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
2008; Elsevier BV; Volume: 10; Issue: 2 Linguagem: Inglês
10.2353/jmoldx.2008.070125
ISSN1943-7811
AutoresFerenc Pintér, Judit Pápay, Andrea Almási, Zoltàn Sápi, Edit Szabó, Melinda Kánya, Anna Tamási, Balázs Jóri, Edit Várkondi, Judit Moldvay, Klára Szondy, Gÿorgý Kéri, Massimo Dominici, Pierfranco Conté, Sándor Eckhardt, László Kopper, Richárd Schwab, István Peták,
Tópico(s)HER2/EGFR in Cancer Research
ResumoThe purpose of this study was to investigate whether detectable protein biomarker overexpression is a prerequisite for the presence of increased gene copy number or activating mutations and responsiveness to the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib in patients with lung adenocarcinomas. EGFR status was prospectively analyzed in tumor biopsy samples by three methods: protein expression (n = 117) by standardized immunohistochemistry (IHC), gene copy number (n = 97) by fluorescent in situ hybridization (FISH), and mutation analysis by sequencing (n = 126). Fifty-nine percent of the samples were positive by IHC, 40% were positive by FISH, and 13.5% contained activating kinase domain mutations. Thirty-four percent of the FISH-positive and 27% of the mutant samples were also IHC-negative. All EGFR mutant patients had major clinical responses (five complete response and five partial response) to gefitinib or erlotinib treatment, although three of these tumors were IHC-negative and four were FISH-negative. In a retrospective analysis of samples from nine patients with excellent therapeutic responses (three complete response, five partial response, one stable disease) to erlotinib or gefitinib, mutations were identified in eight cases, but IHC was negative in four of these tumors. These results indicate that molecular diagnostic methods appear to be most important for the identification of lung adenocarcinoma patients who may benefit from EGFR inhibitor treatments. The purpose of this study was to investigate whether detectable protein biomarker overexpression is a prerequisite for the presence of increased gene copy number or activating mutations and responsiveness to the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib in patients with lung adenocarcinomas. EGFR status was prospectively analyzed in tumor biopsy samples by three methods: protein expression (n = 117) by standardized immunohistochemistry (IHC), gene copy number (n = 97) by fluorescent in situ hybridization (FISH), and mutation analysis by sequencing (n = 126). Fifty-nine percent of the samples were positive by IHC, 40% were positive by FISH, and 13.5% contained activating kinase domain mutations. Thirty-four percent of the FISH-positive and 27% of the mutant samples were also IHC-negative. All EGFR mutant patients had major clinical responses (five complete response and five partial response) to gefitinib or erlotinib treatment, although three of these tumors were IHC-negative and four were FISH-negative. In a retrospective analysis of samples from nine patients with excellent therapeutic responses (three complete response, five partial response, one stable disease) to erlotinib or gefitinib, mutations were identified in eight cases, but IHC was negative in four of these tumors. These results indicate that molecular diagnostic methods appear to be most important for the identification of lung adenocarcinoma patients who may benefit from EGFR inhibitor treatments. The detectable expression of the target protein of novel molecular targeted drugs is often assumed to both sufficient and necessary for predicting the efficacy of these novel drugs. The clinical experience with anti-epidermal growth factor receptor (EGFR) antibody therapy cetuximab in the treatment of colon carcinoma patients revealed that not only not all EGFR-expressing tumors responded, but more surprisingly, tumors without detectable EGFR expression by standard immunohistochemistry (IHC) had similar clinical response to anti-EGFR therapy.1Chung KY Shia J Kemeny NE Shah M Schwartz GK Tse A Hamilton A Pan D Schrag D Schwartz L Klimstra DS Fridman D Kelsen DP Saltz LB Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry.J Clin Oncol. 