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Electric Field–Induced Release and Measurement (EFIRM)

2020; Elsevier BV; Volume: 22; Issue: 8 Linguagem: Inglês

10.1016/j.jmoldx.2020.05.005

1943-7811

Michael Tu, Jordan Cheng, Yi-Lin Chen, Wen-Chien Jea, Wan-Li Chen, Chien-Jung Chen, Chung‐Liang Ho, Wei-Lun Huang, Chien‐Chung Lin, Wu‐Chou Su, Qianlin Ye, Josh Deignan, Wayne W. Grody, Feng Li, David Chia, Fang Wei, Wei Liao, David T. Wong, Charles M. Strom,

Nanopore and Nanochannel Transport Studies

Electric field–induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non–small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma. Electric field–induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non–small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma. The detection and analysis of cell-free circulating tumor DNA (ctDNA) is becoming a useful tool in the care of patients with cancer.1Poulet G. Massias J. Taly V. Liquid biopsy: general concepts.Acta Cytol. 2019; 63: 449-455Crossref PubMed Scopus (129) Google Scholar, 2Xu C. Offin M. Paik P.K. Li B.T. Liquid biopsy guided precision therapy for lung cancers.J Thorac Dis. 2018; 10 Suppl 33: S4173-S4175Crossref Scopus (3) Google Scholar, 3Rolfo C. Mack P.C. Scagliotti G.V. Baas P. Narlesi F. Bivona T.G. Herbst R.S. Mok T.S. Peled N. Pirker R. Raez L.E. Reck M. Reiss J.W. Sequist L.V. Shepherd F.A. Sholl L.M. Tan D.S.W. Wakelee H.A. Wistuba I.I. Wynes M.W. Carbone D.P. Hirsch F.R. Gandara D.R. Liquid biopsy for advanced non-small cell lung cancer (NSCLC): a statement paper from the IASLC.J Thorac Oncol. 2018; 13: 1248-1268Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 4Schwaederle M. Husain H. Fanta P.T. Piccioni D.E. Kesari S. Schwab R.B. Patel S.P. Harismendy O. Ikeda M. Parker B.A. Kurzrock R. Use of liquid biopsies in clinical oncology: pilot experience in 168 patients.Clin Cancer Res. 2016; 22: 5497-5505Crossref PubMed Scopus (99) Google Scholar, 5Almodovar K. Iams W.T. Meador C.B. Zhao Z. York S. Horn L. Yan Y. Hernandez J. Chen H. Shyr Y. Lim L.P. Raymond C.K. Lovly C.M. Longitudinal cell-free DNA analysis in patients with small cell lung cancer reveals dynamic insights into treatment efficacy and disease relapse.J Thorac Oncol. 2018; 13: 112-123Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Commonly referred to as liquid biopsy (LB), this process has both advantages and disadvantages when compared with the gold standard of tissue biopsy.1Poulet G. Massias J. Taly V. Liquid biopsy: general concepts.Acta Cytol. 2019; 63: 449-455Crossref PubMed Scopus (129) Google Scholar Because LB provides no histologic staining or spatial analysis, staging is currently possible only by conventional tissue biopsy. However, when tissue is unavailable, LB may be the only possible source of information regarding the genetic makeup of a solid tumor.1Poulet G. Massias J. Taly V. Liquid biopsy: general concepts.Acta Cytol. 2019; 63: 449-455Crossref PubMed Scopus (129) Google Scholar,3Rolfo C. Mack P.C. Scagliotti G.V. Baas P. Narlesi F. Bivona T.G. Herbst R.S. Mok T.S. Peled N. Pirker R. Raez L.E. Reck M. Reiss J.W. Sequist L.V. Shepherd F.A. Sholl L.M. Tan D.S.W. Wakelee H.A. Wistuba I.I. Wynes M.W. Carbone D.P. Hirsch F.R. Gandara D.R. Liquid biopsy for advanced non-small cell lung cancer (NSCLC): a statement paper from the IASLC.J Thorac Oncol. 2018; 13: 1248-1268Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar In addition, LB samples can be obtained by minimally invasive (venipuncture) or noninvasive (saliva collection) techniques and can therefore be used for serial monitoring. LBs are also less affected by tumor heterogeneity than tissue biopsy.1Poulet G. Massias J. Taly V. Liquid biopsy: general concepts.Acta Cytol. 