Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing: results of the RING observational trial

Atocha Romero, Eloisa Jantus-Lewintre, Beatriz García-Peláez, Ana Royuela, Amelia Insa, Patricia Cruz, Ana Collazo, Javier Pérez Altozano, Oscar Juan Vidal, Pilar Diz, Manuel Cobo, Berta Hernández, Sergio Vázquez Estevez, Gretel Benítez, Maria Guirado, Margarita Majem, Reyes Bernabé, Ana Laura Ortega, Ana Blasco, Joaquim Bosch-Barrera, Jose M Jurado, Jorge García González, Santiago Viteri, Carlos Garcia Giron, Bartomeu Massutí, Ana Lopez Martín, Alejandro Rodriguez-Festa, Silvia Calabuig-Fariñas, Miguel Ángel Molina-Vila, Mariano Provencio, Atocha Romero, Eloisa Jantus-Lewintre, Beatriz García-Peláez, Ana Royuela, Amelia Insa, Patricia Cruz, Ana Collazo, Javier Pérez Altozano, Oscar Juan Vidal, Pilar Diz, Manuel Cobo, Berta Hernández, Sergio Vázquez Estevez, Gretel Benítez, Maria Guirado, Margarita Majem, Reyes Bernabé, Ana Laura Ortega, Ana Blasco, Joaquim Bosch-Barrera, Jose M Jurado, Jorge García González, Santiago Viteri, Carlos Garcia Giron, Bartomeu Massutí, Ana Lopez Martín, Alejandro Rodriguez-Festa, Silvia Calabuig-Fariñas, Miguel Ángel Molina-Vila, Mariano Provencio

Abstract

Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next-generation sequencing (NGS)-based methods, three high-sensitivity PCR-based platforms, and two FDA-approved methods were compared using 72 plasma samples, from EGFR-mutant non-small-cell lung cancer (NSCLC) patients progressing on a first-line tyrosine kinase inhibitor (TKI). NGS platforms as well as high-sensitivity PCR-based methodologies showed excellent agreement for EGFR-sensitizing mutations (K = 0.80-0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86-0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false-positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs.

Trial registration: ClinicalTrials.gov NCT03363139.

Keywords: NGS; circulating free DNA; epidermal growth factor receptor; non-small-cell lung cancer; osimertinib; tyrosine kinase inhibitor.

Conflict of interest statement

The authors have declared no conflict of interest at individual level. AstraZeneca supported this work. All reagents, necessary for the study, were funded by AstraZeneca. The study protocol and the final version of the manuscript were revised and approved by AstraZenca.

© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Figures

Fig. 1
Fig. 1
Venn diagrams showing concordance among NGS‐based methodologies and PCR‐based platforms for T790M detection. L2 NGS: Oncomine™ Pan‐Cancer Cell‐Free Assay performed in laboratory 2. L3 NGS: GeneRead™ QIAact Lung DNA UMI Cancer Panel performed in laboratory 3. L2 dPCR: QuantStudio®3D Digital PCR System, performed in laboratory 2. L3 TaqMan in‐house 5‐nuclease real‐time PCR assay in presence of PNA carried out in laboratory 3. L1 BEAMing OncoBEAMEGFRperformed in laboratory 1.
Fig. 2
Fig. 2
Comparison of MAFs obtained by NGS‐based platforms. (A) Passing‐Bablok regression showing close concordance between the two methods for the assessment of T790M MAFs and Bland–Altman plot showing low level of bias between both methods for quantifying T790M allele frequency. (B) Passing–Bablok regression and Bland–Altman plot showing the agreement between NGS‐based platforms for the quantification ofEGFR‐sensitizing mutations.

