Skip to main content
SpringerLink
Log in

Rapid and Efficient Detection of EGFR Mutations in Problematic Cytologic Specimens by High-Resolution Melting Analysis

  • Original Research Article
  • Published:
Molecular Diagnosis & Therapy Aims and scope Submit manuscript

Abstract

Background and Objective: Chemotherapy for advanced non-small-cell lung cancer (NSCLC) remains marginally effective, with a 5-year overall survival rate of approximately 5%. Recently, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib was approved in Slovakia for the treatment of metastatic NSCLC. Gefitinib is a selective EGFR inhibitor that binds to the adenosine triphosphate binding pocket of the kinase domain and blocks downstream signaling pathways. Mutations of the EGFR gene, particularly an in-frame 15 bp deletion (delE746_A750) in exon 19 and the L858R mutation in exon 21, correlate with enhanced clinical responsiveness to EGFR tyrosine kinase inhibitors. However, the detection of these mutations and thereby prediction of the therapy outcome is sometimes unreliable due to the low sensitivity of direct sequencing if the proportion of tumor cells in the tissue is less than 25%. Therefore we decided to test the applicability of other methods, particularly high-resolution melting analysis (HRMA), for detection of these mutations in clinical samples.

Methods: We analyzed 53 archival cytologic specimens for the presence of EGFR mutations, using the HRMA method. Results were verified by direct sequencing. For samples containing less than 25% tumor cells, we used mutant-enriched PCR before sequencing. We also performed a titration assay to establish the lower limit of the proportion of tumor cells for detection of EGFR mutations.

Results: EGFR mutations were detected in 13 cases (24%). In-frame deletions in exon 19 were detected in eight cases (15%) and the L858R mutation in exon 21 was detected in five cases (9%). The positive results of the HRMA were confirmed by direct sequencing only in five of 13 cases. In the remaining eight positive samples, HRMA results were confirmed by sequencing analysis after mutant-DNA enrichment. The titration assay established that the lower limit for detection of EGFR mutations by HMRA was 1% tumor cells in the clinical sample.

Conclusion: Our results indicated that HRMA in combination with mutant-enriched PCR represents a sensitive method for detection of EGFR mutations from cytologic specimens. When properly executed, this protocol allows identification of EGFR mutations in specimens containing a minimal percentage of tumor cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Table I
Fig. 1
Table II
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. National Health Information Center. Health statistics yearbook of the Slovak Republic 2008. Bratislava: National Health Information Center, 2009: 208

    Google Scholar 

  2. Uddin D. Non-small cell lung cancer (NSCLC): an update. TAJ: Journal of Teachers Association 2002; 15: 115-8 [online]. Available from URL: http://www.banglajol.info/index.php/TAJ/article/view/3923 [Accessed 2011 Mar 1]

    Google Scholar 

  3. Jemal A, Siegel R, Ward E. Cancer statistics. CA Cancer J Clin 2007; 57:43–66

    Article  PubMed  Google Scholar 

  4. Uramoto H, Mitsudomi T. Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer? Br J Cancer 2007; 96: 857–63

    Article  PubMed  CAS  Google Scholar 

  5. Katzel JA, Fanutcchi MP, Li Z. Recent advances of novel targeted therapy in non-small cell lung cancer. J Hematol Oncol 2009; 2: 1–18

    Article  Google Scholar 

  6. Gazdar AF, Minna JD. Deregulated EGFR signaling during lung cancer progression: mutations, amplicons and autocrine loops. Cancer Prev Res 2008; 1: 156–60

    Article  CAS  Google Scholar 

  7. Nair P. Epidermal growth factor receptor family and its role in cancer progression. Current Science 2005; 88: 890–8

    Google Scholar 

  8. Neal JW, Sequist V. Targeted therapies: optimal first-line therapy for NSCLC with EGFR mutations. Nat Rev Clin Oncol 2010; 7: 71–2

    Article  PubMed  CAS  Google Scholar 

  9. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation and clinical response to gefitinib therapy. Science 2004; 304: 1497–500

    Article  PubMed  CAS  Google Scholar 

  10. Yun CH, Boggon TJ, Li Y, et al. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 2007; 11: 217–27

    Article  PubMed  CAS  Google Scholar 

  11. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor underlying responsiveness of NSCLC to gefitinib. N Engl J Med 2004; 350: 2129–39

    Article  PubMed  CAS  Google Scholar 

  12. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 2004; 101: 13306–11

    Article  PubMed  CAS  Google Scholar 

  13. Reed GH, Kent JO, Wittwer CT. High resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 2007; 8: 597–608

    Article  PubMed  CAS  Google Scholar 

  14. Wittwer CT, Reed GH, Gundry CN, et al. High resolution genotyping by amplicon melting analysis using LC Green. Clin Chem 2003; 49: 853–60

    Article  PubMed  CAS  Google Scholar 

  15. Kimura H, Fijiwara Y, Sone T, et al. High sensitivity detection of EGFR mutations in the pleural effusion of NSCLC patients. Cancer Sci 2006; 97: 642–8

