Abstract As with many epithelial cancers, presence of nodal metastases in patients with Head and Neck Squamous Cell Carcinoma (HNSCC) predicts early recurrence and poor survival. Determination of occult metastases in the clinically N-zero (cN0) patient remains problematic and therefore prophylactic neck dissections are routinely performed for high risk cN0 patients. A cohort of 7 cN0 HNSCC patients who subsequently underwent planned neck dissection as part of their treatment was selected. Snap frozen tissue was enriched for tumor and adjacent normal tissue by laser capture microdissection. Total protein was extracted and separated by saturation-labeling 2D difference in-gel electrophoresis (2D-DIGE). Significance analysis of Microarray (SAM) method was used to evaluate for differential protein expression between patients with (n=3) and without (n=4) occult nodal metastases on neck dissection pathologic evaluation. Spot maps were generated for 14 gels (7 HNSCC, 7 normal) and relative abundance values calculated for each of 662 protein spots common to >90% of gels. A 5% false discovery rate and 2-fold change were used as criteria for significance in the defintion of candidate biomarkers. Protein spots meeting these criteria were then extracted, digested and analyzed by liquid chromatography and tandem mass spectrometry to obtain protein identifications. For tumor tissue, there was no significant difference between groups. There were considerable differences in protein expression for normal adjacent tissue between patients with and without occult metastases, with 60 spots meeting criteria for significance (FDR<0.05). There were 31 proteins underexpressed in occult metastases patients, with the top candidate being 11.9 fold lower in the occult metastasis group (adjusted q<0.001). There were 29 proteins overexpressed, with the top candidate being 6.6 fold higher in the occult metastasis group (adjusted q<0.001). This protein was identified as Cornulin, a 53 kDa calcium-binding protein of the s100 family recently identified as a novel HNSCC biomarker. Cornulin plays a known role as a cornification chaperone that is protective against acid stress in squamous epithelium. It is possible that elevated cornulin in surrounding normal tissue may reflect ongoing epithelial injury, predisposing developing tumors to aggressive behavior. In conclusion, we have identified overexpression of cornulin in adjacent normal tissue as a novel biomarker for tumors with occult metastases in the cN0 HNSCC patient population. Additionally, these data highlight the role that tumor-stroma interactions may play in the development of early nodal metastases. 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 3286.