References

    1. Sequist LV, Martins RG, Spigel D, Grunberg SM, Spira A, Jänne PA, Joshi VA, McCollum D, Evans TL, Muzikansky A et al (2008) First‐line gefitinib in patients with advanced non‐small‐cell lung cancer harboring somatic EGFR mutations. J Clin Oncol 26, 2442–2449.
    1. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y et al (2009) Gefitinib or carboplatin‐paclitaxel in pulmonary adenocarcinoma. N Engl J Med 361, 947–957.
    1. Rosell R, Carcereny E, Gervais R, Vergnenegre A, Massuti B, Felip E, Palmero R, Garcia‐Gomez R, Pallares C, Sanchez JM et al (2012) Erlotinib 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 13, 239–246.
    1. Park K, Tan E‐H, O'Byrne K, Zhang L, Boyer M, Mok T, Hirsh V, Yang JC‐H, Lee KH, Lu S et al (2016) Afatinib versus gefitinib as first‐line treatment of patients with EGFR mutation‐positive non‐small‐cell lung cancer (LUX‐Lung 7): a phase 2B, open‐label, randomised controlled trial. Lancet Oncol 17, 577–589.
    1. Mok TS, Wu Y‐L, Ahn M‐J, Garassino MC, Kim HR, Ramalingam SS, Shepherd FA, He Y, Akamatsu H, Theelen WS et al (2017) Osimertinib or platinum‐pemetrexed in EGFR T790M–positive lung cancer. N Engl J Med 376, 629–640.
    1. Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH, Dechaphunkul A, Imamura F, Nogami N, Kurata T et al (2018) Osimertinib in untreated EGFR‐mutated advanced non‐small‐cell lung cancer. N Engl J Med 378, 113–125.
    1. Rolfo C, Mack PC, Scagliotti GV, Baas P, Barlesi F, Bivona TG, Herbst RS, Mok TS, Peled N, Pirker R et al (2018) Liquid biopsy for advanced non‐small cell lung cancer (NSCLC): a statement paper from the IASLC. J Thorac Oncol 13, 1248–1268.
    1. Leighl NB, Page RD, Raymond VM, Daniel DB, Divers SG, Reckamp KL, Villalona‐Calero MA, Dix D, Odegaard JI, Lanman RB et al (2019) Clinical utility of comprehensive cell‐free DNA analysis to identify genomic biomarkers in patients with newly diagnosed metastatic non‐small cell lung cancer. Clin Cancer Res 25, 4691–4700.
    1. Lung Cancer Metastatic. Available online at NCCN guidelines for patients non‐small cell lung cancer.
    1. Mayo‐de‐las‐Casas C, Jordana‐Ariza N, Garzón‐Ibañez M, Balada‐Bel A, Bertrán‐Alamillo J, Viteri‐Ramírez S, Reguart N, Muñoz‐Quintana MA, Lianes‐Barragan P, Camps C et al (2017) Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions. Ann Oncol 28, 2248–2255.
    1. Oxnard GR, Paweletz CP, Kuang Y, Mach SL, O'Connell A, Messineo MM, Luke JJ, Butaney M, Kirschmeier P, Jackman DM et al (2014) 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.
    1. Jenkins S, Yang JCH, Ramalingam SS, Yu K, Patel S, Weston S, Hodge R, Cantarini M, Jänne PA, Mitsudomi T et al (2017) Plasma ctDNA analysis for detection of the EGFR T790M mutation in patients with advanced non‐small cell lung cancer. J Thorac Oncol 12, 1061–1070.
    1. Provencio M, Torrente M, Calvo V, Pérez‐Callejo D, Gutiérrez L, Franco F, Pérez‐Barrios C, Barquín M, Royuela A, García‐García F et al (2018) Prognostic value of quantitative ctDNA levels in non small cell lung cancer patients. Oncotarget 9, 488–494.
    1. Karachaliou N, Mayo‐De Las Casas C, Queralt C, De Aguirre I, Melloni B, Cardenal F, Garcia‐Gomez R, Massuti B, Sánchez JM, Porta R et al (2015) Association of EGFR L858R mutation in circulating free DNA with survival in the EURTAC trial. JAMA Oncol 1, 149–157.
    1. Friedman M (1937) The use of ranks to avoid the assumption of normality implicit in the analysis of variance. J Am Stat Assoc 32, 675–701.
    1. Landis JR & Koch GG (1977) The measurement of observer agreement for categorical data. Biometrics 33, 159.
    1. Lin LI‐K (1989) A concordance correlation coefficient to evaluate reproducibility. Biometrics 45, 255.