2019; 63: 449-455Crossref PubMed Scopus (129) Google Scholar Liquid biopsy has been applied to many solid tumors,4Schwaederle M. Husain H. Fanta P.T. Piccioni D.E. Kesari S. Schwab R.B. Patel S.P. Harismendy O. Ikeda M. Parker B.A. Kurzrock R. Use of liquid biopsies in clinical oncology: pilot experience in 168 patients.Clin Cancer Res. 2016; 22: 5497-5505Crossref PubMed Scopus (99) Google Scholar including lung,3Rolfo C. Mack P.C. Scagliotti G.V. Baas P. Narlesi F. Bivona T.G. Herbst R.S. Mok T.S. Peled N. Pirker R. Raez L.E. Reck M. Reiss J.W. Sequist L.V. Shepherd F.A. Sholl L.M. Tan D.S.W. Wakelee H.A. Wistuba I.I. Wynes M.W. Carbone D.P. Hirsch F.R. Gandara D.R. Liquid biopsy for advanced non-small cell lung cancer (NSCLC): a statement paper from the IASLC.J Thorac Oncol. 2018; 13: 1248-1268Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar,5Almodovar K. Iams W.T. Meador C.B. Zhao Z. York S. Horn L. Yan Y. Hernandez J. Chen H. Shyr Y. Lim L.P. Raymond C.K. Lovly C.M. Longitudinal cell-free DNA analysis in patients with small cell lung cancer reveals dynamic insights into treatment efficacy and disease relapse.J Thorac Oncol. 2018; 13: 112-123Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,6Ma C. YangX Xing W. Yu H. Si T. Guo Z. Detection of circulating tumor DNA from non-small cell lung cancer brain metastasis in cerebrospinal fluid samples.Thorac Cancer. 2020; 11: 588-593Crossref PubMed Scopus (34) Google Scholar breast,7Fernandez-Garcia D. Hills A. Page K. Gastings R.K. Toghill B. Goddard K.S. Ion C. Ogle C. Boydell A.R. Gleason K. Rutherford M. Lim A. Guttery D.S. Coombes R.C. Shaw J.A. Plasma cell-free DNA (cfDNA) as a predictive and prognostic marker in patients with metastatic breast cancer.Breast Cancer Res. 2019; 21: 149Crossref PubMed Scopus (57) Google Scholar pancreas,8Zhu Y. Zhang H. Chen N. Hao J. Jin H. Ma X. Diagnostic value of various liquid biopsy methods for pancreatic cancer: a systematic review and meta-analysis.Medicine (Baltimore). 2020; 99: e18581Crossref PubMed Scopus (31) Google Scholar melanoma,9Alrabadi N. Haddad R. Alomari A.K. Detection of gene mutations in liquid biopsy of melanoma patients: overview and future perspectives.Curr Treat Options Oncol. 2020; 21: 19-26Crossref PubMed Scopus (3) Google Scholar and prostate cancer.10Stuopelyte K. Sabaliauskaite R. Bakavicius A. Hadliadottir B.S. Visakorpi T. Vaananen R.M. Patel C. Danila D.C. Lilja H. Lazutka J.R. Ulys A. Jankevicius J.F. Jarmalaite S. Analysis of AR-FL and AR-V1 in whole blood of patients with castration resistant prostate cancer as a tool for predicting response to abiraterone acetate.J Urol. 2020; 204: 71-78Crossref PubMed Scopus (6) Google Scholar Heretofore, tissue samples obtained by biopsy or at the time of surgery were the only available specimen source. As such, the amount of tissue available was often limiting. More recently, technology has allowed us to detect and measure cell-free DNA in the blood11Wei F. Strom C.M. Cheng J. Lin C.-C. Hsu C.-Y. Soo Hoo G.W. Chia D. Kim Y. Li F. Elashoff D. Grognan T. Tu M. Liao W. Xian R. Grody W.W. Su W.-C. Wong D.T.W. Electric field–induced release and measurement liquid biopsy for noninvasive early lung cancer assessment.J Mol Diagn. 2018; 20: 738-742Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar and saliva,12Wei F. Lin C.-C. Joon A. Feng Z. Troche G. Lira M.E. Chia D. Mao M. Ho C.-L. Su W.-C. Wong D.T.W. Noninvasive saliva-based EGFR gene mutation detection in patients with lung cancer.Am J Respir Crit Care Med. 2014; 190: 1117-1126Crossref PubMed Scopus (115) Google Scholar,13Pu D. Liang H. Wei F. Akin D. Feng Z. Yan Q. Li Y. Zhen Y. Xu L. Dong G. Wan H. Dong J. Qiu X. Qin C. Zhu D. Wang X. Sun T. Zhang W. Li C. Tang X. Qiao Y. Wong D.T.W. Zhou Q. Evaluation of a novel saliva-based epidermal growth factor receptor mutation detection for lung cancer: a pilot study: saliva-based EGFR mutation detection.Thorac Cancer. 