2005; 23: 1803-1810Crossref PubMed Scopus (1059) Google Scholar The oncogenic dependence on a signal transduction molecule may be more dependent on the genetic changes in the target gene itself or downstream signal proteins like RAS, in the case of anti-EGFR therapies.2Lièvre A Bachet JB Le Corre D Boige V Landi B Emile JF Côté JF Tomasic G Penna C Ducreux M Rougier P Penault-Llorca F Laurent-Puig P KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer.Cancer Res. 2006; 66: 3992-3995Crossref PubMed Scopus (1874) Google Scholar Gefitinib and erlotinib are small-molecule inhibitors of the tyrosine kinase domain (TKI) of the EGFR. These EGFR TKIs have an objective response rate of 9 to 19%, mild side effects, and in some patients there was rapid and dramatic tumor shrinkage.3Uramoto H Mitsudomi T Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer.Br J Cancer. 2007; 96: 857-863Crossref PubMed Scopus (98) Google Scholar4Kris MG Natale RB Herbst RS Lynch Jr, TJ Prager D Belani CP Schiller JH Kelly K Spiridonidis H Sandler A Albain KS Cella D Wolf MK Averbuch SD Ochs JJ Kay AC Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial.JAMA. 2003; 290: 2149-2158Crossref PubMed Scopus (2468) Google Scholar5Fukuoka M Yano S Giaccone G Tamura T Nakagawa K Douillard JY Nishiwaki Y Vansteenkiste J Kudoh S Rischin D Eek R Horai T Noda K Takata I Smit E Averbuch S Macleod A Feyereislova A Dong RP Baselga J Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial).J Clin Oncol. 2003; 21: 2237-2246Crossref PubMed Scopus (2843) Google Scholar6Shepherd FA Rodrigues Pereira J Ciuleanu T Tan EH Hirsh V Thongprasert S Campos D Maoleekoonpiroj S Smylie M Martins R van Kooten M Dediu M Findlay B Tu D Johnston D Bezjak A Clark G Santabarbara P Seymour L National Cancer Institute of Canada Clinical Trials Group Erlotinib in previously treated non-small-cell lung cancer.N Engl J Med. 2005; 353: 123-132Crossref PubMed Scopus (5039) Google Scholar7Schwab R Pinter F Moldavy J Papay J Strausz J Kopper L Keri G Pap A Petak I Oreskovich K Mangel L Amplification and mutation of the epidermal growth factor receptor in metastatic lung cancer with remission from gefitinib.J Clin Oncol. 2005; 23: 7736-7738Crossref PubMed Scopus (11) Google Scholar8Thatcher N Chang A Parikh P Rodrigues Pereira J Ciuleanu T von Pawel J Thongprasert S Tan EH Pemberton K Archer V Carroll K Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer).Lancet. 2005; 366: 1527-1537Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar9US Food and Drug Administration FDA public health advisory: new labeling and distribution program for gefitinib (Iressa). US Food and Drug Administration, Washington, DC2005 June 17Google Scholar Biomarkers and clinical characteristics with reliable predictive value remain the focus of several investigations. Adenocarcinoma histology, nonsmoking history, Asian race, and female gender were the characteristics that were associated with increased response to both EGFR TKIs.3Uramoto H Mitsudomi T Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer.Br J Cancer. 