    1. Steichen TJ & Cox NJ (2002) A note on the concordance correlation coefficient. Stata J Promot Commun Stat Stata 2, 183–189.
    1. McBride GB (2005) A proposal for strength‐of‐agreement criteria for Lin's concordance correlation coefficient. NIWA Client Report, HAM2005‐062.
    1. Passing H & Bablok W (1983) A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in Clinical Chemistry. Part I. J Clin Chem Clin Biochem 21, 709–720.
    1. Martin Bland J & Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 327, 307–310.
    1. Thress KS, Brant R, Carr TH, Dearden S, Jenkins S, Brown H, Hammett T, Cantarini M and Barrett JC (2015) EGFR mutation detection in ctDNA from NSCLC patient plasma: a cross‐platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer 90, 509–515.
    1. Papadimitrakopoulou VA, Han JY, Ahn MJ, Ramalingam SS, Delmonte A, Hsia TC, Laskin J, Kim S‐W, He Y, Tsai C‐M et al (2020) Epidermal growth factor receptor mutation analysis in tissue and plasma from the AURA3 trial: osimertinib versus platinum‐pemetrexed for T790M mutation‐positive advanced non–small cell lung cancer. Cancer 126, 373–380.
    1. O'Leary B, Hrebien S, Beaney M, Fribbens C, Garcia‐Murillas I, Jiang J, Li Y, Huang Bartlett C, André F, Loibl S et al (2019) Comparison of beaming and droplet digital PCR for circulating tumor DNA analysis. Clin Chem 65, 1405–1413.
    1. Li BT, Janku F, Jung B, Hou C, Madwani K, Alden R, Razavi P, Reis‐Filho JS, Shen R, Isbell JM et al (2019) Ultra‐deep next‐generation sequencing of plasma cell‐free DNA in patients with advanced lung cancers: results from the actionable genome consortium. Ann Oncol 30, 597–603.
    1. Provencio M, Pérez‐Barrios C, Barquin M, Calvo V, Franco F, Sánchez E, Sánchez R, Marsden D, Cristóbal Sánchez J, Martin Acosta P et al (2019) Next‐generation sequencing for tumor mutation quantification using liquid biopsies. Clin Chem Lab 58, 306–313.
    1. Barquín M, Maximiano C, Pérez‐Barrios C, Sanchez‐Herrero E, Soriano M, Colmena M, García‐Espantaleón M, Tejerina González E, Gutierrez L, Sánchez Ruiz AC et al (2019) Peritoneal washing is an adequate source for somatic BRCA1/2 mutation testing in ovarian malignancies. Pathol Res Pract 215, 392–394.
    1. Torga G & Pienta KJ (2018) Patient‐paired sample congruence between 2 commercial liquid biopsy tests. JAMA Oncol 4, 868–870.
    1. Gupta R, Othman T, Chen C, Sandhu J, Ouyang C & Fakih M (2020) Guardant360 circulating tumor DNA assay is concordant with FoundationOne next‐generation sequencing in detecting actionable driver mutations in anti‐EGFR naive metastatic colorectal cancer. Oncologist 25, 235–243.
    1. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM et al (2014) Detection of circulating tumor DNA in early‐ and late‐stage human malignancies. Sci Transl Med 6, 224ra24.
    1. Villatoro S, Mayo‐de‐las‐Casas C, Jordana‐Ariza N, Viteri‐Ramírez S, Garzón‐Ibañez M, Moya‐Horno I, García‐Peláez B, González‐Cao M, Malapelle U, Balada‐Bel A et al (2019) Prospective detection of mutations in cerebrospinal fluid, pleural effusion, and ascites of advanced cancer patients to guide treatment decisions. Mol Oncol 13, 2633–2645.
    1. Ptashkin RN, Mandelker DL, Coombs CC, Bolton K, Yelskaya Z, Hyman DM, Solit DB, Baselga J, Arcila ME, Ladanyi M et al (2018) Prevalence of clonal hematopoiesis mutations in tumor‐only clinical genomic profiling of solid tumors. JAMA Oncol 4, 1589–1593.
    1. Coombs CC, Gillis NK, Tan X, Berg JS, Ball M, Balasis ME, Montgomery ND, Bolton KL, Parker JS, Mesa TE et al (2018) Identification of clonal hematopoiesis mutations in solid tumor patients undergoing unpaired next‐generation sequencing assays. Clin Cancer Res 24, 5918–5924.

Source: PubMed

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