Journal of Cancer Research and Clinical Oncology

, Volume 142, Issue 10, pp 2209–2216 | Cite as

MassARRAY, pyrosequencing, and PNA clamping for EGFR mutation detection in lung cancer tissue and cytological samples: a multicenter study

  • Kyueng-Whan Min
  • Wan-Seop KimEmail author
  • Se Jin Jang
  • Yoo Duk Choi
  • Sunhee Chang
  • Soon Hee Jung
  • Lucia Kim
  • Mee-Sook Roh
  • Choong Sik Lee
  • Jung Weon Shim
  • Mi Jin Kim
  • Geon Kook Lee
  • Korean Cardiopulmonary Pathology Study Group
Original Article – Clinical Oncology



Testing for epidermal growth factor receptor (EGFR) mutation is an important process in the therapeutic plan of patients with lung cancer. Recently, MassARRAY, based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, has been shown to be a useful method for somatic mutation analysis with pyrosequencing and peptide nucleic acid clamping (PNAc).


A total of 107 tissues and 67 cytological samples, which were confirmed to have lung adenocarcinoma at nine hospitals in Korea, were collected. Among the MassARRAY, pyrosequencing, and PNAc, the concordance rates and sensitivity of EGFR mutation detection were analyzed and validated in comparative tissue and cytological specimens.


The concordance rate between pyrosequencing and PNAc was higher than that between MassARRAY and either of the pyrosequencing and PNAc in both tissue and cytological samples. In a comparison of diagnostic performance, MassARRAY (sensitivity: 85.7 %) was higher than pyrosequencing (74.3 %) and PNAc (70 %) in tissue, although pyrosequencing (80.5 %) was more highly sensitive, compared to MassARRAY (70.7 %) and PNAc (70.7 %) in terms of cytology. Unexpectedly, use of MassARRAY resulted in a significantly different EGFR mutation detection rate between tissue and cytological samples.


When used for the detection of EGFR mutations, MassARRAY was more sensitive than pyrosequencing or PNA clamping in tissue, but not in cytological samples. In EGFR mutation detection between tissues and cytology, PNAc showed relatively higher concordance than MassARRAY or pyrosequencing.


EGFR mutation Mass spectrometry, pyrosequencing Peptide nucleic acid Lung adenocarcinoma 



The authors thank all members of the Korean Cardio-Pulmonary Pathology Study Group for their support and their excellent input.


This research was conducted with the support of AstraZeneca (ISSIRES0079).

Compliance with ethical standards

Conflict of interest

There is no actual or potential conflict of interest to declare.

Ethical standard

This study was conducted under the approval of the Institutional Review Board of the Konkuk University Medical Center (KUH 1210016).

Supplementary material

432_2016_2211_MOESM1_ESM.docx (43 kb)
Supplementary material 1 (DOCX 42 kb)
432_2016_2211_MOESM2_ESM.docx (42 kb)
Supplementary material 2 (DOCX 41 kb)


