This is a competing renewal proposal of translational research intended to advance the clinical use of molecular markers in squamous cell carcinoma of the head and neck. The centerpiece of the project is a large well characterized collection of tumor and histologically clear margin tissue collected in an Intergroup laboratory study. Subject accrual to this study is complete, but laboratory analysis and clinical data collection are ongoing. The primary goal of the study (specific aim #1) is to verify the utility of p53 molecular margin testing to assess risk of recurrence. To be clinically useful, molecular margin analysis must be rapid and applicable to all cases. We propose that high through-put p53 margin analysis using a novel Gap ligase chain reaction (GLCR) can be combined with methylation-specific PCR (MSP) for detection of tumor-specific promoter hypermethylation of target genes for tumors lacking p53 alterations make this possible. We will complete molecular assessment of all tumors in the cohort identifying both p53 mutations and methylation markers. Cases with tumors displaying p53 mutation will then undergo margin analysis using quantitative GLCR. Methylation markers will be tested in the p53 mutated cases to assess the concordance of the two markers in margin samples and to compare their predictive value. MSP will then be used to assess margins in p53 wild-type cases. A tissue microarray will be constructed to screen for methylation markers and explore p53-related molecular pathways (aim 2). The spectrum of target gene methylation and p53 mutations together with their downstream expression profiles will then be examined to refine their clinical predictive value. Expected correlations between clinical outcome and the presence of alterations in tumor and the detection of molecular residual disease in "normal" margin and lymph node tissue will be tested (aim 3). This work makes maximal use of this invaluable collection of HNSCC specimens opening the door to accurate predictive assessment and a deeper understanding of tumor behavior.