R. Massey D. Zazulina V. Shahidi M. Schuler M. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations.J Clin Oncol. 2013; 31: 3327-3334Crossref PubMed Scopus (2553) Google Scholar EGFR mutation testing is therefore a critical step in identifying the right patients for EGFR-TKI treatment.5Lindeman N.I. Cagle P.T. Beasley M.B. Chitale D.A. Dacic S. Giaccone G. Jenkins R.B. Kwiatkowski D.J. Saldivar J.S. Squire J. Thunnissen E. Ladanyi M. Collage of American Pathologists, International Association for the Study of Lung Cancer and Association for Molecular PathologyMolecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.J Mol Diagn. 2013; 15: 415-453Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 6Fukuoka M. Wu Y.L. Thongprasert S. Sunpaweravong P. Leong S.S. Sriuranpong V. Chao T.Y. Nakagawa K. Chu D.T. Saijo N. Duffield E.L. Rukazenkov Y. Speake G. Jiang H. Armour A.A. To K.F. Yang J.C. Mok T.S. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).J Clin Oncol. 2011; 29: 2866-2874Crossref PubMed Scopus (1197) Google Scholar Although tumor tissue samples and tumor cytologic samples are accepted as appropriate sample types for EGFR mutation detection, these samples are not always available on or after diagnosis, and, even when available, they may be of insufficient quality or quantity for mutation testing. Plasma cell-free DNA (cfDNA) as a liquid biopsy was studied as a potentially valuable surrogate specimen for detecting tumor-specific aberrations.7Schwarzenbach H. Hoon D.S. Pantel K. Cell-free nucleic acids as biomarkers in cancer patients.Nat Rev Cancer. 2011; 11: 426-437Crossref PubMed Scopus (2019) Google Scholar, 8Gormally E. Caboux E. Vineis P. Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance.Mutat Res. 2007; 635: 105-117Crossref PubMed Scopus (365) Google Scholar, 9Sozzi G. Conte D. Mariani L. Lo Vullo S. Roz L. Lombardo C. Pierotti M.A. Tavecchio L. Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients.Cancer Res. 2001; 61: 4675-4678PubMed Google Scholar, 10Jahr S. Hentze H. Englisch S. Hardt D. Fackelmayer F.O. Hesch R.D. Knippers R. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res. 2001; 61: 1659-1665PubMed Google Scholar The advantages of using cfDNA for tumor mutation detection include i) noninvasive collection, ii) availability at any time during disease course, iii) real-time detection and monitoring of dynamics of the biomarkers, and iv) potentially fewer heterogeneity issues than tumor tissue testing.11Diaz Jr., L.A. Bardelli A. Liquid biopsies: genotyping circulating tumor DNA.J Clin Oncol. 2014; 32: 579-586Crossref PubMed Scopus (1527) Google Scholar In fact, EGFR-activating mutation status in cfDNA has proved to be able to predict efficacy of EGFR-TKI in the treatment of aNSCLC.12Kimura H. Kasahara K. Kawaishi M. Kunitoh H. Tamura T. Holloway B. Nishio K. Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer.Clin Cancer Res. 2006; 12: 3915-3921Crossref PubMed Scopus (296) Google Scholar, 13Mack P.C. Holland W.S. Burich R.A. Sangha R. Solis L.J. Li Y. Beckett L.A. Lara Jr., P.N. Davies A.M. Gandara D.R. EGFR mutations detected in plasma are associated with patient outcomes in erlotinib plus docetaxel-treated non-small cell lung cancer.J Thorac Oncol. 2009; 4: 1466-1472Crossref PubMed Scopus (60) Google Scholar, 14Bai H. Mao L. Wang H.S. Zhao J. Yang L. An T.T. Wang X. Duan C.J. Wu N.M. Guo Z.Q. Liu Y.X. Liu H.N. Wang Y.Y. Wang J. Epidermal growth factor receptor mutations in plasma DNA samples predict tumor response in Chinese patients with stages IIIB to IV non-small-cell lung cancer.J Clin Oncol. 