  1. Ahmadian A, Ehn M, Hober S (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363:83–94CrossRefPubMedGoogle Scholar
  2. Azzoli CG et al (2010) American society of clinical oncology clinical practice guideline update on chemotherapy for stage IV non-small-cell lung cancer. Zhongguo fei ai za zhi 13:171–189PubMedGoogle Scholar
  3. Basu P, Chandna P, Bamezai RN, Siddiqi M, Saranath D, Lear A, Ratnam S (2011) MassARRAY spectrometry is more sensitive than PreTect HPV-Proofer and consensus PCR for type-specific detection of high-risk oncogenic human papillomavirus genotypes in cervical cancer. J Clin Microbiol 49:3537–3544CrossRefPubMedPubMedCentralGoogle Scholar
  4. Buttitta F et al (2013) Effective assessment of egfr mutation status in bronchoalveolar lavage and pleural fluids by next-generation sequencing. Clin Cancer Res 19:691–698CrossRefPubMedGoogle Scholar
  5. da Cunha Santos G, Saieg MA, Geddie W, Leighl N (2011) EGFR gene status in cytological samples of nonsmall cell lung carcinoma: controversies and opportunities. Cancer Cytopathol 119:80–91. doi: 10.1002/cncy.20150 CrossRefPubMedGoogle Scholar
  6. Deeb KK, Hohman CM, Risch NF, Metzger DJ, Starostik P (2015) Routine clinical mutation profiling of non-small cell lung cancer using next-generation sequencing. Arch Pathol Lab Med 139:913–921CrossRefPubMedGoogle Scholar
  7. Dufort S, Richard MJ, de Fraipont F (2009) Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Anal Biochem 391:166–168CrossRefPubMedGoogle Scholar
  8. Dufort S, Richard MJ, Lantuejoul S, de Fraipont F (2011) Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC. J Exp Clin Cancer Res 30:57CrossRefPubMedPubMedCentralGoogle Scholar
  9. Fassina A, Gazziero A, Zardo D, Corradin M, Aldighieri E, Rossi GP (2009) Detection of EGFR and KRAS mutations on trans-thoracic needle aspiration of lung nodules by high resolution melting analysis. J Clin Pathol 62:1096–1102CrossRefPubMedGoogle Scholar
  10. Goto K et al (2012) An evaluation study of EGFR mutation tests utilized for non-small-cell lung cancer in the diagnostic setting. Ann Oncol 23:2914–2919CrossRefPubMedGoogle Scholar
  11. Jones ES, Sullivan H, Bhattramakki D, Smith JS (2007) A comparison of simple sequence repeat and single nucleotide polymorphism marker technologies for the genotypic analysis of maize (Zea mays L.). Theor Appl Genet 115:361–371CrossRefPubMedGoogle Scholar
  12. Khode R et al (2013) Comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas. Cancer Cytopathol 121:361–369CrossRefPubMedGoogle Scholar
  13. Kim SK et al (2008) Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules. Diagn Mol Pathol 17:118–125CrossRefPubMedGoogle Scholar
  14. Kim HJ et al (2012) Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer. Lung Cancer 75:321–325CrossRefPubMedGoogle Scholar
  15. Kriegsmann M, Arens N, Endris V, Weichert W, Kriegsmann J (2015) Detection of KRAS, NRAS and BRAF by mass spectrometry—a sensitive, reliable, fast and cost-effective technique. Diagn Pathol 10:132CrossRefPubMedPubMedCentralGoogle Scholar
  16. Langaee T, Ronaghi M (2005) Genetic variation analyses by pyrosequencing. Mutat Res 573:96–102CrossRefPubMedGoogle Scholar
  17. Lee SH et al (2015) Analysis of mutations in epidermal growth factor receptor gene in Korean patients with non-small cell lung cancer: summary of a nationwide survey. J Pathol Transl Med 49:481–488CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lozano MD et al (2011) Assessment of epidermal growth factor receptor and K-ras mutation status in cytological stained smears of non-small cell lung cancer patients: correlation with clinical outcomes. Oncologist 16:877–885CrossRefPubMedPubMedCentralGoogle Scholar
  19. MacConaill LE et al (2009) Profiling critical cancer gene mutations in clinical tumor samples. PLoS ONE 4:e7887CrossRefPubMedPubMedCentralGoogle Scholar