2016; 7: 428-436Crossref PubMed Scopus (55) Google Scholar providing ready access to a limitless supply of specimens. Although liquid biopsy is still in its infancy as a clinical tool, it has already demonstrated value in detecting EGFR mutations in patients with non–small-cell lung cancer (NSCLC) at the time of presentation and relapse,14Elazezy M. Joosse S.A. Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management.Comput Struct Biotechnol J. 2018; 16: 370-378Crossref PubMed Scopus (179) Google Scholar, 15Takahama T. Azuma K. Shimokawa M. Takeda M. Ishii H. Kato T. Saito H. Daga H. Tsuboguchi Y. Okamato I. Ossubo K. Akamatsu H. Teraoka S. Takahashi T. Ono A. Ohira T. Yokoyama T. Sakai K. Yamamoto N. Nishio K. Nagagawa K. Plasma screening for the T790M mutation of EGFR and phase 2 study of osimertinib efficacy in plasma T790M-positive non-small cell lung cancer: West Japan Oncology Group 8815L/LPS study.Cancer. 2020; 126: 1940-1948Crossref PubMed Scopus (10) Google Scholar, 16Papadopoulou E. Tsoulos N. Tsantikidi K. Metaxa-Mariatou V. Stamou P.E. Kladi-Skandali A. Kapeni E. Tsaousis G. Pentheroudakis G. Petrakis D. Lampropoulou S.I. Aravantinos G. Varthalitis G. Kesesis G. Boukovinas I. Papakotoulas P. Katirtzoglou N. Athanasiadis E. Stavridu F. Christodoulou C. Koumarianou A. Eralp Y. Nasioulas G. Clinical feasibility of NGS liquid biopsy analysis in NSCLC patients.PLoS One. 2019; 14: 1371-1378Crossref Scopus (27) Google Scholar, 17Zhang Y.-C. Zhou Q. Wu Y.-L. The emerging roles of NGS-based liquid biopsy in non-small cell lung cancer.J Hematol Oncol. 2017; 10: 167Crossref PubMed Scopus (44) Google Scholar, 18Offin M. Chabon J.J. Razavi P. Isbell J.M. Rudin C.M. Diehn M. Li B.T. Capturing genomic evolution of lung cancers through liquid biopsy for circulating tumor DNA.J Oncol. 2017; 2017: 4517834Crossref PubMed Scopus (19) Google Scholar, 19Karlovich C. Goldman J.W. Sun J.M. Mann E. Sequist L.V. Konopa K. Wen W. Angendendt P. Horn L. Spiegel D. Soria J.-C. Solomon B. Camidge D.R. Gadgeel S. Paweletz C. Wu L. Chien S. O'Donnell P. Matheny S. Despain D. Rolfe L. Raponi M. Allen A.R. Park K. Wakelee H. Assessment of EGFR mutation status in matched plasma and tumor tissue of NSCLC patients from a phase 1 study of Rociletimib (CO-1686).Clin Cancer Res. 2016; 22: 2386-2395Crossref PubMed Scopus (159) Google Scholar, 20Wu Y.L. Sequist L.V. Hu C.P. Feng J. Lu S. Huang Y. Li W. Hou M. Schuler M. Mok T. Yamamoto N. O'Byrne K. Hirsh V. Gibson N. Massey D. Kim M. Yang J.C.-H. EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: analysis of LUX-lung 3 and 6.Br J Cancer. 2017; 116: 175-185Crossref PubMed Scopus (65) Google Scholar prompting Roche Molecular Systems (Pleasanton, CA) to commercially release the Cobas EGFR Mutation Test in 2017. This assay was specifically validated for plasma testing. There is, as yet, no consensus regarding the clinical utility of LB in NSCLC or other solid tumors. A joint review by the American Society of Clinical Oncology and the College of Molecular Pathologists, published in 2018, asserted that there was insufficient evidence of clinical validity to recommend ctDNA analysis in the routine clinical setting.21Merker J. Oxnard G. Compton C. Diehn M. Hurley P. Lazar A.J. Lindeman N. Lockwood C.M. Rai A.J. Schilsky R.L. Tsimberidou A.M. Vasalos P. Billman B.L. Oliver T.K. Bruinooge S.S. Hayes D.F. Turner N.C. Circulating tumor DNA analysis in patients with cancer: American Society of Clinical Oncology and College of American Pathologists joint review.J Clin Oncol. 2018; 36: 1631-1641Crossref PubMed Scopus (521) Google Scholar In contrast, a statement article from the International Association for the Study of Lung Cancer that same year concluded that immediate implementation of LB in the clinic is justified in several therapeutic settings relevant to NSCLC.22Forbes S.A. Bhamra G. Bamford S. Dawson E. Kok C. Clements J. Menzies A. Teague J.W. Futreal P.A. Stratton M.R. The catalogue of somatic mutations in cancer (COSMIC).Curr Protoc Hum Genet. 