title: Assessment of gefitinib- and CI-1040-mediated changes in epidermal growth factor receptor signaling in HuCCT-1 human cholangiocarcinoma by serial fine needle aspiration.
abstract: One specific limitation to the clinical development of targeted cancer therapeutics is the lack of well-validated pharmacodynamic markers. Such tools might conceivably provide a framework within which to better evaluate the selection of specific molecules as therapeutic targets. Nevertheless, the practical application of this hypothesis in clinical development remains elusive. In this study, we present a minimally invasive pharmacodynamic assay for monitoring therapy-mediated changes in the activity of target signaling pathways by using fine needle aspiration (FNA) samples and quantitative ELISA methods. To this end, we used the HuCCT-1 cholangiocarcinoma cell line treated with gefitinib (ZD1839, Iressa), a selective blocker of the epidermal growth factor receptor (EGFR), and CI-1040, a selective inhibitor of the mitogen extracellular regulated kinase [mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase 1/2]. HuCCT-1 cells were resistant to gefitinib and CI-1040 alone but susceptible to the combination of these drugs in vitro and in vivo. This effect was associated with a greater inhibition of ERK1/2 activation, a downstream mediator in the EGFR-mitogen-activated protein/ERK kinase pathway. Using this model, we sought to assess whether FNA-obtained tumor biopsies could be used to measure signaling pathway activation. Cellular extracts prepared from FNA samples yielded adequately cellular, high-quality samples to assess therapy-mediated changes in EGFR and ERK1/2 phosphorylation by Western blotting and quantitative ELISA assays. Treatment with gefitinib alone effectively inhibited EGFR activation but failed to block ERK1/2 phosphorylation and tumor growth. Blocking was achieved by the addition of CI-1040 to the treatment regimen. These results show that the combination of serial FNA sampling with highly sensitive quantitative ELISA assays permits assessment of therapy-mediated changes in signaling pathways, which correlate well with antitumor effects. This assay is simple to implement and broadly applicable to diverse tumor types in clinical studies with cancer patients and may be useful in the development of targeted anticancer agents.
authors.affiliation: Department of Pathology. Johns Hopkins School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Hidalgo
authors.forename: Maria Luz
authors.initials: ML
authors.lastname: Amador
authors.forename: Antonio
authors.initials: A
authors.lastname: Jimeno
authors.forename: Heather
authors.initials: H
authors.lastname: Mezzadra
authors.forename: Pina
authors.initials: P
authors.lastname: Patel
authors.forename: Audrey
authors.initials: A
authors.lastname: Chan
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Nielsen
authors.forename: Anirban
authors.initials: A
authors.lastname: Maitra
authors.forename: Soner
authors.initials: S
authors.lastname: Altiok
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D001549:Benzamides / Q000494:pharmacology / Q000627:therapeutic use*; D001650:Bile Duct Neoplasms / Q000188:drug therapy / Q000473:pathology*; D001653:Bile Ducts, Intrahepatic / Q000187:drug effects / Q000473:pathology*; D044963:Biopsy, Fine-Needle; D018281:Cholangiocarcinoma / Q000188:drug therapy / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005260:Female; D000077156:Gefitinib; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011799:Quinazolines / Q000494:pharmacology / Q000627:therapeutic use*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D000970:Antineoplastic Agents; D001549:Benzamides; D011799:Quinazolines; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000077156:Gefitinib