2007; 96: 857-863Crossref PubMed Scopus (98) Google Scholar4Kris MG Natale RB Herbst RS Lynch Jr, TJ Prager D Belani CP Schiller JH Kelly K Spiridonidis H Sandler A Albain KS Cella D Wolf MK Averbuch SD Ochs JJ Kay AC Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial.JAMA. 2003; 290: 2149-2158Crossref PubMed Scopus (2468) Google Scholar5Fukuoka M Yano S Giaccone G Tamura T Nakagawa K Douillard JY Nishiwaki Y Vansteenkiste J Kudoh S Rischin D Eek R Horai T Noda K Takata I Smit E Averbuch S Macleod A Feyereislova A Dong RP Baselga J Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial).J Clin Oncol. 2003; 21: 2237-2246Crossref PubMed Scopus (2843) Google Scholar6Shepherd FA Rodrigues Pereira J Ciuleanu T Tan EH Hirsh V Thongprasert S Campos D Maoleekoonpiroj S Smylie M Martins R van Kooten M Dediu M Findlay B Tu D Johnston D Bezjak A Clark G Santabarbara P Seymour L National Cancer Institute of Canada Clinical Trials Group Erlotinib in previously treated non-small-cell lung cancer.N Engl J Med. 2005; 353: 123-132Crossref PubMed Scopus (5039) Google Scholar,8Thatcher N Chang A Parikh P Rodrigues Pereira J Ciuleanu T von Pawel J Thongprasert S Tan EH Pemberton K Archer V Carroll K Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer).Lancet. 2005; 366: 1527-1537Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar Mutations in the tyrosine kinase domain of EGFR were reported in the majority of tumors with dramatic responses to gefitinib and erlotinib,10Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Brannigan BW Harris PL Haserlat SM Supko JG Haluska FG Louis DN Christiani DC Settleman J Haber DA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9823) Google Scholar11Paez JG Jänne PA Lee JC Tracy S Greulich H Gabriel S Herman P Kaye FJ Lindeman N Boggon TJ Naoki K Sasaki H Fujii Y Eck MJ Sellers WR Johnson BE Meyerson M EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8337) Google Scholar12Pao W Miller V Zakowski M Doherty J Politi K Sarkaria I Singh B Heelan R Rusch V Fulton L Mardis E Kupfer D Wilson R Kris M Varmus H 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 USA. 2004; 101: 13306-13311Crossref PubMed Scopus (3835) Google Scholar and in some series, the presence of mutations was associated with improved survival.13Han SW Kim TY Hwang PG Jeong S Kim J Choi IS Oh DY Kim JH Kim DW Chung DH Im SA Kim YT Lee JS Heo DS Bang YJ Kim NK 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: 2493-2501Crossref PubMed Scopus (721) Google Scholar14Cortes-Funes H Gomez C Rosell R Valero P Garcia-Giron C Velasco A Izquierdo A Diz P Camps C Castellanos D Alberola V Cardenal F Gonzalez-Larriba JL Vieitez JM Maeztu I Sanchez JJ Queralt C Mayo C Mendez P Moran T Taron M Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients.Ann Oncol. 2005; 16: 1081-1086Crossref PubMed Scopus (228) Google Scholar15Mitsudomi T Kosaka T Endoh H Horio Y Hida T Mori S Hatooka S Shinoda M Takahashi T Yatabe Y 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. 2005; 23: 2513-2520Crossref PubMed Scopus (892) Google Scholar16Taron M Ichinose Y Rosell R Mok T Massuti B Zamora L Mate JL Manegold C Ono M Queralt C Jahan T Sanchez JJ Sanchez-Ronco M Hsue V Jablons D Sanchez JM Moran T Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.Clin Cancer Res. 2005; 11: 5785-5878Crossref Scopus (316) Google Scholar17Chou TY Chiu CH Li LH Hsiao CY Tzen CY Chang KT Chen YM Perng RP Tsai SF Tsai CM Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.Clin Cancer Res. 2005; 11: 3750-3757Crossref PubMed Scopus (290) Google Scholar18Takano T Ohe Y Tsuta K Fukui T Sakamoto H Yoshida T Tateishi U Nokihara H Yamamoto N Sekine I Kunitoh H Matsuno Y Furuta K Tamura T 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; 28: 6829-6837Crossref Scopus (672) Google Scholar EGFR mutations were more common in patients with the same clinical characteristics as those associated with better treatment response. The latest advances in research of biological and clinical relevance of activating mutations have been reviewed recently.3Uramoto H Mitsudomi T Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer.Br J Cancer. 