PROJECT SUMMARY Nearly 13,000 new cases of laryngeal cancer are diagnosed in the U.S. annually. Transoral laser microsurgery (TLM) has become the primary surgical approach for early-stage endolaryngeal tumors as TLM provides tumor control while allowing maximal salvage of normal tissue to preserve post-treatment laryngeal function (i.e. voice and swallow). However, no reliable intraoperative approaches are approved to aid the surgeon's subjective visual assessment of tumor borders during TLM, leading to post-TLM recurrence of disease in a substantial proportion of patients. Panitumumab-IRDye800 has been used in multiple clinical trials for near-infrared fluorescence (NIRF) imaging during surgery of various malignancies. Current fluorescence guided surgery (FGS) clinical trials using panitumumab-IRDye800 require intraoperative pathological confirmation of cancer before further surgical actions can be made beyond the standard of care. Marginal biopsies during TLM are limited in volume and number, which confounds pathological assessment and intraoperative attempts to identify clinically occult invasive margins. The lack of FGS clinical studies during TLM in patients with laryngeal cancer has been a barrier in determining if current metrics to guide FGS would be applicable during TLM procedures. Addressing this clinical barrier is a critical step in determining the benefit of intraoperative NIRF to positively impact both TLM surgery and outcomes in patients with laryngeal cancer. We propose a clinical study to demonstrate feasibility for a novel combination of intraoperative optical imaging approaches to detect occult tumor margins in patients with laryngeal cancer. Objective 1 will establish metrics of intraoperative optical imaging that define pathology- confirmed occult laryngeal cancer margins. This objective will be accomplished by correlating intraoperative imaging during total laryngectomy procedures with post-resection tissue analyses including histopathology as the gold standard for true positive and true negative occult disease. Objective 2 will test the accuracy of intraoperative optical imaging metrics to identify occult laryngeal cancer margins during TLM. This objective will be accomplished using imaging metrics to identify positive optical signals beyond the region of treatment planned in patients who undergo TLM. Outcomes from these studies will be used to design a randomized phase 2 trial to test the combined imaging approach to guide TLM in patients with laryngeal cancer. The long-term outcomes of these studies are anticipated to positively impact intraoperative identification of occult malignancy while minimizing loss of critical laryngeal function.