2009; 27: 2653-2659Crossref PubMed Scopus (273) Google Scholar, 15Liu Y. Liu B. Li X.Y. Li J.J. Qin H.F. Tang C.H. Guo W.F. Hu H.X. Li S. Chen C.J. Liu B. Gao H.J. Liu X.Q. A comparison of ARMS and direct sequencing for EGFR mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non-small-cell lung cancer patients.J Exp Clin Cancer Res. 2011; 30: 111Crossref PubMed Scopus (65) Google Scholar, 16Goto K. Ichinose Y. Ohe Y. Yamamoto N. Negoro S. Nishio K. Itoh Y. Jiang H. Duffield E. McCormack R. Saijo N. Mok T. Fukuoka M. Epidermal growth factor receptor mutation status in circulating free DNA in serum: from IPASS, a phase III study of gefitinib or carboplatin/paclitaxel in non-small cell lung cancer.J Thorac Oncol. 2012; 7: 115-121Crossref PubMed Scopus (200) Google Scholar, 17Douillard J.Y. Ostoros G. Cobo M. Ciuleanu T. McCormack R. Webster A. Milenkova T. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.Br J Cancer. 2014; 110: 55-62Crossref PubMed Scopus (313) Google Scholar However, technical challenges remain in testing cfDNA for tumor EGFR mutations. The presence and/or concentration of cfDNA from individual patients could be variable and, in general, tends to be low.18Crowley E. Di Nicolantonio F. Loupakis F. Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood.Nat Rev Clin Oncol. 2013; 10: 472-484Crossref PubMed Scopus (1213) Google Scholar The fragments of the cfDNA are relatively small with peak size of approximately 180 bp.10Jahr S. Hentze H. Englisch S. Hardt D. Fackelmayer F.O. Hesch R.D. Knippers R. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res. 2001; 61: 1659-1665PubMed Google Scholar The percentage of tumor-derived DNA fraction in total cfDNA is individually variable and often too low for detection.18Crowley E. Di Nicolantonio F. Loupakis F. Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood.Nat Rev Clin Oncol. 2013; 10: 472-484Crossref PubMed Scopus (1213) Google Scholar Finally, previous studies only reported 43% to 66% sensitivity for EGFR mutation detection in cfDNA compared with tumor tissue testing.16Goto K. Ichinose Y. Ohe Y. Yamamoto N. Negoro S. Nishio K. Itoh Y. Jiang H. Duffield E. McCormack R. Saijo N. Mok T. Fukuoka M. Epidermal growth factor receptor mutation status in circulating free DNA in serum: from IPASS, a phase III study of gefitinib or carboplatin/paclitaxel in non-small cell lung cancer.J Thorac Oncol. 2012; 7: 115-121Crossref PubMed Scopus (200) Google Scholar, 17Douillard J.Y. Ostoros G. Cobo M. Ciuleanu T. McCormack R. Webster A. Milenkova T. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.Br J Cancer. 2014; 110: 55-62Crossref PubMed Scopus (313) Google Scholar Therefore, the development of a method with high sensitivity for EGFR mutation detection in cfDNA is important. Several different methods were reported to be able to detect EGFR mutations from cfDNA. However, the detection sensitivities are variable among different laboratories with different methods and thus are not conclusive and satisfactory for clinical adoption as routine practice.12Kimura H. Kasahara K. Kawaishi M. Kunitoh H. Tamura T. Holloway B. Nishio K. Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer.Clin Cancer Res. 2006; 12: 3915-3921Crossref PubMed Scopus (296) Google Scholar, 14Bai H. Mao L. Wang H.S. Zhao J. Yang L. An T.T. Wang X. Duan C.J. Wu N.M. Guo Z.Q. Liu Y.X. Liu H.N. Wang Y.Y. Wang J. Epidermal growth factor receptor mutations in plasma DNA samples predict tumor response in Chinese patients with stages IIIB to IV non-small-cell lung cancer.J Clin Oncol. 2009; 27: 2653-2659Crossref PubMed Scopus (273) Google Scholar, 16Goto K. Ichinose Y. Ohe Y. Yamamoto N. Negoro S. Nishio K. Itoh Y. Jiang H. Duffield E. McCormack R. Saijo N. Mok T. Fukuoka M. Epidermal growth factor receptor mutation status in circulating free DNA in serum: from IPASS, a phase III study of gefitinib or carboplatin/paclitaxel in non-small cell lung cancer.J Thorac Oncol. 