2007; 96: 857-863Crossref PubMed Scopus (98) Google Scholar The frequencies of mutations in lung adenocarcinomas were 22 to 67% in Asia, 3 to 25% in North America, and 10 to 24% in South Europe.11Paez JG Jänne PA Lee JC Tracy S Greulich H Gabriel S Herman P Kaye FJ Lindeman N Boggon TJ Naoki K Sasaki H Fujii Y Eck MJ Sellers WR Johnson BE Meyerson M EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8337) Google Scholar,13Han SW Kim TY Hwang PG Jeong S Kim J Choi IS Oh DY Kim JH Kim DW Chung DH Im SA Kim YT Lee JS Heo DS Bang YJ Kim NK 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: 2493-2501Crossref PubMed Scopus (721) Google Scholar14Cortes-Funes H Gomez C Rosell R Valero P Garcia-Giron C Velasco A Izquierdo A Diz P Camps C Castellanos D Alberola V Cardenal F Gonzalez-Larriba JL Vieitez JM Maeztu I Sanchez JJ Queralt C Mayo C Mendez P Moran T Taron M Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients.Ann Oncol. 2005; 16: 1081-1086Crossref PubMed Scopus (228) Google Scholar15Mitsudomi T Kosaka T Endoh H Horio Y Hida T Mori S Hatooka S Shinoda M Takahashi T Yatabe Y 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. 2005; 23: 2513-2520Crossref PubMed Scopus (892) Google Scholar,17Chou TY Chiu CH Li LH Hsiao CY Tzen CY Chang KT Chen YM Perng RP Tsai SF Tsai CM Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer.Clin Cancer Res. 2005; 11: 3750-3757Crossref PubMed Scopus (290) Google Scholar18Takano T Ohe Y Tsuta K Fukui T Sakamoto H Yoshida T Tateishi U Nokihara H Yamamoto N Sekine I Kunitoh H Matsuno Y Furuta K Tamura T 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; 28: 6829-6837Crossref Scopus (672) Google Scholar19Tokumo M Toyooka S Kiura K Shigematsu H Tomii K Aoe M Ichimura K Tsuda T Yano M Tsukuda K Tabata M Ueoka H Tanimoto M Date H Gazdar AF Shimizu N The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers.Clin Cancer Res. 2005; 11: 1167-1173PubMed Google Scholar20Shigematsu H Lin L Takahashi T Nomura M Suzuki M Wistuba II Fong KM Lee H Toyooka S Shimizu N Fujisawa T Feng Z Roth JA Herz J Minna JD Gazdar AF Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers.J Natl Cancer Inst. 2005; 97: 339-346Crossref PubMed Scopus (2033) Google Scholar21Marchetti A Martella C Felicioni L Barassi F Salvatore S Chella A Camplese PP Iarussi T Mucilli F Mezzetti A Cuccurullo F Sacco R Buttitta F 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: 857-865Crossref PubMed Scopus (767) Google Scholar The prevalence of EGFR gene mutations and copy number alterations in Eastern and Central Europe has not been published. EGFR gene copy number, detected by fluorescent in situ hybridization (FISH), is also associated with response to gefitinib. Gefitinib-treated patients carrying EGFR gene amplification or high polysomy (FISH+) had a statistically significant improvement in response, time to progression, and survival compared with patients with no or low genomic gain for EGFR.22Cappuzzo F Hirsch FR Rossi E Bartolini S Ceresoli GL Bemis L Haney J Witta S Danenberg K Domenichini I Ludovini V Magrini E Gregorc V Doglioni C Sidoni A Tonato M Franklin WA Crino L Bunn Jr, PA Varella-Garcia M Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer.J Natl Cancer Inst. 2005; 97: 643-655Crossref PubMed Scopus (1475) Google Scholar The efficiency of this molecular predictive marker was confirmed on a subgroup of samples of phase II study of gefitinib (S0126) and on subgroup of specimens of phase III study of erlotinib (BR.21).23Hirsch FR Varella-Garcia M McCoy J West H Xavier AC Gumerlock P Bunn Jr, PA Franklin WA Crowley J Gandara DR Southwest Oncology Group Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchoalveolar carcinoma subtypes: a Southwest Oncology Group Study.J Clin Oncol. 