Assessment of gefitinib- and CI-1040-mediated changes in epidermal growth factor receptor signaling in HuCCT-1 human cholangiocarcinoma by serial fine needle aspiration Manuel Hidalgo, 1 Maria Luz Amador, 1 Antonio Jimeno, 1 Heather Mezzadra, 2 Pina Patel, 2 Audrey Chan, 1 Matthew E. Nielsen, 2,3 Anirban Maitra, 2 and Soner Altiok 2 1 The Sidney Kimmel Comprehensive Cancer Center and Departments of 2 Pathology and 3 Urology, The Johns Hopkins School of Medicine, Baltimore, Maryland 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 suscep- tible 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 path- way. 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 quanti- tative 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. [Mol Cancer Ther 2006; 5(7):1895–903] Introduction The significant advances in molecular biology of cancer have led to the elucidation of a large number of novel molecular targets in almost every pathway important for cancer development, progression, and dissemination. This has been followed by the discovery of a large number of novel anticancer agents, many of which are very specific at inhibiting cancer-relevant targets (1, 2). One of such class of targets is protein kinases. Studies have shown that protein kinases are frequently mutated in cancer and play an important role in the genesis, maintenance, and progres- sion of such cancers (3). Tumors with mutated kinases are thought to be dependent on such kinases so that their pharmacologic inhibition results in antitumor effects (4). This is illustrated by the success of imatinib mesylate in patients with chronic myelogenous mutations and gastro- intestinal stromal tumors and, more recently, epidermal growth factor (EGF) receptor (EGFR) inhibitors in patients with activating mutations in the EGFR gene (5–7). It thus seems that targeted anticancer agents are more likely to be active in tumors in which the targeted pathway is relevant and the drugs are administered at doses and schedules that result in adequate and sustained target inhibition (2, 8, 9). There are at least two key components in this statement. One is the selection of tumors in which the target is important for tumor growth. This is illustrated by studies with trastuzumab in Her-2-positive breast cancer, with imatinib mesylate in chronic myelogenous leukemia and gastrointestinal stromal tumors with bcr/abl transloca- tion and c-kit mutations, respectively, with gefitinib in the treatment of non–small cell lung cancer patients with EGFR mutation and perhaps with cetuximab in tumors with amplification of the EGFR gene (5, 6, 10–12). A second equally important question is whether the drug inhibits the Received 12/14/05; revised 5/15/06; accepted 5/23/06. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Soner Altiok, Department of Pathology. Johns Hopkins School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287. Phone: 443-287-4638; Fax: 410-614-9556. E-mail: saltiok1@jhmi.edu Copyright C 2006 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0525 1895 Mol Cancer Ther 2006;5(7). July 2006 Downloaded from http://aacrjournals.org/mct/article-pdf/5/7/1895/1873523/1895.pdf by guest on 17 June 2022