Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines

Background Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs. Methodology/Principal Findings We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results:  1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance.  2. mKRAS was associated with increased in vitro resistance to gefitinib. Conclusions/Significance Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.

[1]  W. Lam,et al.  PIK3CA mutations and copy number gains in human lung cancers. , 2008, Cancer research.

[2]  John D. Minna,et al.  Deregulated EGFR Signaling during Lung Cancer Progression: Mutations, Amplicons, and Autocrine Loops , 2008, Cancer Prevention Research.

[3]  Jie Zhang,et al.  Intratumoral Epiregulin Is a Marker of Advanced Disease in Non–Small Cell Lung Cancer Patients and Confers Invasive Properties on EGFR-Mutant Cells , 2008, Cancer Prevention Research.

[4]  I. Wistuba,et al.  Epidermal Growth Factor Receptor Abnormalities in the Pathogenesis and Progression of Lung Adenocarcinomas , 2008, Cancer Prevention Research.

[5]  M. Sporn,et al.  Clinical Prevention of Recurrence of Colorectal Adenomas by the Combination of Difluoromethylornithine and Sulindac: An Important Milestone , 2008, Cancer Prevention Research.

[6]  E. Hovig,et al.  GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data , 2008, Genome Biology.

[7]  Hiromasa Yamamoto,et al.  Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung , 2008, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[8]  S. Knuutila,et al.  Single nucleotide polymorphism microarray analysis of karyotypically normal acute myeloid leukemia reveals frequent copy number neutral loss of heterozygosity , 2008, Haematologica.

[9]  Y. Yatabe,et al.  Epidermal growth factor receptor gene amplification is acquired in association with tumor progression of EGFR-mutated lung cancer. , 2008, Cancer research.

[10]  A. Jemal,et al.  Cancer Statistics, 2008 , 2008, CA: a cancer journal for clinicians.

[11]  Steven P Gygi,et al.  Signaling networks assembled by oncogenic EGFR and c-Met , 2008, Proceedings of the National Academy of Sciences.

[12]  M. McDevitt,et al.  SNP Array Karyotyping Allows for the Detection of Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MPD , 2007, PloS one.

[13]  A. Gazdar,et al.  Lung cancer in never smokers — a different disease , 2007, Nature Reviews Cancer.

[14]  F. Shepherd,et al.  Weighing Tumor Biology in Treatment Decisions for Patients with Non-small Cell Lung Cancer , 2007, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[15]  R. Wilson,et al.  Mutational Analysis of EGFR and Related Signaling Pathway Genes in Lung Adenocarcinomas Identifies a Novel Somatic Kinase Domain Mutation in FGFR4 , 2007, PloS one.

[16]  Ziding Feng,et al.  Memorial Sloan-Kettering Cancer , 2006 .

[17]  Daniel A. Haber,et al.  Epidermal growth factor receptor mutations in lung cancer , 2007, Nature Reviews Cancer.

[18]  T. Mitsudomi,et al.  Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer? , 2007, British Journal of Cancer.

[19]  Calum MacAulay,et al.  Whole genome tiling path array CGH analysis of segmental copy number alterations in cervical cancer cell lines , 2007, International journal of cancer.

[20]  T. Cloughesy,et al.  PTEN-Mediated Resistance to Epidermal Growth Factor Receptor Kinase Inhibitors , 2007, Clinical Cancer Research.

[21]  Chan Zeng,et al.  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 , 2006, Clinical Cancer Research.

[22]  Bradley P. Coe,et al.  Differential disruption of cell cycle pathways in small cell and non-small cell lung cancer , 2006, British Journal of Cancer.

[23]  W. Park,et al.  Somatic mutations of the ERBB4 kinase domain in human cancers , 2006, International journal of cancer.

[24]  A. Gazdar,et al.  Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers , 2006, International journal of cancer.

[25]  J. Minna,et al.  Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. , 2006, Journal of the National Cancer Institute.

[26]  F. Cappuzzo,et al.  HER3 genomic gain and sensitivity to gefitinib in advanced non-small-cell lung cancer patients , 2005, British Journal of Cancer.

[27]  M. Ostland,et al.  Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[28]  Elisa Rossi,et al.  Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[29]  Renato Martins,et al.  Erlotinib in previously treated non-small-cell lung cancer. , 2005, The New England journal of medicine.

[30]  F. Hirsch,et al.  O-191 HER1-HER2-HER3 gene amplification and akt activation inpatients with advanced non-small cell lung cancer (NSCLC) treated with gefitinib , 2005 .

[31]  J. Minna,et al.  Somatic mutations of the HER2 kinase domain in lung adenocarcinomas. , 2005, Cancer research.

[32]  H. Varmus,et al.  KRAS Mutations and Primary Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib , 2005, PLoS medicine.

[33]  J. Pollack,et al.  Immortalization of Human Bronchial Epithelial Cells in the Absence of Viral Oncoproteins , 2004, Cancer Research.

[34]  Elena Marchiori,et al.  Breakpoint identification and smoothing of array comparative genomic hybridization data , 2004, Bioinform..

[35]  Joachim Herz,et al.  Mutations and addiction to EGFR: the Achilles 'heal' of lung cancers? , 2004, Trends in molecular medicine.

[36]  R. Wilson,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. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[37]  S. Gabriel,et al.  EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy , 2004, Science.

[38]  Patricia L. Harris,et al.  Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. , 2004, The New England journal of medicine.

[39]  J. Ptak,et al.  High Frequency of Mutations of the PIK3CA Gene in Human Cancers , 2004, Science.

[40]  E. Rowinsky.  The erbB family: targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibodies and tyrosine kinase inhibitors. , 2004, Annual review of medicine.

[41]  David Cella,et al.  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. , 2003, JAMA.

[42]  Chan Zeng,et al.  Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[43]  Masahiro Fukuoka,et al.  Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[44]  M. Varella‐Garcia,et al.  Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. , 2003, The oncologist.

[45]  M. Meyerson,et al.  Missense mutations of the BRAF gene in human lung adenocarcinoma. , 2002, Cancer research.

[46]  R. Nicholson,et al.  EGFR and cancer prognosis. , 2001, European journal of cancer.

[47]  Ming You,et al.  Wildtype Kras2 can inhibit lung carcinogenesis in mice , 2001, Nature Genetics.

[48]  Seung-Ho Kang,et al.  Sample size calculations for clustered binary data , 2001, Statistics in medicine.

[49]  I. Bièche,et al.  Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications. , 1999, Clinical chemistry.

[50]  H. Tanke,et al.  Comparative genomic hybridization in hypotriploid/hyperdiploid tumors. , 1997, Cytometry.

[51]  E. Galanis,et al.  Extrapulmonary small cell carcinoma , 1997, Cancer.

[52]  J. Minna,et al.  NCI‐navy medical oncology branch cell line data base , 1996, Journal of cellular biochemistry. Supplement.

[53]  T. Pawson.  Regulation of the Ras signalling pathway by protein-tyrosine kinases. , 1994, Biochemical Society transactions.

[54]  Brian Everitt,et al.  Statistical Methods for Medical Investigations , 1990 .

[55]  N. Dubrawsky.  Cancer statistics , 1989, CA: a cancer journal for clinicians.

[56]  R. Tsien,et al.  Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor , 1987, Nature.

[57]  V. Barnett,et al.  Applied Linear Statistical Models , 1975 .