  20. Maemondo M et al (2010) Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 362:2380–2388CrossRefPubMedGoogle Scholar
  21. Nakajima T, Yasufuku K, Nakagawara A, Kimura H, Yoshino I (2011) Multigene mutation analysis of metastatic lymph nodes in non-small cell lung cancer diagnosed by endobronchial ultrasound-guided transbronchial needle aspiration. Chest 140:1319–1324CrossRefPubMedGoogle Scholar
  22. Peng J, Yang F, Xiong Z, Guo J, Du J, Hu Y, Jin Q (2013) Sensitive and rapid detection of viruses associated with hand foot and mouth disease using multiplexed MALDI-TOF analysis. J Clin Virol 56:170–174CrossRefPubMedGoogle Scholar
  23. Sakurada A, Lara-Guerra H, Liu N, Shepherd FA, Tsao MS (2008) Tissue heterogeneity of EGFR mutation in lung adenocarcinoma. J Thorac Oncol 3:527–529CrossRefPubMedGoogle Scholar
  24. Tsai TH et al (2012) Effusion immunocytochemistry as an alternative approach for the selection of first-line targeted therapy in advanced lung adenocarcinoma. J Thorac Oncol 7:993–1000CrossRefPubMedGoogle Scholar
  25. van Eijk R et al (2011) Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qPCR. PLoS ONE 6:e17791CrossRefPubMedPubMedCentralGoogle Scholar
  26. Vincek V, Nassiri M, Nadji M, Morales AR (2003) A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples. Lab Investig 83:1427–1435CrossRefPubMedGoogle Scholar
  27. Vivante A, Amariglio N, Koren-Michowitz M, Ashur-Fabian O, Nagler A, Rechavi G, Cohen Y (2007) High-throughput, sensitive and quantitative assay for the detection of BCR-ABL kinase domain mutations. Leukemia 21:1318–1321CrossRefPubMedGoogle Scholar
  28. Wang S, Yu B, Ng CC, Mercorella B, Selinger CI, O’Toole SA, Cooper WA (2015) The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer. Transl Lung Cancer Res 4:119–125PubMedPubMedCentralGoogle Scholar
  29. Yang JC et al (2014) Epidermal growth factor receptor mutation analysis in previously unanalyzed histological samples and cytology samples from the phase III Iressa Pan-ASia Study (IPASS). Lung Cancer 83:174–181CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Kyueng-Whan Min
    • 1
  • Wan-Seop Kim
    • 2
    Email author
  • Se Jin Jang
    • 3
  • Yoo Duk Choi
    • 4
  • Sunhee Chang
    • 5
  • Soon Hee Jung
    • 6
  • Lucia Kim
    • 7
  • Mee-Sook Roh
    • 8
  • Choong Sik Lee
    • 9
  • Jung Weon Shim
    • 10
  • Mi Jin Kim
    • 11
  • Geon Kook Lee
    • 12
  • Korean Cardiopulmonary Pathology Study Group
  1. 1.Department of PathologyHanyang University Guri Hospital, Hanyang University College of MedicineGuri-siSouth Korea
  2. 2.Department of PathologyKonkuk University Medical Center, Konkuk University School of MedicineSeoulSouth Korea
  3. 3.Department of PathologyAsan Medical Center, University of Ulsan College of MedicineSeoulSouth Korea
  4. 4.Department of PathologyChonnam National University Hospital, Chonnam National University Medical SchoolGwangjuSouth Korea
  5. 5.Department of PathologyInje Unversity Ilsan Paik Hospital, Inje University School of MedicineGoyang-siSouth Korea
  6. 6.Department of PathologyWonju Severance Christian Hospital, Yonsei University Wonju College of MedicineWonju-siSouth Korea
  7. 7.Department of PathologyInha University Hospital, Inha University School of MedicineIncheonSouth Korea
  8. 8.Department of PathologyDong-A University Hospital, Dong-A University College of MedicineBusanSouth Korea
  9. 9.Department of PathologyChungnam National University Hospital, Chungnam National University College of MedicineDaejeonSouth Korea
  10. 10.Department of PathologyHallym University Dongtan Sacred Heart Hospital, Hallym University College of MedicineHwaseong-siSouth Korea
  11. 11.Department of PathologyYeungnam University Medical Center, Yeungnam University College of MedicineDaeguSouth Korea
  12. 12.Department of PathologyNational Cancer CenterGoyang-siSouth Korea

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