    Article  PubMed  CAS  Google Scholar 

  16. Cohen V, Aqulnik JS, Jarry J, et al. Evaluation of denaturating high-performance liquid chromatography as a rapid detection method for identification of EGFR mutations in NSCLC. Cancer 2006; 107: 2858–65

    Article  PubMed  CAS  Google Scholar 

  17. Asano H, Toyooka S, Tokumo M, et al. Detection of EGFR gene mutation in lung cancer by mutant-enriched PCR polymerase chain reaction assay. Clin Cancer Res 2006; 12: 43–8

    Article  PubMed  CAS  Google Scholar 

  18. Nomoto K, Tsuta K, Takano T, et al. Detection of EGFR mutations in archieved cytologic specimens of NSCLC using HRMA. Am J Clin Pathol 2006; 126: 608–15

    Article  PubMed  CAS  Google Scholar 

  19. Fukui T, Ohe Y, Tsuta K, et al. Prospective study of the accuracy of EGFR mutational analysis by high-resolution melting analysis in small samples obtained from patients with non-small cell lung cancer. Clin Cancer Res 2008; 14:4751–7

    Article  PubMed  CAS  Google Scholar 

  20. Heideman DAM, Thunnisen FB, Doeleman M, et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes. Cellular Oncology 2009; 31: 329–33

    PubMed  CAS  Google Scholar 

  21. Fassina A, Gazziero A, Zardo D, et al. Detection of EGFR and KRAS mutations on trans-thoracic needle aspiration of lung nodules by high resolution melting analysis. J Clin Pathol 2009; 62: 1096–102

    Article  PubMed  CAS  Google Scholar 

  22. Takano T, Ohe Y, Fukui T, et al. Epidermal growth factor receptor mutation detection using high-resolution melting analysis predicts outcomes in patients with advanced non-small cell lung cancer treated with gefitinib. Clin Cancer Res 2007; 13: 5385–90

    Article  PubMed  CAS  Google Scholar 

  23. Chin TM, Anuar D, Soo R, et al. Detection of epidermal growth factor receptor variations by partially denaturing HPLC. Clin Chem 2007; 53: 62–70

    Article  PubMed  CAS  Google Scholar 

  24. Janne PA, Borras AM, Kuang Y, et al. A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res 2006; 12: 751–8

    Article  PubMed  Google Scholar 

  25. Takano T, Ohe T, Sakamoto H, et al. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005; 23: 6829–37

    Article  PubMed  CAS  Google Scholar 

  26. Marchetti A, Martella C, Felicioni L, et al. EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 2005; 23: 857–65

    Article  PubMed  CAS  Google Scholar 

  27. Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn 2005; 7: 396–403

    Article  PubMed  CAS  Google Scholar 

  28. Miyazawa H, Tanaka T, Nagai Y, et al. Peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based detection test for gefitinib-refractory T790M epidermal growth factor receptor mutation. Cancer Science 2008; 99: 595–600

    Article  PubMed  CAS  Google Scholar 

  29. Endo K, Konishi A, Sasaki H, et al. Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay. Lung Cancer 2005; 50: 375–84

    Article  PubMed  Google Scholar 

  30. Kimura H, Kasahara K, Kawaishi M, et al. Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer. Clin Cancer Res 2006; 12: 3915–21

    Article  PubMed  CAS  Google Scholar 

  31. Do H, Krypuy M, Mitchell PL, et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies. BMC Cancer 2008; 8: 1–14

    Article  Google Scholar 

  32. Do H, Dobrovic A. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies. Mol Cancer 2009; 8: 1–11

    Article  Google Scholar 

  33. Quach N, Goodman F, Shibata D. In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol 2004; 4: 1

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Jana Tinakova for her help with figure design and Jan Marcus for review and approval of the manuscript. The authors have no conflicts of interest that are directly relevant to the content of this study.

Author information

Authors and Affiliations

  1. Department of Cancer Genetics, National Cancer Institute, Bratislava, Slovakia

    Katarina Hlinkova

  2. Department of Pathology, Faculty of Medicine, Comenius University, Bratislava, Slovakia

    Pavel Babál

  3. Department of Oncology, Specialized Hospital of St. Zoerardus Zobor, Teaching Base of the Slovak Medical University, Nitra, Slovakia

    Peter Berzinec

  4. Department of Pneumology and Phthisiology, Faculty of Medicine Comenius University, Bratislava, Slovakia

    Ivan Majer

  5. Pediatric Department, Children’s University Hospital, Comenius University, Limbova 1, 833 40, Bratislava, Slovakia

    Denisa Ilencikova MD, PhD (Associate Professor)

Authors
  1. Katarina Hlinkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pavel Babál
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter Berzinec
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ivan Majer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Denisa Ilencikova MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Denisa Ilencikova MD, PhD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hlinkova, K., Babál, P., Berzinec, P. et al. Rapid and Efficient Detection of EGFR Mutations in Problematic Cytologic Specimens by High-Resolution Melting Analysis. Mol Diagn Ther 15, 21–29 (2011). https://doi.org/10.1007/BF03257190

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03257190

Keywords

Search

Navigation