2005; 23: 6838-6845Crossref PubMed Scopus (548) Google Scholar,24Tsao MS Sakurada A Cutz JC Zhu CQ Kamel-Reid S Squire J Lorimer I Zhang T Liu N Daneshmand M Marrano P da Cunha Santos G Lagarde A Richardson F Seymour L Whitehead M Ding K Pater J Shepherd FA Erlotinib in lung cancer: molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1716) Google Scholar Although initial retrospective studies had suggested that the protein expression is not associated with gefitinib response,25Bailey LR Kris M Wolf M Kay A Averbuch S Askaa J Jans M Schmidt K Fukuoka M Tumor EGFR membrane staining is not clinically relevant for predicting response in patients receiving gefitinib (“Iressa” ZD1839) monotherapy for pretreated advanced non-small-cell lung cancer: iDEAL 1 and 2 (abstr LB-170).Proc Am Assoc Cancer Res. 2003; 44: 1362Google Scholar26Cappuzzo F Gregorc V Rossi E Cancellieri A Magrini E Paties CT Ceresoli G Lombardo L Bartolini S Calandri C de Rosa M Villa E Crino L Gefitinib in pretreated non-small-cell lung cancer (NSCLC): analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC.J Clin Oncol. 2003; 21: 2658-2663Crossref PubMed Scopus (196) Google Scholar27Parra HS Cavina R Latteri F Campagnoli E Morenghi E Torri W Brambilla G Alloisio M Santoro A 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. 2004; 91: 208-212PubMed Google Scholar two subsequent studies reported longer survival among TKI-treated patients with protein overexpression detected by IHC.23Hirsch FR Varella-Garcia M McCoy J West H Xavier AC Gumerlock P Bunn Jr, PA Franklin WA Crowley J Gandara DR Southwest Oncology Group Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchoalveolar carcinoma subtypes: a Southwest Oncology Group Study.J Clin Oncol. 2005; 23: 6838-6845Crossref PubMed Scopus (548) Google Scholar,24Tsao MS Sakurada A Cutz JC Zhu CQ Kamel-Reid S Squire J Lorimer I Zhang T Liu N Daneshmand M Marrano P da Cunha Santos G Lagarde A Richardson F Seymour L Whitehead M Ding K Pater J Shepherd FA Erlotinib in lung cancer: molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1716) Google Scholar In the BR.21 study, survival among patients with protein overexpression (50–55%) was longer in the erlotinib group than in the placebo group, but there was no survival advantage among patients with EGFR IHC-negative tumors, although the P value for interaction was 0.25, which indicates very low level of statistical significance.24Tsao MS Sakurada A Cutz JC Zhu CQ Kamel-Reid S Squire J Lorimer I Zhang T Liu N Daneshmand M Marrano P da Cunha Santos G Lagarde A Richardson F Seymour L Whitehead M Ding K Pater J Shepherd FA Erlotinib in lung cancer: molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1716) Google Scholar In addition, recent preclinical studies in cell lines did not find correlation between EGFR sensitivity and EGFR protein expression.28Helfrich BA Raben D Varella-Garcia M Gustafson D Chan DC Bemis L Coldren C Barón A Zeng C Franklin WA Hirsch FR Gazdar A Minna J Bunn Jr, PA Antitumor activity of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in non-small cell lung cancer cell lines correlates with gene copy number and EGFR mutations but not EGFR protein levels.Clin Cancer Res. 2006; 12: 7117-7125Crossref PubMed Scopus (117) Google Scholar In 2004 our group identified the simultaneous presence of amplification and mutation of the EGFR gene and overexpression of the EGFR protein in primary non-small cell lung cancer (NSCLC) with complete regression of brain and lung metastases in response to gefitinib.7Schwab R Pinter F Moldavy J Papay J Strausz J Kopper L Keri G Pap A Petak I Oreskovich K Mangel L Amplification and mutation of the epidermal growth factor receptor in metastatic lung cancer with remission from gefitinib.J Clin Oncol. 