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Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcinoma

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Abstract

Specific inhibitors targeting the epidermal growth factor receptor (EGFR) can increase survival rates in certain lung adenocarcinoma patients with mutations in the EGFR gene. Although such EGFR-targeted therapies have been approved for use, there is no general consensus among surgical pathologists on how the EGFR status should be tested in lung adenocarcinoma tissues and whether the results of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mutational analysis by molecular methods correlate. We evaluated the EGFR status in 61 lung adenocarcinomas by IHC (using total and mutant-specific antibodies against EGFR), by FISH analysis on tissue microarrays (TMAs), and by direct sequencing. The results of each method were compared using χ 2 and κappa statistics. The sensitivity and negative predictive value estimating the presence of abnormal EGFR for each test was calculated. The results show that, with respect to expression patterns and clinicopathological parameters, the total and mutant-specific EGFR detected by immunohistochemistry and FISH analysis on TMAs are valid and are equivalent to conventional methods performed on whole-tissue sections. Abnormal EGFR was detected in 52.4% of patients by IHC, FISH, and sequencing. The best sensitivity (100%) and negative predictive value (100%) was determined by evaluating the EGFR status with all methods. Testing for molecular changes in EGFR using a single test is likely to underestimate the presence of EGFR abnormalities. Taken together, these results demonstrate the high potential of TMAs to test for the major mechanisms of EGFR activation in patients with lung adenocarcinoma.

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References

  1. Hirsch FR, Witta S (2005) Biomarkers for prediction of sensitivity to EGFR inhibitors in non-small cell lung cancer. Curr Opin Oncol 17:118–122

    Article  CAS  PubMed  Google Scholar 

  2. Reck M (2009) Gefitinib in the treatment of advanced non-small-cell lung cancer. Expert Rev Anticancer Ther 9:401–412. doi:10.1586/era.09.1

    Article  CAS  PubMed  Google Scholar 

  3. Mok TS, Wu YL, Thongprasert S et al (2009) Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 361:947–957

    Article  CAS  PubMed  Google Scholar 

  4. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW (2003) Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 284:31–53. doi:10.1016/S0014-4827(02)00098-8

    Article  CAS  PubMed  Google Scholar 

  5. Sato M, Shames DS, Gazdar AF, Minna JD (2007) A translational view of the molecular pathogenesis of lung cancer. J Thorac Oncol 2:327–343. doi:10.1097/01.JTO.0000263718.69320.4c

    Article  PubMed  Google Scholar 

  6. Ono M, Kuwano M (2006) Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 12:7242–7251. doi:10.1158/1078-0432.CCR-06-0646

    Article  CAS  PubMed  Google Scholar 

  7. Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359:1367–1380. doi:10.1056/NEJMra0802714

    Article  CAS  PubMed  Google Scholar 

  8. Mendelsohn J, Baselga J (2003) Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 21:2787–2799

    Article  CAS  PubMed  Google Scholar 

  9. Ciardiello F, Tortora G (2008) EGFR antagonists in cancer treatment. N Engl J Med 358:1160–1174. doi:10.1056/NEJMra0707704

    Article  CAS  PubMed  Google Scholar 

  10. Sequist LV, Lynch TJ (2008) EGFR tyrosine kinase inhibitors in lung cancer: an evolving story. Annu Rev Med 59:429–442. doi:10.1146/annurev.med.59.090506.202405

    Article  CAS  PubMed  Google Scholar 

  11. Cappuzzo F, Hirsch FR, Rossi E et al (2005) Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 97:643–655. doi:10.1093/jnci/dji112

    Article  CAS  PubMed  Google Scholar 

  12. Parra HS, Cavina R, Latteri F, Zucali PA, Campagnoli E, Morenghi E, Grimaldi GC, Roncalli M, Santoro A (2004) Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib (‘Iressa’, ZD1839) in non-small-cell lung cancer. Br J Cancer 91:208–212. doi:10.1038/sj.bjc.6601923

    CAS  PubMed  Google Scholar 

  13. Perez-Soler R, Chachoua A, Hammond LA et al (2004) Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer. J Clin Oncol 22:3238–3247. doi:10.1200/JCO.2004.11.057

    Article  CAS  PubMed  Google Scholar 

  14. Hirsch FR, Varella-Garcia M, Bunn PA Jr et al (2006) Molecular predictors of outcome with gefitinib in a phase III placebo-controlled study in advanced non-small-cell lung cancer. J Clin Oncol 24:5034–5042. doi:10.1200/JCO.2006.06.3958

    Article  CAS  PubMed  Google Scholar 

  15. Tsao MS, Sakurada A, Cutz JC et al (2005) Erlotinib in lung cancer—molecular and clinical predictors of outcome. N Engl J Med 353:133–144

