5528 Background: Human papilloma virus infection (HPV) is a powerful prognostic biomarker in LASCCHN and can be indirectly identified by p16 immunohistochemistry (IHC). Increased EGFR gene copy number has been reported to be a negative predictor of outcome in LASCCHN. In this study the relationship of p16 to EGFR gene copy number in patients treated with chemoradiation was assessed. Methods: Formalin-fixed paraffin embedded tissue was obtained from 110 patients with LASCCHN treated on three clinical trials with conventional chemoradiation or with a tirapazamine containing regimen. EGFR gene copy number was assessed by Fluorescence in situ hybridization (FISH) using LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probes (Vysis/Abbott). Tumors with high polysomy or amplification of EGFR were scored as positive. p16 was assessed by IHC using a validated antibody (Neomarkers). Markers were correlated with outcome i.e., failure-free survival (FFS) and overall survival (OS) using Cox regression, adjusting for trial. Results: Results for both EGFR FISH and p16 IHC were obtained in 97 patients. EGFR FISH was positive in 21/97 (22%) tumors including 12/66 (18%) oropharyngeal tumors. p16 was positive in 40/97 (41%) tumors including 36/66 (55%) oropharyngeal tumours. Only 1/40 p16 positive tumors were EGFR FISH positive compared with 20/57 p16 negative tumors (p<0.001). EGFR FISH positivity was associated with inferior FFS (HR 2.48 (95%CI 1.14 to 5.38), p = 0.022) and OS (HR 2.33 (95%CI 0.98 to 5.57), p = 0.057). p16 positivity was associated with improved FFS (HR 0.19 (95%CI 0.07 to 0.56), p = 0.002) and OS (HR 0.18 (95%CI 0.05 to 0.62), p = 0.006). On multivariable Cox regression analysis only p16 was independently predictive of outcome (p = 0.007 and 0.0014 for FFS and OS respectively). Conclusions: EGFR FISH positivity and p16 expression were found to be almost mutually exclusive in LASCCHN with only 1 of 97 tumors positive for both markers suggesting that increased EGFR copy number is largely restricted to HPV-negative LASCCHN. EGFR FISH and p16 were predictive of outcome in univariate analyses, but only p16 remained predictive on multivariable analysis. No significant financial relationships to disclose.