2005; 23: 7736-7738Crossref PubMed Scopus (11) Google Scholar Due to the lack of consensus on the significance of predictive diagnostic tests, in particular the mutation tests, clinical oncologists both in the United States and the European Union most often rely on the IHC of EGFR for patient selection. This decision is based on the assumption that detection of the molecular target protein is the most reliable way to use a molecular targeted therapeutic drug. In addition, it is also assumed that the IHC-positive population includes the smaller patient populations of FISH-positive and tumors with activating EGFR mutations. In this study we used the standardized PharmDx (Dako) IHC kit to analyze EGFR expression by IHC in large set of NSCLC samples. This is the most commonly used IHC test for EGFR, and this was the clinical trial assay in the BR21 study that led to the market authorization of erlotinib. We also analyzed gene copy number by FISH using the most standard probes (Vysis) and the presence of activating mutations by the gold standard method of bidirectional sequencing. The clinical significance of EGFR mutations in response to gefitinib has been far more studied (over 20 publications) than in response to erlotinib (three publications).3Uramoto H Mitsudomi T Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer.Br J Cancer. 2007; 96: 857-863Crossref PubMed Scopus (98) Google Scholar The BR21 clinical trial included only eight erlotinib-treated patients with classic EGFR mutations. In this study we provide clinical data of 14 erlotinib-treated patients with EGFR mutations both IHC-positive and -negative. Our study revealed that the positive patient populations of these common EGFR biomarkers do not overlap. Almost half of the FISH-positive and mutant samples were IHC-negative. We also found examples of NSCLC patients with IHC-negative but EGFR mutant NSCLC tumors who had complete responses to erlotinib treatment. One hundred twenty-seven primary NSCLC tissues prospectively before and a further nine samples retrospectively after EGFR TKI therapy were examined in the same laboratory throughout a 1-year period. Each specimen was reviewed by a pathologist (P.J.), and only those with ≥30% tumor component were used for DNA mutation analysis in the prospective examination. In retrospective analysis two specimens with <30% tumor component were reanalyzed by mutant-enriched polymerase chain reaction (PCR) and sequencing. For treatment, 250 mg of gefitinib or 150 mg of erlotinib was administered daily. Tumor response was evaluated in accordance with Response Evaluation Criteria in Solid Tumors (RECIST). After DNA extraction from the paraffin-embedded specimens and biopsy smears, exons 18, 19, and 21 of the EGFR gene were amplified using nested PCR with “touch-down” protocol. Primers are shown in Table 1. The universal amplification protocol was as follows: 95°C for 2 minutes, 10 cycles of 94°C for 30 seconds, 62°C decreased with 1°C in each cycle for 30 seconds and 72°C for 45 seconds, 30 cycles of 94°C for 30 seconds, 52°C for 30 seconds and 72°C for 45 seconds, closing cycle of 72°C for 10 minutes. After purification and bidirectional sequencing reactions using the second step primers, sequencing fragments were detected with ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). High-quality sequence variations were confirmed from both directions in two independent PCR reactions