2012; 7: 115-121Crossref PubMed Scopus (200) Google Scholar, 19Yung T.K. Chan K.C. Mok T.S. Tong J. To K.F. Lo Y.M. Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients.Clin Cancer Res. 2009; 15: 2076-2084Crossref PubMed Scopus (358) Google Scholar, 20Brevet M. Johnson M.L. Azzoli C.G. Ladanyi M. Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors.Lung Cancer. 2011; 73: 96-102Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Taniguchi K. Uchida J. Nishino K. Kumagai T. Okuyama T. Okami J. Higashiyama M. Kodama K. Imamura F. Kato K. Quantitative detection of EGFR mutations in circulating tumor DNA derived from lung adenocarcinomas.Clin Cancer Res. 2011; 17: 7808-7815Crossref PubMed Scopus (281) Google Scholar, 22Yam I. Lam D.C. Chan K. Chung-Man Ho J. Ip M. Lam W.K. Chan T.K. Chan V. EGFR array: uses in the detection of plasma EGFR mutations in non-small cell lung cancer patients.J Thorac Oncol. 2012; 7: 1131-1140Crossref PubMed Scopus (40) Google Scholar, 23Nakamura T. Sueoka-Aragane N. Iwanaga K. Sato A. Komiya K. Kobayashi N. Hayashi S. Hosomi T. Hirai M. Sueoka E. Kimura S. Application of a highly sensitive detection system for epidermal growth factor receptor mutations in plasma DNA.J Thorac Oncol. 2012; 7: 1369-1381Crossref PubMed Scopus (67) Google Scholar, 24Kim H.R. Lee S.Y. Hyun D.S. Lee M.K. Lee H.K. Choi C.M. Yang S.H. Kim Y.C. Lee Y.C. Kim S.Y. Jang S.H. Lee J.C. Lee K.Y. Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping.J Exp Clin Cancer Res. 2013; 32: 50Crossref PubMed Scopus (87) Google Scholar, 25Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar The weakness of these studies included relatively small sample size, poor reproducibility, or complexity of the method. Droplet digital PCR (ddPCR) is a new generation of PCR technique, which allows the independent amplification and fluorescence reading of tens of thousands of individual droplets in one well. It enables the highly sensitive genotyping and the absolute quantification of mutant genes. Oxnard et al26Oxnard G.R. Paweletz C.P. Kuang Y. Mach S.L. O'Connell A. Messineo M.M. Luke J.J. Butaney M. Kirschmeier P. Jackman D.M. Janne P.A. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA.Clin Cancer Res. 2014; 20: 1698-1705Crossref PubMed Scopus (627) Google Scholar recently reported the development of ddPCR assays for detecting EGFR mutations with the focus on optimizing the assay specificity.26Oxnard G.R. Paweletz C.P. Kuang Y. Mach S.L. O'Connell A. Messineo M.M. Luke J.J. Butaney M. Kirschmeier P. Jackman D.M. Janne P.A. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA.Clin Cancer Res. 2014; 20: 1698-1705Crossref PubMed Scopus (627) Google Scholar By testing plasma samples from 23 patients, their assays for exon19 deletions (E19-Dels) and L858R mutations reached 100% specificity, whereas the sensitivity was only 66.7%. With the goal to efficiently identify the EGFR mutant DNA in plasma, usually known to be low in prevalence, we developed the highly sensitive and specific ddPCR assays for detecting EGFR-activating mutations and proved significantly increased clinical testing sensitivity up to >80%. All of the patients and samples included in this analysis were collected from a previous method comparison study.25Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar Briefly, 86 patients diagnosed with aNSCLC at the Affiliated Hospital of Academy of Military Medical Science (Beijing, China) during January 2008 to March 2012 were recruited into the study. Demographic characteristics were as follows: 56 (65%) were male and 30 (35%) were female; the age of the patients