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Performance Characteristics of a Real-Time Polymerase Chain Reaction Assay for the Detection of Epidermal Growth Factor Receptor (EGFR) Mutations in Plasma Samples of Non-Small Cell Lung Cancer (NSCLC) Patients

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Abstract

Background

Circulating free DNA in plasma is an alternative source of tumor-derived DNA that can be a surrogate for tissue epidermal growth factor receptor (EGFR) testing.

Objective

We evaluated the analytical performance of the cobas® EGFR Mutation Test v2 (cobas test), a real-time polymerase chain reaction assay designed to detect defined EGFR gene mutations in plasma from patients with advanced non-small cell lung cancer (NSCLC).

Methods

We used K2-ethylenediaminetetraacetic acid plasma samples from NSCLC patients and healthy donors (HDs), along with cell line DNA. Results from a complete technical performance evaluation are described, including a comparison between NSCLC and HD plasma to support the use of surrogate samples and an independent confirmation of the limit of detection (LoD).

Results

The cobas test reported an overall percent agreement of approximately 88% for plasma samples when compared with a next-generation sequencing method. The LoD for all EGFR mutations was ≤ 100 copies/mL for plasma samples. An external study confirmed the LoD for exon 19 deletion, L858R, and T790M at ≤ 100 copies/mL using samples derived from NSCLC patient specimens. The cobas test showed linearity between at least 50 and 10,000 copies/mL for plasma samples. An internal repeatability study reported a correct call accuracy of 99.2% for plasma samples. The performance of the cobas test is equivalent when using sheared or intact cell line DNA diluted into either HD plasma or NSCLC patient plasma.

Conclusions

The cobas test is a sensitive, robust, and accurate assay that delivers reproducible results.

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Associated datasets will not be deposited.

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Acknowledgements

We would like to acknowledge Sweta Shah, Johnny Shyu, Jingchuan Li, Robert Current, Taraneh Rehage, Misgana Bogale, and Yiqiao Wu (all of Roche Molecular Systems) for their contributions during the execution of this study. Funding for this study was provided by Roche Molecular Systems (Pleasanton, CA, USA). COBAS is a trademark of Roche. All other trademarks are the property of their respective owners.

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Authors and Affiliations

  1. Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, CA, 94588, USA

    Patrick O’Donnell, Theresa May, Kelli DeMartin, Harkanwal Halait, Wen Wei, Karen Yu & Sid Scudder

  2. Thermo Fisher Scientific, 46500 Kato Road, Fremont, CA, 94538, USA

    Jane Ferguson

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Contributions

All authors contributed to the visualization, writing, review, and editing of the manuscript. PO was responsible for conceptualization, methodology, investigation, validation, formal analysis, and data curation. TM, KDM, and JF were responsible for methodology, validation, investigation, formal analysis, and data curation. HH was responsible for conceptualization, investigation, and data curation. WW was responsible for conceptualization, methodology, validation, investigation, and data curation. KY was responsible for conceptualization, validation, and data curation. SS was responsible for conceptualization. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Patrick O’Donnell.

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Funding

This study was funded by Roche Molecular Solutions, Pleasanton, CA, USA.

Conflict of interest

Patrick O’Donnell is an employee of Roche Molecular Solutions. Theresa May is an employee of Roche Molecular Solutions. Kelli DeMartin is an employee of Roche Molecular Solutions. Jane Ferguson was previously an employee of Roche Molecular Systems until September 2018. Harkanwal Halait is an employee of Roche Molecular Solutions. Wei Wen is an employee of Roche Molecular Solutions and has previously received patent authorization for patents related to the cobas test. Karen Yu is an employee of Roche Molecular Solutions. Sid Scudder is an employee of Roche Molecular Solutions and owns stock in F. Hoffmann-La Roche AG.

Ethics approval

K2-EDTA plasma specimens from NSCLC patients and healthy donors were obtained from commercial vendors and collaborators with appropriate informed consent.

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O’Donnell, P., May, T., DeMartin, K. et al. Performance Characteristics of a Real-Time Polymerase Chain Reaction Assay for the Detection of Epidermal Growth Factor Receptor (EGFR) Mutations in Plasma Samples of Non-Small Cell Lung Cancer (NSCLC) Patients. Mol Diagn Ther 24, 451–460 (2020). https://doi.org/10.1007/s40291-020-00458-7

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