    Article  CAS  PubMed  Google Scholar 

  16. Hirsch FR, Varella-Garcia M, McCoy J et al (2005) Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J Clin Oncol 23:6838–6845. doi:10.1200/JCO.2005.01.2823

    Article  CAS  PubMed  Google Scholar 

  17. Bell DW, Lynch TJ, Haserlat SM et al (2005) Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials. J Clin Oncol 23:8081–8092. doi:10.1200/JCO.2005.02.7078

    Article  CAS  PubMed  Google Scholar 

  18. Han SW, Kim TY, Hwang PG et al (2005) Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol 23:2493–2501. doi:10.1200/JCO.2005.01.388

    Article  CAS  PubMed  Google Scholar 

  19. Morita S, Okamoto I, Kobayashi K et al (2009) Combined survival analysis of prospective clinical trials of gefitinib for non-small cell lung cancer with EGFR mutations. Clin Cancer Res 15:4493–4498. doi:10.1158/1078-0432.CCR-09-0391

    Article  CAS  PubMed  Google Scholar 

  20. Sharma SV, Bell DW, Settleman J, Haber DA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7:169–181. doi:10.1038/nrc2088

    Article  CAS  PubMed  Google Scholar 

  21. Ellis PM, Morzycki W, Melosky B et al (2009) The role of the epidermal growth factor receptor tyrosine kinase inhibitors as therapy for advanced, metastatic, and recurrent non-small-cell lung cancer: a Canadian national consensus statement. Curr Oncol 16:27–48

    Article  CAS  PubMed  Google Scholar 

  22. Yu J, Kane S, Wu J et al (2009) Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clin Cancer Res 15:3023–3028. doi:10.1158/1078-0432.CCR-08-2739

    Article  CAS  PubMed  Google Scholar 

  23. Hirsch FR, Varella-Garcia M, Cappuzzo F et al (2007) Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib. Ann Oncol 18:752–760. doi:10.1093/annonc/mdm003

    Article  CAS  PubMed  Google Scholar 

  24. Hirsch FR, Varella-Garcia M, Bunn PA Jr, Di Maria MV, Veve R, Bremmes RM, Baron AE, Zeng C, Franklin WA (2003) Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798–3807. doi:10.1200/JCO.2003.11.069

    Article  CAS  PubMed  Google Scholar 

  25. Li AR, Chitale D, Riely GJ, Pao W, Miller VA, Zakowski MF, Rusch V, Kris MG, Ladanyi M (2008) EGFR mutations in lung adenocarcinomas: clinical testing experience and relationship to EGFR gene copy number and immunohistochemical expression. J Mol Diagn 10:242–248. doi:10.2353/jmoldx.2008.070178

    Article  CAS  PubMed  Google Scholar 

  26. Gupta R, Dastane AM, Forozan F, Riley-Portuguez A, Chung F, Lopategui J, Marchevsky AM (2009) Evaluation of EGFR abnormalities in patients with pulmonary adenocarcinoma: the need to test neoplasms with more than one method. Mod Pathol 22:128–133. doi:10.1038/modpathol.2008.182

    Article  CAS  PubMed  Google Scholar 

  27. El-Zammar OA, Zhang S, Katzenstein AL (2009) Comparison of FISH, PCR, and immunohistochemistry in assessing EGFR status in lung adenocarcinoma and correlation with clinicopathologic features. Diagn Mol Pathol 18:133–137. doi:10.1097/PDM.0b013e3181857ea9

    Article  CAS  PubMed  Google Scholar 

  28. Gupta R, Dastane AM, McKenna R Jr, Marchevsky AM (2009) The predictive value of epidermal growth factor receptor tests in patients with pulmonary adenocarcinoma: review of current “best evidence” with meta-analysis. Hum Pathol 40:356–365. doi:10.1016/j.humpath.2008.08.008

    Article  CAS  PubMed  Google Scholar 

  29. Eberhard DA, Giaccone G, Johnson BE (2008) Biomarkers of response to epidermal growth factor receptor inhibitors in non-small-cell lung cancer working group: standardization for use in the clinical trial setting. J Clin Oncol 26:983–994. doi:10.1200/JCO.2007.12.9858

    Article  PubMed  Google Scholar 

  30. Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC (2004) World health organization classification of tumours. Pathology and genetics of tumours of the lung, pleura, thymus and heart. IARC Press, Lyon

    Google Scholar 

  31. Mountain CF (1997) Revisions in the international system for staging lung cancer. Chest 111:1710–1717. doi:10.1378/chest.111.6.1710

    Article  CAS  PubMed  Google Scholar 

  32. Betsou F, Lehmann S, Ashton G et al (2010) Standard preanalytical coding for biospecimens: defining the sample PREanalytical code. Cancer Epidemiol Biomark Prev 19:1004–1011. doi:10.1158/1055-9965.EPI-09-1268

