Abstract 5474: Epidermal growth factor inhibition of HPV positive head and neck cancer cells and primary tumorgrafts results in significant growth inhibition mediated by apoptosis.
Authors: Blitzer Grace C. , Smith Molly A. , Torres Alexandra D. , Armstrong Eric A. , Harari Paul M. , Lambert Paul F. , Kimple Randall J. 
Published: August 2014

Abstract
Purpose: Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), is approved for use in the treatment of head and neck cancer (HNC). One recent study (SPECTRUM) suggested that patient with recurrent or metastatic HPV+ HNC do not benefit from treatment with EGFR targeted therapy. However, most studies of EGFR inhibition have been performed with HPV-negative cells and clinical trials have been performed without regard to HPV status. We performed this study to determine whether HPV+ HNC cells and patient-derived tumorgrafts respond to cetuximab and to examine the mechanisms through which cetuximab affects HPV+ HNC.
Methods: Four HPV+ cell lines (UD-SCC2, UM-SCC47, UPCI-SCC90, 93-VU-147T) were assessed for EGFR expression by western blot. Sensitivity to cetuximab was tested by assessing cell density 2, 4, 6, and 8 days after cetuximab treatment and by assessing colony formation 2 weeks after plating. Apoptosis was measured by caspase activation, flow cytometry for Annexin V and propidium iodide staining, and immunoblot. Cell cycle was assessed by immunoblot for cyclin D1, cyclin B1, and p27Kip-1 and confirmed by flow cytometry for propidium iodide stained cells. Subcutaneous flank xenografts and tumorgrafts were performed in Hsd:athymic Nude-Foxn1nu female mice treated with intraperitoneal cetuximab (0.2mg/mouse) delivered twice weekly for 2 weeks. Time to tumor quadrupling from baseline was assessed by Kaplan-Meier method.
Results: Significant variation in EGFR expression was seen in HPV+ cells. Cetuximab treatment resulted in significant delay in cell proliferation (p&lt;0.005 for all lines) and decrease in colony formation (p&lt;0.04 for all lines). In these cell lines, cetuximab caused an increase in apoptosis as measured by caspase activity, Bax activation, and Annexin V labeling. In addition, as previously seen in HPV- cell lines, cetuximab increased p27Kip-1 and decreased cyclin B1 and cyclin D1 coinciding with in a G1 cell cycle arrest. Using both a cell line xenograft model and a direct-from-patient tumorgraft model of HPV+ HNCs, cetuximab resulted in significant tumor growth delay (median time to tumor quadrupling: 15 vs. 24 days, p=0.02; and 42 vs. 89 days, p=0.0001, respectively).
Conclusions: Epidermal growth factor receptor inhibition by cetuximab appears to be effective in slowing proliferation and inducing apoptosis in HPV+ HNC. The proposed mechanism of action appears to be similar to that shown over a decade ago in HPV- HNC. These results suggest that cetuximab may play a role in the management of patients with HPV+ HNC.
Citation Format: Grace C. Blitzer, Molly A. Smith, Alexandra D. Torres, Eric A. Armstrong, Paul M. Harari, Paul F. Lambert, Randall J. Kimple. Epidermal growth factor inhibition of HPV positive head and neck cancer cells and primary tumorgrafts results in significant growth inhibition mediated by apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2013-5474