Mutational Analysis of EGFR and Related Signaling Pathway Genes in Lung Adenocarcinomas Identifies a Novel Somatic Kinase Domain Mutation in FGFR4

Background Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis. Methodology/Principal Findings We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line. Conclusions/Significance This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.

[1]  G. Giaccone,et al.  Erlotinib for Frontline Treatment of Advanced Non–Small Cell Lung Cancer: a Phase II Study , 2006, Clinical Cancer Research.

[2]  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.

[3]  Rameen Beroukhim,et al.  Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. , 2004, Nucleic acids research.

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

[5]  M. Stratton,et al.  COSMIC 2005 , 2006, British Journal of Cancer.

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

[7]  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.

[8]  Young Tae Kim,et al.  Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[9]  Andrew D. Yates,et al.  Somatic mutations of the protein kinase gene family in human lung cancer. , 2005, Cancer research.

[10]  J. Pollack,et al.  Amplification of whole tumor genomes and gene-by-gene mapping of genomic aberrations from limited sources of fresh-frozen and paraffin-embedded DNA. , 2005, The Journal of molecular diagnostics : JMD.

[11]  V. P. Eswarakumar,et al.  Cellular signaling by fibroblast growth factor receptors. , 2005, Cytokine & growth factor reviews.

[12]  A. Nicholson,et al.  Mutations of the BRAF gene in human cancer , 2002, Nature.

[13]  M. Kris,et al.  Clinical pattern and pathologic stage but not histologic features predict outcome for bronchioloalveolar carcinoma. , 2002, The Annals of thoracic surgery.

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

[15]  Andrew D. Yates,et al.  Athletics: Momentous sprint at the 2156 Olympics? , 2004, Nature.

[16]  H. Varmus,et al.  Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain , 2005, PLoS medicine.

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

[18]  D. Nickerson,et al.  PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. , 1997, Nucleic acids research.

[19]  M. Gerretsen,et al.  A phase III randomised trial of cisplatinum, methotrextate, cisplatinum + methotrexate and cisplatinum + 5-FU in end stage squamous carcinoma of the head and neck. Liverpool Head and Neck Oncology Group. , 1990, British Journal of Cancer.

[20]  J. Tichelaar,et al.  Conditional Expression of Fibroblast Growth Factor-7 in the Developing and Mature Lung* , 2000, The Journal of Biological Chemistry.

[21]  Ken Chen,et al.  PolyScan: an automatic indel and SNP detection approach to the analysis of human resequencing data. , 2007, Genome research.

[22]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[23]  Rodrigo Lopez,et al.  Multiple sequence alignment with the Clustal series of programs , 2003, Nucleic Acids Res..

[24]  Tatiana A. Tatusova,et al.  Entrez Gene: gene-centered information at NCBI , 2004, Nucleic Acids Res..

[25]  E. Gabrielson.  Worldwide trends in lung cancer pathology , 2006, Respirology.

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

[27]  B. Stripp,et al.  Phenotypic consequences of lung-specific inducible expression of FGF-3 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Richard Wooster,et al.  BRAF and RAS mutations in human lung cancer and melanoma. , 2002, Cancer research.

[29]  Joseph Schlessinger,et al.  Structure of the FGF Receptor Tyrosine Kinase Domain Reveals a Novel Autoinhibitory Mechanism , 1996, Cell.

[30]  Narmada Thanki,et al.  CDD: a conserved domain database for interactive domain family analysis , 2006, Nucleic Acids Res..

[31]  M. Meyerson,et al.  EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. , 2005, The New England journal of medicine.

[32]  Y. Yatabe,et al.  PTEN and PIK3CA Expression Is Associated with Prolonged Survival after Gefitinib Treatment in EGFR-Mutated Lung Cancer Patients , 2006, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

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

[34]  A. Jemal,et al.  Cancer Statistics, 2006 , 2006, CA: a cancer journal for clinicians.

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

[36]  G. Parmigiani,et al.  The Consensus Coding Sequences of Human Breast and Colorectal Cancers , 2006, Science.

[37]  J. Tichelaar,et al.  FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo. , 2001, American journal of physiology. Lung cellular and molecular physiology.

[38]  W. Travis,et al.  The new World Health Organization classification of lung tumours , 2001, European Respiratory Journal.

[39]  H. Sasaki,et al.  PIK3CA mutation status in Japanese lung cancer patients. , 2006, Lung cancer.