2008; 57: 10.11.1-10.11.26Google Scholar As more data accumulate, LB is rapidly gaining acceptance by clinicians and insurers in certain clinical situations. Commercial laboratories now offer single or panel laboratory-developed mutation tests using cell-free DNA obtained by liquid biopsy for patients with NSCLC.3Rolfo C. Mack P.C. Scagliotti G.V. Baas P. Narlesi F. Bivona T.G. Herbst R.S. Mok T.S. Peled N. Pirker R. Raez L.E. Reck M. Reiss J.W. Sequist L.V. Shepherd F.A. Sholl L.M. Tan D.S.W. Wakelee H.A. Wistuba I.I. Wynes M.W. Carbone D.P. Hirsch F.R. Gandara D.R. Liquid biopsy for advanced non-small cell lung cancer (NSCLC): a statement paper from the IASLC.J Thorac Oncol. 2018; 13: 1248-1268Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar,16Papadopoulou E. Tsoulos N. Tsantikidi K. Metaxa-Mariatou V. Stamou P.E. Kladi-Skandali A. Kapeni E. Tsaousis G. Pentheroudakis G. Petrakis D. Lampropoulou S.I. Aravantinos G. Varthalitis G. Kesesis G. Boukovinas I. Papakotoulas P. Katirtzoglou N. Athanasiadis E. Stavridu F. Christodoulou C. Koumarianou A. Eralp Y. Nasioulas G. Clinical feasibility of NGS liquid biopsy analysis in NSCLC patients.PLoS One. 2019; 14: 1371-1378Crossref Scopus (27) Google Scholar In NSCLC, there are some data to suggest that liquid biopsy is preferable to tissue-based diagnosis for EGFR mutations in some situations, and indications for the use of liquid biopsy in NSCLC are increasing.3Rolfo C. Mack P.C. Scagliotti G.V. Baas P. Narlesi F. Bivona T.G. Herbst R.S. Mok T.S. Peled N. Pirker R. Raez L.E. Reck M. Reiss J.W. Sequist L.V. Shepherd F.A. Sholl L.M. Tan D.S.W. Wakelee H.A. Wistuba I.I. Wynes M.W. Carbone D.P. Hirsch F.R. Gandara D.R. Liquid biopsy for advanced non-small cell lung cancer (NSCLC): a statement paper from the IASLC.J Thorac Oncol. 2018; 13: 1248-1268Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar,19Karlovich C. Goldman J.W. Sun J.M. Mann E. Sequist L.V. Konopa K. Wen W. Angendendt P. Horn L. Spiegel D. Soria J.-C. Solomon B. Camidge D.R. Gadgeel S. Paweletz C. Wu L. Chien S. O'Donnell P. Matheny S. Despain D. Rolfe L. Raponi M. Allen A.R. Park K. Wakelee H. Assessment of EGFR mutation status in matched plasma and tumor tissue of NSCLC patients from a phase 1 study of Rociletimib (CO-1686).Clin Cancer Res. 2016; 22: 2386-2395Crossref PubMed Scopus (159) Google Scholar,20Wu Y.L. Sequist L.V. Hu C.P. Feng J. Lu S. Huang Y. Li W. Hou M. Schuler M. Mok T. Yamamoto N. O'Byrne K. Hirsh V. Gibson N. Massey D. Kim M. Yang J.C.-H. EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: analysis of LUX-lung 3 and 6.Br J Cancer. 2017; 116: 175-185Crossref PubMed Scopus (65) Google Scholar Our earlier work describes a new method for cell-free DNA analysis that demonstrated both superior sensitivity and specificity to existing PCR-based or next-generation sequencing (NGS) methods in patients with both early-stage (stages I and II) and late-stage NSCLC.11Wei F. Strom C.M. Cheng J. Lin C.-C. Hsu C.-Y. Soo Hoo G.W. Chia D. Kim Y. Li F. Elashoff D. Grognan T. Tu M. Liao W. Xian R. Grody W.W. Su W.-C. Wong D.T.W. Electric field–induced release and measurement liquid biopsy for noninvasive early lung cancer assessment.J Mol Diagn. 2018; 20: 738-742Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 12Wei F. Lin C.-C. Joon A. Feng Z. Troche G. Lira M.E. Chia D. Mao M. Ho C.-L. Su W.-C. Wong D.T.W. Noninvasive saliva-based EGFR gene mutation detection in patients with lung cancer.Am J Respir Crit Care Med. 2014; 190: 1117-1126Crossref PubMed Scopus (115) Google Scholar, 13Pu D. Liang H. Wei F. Akin D. Feng Z. Yan Q. Li Y. Zhen Y. Xu L. Dong G. Wan H. Dong J. Qiu X. Qin C. Zhu D. Wang X. Sun T. Zhang W. Li C. Tang X. Qiao Y. Wong D.T.W. Zhou Q. Evaluation of a novel saliva-based epidermal growth factor receptor mutation detection for lung cancer: a pilot study: saliva-based EGFR mutation detection.Thorac Cancer. 