Purpose: The genetic differences between human papilloma virus(HPV)-positive and-negative head and neck squamous cell carcinomas(HNSCC)remain largely unknown.To identify differential biology and novel therapeutic targets for both entities,we determined mutations and copy-number aberrations in a large cohort of locoregionally advanced HNSCC.Experimental Design: We performed massively parallel sequencing of 617 cancer-associated genes in 120 matched tumor/normal samples(42.5%HPV-positive).Mutations and copy-number aberrations were determined and results validated with a secondary method.Results: The overall mutational burden in HPV-negative and HPV-positive HNSCC was similar with an average of 15.2 versus 14.4 somatic exonic mutations in the targeted cancer-associated genes.HPV-negative tumors showed a mutational spectrum concordant with published lung squamous cell carcinoma analyses with enrichment for mutations in TP53,CDKN2A,MLL2,CUL3,NSD1,PIK3CA,and NOTCH genes.HPV-positive tumors showed unique mutations in DDX3X,FGFR2/3 and aberrations in PIK3CA,KRAS,MLL2/3,and NOTCH1 were enriched in HPVpositive tumors.Currently targetable genomic alterations were identified in FGFR1,DDR2,EGFR,FGFR2/3,EPHA2,and PIK3CA.EGFR,CCND1,and FGFR1 amplifications occurred in HPV-negative tumors,whereas 17.6%of HPV-positive tumors harbored mutations in fibroblast growth factor receptor genes(FGFR2/3),including six recurrent FGFR3 S249C mutations.HPV-positive tumors showed a 5.8%incidence of KRAS mutations,and DNArepair gene aberrations,including 7.8%BRCA1/2 mutations,were identified.Conclusions: The mutational makeup of HPV-positive and HPV-negative HNSCC differs significantly,including targetable genes.HNSCC harbors multiple therapeutically important genetic aberrations,including frequent aberrations in the FGFR and PI3K pathway genes.