title: The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer.
abstract: OBJECTIVE
  The epidermal growth factor receptor (EGFR) overexpressed in approximately 80% of non-small cell lung cancers (NSCLC) is a target for novel therapeutics. Concurrent chemoradiation is the current standard of care for treatment of patients with locally advanced NSCLC. However, < 20% of patients remain disease-free at 5 years despite this aggressive treatment. Cetuximab is a humanized monoclonal antibody that recognizes the human EGFR, and in previous studies, inhibited the growth of EGFR-expressing human cancer cell lines. In this report, we investigated the cellular and molecular effects of cetuximab alone and in combination with radiation and/or chemotherapy in human NSCLC cell lines with varying levels of EGFR overexpression in vitro and in vivo.
  
  
  METHODS
  We evaluated the EGFR status of a panel of human NSCLC cancer cell lines by immunohistochemistry and flow cytometry. We then evaluated cetuximab effects on growth, cell cycle distribution, and downstream intracellular signaling molecules in this panel of NSCLC cancer cell lines. NSCLC cell lines were treated with cetuximab alone or in combination with radiation, chemotherapy, or chemoradiation to determine the cooperative effects of cetuximab both in vitro and in vivo in athymic nude mice bearing NSCLC xenografts.
  
  
  RESULTS
  Cetuximab alone inhibited the in vitro growth of some but not all EGFR-expressing NSCLC cell lines in a dose-dependent manner. Flow cytometric analysis of cell cycle distribution after 24 hours of cetuximab treatment revealed a shift into the G(0)/G(1) phase of the cell cycle in cetuximab-sensitive EGFR-expressing cell lines and at concentrations that were growth-inhibitory. There were no cell cycle changes in the EGFR-negative cell lines. After 4 hours of exposure, cetuximab reduced epidermal growth factor (EGF)-induced phosphorylation of EGFR (pEGFR) and HER-2 (pHER2) in cetuximab-sensitive cell lines but not in cetuximab-resistant cell lines. Cetuximab reduced EGF-induced phosphorylation of extracellular signal-regulated kinase-1/2 (pERK) in all EGFR-expressing cell lines. In the absence of EGF, cetuximab alone increased the level of pEGFR and pHER2 above that seen in untreated control cells in both sensitive and resistant cell lines that were EGFR- and HER2-positive, but not in EGFR- or HER2-negative lines. Despite the cetuximab-induced increase in phosphorylation of EGFR and HER2, peak EGF-induced levels of pEGFR and pHER2 were reduced by cetuximab in the cetuximab-sensitive lines but not in the resistant lines. Cooperative (combination index values < 1.0) growth inhibitory effects were observed in vitro combination assays with cetuximab and radiation only in cetuximab-sensitive NSCLC cell lines. A lack of cooperation was seen in cetuximab-insensitive NSCLC cell lines. Similar findings were observed with in vitro combination studies of cetuximab plus cisplatin or paclitaxel. In nude mice bearing EGFR-expressing, cetuximab-sensitive, NSCLC cell line xenografts, cetuximab plus radiation induced a marked improvement in tumor growth inhibition over either agent alone. The growth inhibitory effects of cetuximab-radiation were similar to the growth inhibitory effects of concurrent chemoradiation. Triple combination therapy of cetuximab and chemoradiation yielded a nonsignificant advantage in tumor growth control over doublet combinations (cetuximab and radiation or chemoradiation) in vivo.
  
  
  CONCLUSIONS
  Similar results in tumor growth inhibition observed in mice treated with cetuximab-radiation and cisplatin-radiation provide a rationale for the clinical investigation of cetuximab with concurrent radiation in selected patients with locally advanced NSCLC. Local tumor control and treatment toxicity should be evaluated between cetuximab-radiation and chemoradiation regimens. Proper patient selection will be critical to the success of such trials and further studies are needed to identify optimal patient selection criteria.
authors.affiliation: Tobacco Related Malignancy Program, Department of Radiation Oncology, Anschutz Cancer Pavilion, University of Colorado Health Sciences Center, 1165 North Ursula Street F706, Aurora, CO 80010-0510, USA. david.raben@uchsc.edu
authors.forename: David
authors.initials: D
authors.lastname: Raben
authors.forename: Barb
authors.initials: B
authors.lastname: Helfrich
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Chan
authors.forename: Fortunato
authors.initials: F
authors.lastname: Ciardiello
authors.forename: LiMin
authors.initials: L
authors.lastname: Zhao
authors.forename: Wilbur
authors.initials: W
authors.lastname: Franklin
authors.forename: Anna E
authors.initials: AE
authors.lastname: Barón
authors.forename: Chan
authors.initials: C
authors.lastname: Zeng
authors.forename: Tim K
authors.initials: TK
authors.lastname: Johnson
authors.forename: Paul A
authors.initials: PA
authors.lastname: Bunn
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism / Q000532:radiotherapy; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D000068818:Cetuximab; D003131:Combined Modality Therapy; D019008:Drug Resistance, Neoplasm; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000532:radiotherapy; D051379:Mice; D008819:Mice, Nude; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011839:Radiation, Ionizing; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D014183:Transplantation, Heterologous
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D000068818:Cetuximab; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases