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Objective: To evaluate expression of the potential molecular imaging targets glycoprotein nonmetastatic melanoma protein B (GPNMB) and vascular endothelial growth factor (VEGF) in comparison to classical epidermal growth factor receptor (EGFR) in previously untreated head and neck cancer primary tumors and corresponding lymph node metastases, and in lymph node metastases after initial (chemo)radiotherapy. Methods: Primary tumors and lymph node metastases of 38 patients who underwent primary resection of head and neck squamous cell carcinoma (HNSCC) were selected. In addition, lymph node metastases from salvage surgery after (chemo)radiotherapy from 22 patients with HNSCC were selected. Immunohistochemical staining for EGFR, VEGF and GPNMB was performed, after which protein expression was scored using H-scores (percentage of positive tumor cells multiplied by staining intensity ranging from 0 to 2). Primary tumors and lymph node metastases with an H-score ≥5 were considered positive, after which sensitivity rates were calculated. Groups were compared using the Mann-Whitney U test for non-parametric data with post-hoc Bonferroni corrections. Results: In untreated primary HNSCC the sensitivity was 86.8% for EGFR (median H-score 60), 92.1% for VEGF (median 60), and 100% for GPNMB (median 100). In previously untreated lymph node metastases the sensitivity was 81.6% for EGFR (median 30), 86.8% for VEGF (median 65), and 100% for GPNMB (median 118). In lymph node metastases after initial (chemo)radiotherapy, sensitivity was 86.4% for EGFR (median 95) and 100% for GPMB (median 100). VEGF sensitivity could not reliably be evaluated in these lymph nodes due to nonspecific staining in all tissue slides. GPNMB expression was significantly higher compared to EGFR in untreated primary HNSCC (p = .009 and p < .001, respectively) and lymph node metastases (p < .001 for both). Conclusion: GPNMB shows better tumor-specific receptor expression in both sensitivity and H-scores compared to EGFR and VEGF in immunohistochemistry. VEGF performed comparable to EGFR in untreated HNSCC and lymph node metastases. GPNMB could therefore be a new potential imaging target based on these immunohistochemical results. Citation Format: Jeroen E. van Schaik, Bert van der Vegt, Saskia H. Hanemaaijer, Gyorgy B. Halmos, Max J. Witjes, Bernard F.A.M. van der Laan, Rudolf S. Fehrmann, Sjoukje F. Oosting, Boudewijn E. Plaat. Comparison of glycoprotein nonmetastatic melanoma protein b, epidermal growth factor receptor and vascular endothelial growth factor expression in head and neck squamous cell carcinoma, and untreated and residual lymph node metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6261.