Sequenziamento mediante pyrosequencing: un’analisi rapida e sensibile per la determinazione delle mutazioni di EGFR nel carcinoma polmonare non a piccole cellule

  • Giovanna Dott.ssa Maglio
  • F. Rosa
  • M. Ferruggia
  • E. Masiero
  • S. Pizzolitto
  • G. M. Guarrera
  • C. Favaretti
Articolo originale


Lung cancer is the most common tumor and one of the most aggressive, with rates of survival at 5 years of about 10% in most countries. Studies reported EGFR (Epidermal Growth Factor Receptor) gene activating mutations in 10–15% of NSCLC (Non Small Cell Lung Cancer) in the European population.

Molecular assessment of EGFR mutations became mandatory for gefitinib administration in NSCLC patients. Only tumors harboring at least one activating mutation in either exon 18, 19 or 21 are eligible for therapy with this drug.

Several methods are available in order to study EGFR mutations, such as direct sequencing and Real Time PCR. Pyrosequencing is a DNA sequencing technique based on the principle of sequencing-by-synthesis and it is characterized by a greater sensitivity compared to direct sequencing. We compared pyrosequencing with Real Time PCR, both combined with CE-IVD approved commercially available kits for EGFR analysis, in terms of effectiveness, economic and logistic impact.

