Abstract 4646: Evaluation of EGFR gene amplification status, mRNA, protein, and phosphoprotein levels expression in head and neck cancer patient tissues
Authors: Wheeler Sarah , Seethala Raja , Siwak Doris , Cieply Kathleen , Sherer Carol , Mills Gordon , Grandis Jennifer R. , Egloff Ann Marie 
Published: February 2011

Abstract
Purpose: Evaluate EGFR gene amplification status and EGFR mRNA and protein/phosphoprotein levels in tumors from patients with head and neck squamous cell carcinoma (HNSCC) surgically treated with curative intent from 2000-4 to define relationships between and assess the prognostic values of these biomarkers.
Background: EGFR has been reported to be overexpressed in HNSCC, and high levels of EGFR protein and EGFR gene amplification have been independently reported to be associated with poorer prognosis. To our knowledge, tumor levels of the known site-specific phosphorylated EGFR (Y992, Y1068) have not been reported as prognostic indicators for HNSCC.
Methods: 58 tumor specimens and 50 paired adjacent mucosal tissues from patients surgically treated for HNSCC were collected through an Early Detection Research Network (EDRN) study. Tissue microarrays (TMAs) were constructed using these paired patient samples and 30 oral mucosal specimens from cancer-free controls. Three cores from each specimen were arrayed on two TMAs and evaluated for EGFR gene amplification by dual-color FISH and for EGFR by IHC (EGFR levels in these IHC specimens have been previously reported). Corresponding fresh-frozen tumor specimens were evaluated by reverse-phase protein array (RPPA) for total EGFR, EGFR P-Y992 and P-Y1068 levels and by quantitative RT-PCR for EGFR mRNA levels. Progression-free survival (PFS) was calculated using the University of Pittsburgh Head and Neck Cancer Registry data as months from date of surgery to date of first cancer recurrence, new primary, or death. Hazard Ratios were determined using Cox proportional hazards models.
Results: Tumors with high level EGFR gene amplification expressed significantly higher levels of EGFR protein as assessed by IHC than tumors without EGFR gene amplification (P&lt;0.001). Increased EGFR levels were also detected in 11.4% of tumors without EGFR gene amplification. EGFR gene amplification was not associated reduced PFS (H.R.=1.64, P=0.16). High EGFR protein levels and elevated tumor levels of EGFR P-Y992 as determined by RPPA were not associated with reduced PFS. However, high tumor levels of EGFR P-Y1068 were significantly associated with reduced PFS (H.R.=3.34, P=0.008).
Conclusions: Similar to previous findings, we find that patients with tumor EGFR gene amplification tend to have shorter PFS than do patients without EGFR gene amplification. We find that tumors with EGFR gene amplification have significantly higher EGFR protein levels as assessed by IHC. Though increased EGFR protein levels in tumors are not always associated with gene amplification, this may be an important mechanism for increased EGFR expression. Differential levels of site-specific EGFR phosphorylation at Y992 and Y1068 indicate that signaling pathways associated with these sites may play different roles in disease progression.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4646.