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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter

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Volume 55, Issue 12


Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients

Saeam Shin
  • Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
  • Department of Laboratory Medicine, Hallym University College of Medicine, Kangnam Sacred Heart Hospital, Seoul, Republic of Korea
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/ Juwon Kim
  • Department of Laboratory Medicine, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea
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/ Yoonjung Kim
  • Department of Laboratory Medicine, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea
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/ Sun-Mi Cho
  • Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
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/ Kyung-A Lee
  • Corresponding author
  • Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
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Published Online: 2017-03-17 | DOI: https://doi.org/10.1515/cclm-2016-0851



EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples.


A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples.


All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%.


We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Keywords: cobas; EGFR; malignant pleural effusion; non-small-cell lung cancer; real-time PCR


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About the article

Corresponding author: Kyung-A Lee, MD, PhD, Department of Laboratory Medicine, Yonsei University College of Medicine, 211, Eonju-ro, Gangnam-gu, Seoul, 06273, Republic of Korea, Phone: +82-2-2019-3531, Fax: +82-2-2019-4822

aSaeam Shin and Juwon Kim contributed equally to this work.

Received: 2016-09-20

Accepted: 2017-02-07

Published Online: 2017-03-17

Published in Print: 2017-10-26

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This study was supported by grants from the Korean Health Technology R&D Project of the Ministry of Health & Welfare (A120030), as well as the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A13053986).

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 55, Issue 12, Pages 1962–1969, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2016-0851.

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