2016; 7: 428-436Crossref PubMed Scopus (55) Google Scholar The electric field–induced release and measurement (EFIRM) LB (eLB) method uses untreated plasma or saliva as input. As no special specimen preparation is required, pre-analytic variables are few. In addition, the eLB platform is in a 96-well microtiter plate format, increasing the opportunities for automation and greatly increasing specimen throughput and reducing turnaround time to as little as 3 hours. Furthermore, the 96-well format allows a dramatic reduction in assay cost to as low as ≤$100/mutation. In this article, we describe the eLB process and experiments to determine what eLB is measuring, followed by the technical development and validation of four separate eLB assays for six clinically actionable variants in the EGFR gene and four variants in g.exon19del, p.L858R, and p.T790M. There are qualitative assays for both plasma and saliva for potential screening purposes or recurrence detection and quantitative assays for plasma and saliva for the purpose of monitoring disease progression or drug response. We will present data, including reference range determinations, for the quantitative assay for both plasma and serum and the precision, sensitivity, specificity, level of detection, and intra-assay and interassay reproducibility. We also examine the nature of the nucleic acid sequences that are targeted by eLB. Figure 1 represents a schematic of the EFIRM method. Initially, a single allele-specific capture probe is added to a polypyrrole solution and added to a microtiter plate containing a gold electrode at the bottom of each well. After an electric field has been applied, the liquid becomes polymerized into a conducting gel and the capture oligonucleotides are anchored in the gel. Subsequently, the biological sample is applied and electrically facilitated allele-specific hybridization is performed. In this step, the kinetic energy imparted to the system increases the specificity of the hybridization. A biotinylated detector probe, whose sequence continues immediately after the capture probe sequence ends, is added and another electrically aided hybridization is performed. Subsequently, signal amplification and signal production are accomplished using standard methods. The final reaction generates an nA current that is measured by the EFIRM reader. A step-by-step workflow is as follows. Initially, a monomer solution is generated, consisting of 0.3 mol/L KCl (number 60137; Sigma Aldrich, St. Louis, MO) and 144.1 mmol/L pyrrole (W336805; Sigma Aldrich). An appropriate capture probe (Integrated DNA Technologies, Coralville, IA) is added to a final concentration of 2.5 μmol/L. The solution is transferred into a microcentrifuge tube and vortexed to thoroughly mix the contents. Subsequently, 60 μL of monomeric solution is transferred into each of 96 wells of an EFIRM E-Plate (EZLife Bio Inc., Los Angeles, CA). The E-Plate is then mounted into the EFIRM E-Reader (EZLife Bio Inc.) and a cyclic-square wave consisting of 350 mV and 1100 mV for 1 second each is applied for a total of 8 seconds. This step causes the monomer to polymerize into a conducting gel, coating the surface of the gold electrode while anchoring the capture probe to the gel. The plate is then removed from the E-Reader and placed into a 96-well plate washer (model 405LS; BioTek, Winooski, VT). A single wash is then performed using a 2× standard saline citrate 0.5% SDS solution. The plate is now ready for the addition of the clinical sample. Table 1 lists the capture probe/signal probe combinations used is this assay.Table 1Capture and Detection Oligonucleotides for eLB EGFR AssayVariantProbesCapture ProbesExon 19del5′-TGTTGCTTCCTTG-3′p.L858R5′-GTTTGACCCGCCCA-3′p.T790M5′-GAGCGGCATGATGA-3′Detector Probes∗Detector probes are biotin labeled at the terminal 3′ nucleotide.Exon 19del5′-ATAGCGACGGGAATTTTAACTTTCTCACCT-3′p.L858R5′-AAAATCTGTGATCTTGACATGCTGCGGTGTTTTGTGCAG-3′p.T790M5′-GCTGCACGGTGGAGGTGAGGCAGATGCCCAGC-3′eLB, electric field–induced release and measurement liquid biopsy.