Advertisement

Tumor Biology

, Volume 36, Issue 2, pp 693–700 | Cite as

Immunohistochemistry with a novel mutation-specific monoclonal antibody as a screening tool for the EGFR L858R mutational status in primary lung adenocarcinoma

  • Wei Ping
  • Chunjiao Xia
  • Shengling Fu
  • Yixin Cai
  • Yu Deng
  • Wei Sun
  • Cuiping Dong
  • Xiangning Fu
Research Article

Abstract

Epidermal growth factor receptor (EGFR) mutation status is the best predictor of patient response to treatments with tyrosine kinase inhibitors in primary lung adenocarcinoma and is typically analyzed by DNA-based techniques, such as direct DNA sequencing and allele-specific PCR. Recently, however, two mutation-specific antibodies against delE746-A750 in exon 19 and L858R in exon 21 have opened the door for a more convenient and more efficient strategy to determine EGFR mutation status. To evaluate the clinical application of a new mutation-specific mouse monoclonal antibody for EGFR (L858R), we performed immunohistochemistry (IHC) studies with tumor samples from primary lung adenocarcinoma in retrospective and validation settings. A total of 215 cases of primary lung adenocarcinoma were examined and compared using a combination of DNA-based techniques (direct DNA sequencing and/or allele-specific PCR) and protein-based IHC. IHC staining was assessed on a 0 to 3+ score scale, and a cutoff value of 2+ was used as positive by IHC. In the retrospective setting, statistical analyses of the data showed that the sensitivity of IHC was 90.9 % and the specificity was 96.8 %. Findings from the validation study demonstrated that the sensitivity and specificity of IHC were 88.2 % and 100 %, respectively. IHC with the novel mutation-specific antibody could be used as a screening method to assess the EGFR L858R mutation status in primary lung adenocarcinoma.

Keywords

EGFR-L858R Immunohistochemistry Primary lung adenocarcinoma 

Notes

Acknowledgments

We would like to acknowledge NewEast Biosciences for providing the antibody used in this study. This work was supported by the National Natural Science Foundation of China (no. 81301505) and the Natural Science Foundation of Hubei Province (no. 2011CDB561).