of the original DNA samples.Table 1Primers Used for PCR Amplification of Exons 18, 19, and 21 of the EGFR GeneNameSequence18externalF*See Lynch et al.105′-CAAGTGCCGTGTCCTGGCACCCAAGC-3′18externalR*See Lynch et al.105′-CCAAACACTCAGTGAAACAAAGAG-3′18nestedF5′-GCCATGTCTGGCACTGCTTT-3′18nestedR5′-AGTAGATGATGGAAATATACAGCTTGC-3′19externalF5′-CTGGTAACATCCACCCAGATCACTG-3′19externalR5′-GAGATGAGCAGGGTCTAGAGCAGAG-3′19nestedF5′-CAGATCACTGGGCAGCATGTGG-3′19nestedR5′-CTAGAGCAGAGCAGCTGCCAGACAT-3′21externalF5′-CTGAATTCGGATGCAGAGCTTCTT-3′21externalR5′-CATCCTCCCCTGCATGTGTTAAA-3′21nestedF5′-GATGCAGAGCTTCTTCCCATGAT-3′21nestedR5′-GCATGTGTTAAACAATACAGCTAGTGG-3′F, forward; R, reverse.* See Lynch et al.10Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Brannigan BW Harris PL Haserlat SM Supko JG Haluska FG Louis DN Christiani DC Settleman J Haber DA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9823) Google Scholar Open table in a new tab F, forward; R, reverse. Mutant-enriched PCR is a multistep PCR with intermittent restriction digestion to eliminate wild-type genes selectively, thus enriching the genes with exon 19 deletion or L858R exon 21 point mutation. We used a modified protocol described by Asano et al.29Asano H Toyooka S Tokumo M Ichimura K Aoe K Ito S Tsukuda K Ouchida M Aoe M Katayama H Hiraki A Sugi K Kiura K Date H Shimizu N Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay.Clin Cancer Res. 2006; 12: 43-48Crossref PubMed Scopus (178) Google Scholar Gene copy number per cell was investigated by FISH using the Vysis EGFR probe (Abbott Laboratories, Des Plaines, IL) and semiautomated or manual procedure. In the former procedure the tissue was pretreated by a Discovery Automatic Hybridizator (Ventana, Tucson, AZ). The classification was done according to the six FISH categories defined by Cappuzzo et al22Cappuzzo F Hirsch FR Rossi E Bartolini S Ceresoli GL Bemis L Haney J Witta S Danenberg K Domenichini I Ludovini V Magrini E Gregorc V Doglioni C Sidoni A Tonato M Franklin WA Crino L Bunn Jr, PA Varella-Garcia M Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer.J Natl Cancer Inst. 2005; 97: 643-655Crossref PubMed Scopus (1475) Google Scholar and was also used in the BR.21 study.24Tsao MS Sakurada A Cutz JC Zhu CQ Kamel-Reid S Squire J Lorimer I Zhang T Liu N Daneshmand M Marrano P da Cunha Santos G Lagarde A Richardson F Seymour L Whitehead M Ding K Pater J Shepherd FA Erlotinib in lung cancer: molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1716) Google Scholar Samples with a high EGFR gene copy number (high polysomy or amplification) were considered to be FISH-positive. The expression of EGFR protein was determined by IHC using Dako EGFR PharmDx kits (DakoCytomation), and the slides were counterstained with hematoxylin. For evaluation the same categorization was used as in the BR.21 study; samples with more than 10% tumor cells showing membranous (partial or complete) staining of any intensity were stated as positive for EGFR.21Marchetti A Martella C Felicioni L Barassi F Salvatore S Chella A Camplese PP Iarussi T Mucilli F Mezzetti A Cuccurullo F Sacco R Buttitta F 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: 857-865Crossref PubMed Scopus (767) Google Scholar For semiquantitation we also used the scoring system defined by Capuzzo et al22Cappuzzo F Hirsch FR Rossi E Bartolini S Ceresoli GL Bemis L Haney J Witta S Danenberg K Domenichini I Ludovini V Magrini E Gregorc V Doglioni C Sidoni A Tonato M Franklin WA Crino L Bunn Jr, PA Varella-Garcia M Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer.J Natl Cancer Inst. 2005; 97: 643-655Crossref PubMed Scopus (1475) Google Scholar with the modifica
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