    Article  CAS  Google Scholar 

  33. Hofman V, Ilie M, Gavric-Tanga V et al (2010) Rôle du laboratoire d’anatomie pathologique dans l’approche pré-analytique des examens de biologie moléculaire réalisés en pathologie tumorale. Ann Pathol 30:85–93

    Article  PubMed  Google Scholar 

  34. Hofman P, Butori C, Havet K, Hofman V, Selva E, Guevara N, Santini J, Van Obberghen-Schilling E (2008) Prognostic significance of cortactin levels in head and neck squamous cell carcinoma: comparison with epidermal growth factor receptor status. Br J Cancer 98:956–964. doi:10.1038/sj.bjc.6604245

    Article  CAS  PubMed  Google Scholar 

  35. Brevet M, Arcila M, Ladanyi M (2010) Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. J Mol Diagn 12:169–176. doi:10.2353/jmoldx.2010.090140

    Article  CAS  PubMed  Google Scholar 

  36. Koynova DK, Tsenova VS, Jankova RS, Gurov PB, Toncheva DI (2005) Tissue microarray analysis of EGFR and HER2 oncogene copy number alterations in squamous cell carcinoma of the larynx. J Cancer Res Clin Oncol 131:199–203. doi:10.1007/s00432-004-0627-y

    Article  CAS  PubMed  Google Scholar 

  37. Choong NW, Dietrich S, Seiwert TY et al (2006) Gefitinib response of erlotinib-refractory lung cancer involving meninges—role of EGFR mutation. Nat Clin Pract Oncol 3:50–57. doi:10.1038/ncponc0400

    Article  CAS  PubMed  Google Scholar 

  38. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  39. Park K, Han S, Shin E, Kim HJ, Kim JY (2007) EGFR gene and protein expression in breast cancers. Eur J Surg Oncol 33:956–960. doi:10.1016/j.ejso.2007.01.033

    CAS  PubMed  Google Scholar 

  40. Sauter G, Simon R, Hillan K (2003) Tissue microarrays in drug discovery. Nat Rev Drug Discov 2:962–972

    Article  CAS  PubMed  Google Scholar 

  41. Al-Kuraya K, Schraml P, Torhorst J et al (2004) Prognostic relevance of gene amplifications and coamplifications in breast cancer. Cancer Res 64:8534–8540. doi:10.1158/0008-5472.CAN-04-1945

    Article  CAS  PubMed  Google Scholar 

  42. Giltnane JM, Molinaro A, Cheng H, Robinson A, Turbin D, Gelmon K, Huntsman D, Rimm DL (2008) Comparison of quantitative immunofluorescence with conventional methods for HER2/neu testing with respect to response to trastuzumab therapy in metastatic breast cancer. Arch Pathol Lab Med 132:1635–1647

    PubMed  Google Scholar 

  43. Bhargava R, Lal P, Chen B (2004) Feasibility of using tissue microarrays for the assessment of HER-2 gene amplification by fluorescence in situ hybridization in breast carcinoma. Diagn Mol Pathol 13:213–216

    Article  CAS  PubMed  Google Scholar 

  44. Ilie M, Mazure NM, Hofman V et al (2010) High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer. Br J Cancer 102:1627–1635

    Article  CAS  PubMed  Google Scholar 

  45. Suzuki S, Dobashi Y, Sakurai H, Nishikawa K, Hanawa M, Ooi A (2005) Protein overexpression and gene amplification of epidermal growth factor receptor in non small cell lung carcinomas. An immunohistochemical and fluorescence in situ hybridization study. Cancer 103:1265–1273. doi:10.1002/cncr.20909

    Article  CAS  PubMed  Google Scholar 

  46. Bubendorf L, Nocito A, Moch H, Sauter G (2001) Tissue microarray (TMA) technology: miniaturized pathology archives for high-throughput in situ studies. J Pathol 195:72–79. doi:10.1002/path.893

    Article  CAS  PubMed  Google Scholar 

  47. Sato-Kuwabara Y, Neves JI, Fregnani JH, Sallum RA, Soares FA (2009) Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry. BMC Cancer 9:6. doi:10.1186/1471-2407-9-6

    Article  PubMed  Google Scholar 

  48. Nocito A, Kononen J, Kallioniemi OP, Sauter G (2001) Tissue microarrays (TMAs) for high-throughput molecular pathology research. Int J Cancer 94:1–5. doi:10.1002/ijc.1385

    Article  CAS  PubMed  Google Scholar 

  49. Brabender J, Danenberg KD, Metzger R, Schneider PM, Park J, Salonga D, Holscher AH, Danenberg PV (2001) Epidermal growth factor receptor and HER2-neu mRNA expression in non-small cell lung cancer is correlated with survival. Clin Cancer Res 7:1850–1855