Conflicts of interest

None

References

  1. 1.
    Siegel R, Ward E, Brawley O, et al. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011;61:212–36.CrossRefPubMedGoogle Scholar
  2. 2.
    Molina JR, Yang P, Cassivi SD, et al. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc. 2008;83:584–94.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Verdecchia A, Francisci S, Brenner H, et al. Recent cancer survival in Europe: a 2000–02 period analysis of EUROCARE-4 data. Lancet Oncol. 2007;8:784–96.CrossRefPubMedGoogle Scholar
  4. 4.
    Dancey JE, Freidlin B. Targeting epidermal growth factor receptor—are we missing the mark? Lancet. 2003;362:62–4.CrossRefPubMedGoogle Scholar
  5. 5.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350:2129–39.CrossRefPubMedGoogle Scholar
  7. 7.
    Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304:1497–500.CrossRefPubMedGoogle Scholar
  8. 8.
    Rosell R, Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009;361:958–67.CrossRefPubMedGoogle Scholar
  9. 9.
    Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 2005;97:339–46.CrossRefPubMedGoogle Scholar
  10. 10.
    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.CrossRefPubMedGoogle Scholar
  11. 11.
    Mu XL, Li LY, Zhang XT, et al. Gefitinib-sensitive mutations of the epidermal growth factor receptor tyrosine kinase domain in Chinese patients with non-small cell lung cancer. Clin Cancer Res. 2005;11:4289–94.CrossRefPubMedGoogle Scholar
  12. 12.
    Huang SF, Liu HP, Li LH, et al. High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res. 2004;10:8195–203.CrossRefPubMedGoogle Scholar
  13. 13.
    Han SW, Kim TY, Lee KH, et al. Clinical predictors versus epidermal growth factor receptor mutation in gefitinib-treated non-small-cell lung cancer patients. Lung Cancer. 2006;54:201–7.CrossRefPubMedGoogle Scholar
  14. 14.
    Sharma SV, Bell DW, Settleman J, et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7:169–81.CrossRefPubMedGoogle Scholar
  15. 15.
    Scagliotti GV, Selvaggi G, Novello S, et al. The biology of epidermal growth factor receptor in lung cancer. Clin Cancer Res. 2004;10:4227s–32s.CrossRefPubMedGoogle Scholar
  16. 16.
    Sordella R, Bell DW, Haber DA, et al. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 2004;305:1163–7.CrossRefPubMedGoogle Scholar
  17. 17.
    Ellison G, Zhu G, Moulis A, et al. EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples. J Clin Pathol. 2013;66:79–89.CrossRefPubMedGoogle Scholar
  18. 18.
    Yu J, Kane S, Wu J, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res. 2009;15:3023–8.CrossRefPubMedGoogle Scholar
  19. 19.
    Kato Y, Peled N, Wynes MW, et al. Novel epidermal growth factor receptor mutation-specific antibodies for non-small cell lung cancer: immunohistochemistry as a possible screening method for epidermal growth factor receptor mutations. J Thorac Oncol. 2010;5:1551–8.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kozu Y, Tsuta K, Kohno T, et al. The usefulness of mutation-specific antibodies in detecting epidermal growth factor receptor mutations and in predicting response to tyrosine kinase inhibitor therapy in lung adenocarcinoma. Lung Cancer. 2011;73:45–50.CrossRefPubMedGoogle Scholar
  21. 21.
    Azuma K, Okamoto I, Kawahara A, et al. Association of the expression of mutant epidermal growth factor receptor protein as determined with mutation-specific antibodies in non-small cell lung cancer with progression-free survival after gefitinib treatment. J Thorac Oncol. 2012;7:122–7.CrossRefPubMedGoogle Scholar
  22. 22.
    Liu Y, Liu B, Li XY, et al. A comparison of ARMS and direct sequencing for EGFR mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non-small-cell lung cancer patients. J Exp Clin Cancer Res. 2011;30:111.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Ping W, Jiang WY, Chen WS, et al. Expression and significance of hypoxia inducible factor-1alpha and lysyl oxidase in non-small cell lung cancer. Asian Pac J Cancer Prev. 2013;14:3613–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Sequist LV, Joshi VA, Janne PA, et al. Epidermal growth factor receptor mutation testing in the care of lung cancer patients. Clin Cancer Res. 2006;12:4403s–8s.CrossRefPubMedGoogle Scholar
  25. 25.
    Nagai Y, Miyazawa H, Huqun. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res. 2005;65:7276–82.CrossRefPubMedGoogle Scholar
  26. 26.
    Sakurada A, Lara-Guerra H, Liu N, et al. Tissue heterogeneity of EGFR mutation in lung adenocarcinoma. J Thorac Oncol. 2008;3:527–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361:947–57.CrossRefPubMedGoogle Scholar
  28. 28.
    Angulo B, Garcia-Garcia E, Martinez R, et al. A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinoma. J Mol Diagn. 2010;12:292–9.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Angulo B, Conde E, Suarez-Gauthier A, et al. A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry. PLoS One. 2012;7:e43842.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Buttitta F, Felicioni L, Del Grammastro M, et al. Effective assessment of egfr mutation status in bronchoalveolar lavage and pleural fluids by next-generation sequencing. Clin Cancer Res. 2013;19:691–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Asano H, Toyooka S, Tokumo M, et al. Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin Cancer Res. 2006;12:43–8.CrossRefPubMedGoogle Scholar
  32. 32.
    Takano T, Ohe Y, Tsuta K, 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.CrossRefPubMedGoogle Scholar
  33. 33.
    Pao W, Ladanyi M. Epidermal growth factor receptor mutation testing in lung cancer: searching for the ideal method. Clin Cancer Res. 2007;13:4954–5.CrossRefPubMedGoogle Scholar
  34. 34.
    Ilie MI, Hofman V, Bonnetaud C, et al. Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcinoma. Virchows Arch. 2010;457:483–95.CrossRefPubMedGoogle Scholar
  35. 35.
    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;12:169–76.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Nakamura H, Mochizuki A, Shinmyo T, et al. Immunohistochemical detection of mutated epidermal growth factor receptors in pulmonary adenocarcinoma. Anticancer Res. 2010;30:5233–7.PubMedGoogle Scholar
  37. 37.
    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;8:135.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wu SG, Chang YL, Lin JW, et al. Including total EGFR staining in scoring improves EGFR mutations detection by mutation-specific antibodies and EGFR TKIs response prediction. PLoS One. 2011;6:e23303.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kitamura A, Hosoda W, Sasaki E, et al. Immunohistochemical detection of EGFR mutation using mutation-specific antibodies in lung cancer. Clin Cancer Res. 2010;16:3349–55.CrossRefPubMedGoogle Scholar
  40. 40.
    Kawahara A, Yamamoto C, Nakashima K, et al. Molecular diagnosis of activating EGFR mutations in non-small cell lung cancer using mutation-specific antibodies for immunohistochemical analysis. Clin Cancer Res. 2010;16:3163–70.CrossRefPubMedGoogle Scholar
  41. 41.
    Kawahara A, Azuma K, Sumi A, et al. Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry. Lung Cancer. 2011;74:35–40.CrossRefPubMedGoogle Scholar
  42. 42.
    Hasanovic A, Ang D, Moreira AL, et al. Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma. Lung Cancer. 2012;77:299–305.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  1. 1.Department of Thoracic Surgery, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
  2. 2.Department of PathologyHubei Cancer HospitalWuhanChina

Personalised recommendations