In conclusion, although Real Time PCR has a greater sensitivity, pyrosequencing is an effective and reliable method, with an advantage in detecting new mutations whose clinical impact is yet unknown. More studies are needed in order to define which sensitivity level is really correlated to clinical response.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
  2. 2.
    Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008; 359(13): 1367–80PubMedCrossRefGoogle Scholar
  3. 3.
  4. 4.
    Rusch V, Baselga J, Cordon-Cardo C, et al. Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 1993; 53(10 suppl): 2379–85PubMedGoogle Scholar
  5. 5.
    Keedy VL, Temin S, Somerfield MR, et al. American Society of Clinical Oncology Provisional Clinical Opinion: epidermal growth factor receptor (EGFR) mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol 2011; 29(15): 2121–7PubMedCrossRefGoogle Scholar
  6. 6.
    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(15): 4751–7PubMedCrossRefGoogle Scholar
  7. 7.
    Determinazione AIFA 11/05/2010, G.U. n. 122, 27/05/2010Google Scholar
  8. 8.
    Pirker R, Herth FJ, Kerr KM, et al. Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop. J Thorac Oncol 2010; 5(10): 1706–13PubMedCrossRefGoogle Scholar
  9. 9.
    EGFR TKI Response® (Resistance) — Manuale d’uso versione 2010/10, Diatech Pharmacogenetics (Italy)Google Scholar
  10. 10.
    Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005; 2(3): 225–35CrossRefGoogle Scholar
  11. 11.
    Nguyen KS, Kobayashi S, Costa DB. Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancers dependent on the epidermal growth factor receptor pathway. Clin Lung Cancer 2009; 10(4): 281–9PubMedCrossRefGoogle Scholar
  12. 12.
    Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med 2008; 358(11): 1160–74PubMedCrossRefGoogle Scholar
  13. 13.
    Marchetti A, Normanno N. Raccomandazioni per l’analisi mutazionale del gene EGFR nel carcinoma polmonare. AIOM-SIAPEC-IAP 2008Google Scholar
  14. 14.
    Ahmadian A, Ehn M, Hober S. Pyrosequencing: history, biochemistry and future. Clin Chim Acta 2006; 363(1–2): 83–94PubMedCrossRefGoogle Scholar
  15. 15.
    EGFR TKI Response® (Sensitivity) — Manuale d’uso versione 2010/11, Diatech Pharmacogenetics (Italy)Google Scholar
  16. 16.
    Kosaka T, Yatabe Y, Onozato R, et al. Prognostic implication of EGFR, KRAS, and TP53 gene mutations in a large cohort of Japanese patients with surgically treated lung adenocarcinoma. J Thorac Oncol 2009; 4(1): 22–9PubMedCrossRefGoogle Scholar
  17. 17.
    Schneider CP, Heidegener D, Schott-von-Römer K, et al. Epidermal growth factor receptor-related tumor markers and clinical outcomes with erlotinib in non-small cell lung cancer: an analysis of patients from German centers in the TRUST study. J Thorac Oncol 2008; 3(12): 1446–53PubMedCrossRefGoogle Scholar
  18. 18.
    Sunaga N, Tomizawa Y, Yanagitani N, et al. Phase II prospective study of the efficacy of gefitinib for the treatment of stage III/IV non-small cell lung cancer with EGFR mutations, irrespective of previous chemotherapy. Lung Cancer 2007; 56(3): 383–9PubMedCrossRefGoogle Scholar
  19. 19.
    Cappuzzo F, Hirsch FR, Rossi E, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005; 97(9): 643–55PubMedCrossRefGoogle Scholar
  20. 20.
    Han SW, Kim TY, Hwang PG, et al. Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol 2005; 23(11): 2493–501PubMedCrossRefGoogle Scholar
  21. 21.
    Ellison G, Donald E, McWalter G, et al. A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples. J Exp Clin Cancer Res 2010; 29: 132PubMedCrossRefGoogle Scholar
  22. 22.
    Ohnishi H, Ohtsuka K, Ooide A, et al. A simple and sensitive method for detecting major mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene in non-small-cell lung carcinoma. Diagn Mol Pathol 2006; 15(2): 101–8PubMedCrossRefGoogle Scholar
  23. 23.
    Oh YH, Kim Y, Kim YP, et al. Rapid detection of the epidermal growth factor receptor mutation in non-small-cell lung cancer for analysis of acquired resistance using molecular beacons. J Mol Diagn 2010; 12(5): 644–52PubMedCrossRefGoogle Scholar
  24. 24.
    Cohen V, Agulnik JS, Ang C, et al. Epidermal growth factor receptor mutations detected by denaturing high-performance liquid chromatography in nonsmall cell lung cancer: impact on response to therapy with epidermal growth factor receptor-tyrosine kinase inhibitors. Cancer 2010; 116(18): 4309–17PubMedCrossRefGoogle Scholar
  25. 25.
    Minarik M, Gassman M, Belsanova B, et al. A novel high-resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA. Electrophoresis 2010; 31(21): 3518–24PubMedCrossRefGoogle Scholar
  26. 26.
    Heideman DA, Thunnissen 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. Cell Oncol 2009; 31(5): 329–33PubMedGoogle Scholar
  27. 27.
    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(12): 1096–102PubMedCrossRefGoogle Scholar
  28. 28.
    Wang J, Ramakrishnan R, Tang Z, et al. Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays. Clin Chem 2010; 56(4): 623–32PubMedCrossRefGoogle Scholar
  29. 29.
    Li G, Luo X, He J, et al. A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue. Clin Chem Lab Med2011; 49(2): 191–5PubMedGoogle Scholar
  30. 30.
    Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn 2005; 7(3): 413–21PubMedCrossRefGoogle Scholar
  31. 31.
    Takano T, Ohe Y, 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(28): 6829–37PubMedCrossRefGoogle Scholar
  32. 32.
    Gridelli C, De Marinis F, Di Maio M, et al. Gefitinib as first-line treatment for patients with advanced non-small-cell lung cancer with activating Epidermal Growth Factor Receptor mutation: implications for clinical practice and open issues. Lung Cancer 2011; 72(1): 3–8PubMedCrossRefGoogle Scholar
  33. 33.
    Rho JK, Choi YJ, Lee JK, et al. The role of MET activation in determining the sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors. Mol Cancer Res 2009; 7(10): 1736–43PubMedCrossRefGoogle Scholar
  34. 34.
    Dahse R, Berndt A, Kosmehl H. PCR-based testing for therapy-related EGFR mutations in patients with non-small cell lung cancer. Anticancer Res 2008; 28(48): 2265–70PubMedGoogle Scholar
  35. 35.
    Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989; 17(7): 2503–16PubMedCrossRefGoogle Scholar
  36. 36.
    Thelwell N, Millington S, Solinas A, et al. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res 2000; 28(19): 3752–61PubMedCrossRefGoogle Scholar
  37. 37.
    Whitcombe D, Theaker J, Guy SP, et al. Detection of PCR products using self-probing amplicons and fluorescence. Nature Biotech 1999; 17(8): 804–7CrossRefGoogle Scholar
  38. 38.
    Chen S, Chen Z, Guo A, et al. Detection of epidermal growth factor receptor mutations in non-small cell lung cancer tumor specimens from various ways by denaturing high-performance liquid chromatography (abstract). Zhongguo Fei Ai Za Zhi (Chinese journal of lung cancer) 2010; 13(9): 850AGoogle Scholar
  39. 39.
    Cohen V, Agulnik JS, Jarry J, et al. Evaluation of denaturing high-performance liquid chromatography as a rapid detection method for identification of epidermal growth factor receptor mutations in nonsmall-cell lung cancer. Cancer 2006; 107(12): 2858–65PubMedCrossRefGoogle Scholar
  40. 40.
    Kramer D, Thunnissen FB, Gallegos-Ruiz MI, et al. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations. Cell Oncol 2009; 31(3): 161–7PubMedGoogle Scholar
  41. 41.
    Simonetti S, Molina MA, Queralt C, et al. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer. J Transl Med 2010; 8: 135PubMedCrossRefGoogle Scholar
  42. 42.
    Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. J Mol Diagn 2010; 12(2): 169–76PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2012

Authors and Affiliations

  • Giovanna Dott.ssa Maglio
    • 1
  • F. Rosa
    • 2
  • M. Ferruggia
    • 2
  • E. Masiero
    • 1
  • S. Pizzolitto
    • 1
  • G. M. Guarrera
    • 2
  • C. Favaretti
    • 3
  1. 1.SOC Anatomia PatologicaAzienda Ospedaliero Universitaria Santa Maria della MisericordiaUdine
  2. 2.Unità di Valutazione delle Tecnologie SanitarieAzienda Ospedaliero Universitaria Santa Maria della MisericordiaUdine
  3. 3.Direttore GeneraleAzienda Ospedaliero Universitaria Santa Maria della MisericordiaUdine

Personalised recommendations