∗ Detector probes are biotin labeled at the terminal 3′ nucleotide. Open table in a new tab eLB, electric field–induced release and measurement liquid biopsy. To prepare clinical samples for testing, 20 to 30 μL of either plasma or saliva is diluted 1:2 in Ultrahyb Oligo Hybridization buffer (Thermo Fisher Scientific, Waltham, MA). Subsequently, 25 μL of the mixture is transferred to the bottom of each well. Once all samples have been added, the E-Plate is placed back into the E-Reader and a cyclic-square wave consisting of 300 mV and 500 mV for 1 second each is applied for a total of 300 seconds, followed by a 30-minute room temperature incubation in the E-Reader. The plate is then removed from the instrument and washed with 2× standard saline citrate 0.5% SDS in the plate washer. After this wash, 25 μL of a 100 nmol/L solution of detector probe (Integrated DNA Technologies) in casein/phosphate-buffered saline (PBS; 37528; Thermo Fisher Scientific) is added to each, the plate is returned to the E-Reader, and hybridization is performed using a cyclic-square wave consisting of −300 mV and 500 mV for 1 second each for a total of 300 seconds, followed by a 30-minute incubation at room temperature in the E-Reader. The plate is then returned to the plate washer and washed with 2× standard saline citrate 0.5% SDS. The plate is then removed from the plate washer, and a streptavidin poly–horseradish peroxidase (Thermo Fisher Scientific) solution is prepared by diluting the reagent 1:1000 in casein/PBS and then 60 μL is added to each well and incubated at room temperature for 30 minutes. The plate is then washed again using 1× PBS with 0.05% Tween-20. Biotinylated Anti-Streptavidin Antibody (BA-0500; Vector Laboratories, Burlingame, CA) is diluted 1:10,000 in casein/PBS and 60 μL is pipetted into each well, followed by a 30-minute incubation at room temperature and another wash step with 1× PBS with 0.05% Tween-20. A 1:1000 dilution of Poly80HRP-Streptavidin (Fitzgerald Industries, Acton, MA) casein/PBS is made, and 60 μL is pipetted into each electrode well and incubated at room temperature for 30 minutes, followed by a final wash using 1× PBS with 0.05% Tween-20. Finally, 60 μL of 1-Step Ultra TMB substrate (34028; Thermo Fisher Scientific) solution is added to each well, the plate is placed into the E-Reader, and electrochemical current in nA is measured at a potential of −200 mV for 1 minute. The technologist is guided in all steps by the EZL-Reader Software version 1.0 (EZLife Bio, Woodland Hills, CA), which provides sequential directions using a graphical user interface. There are five 30-minute incubations, and combined with the pipetting steps, the assay can be easily completed by a single technologist in 3 hours. Once the clinical samples have been added to the E-Plate, walk-away automation would be possible because all steps are performed with the plate washer, the E-Reader, or routine liquid handling. RNase treatment was performed by adding RNase cocktail reagent, consisting of 0.025 U/μL of RNase A and 1 U/μL of T1 RNase in casein/PBS RNase (AM2286; Thermo Fisher Scientific), and incubating for 30 minutes at room temperature. Washing was performed using 2× standard saline citrate 0.5% SDS wash buffer. This treatment was performed following the sample capture step of the EFIRM protocol. DNase treatment was performed to assess the strandedness of the DNA. First, Proteinase K (P8107S; New England Biolabs, Boston, MA) was added to