    CAS  PubMed  Google Scholar 

  50. Nakamura H, Kawasaki N, Taguchi M, Kabasawa K (2006) Survival impact of epidermal growth factor receptor overexpression in patients with non-small cell lung cancer: a meta-analysis. Thorax 61:140–145. doi:10.1136/thx.2005.042275

    Article  CAS  PubMed  Google Scholar 

  51. Franklin WA, Veve R, Hirsch FR, Helfrich BA, Bunn PA Jr (2002) Epidermal growth factor receptor family in lung cancer and premalignancy. Semin Oncol 29:3–14. doi:10.1053/sonc.2002.31520

    Article  CAS  PubMed  Google Scholar 

  52. Pinter F, Papay J, Almasi A et al (2008) Epidermal growth factor receptor (EGFR) high gene copy number and activating mutations in lung adenocarcinomas are not consistently accompanied by positivity for EGFR protein by standard immunohistochemistry. J Mol Diagn 10:160–168. doi:10.2353/jmoldx.2008.070125

    Article  CAS  PubMed  Google Scholar 

  53. Sone T, Kasahara K, Kimura H et al (2007) Comparative analysis of epidermal growth factor receptor mutations and gene amplification as predictors of gefitinib efficacy in Japanese patients with nonsmall cell lung cancer. Cancer 109:1836–1844. doi:10.1002/cncr.22593

    Article  CAS  PubMed  Google Scholar 

  54. Mitsudomi T, Kosaka T, Endoh H et al (2005) Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol 23:2513–2520. doi:10.1200/JCO.2005.00.992

    Article  CAS  PubMed  Google Scholar 

  55. Sasaki H, Endo K, Okuda K et al (2008) Epidermal growth factor receptor gene amplification and gefitinib sensitivity in patients with recurrent lung cancer. J Cancer Res Clin Oncol 134:569–577

    Article  CAS  PubMed  Google Scholar 

  56. VanMeter AJ, Rodriguez AS, Bowman ED et al (2008) Laser capture microdissection and protein microarray analysis of human non-small cell lung cancer: differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type. Mol Cell Proteomics 7:1902–1924

    Article  CAS  PubMed  Google Scholar 

  57. Yu YY, Zhu ZG (2010) Significance of biological resource collection and tumor tissue bank creation. World J Gastrointest Oncol 2:5–8. doi:10.4251/wjgo.v2.i1.5

    Article  Google Scholar 

  58. Lassalle S, Hofman V, Marius I et al (2009) Assessment of morphology, antigenicity, and nucleic acid integrity for diagnostic thyroid pathology using formalin substitute fixatives. Thyroid 19:1239–1248

    Article  CAS  PubMed  Google Scholar 

  59. Piqueras M, Navarro S, Castel V, Canete A, Llombart-Bosch A, Noguera R (2009) Analysis of biological prognostic factors using tissue microarrays in neuroblastic tumors. Pediatr Blood Cancer 52:209–214

    Article  PubMed  Google Scholar 

  60. Uramoto H, Mitsudomi T (2007) Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer? Br J Cancer 96:857–863. doi:10.1038/sj.bjc.6603665

    Article  CAS  PubMed  Google Scholar 

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Acknowledgement

M.I. Ilie was supported by a fellowship from Institut National du Cancer (INCa). P. Hofman, V. Hofman, and J. Mouroux were supported by a PHRC 2003 Grant (CHU of Nice).

Conflict of interest statement

We declare that we have no conflict of interest.

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Authors and Affiliations

  1. Laboratory of Clinical and Experimental Pathology, Louis Pasteur Hospital, 30 Avenue de la Voie Romaine, 06002, Nice Cedex 01, France

    Marius I. Ilie, Véronique Hofman, Katia Havet & Paul Hofman

  2. INSERM ERI-21/EA 4319, Faculty of Medicine, University of Nice Sophia Antipolis, Nice, France

    Marius I. Ilie, Véronique Hofman, Nicolas Vénissac, Jerôme Mouroux & Paul Hofman

  3. Human Tissue Biobank Unit/CRB INSERM, Louis Pasteur Hospital, Nice, France

    Marius I. Ilie, Véronique Hofman, Christelle Bonnetaud, Virginie Lespinet-Fabre, Céline Coëlle, Virginie Gavric-Tanga & Paul Hofman

  4. Department of Thoracic Surgery, Louis Pasteur Hospital, Nice, France

    Nicolas Vénissac & Jerôme Mouroux

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  1. Marius I. Ilie
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  2. Véronique Hofman
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Correspondence to Paul Hofman.

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Ilie, M.I., 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 457, 483–495 (2010). https://doi.org/10.1007/s00428-010-0963-z

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