ranged from 28 to 81 years with mean age of 55 years; 39 (45%) were never smokers, 10 (12%) were former smokers, and 17 (20%) are current smokers. Four patients (5%) had stage IIIB disease and 82 (95%) had stage IV disease. All of the lung tumors were adenocarcinoma except for one case that was adenosquamous type. The study was approved by the institutional ethics committee of the hospital. All patients gave informed consent for specimen collection, clinical information collection, and biomarker analysis. Collection of tumor tissue sample and matched plasma was mandatory for each patient. All tumor tissue samples were formalin-fixed, paraffin-embedded diagnostic samples and underwent pathologic evaluation to confirm the diagnosis of adenocarcinoma and the tumor content of at least 2%. Four to eight sections (5-μm thickness) of each qualifying tumor tissue sample were used for DNA extraction. Peripheral blood samples were collected into EDTA tubes and centrifuged at 2500 × g for 10 minutes at 4°C within 1 hour to isolate plasma, which was then stored at −80°C until cfDNA extraction. The tumor tissue DNA from formalin-fixed, paraffin-embedded slides was isolated by using QIAamp DNA FFPE (formalin-fixed, paraffin-embedded) Tissue Kit (Qiagen, Hilden, Germany) as advised in the user manual. Plasma cfDNA was extracted from 2 mL of plasma from each patient with the QIAamp Circulating Nucleic Acid kit (Qiagen), following the manufacturer's instructions. Extracted cfDNA from each plasma sample was eluted in 100 μL of Tris-EDTA buffer. Tumor tissue sample analyses for EGFR mutations were conducted in a previous study, in which ADx-ARMS (amplification refractory mutation system) kit (Amoy Diagnostics, Xiamen, China) was used, and all experiments and genotyping calling were performed, following the manufacturer's instructions.25Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar This kit was approved by the Chinese Food and Drug Administration for in vitro diagnostics use, which detects the 29 most-common EGFR mutations so far described in lung cancer. Status of E19-Del and L858R mutations was used for the analysis of this study. ddPCR is a digital PCR platform that allows the partitioning of input DNA into 20,000 droplets. PCR reaction occurs independently in individual droplets, which are subject to fluorescence reading after amplification. Each droplet is assigned as being positive or negative on the basis of the fluorescence signal, which is used to determine the existence of target DNA. The design principle of the ddPCR assays is shown in Figure 1, and sequences of primers and probes are listed in Table 1. A 15-bp peptide nucleic acid (PNA) that targets the common E19-Del region, E746 to A750, was introduced to block the amplification of wild-type EGFR allele (Figure 1). Therefore, the FAM-labeled probe that targeted the exon19 amplicon of both wild-type and mutant EGFR alleles is used to reflect the existence of multiple types of EGFR mutants with deletions within this PNA targeting region. All 19 types of EGFR E19-Dels from the ADx-ARMS kit are covered by this ddPCR assay. A VIC-labeled probe was designed to target EGFR-exon2 for total EGFR gene input control. For L858R assay, two probes that targeted the mutation region with one nucleotide difference were labeled with FAM and VIC, to detect the mutant and wild-type EGFR allele, respectively. These customized primers and fluorescent probes were ordered from Life Technologies (Thermo Fisher Scientific Inc., Boston, MA). The PNA-blocking probe was synthesized by PANAGENE Inc. (Daejeon, Korea).Table 1Sequence Information of the Primers and Probes for the ddPCR AssaysMutationPrimer/probe