achieve a concentration of 2 μg/μL in plasma samples. The samples were then incubated for 30 minutes at 50°C. Following this digest, proteinase K was then heated to 65°C for 10 minutes to deactivate the enzyme and then the solution was cooled to 4°C for 1 hour (using a cooling rate of 0.1°C/second) in a thermocycler. Following this initial proteinase K digest to remove interfering proteins, Exonuclease VII (M0379S; New England Biolabs) was introduced at a concentration of 0.33 units/μL and incubated at 37°C for 30 minutes. Exonuclease was subsequently deactivated by heat treatment at 95°C for 10 minutes, followed by cooling to 4°C for 1 hour (with a ramp rate of 0.1°C/second) to reanneal the double-stranded DNA present in the samples. The final solution was then assayed using the EFIRM protocol described in RNase Treatment Method. To generate genomic DNA reference standards for the point mutations p.L858R and p.T790M, genomic DNA was isolated using a QuickgDNA miniprep (Zymo Research, Irvine, CA) from the NCI-H1975 cell line (ATCC, Manassas, VA). For g.Exon19del testing, genomic DNA from four different cell lines harboring the top four variants of g.Exon19del mutations (COSM6623/6225/12370/12382) was acquired (Applied Stem Cell, Milpitas, CA). DNA extracted from the cell lines was diluted in water to a 50 ng/mL concentration. Shearing was accomplished by heat treatment in a thermocycler at 4°C for 3 minutes, followed by heating to 95°C for 7 minutes at a ramp rate of 4°C/second. Following heat treatment, the temperature was lowered to 4°C for 10 minutes at a ramp rate of 4°C/second. The sheared samples were then serially diluted in water and Ultrahyb Oligo Hybridization buffer, maintaining the same ratio of water/Ultrahyb Oligo buffer in each sample. Finally, 30 μL of each dilution was assayed using the EFIRM platform. The multichannel EFIRM Reader is controlled via a USB 2.0 connection, and parameters are set by a custom software suite called EZL-Reader version 1.0. Data from each EFIRM experiment were exported to the comma-separated value file format and analyzed using the R-Language for statistical analysis. Saliva was collected from healthy individual volunteers at meetings of the American Dental Association between 2006 and 2011. Consent was obtained under institutional review board approval (University of California, Los Angeles Institutional Review Board number 06-05-042). There was a mixture of males/females, mostly non-smokers, between 18 and 80 years of age, and a mixture of ethnicities. All subjects consented before collection. Each subject would expectorate approximately 5 mL of whole saliva in a 50 μL conical tube set on ice. The saliva was processed within 0.5 hours of collection. Samples were centrifuged in a refrigerated centrifuge at 2600 × g for 15 minutes at 4°C. The supernatant (cell-free saliva) was then pipetted into two 2-mL cryotubes and the following reagents were added to preserve the RNA and DNA: 1.1 μL Superase-In/1 mL supernatant (Ambion, Austin, TX). After the additional reagents were added, each tube was inverted to mix. The samples were then frozen on dry ice and later stored in −80°C. One 5-mL tube of blood was collected from consented subjects using a BD Vacutainer Safety-LokBlood Collection Set (367283; BD Biosciences, Franklin Lakes, NJ) and Lavender-top K2 EDTAtubes (367525; BD Biosciences). The tubes were filled and inverted and then centrifuged at 2500 × g at 4°C for 10 minutes within 2 hours of collection. The buffy coat free supernatant (plasma) was then removed, frozen, and stored at −70°C until assayed. The EFIRM technology is capable of detecting either DNA or RNA molecules. It was investigated whether the EGFR variants detected in clinical samples from patients with NSCLC and reported previously8Zhu Y. Zhang H. 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