IDSequenceE19-DelsE19-F5′-GAAAGTTAAAATTCCCGTCGCTAT-3′E19-R5′-ACCCCCACACAGCAAAGC-3′E19-common probe5′-FAM-ACATCGAGGATTTCCTTGT-MGB-3′E19-wt PNA probe5′-GAATTAAGAGAAGCA-3′E2-F5′-GCCAAGGCACGAGTAACAAGC-3′E2-R5′-TCCTCTGGAGGCTGAGAAAATGA-3′E2-common probe5′-VIC-TCACGCAGTTGGGCAC-MGB-3′L858RE21-F5′-CCGCAGCATGTCAAGATCAC-3′E21-R5′-TCCTTCTGCATGGTATTCTTTCTCT-3′E21-Mt probe5′-FAM-TTGGCCCGCCCAA-MGB-3′E21-Wt probe5′-VIC-TTGGCCAGCCCAA-MGB-3′ddPCR, droplet digital PCR; E19-Del, exon 19 deletion; Mt, mutant allele; PNA, peptide nucleic acid; Wt, wild-type allele. Open table in a new tab ddPCR, droplet digital PCR; E19-Del, exon 19 deletion; Mt, mutant allele; PNA, peptide nucleic acid; Wt, wild-type allele. To evaluate the sensitivity of E19-Del assay, DNA from NCI-H1650 cells (harboring E19-Del mutation) was serially diluted to human reference genomic DNA (catalog G1471; Promega Corporation, Madison, WI) to achieve decreasing ratios (1:1 to 1:10,000) of the E19-Del mutant allele versus the wild-type allele. For the L858R assay, DNA from NCI-H1975 cells (harboring the L858R mutation) was serially diluted to human reference genomic DNA to achieve decreasing ratios (1:1 to 1:10,000) of the L858R mutant allele versus the wild-type allele. The final 20 μL of TaqMan PCR reaction mixture was assembled with 1× ddPCR Master mixture (Bio-Rad Laboratories, Hercules, CA), 900 nmol/L of each primer, 450 nmol/L of each probe, and 50 ng of genomic DNA templates. In the assay to detect E19-Dels, 900 nmol/L PNA was included in the reaction mix. Each assembled ddPCR reaction mixture was then loaded into the sample well of an eight-channel droplet generator cartridge (Bio-Rad Laboratories). A volume of 70 μL of droplet generation oil (Bio-Rad Laboratories) was loaded into the oil well for each channel to generate droplets. Theoretically, a maximum of 20,000 droplets could be generated for each sample. The droplets from each of the samples were then manually transferred to each well of a 96-well PCR plate. The plate was heat-sealed with a foil seal and then placed on a thermal cycler for amplification. Thermal cycling profile for E19-Del assay was 10 minutes of incubation at 95°C, followed by 45 cycles of 95°C for 15 seconds, 70°C for 15 seconds, 60°C for 1 minute, and then 4°C hold. Thermal cycling profile for L858R assay was 10 minutes of incubation at 95°C, followed by 45 cycles of 95°C for 15 seconds, 60°C for 1 minute, and then 4°C hold. After PCR, the 96-well PCR plate was loaded on the droplet reader (Bio-Rad Laboratories) to read the droplets from each well of the plate. Analysis of the ddPCR data for allele calling was performed with QuantaSoft version 1.3.2.0 (Bio-Rad Laboratories). Human reference genomic DNA was routinely included as negative control and used to determine the cutoff for allele calling. Single droplet occasionally showed up as random event when non-template control reactions were tested. Therefore, the samples that had at least two droplets in the positive area for FAM signal were counted as positive for the mutation. The reaction mix was prepared as described in the above section, and plasma cfDNA, 8 μL from each, was loaded into the reaction mix. The PCR thermal profile was the same as mentioned in the above section as well. Four wells of negative controls with human reference genomic DNA, two wells of positive controls with 1:2500 ratio of mutant allele to wild-type allele, and two wells of non-template control were always included in every run. The number of positive droplets and sample DNA input follows the Poisson distribution. Calculation of plasma sample DNA input per reaction in the unit of genome equivalents (GEs) per reaction (I, GEs/reaction) was calculated with the following equation: I(GEs/reaction)=-ln(1-p)/V×1000×20(1) where p is the fraction of positive droplets and V is the volume of each droplet (0.91 nL). For L858R assay, I equals to the total copies of EGFR mutant and wild-type DNA templates (FAM and VIC signal). For E19-Del assay, I equals to the copies of EGFR-Exon2 DNA template (VIC signal only). The samples were called positive for target mutations when they contained at least two droplets in the positive area of FAM signal. The fraction of EGFR L858R mutant (F1) was calculated as follows: F1=I(FAM)/(I(FAM)+I(VIC))(2) The fraction of EGFR E19-Del mutant (F2) was calculated as follows: F2=I(FAM)/I(VIC)(3) The sensitivity and specificity of the blood test (detected by ddPCR) was calculated by comparing with the paired tumor tissue result (detected by ARMS). The result consistency between blood and tissue was assessed by Cohen's κ test,27Landis J.R. Koch G.G. The measurement of observer agreement for categorical data.Biometrics. 1977; 33: 159-174Crossref PubMed Scopus (51055) Google Scholar and P < 0.05 was considered significant. The CIs for sensitivity and specificity were computed with the Clopper and Pearson method.28Clopper C.J. Pearson E.S. The use of confidence or fiducial limits illustrated in the case of the binomial.Biometrika. 1934; 26: 404-413Crossref Google Scholar The analysis was performed with R software version 3.02 (http://www.r-project.org). Through testing serial dilutions of EGFR mutants by mixing DNA derived from the positive cell lines (H1650 for E19-Dels, H1975 for L858R) with wild-type DNA from human reference genomic DNA, we were able to stably detect the two major types of EGFR mutations until the ratio of mutant to wild-type was ≥1:2500 (0.04%) (Figure 2). Further diluting the mutant DNA in a mixture of 1:5000 could not reliably detect >1 FAM+ droplet (Figure 2). Therefore, the selective sensitivity of the ddPCR assays for both E19-Dels and L858R was 0.04% in the cell line DNA test. In total, 86 plasma cfDNA samples collected from EGFR-TKI–naive NSCLC patients were tested by the two ddPCR assays for the EGFR-activating mutations, E19-Dels and L858R. Their paired 86 tumor tissue samples were previously tested by using an ARMS method.25Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar For the E19-Del ddPCR assay, 19 plasma samples were identified to be positive, including 18 with E19-Dels alone and 1 with E19-Del and L858R double mutation. The 18 patients positive for E19-Dels alone in the plasma were also positive for E19-Dels in the paired tumor tissues tested by ARMS. The patient with E19-Del and L858R double mutation in plasma only had a L858R mutation detected in the matched tumor tissue. However, four patients who were positive for E19-Dels in the tumor tissues, including one with E19-Del and L858R double mutation, did not have detectable E19-Del mutation in the paired plasma samples. The rest of the 63 patients were negative in both tumor tissue and plasma samples for E19-Del mutation. Put together, the overall concordance rate between plasma and tumor tissue testing was 94.19% (81 of 86; κ = 0.840; 95% CI, 0.704–0.976; P < 0.0001). The sensitivity and specificity for plasma testing of E19-Del mutation by ddPCR were 81.82% (18 of 22; 95% CI, 59.72%–94.81%) and 98.44% (63 of 64; 95% CI, 91.60%–99.96%), respectively (Table 2).Table 2Comparison of EGFR E19-Dels and EGFR L858R Detected in Plasma cfDNA by ddPCR versus the Status Detected in Paired Tumor Tissue by ARMSPlasma cfDNATumor tissueTotal+−EGFR E19-Dels +18119 −46367 Total226486EGFR L858R +12315 −36871 Total157186ARMS, amplification refractory mutation system; cfDNA, cell-free DNA; ddPCR, droplet digital P

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