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Introduction: Surgical resection is the standard of care for Oral Squamous Cell Carcinoma (OSCC) and the surgeon's ability to secure negative resection margins is associated with a reduced risk of local recurrence and improved survival rates.(1) Optimal functional outcomes are dependent on the balance between adequate surgical resection for complete removal of cancer and preservation of normal tissue to preserve essential functions such as swallowing, speaking, and breathing. Currently, histopathologic examination of the surgical resection specimen is the gold standard for margin assessment, yet it is time consuming and does not allow the surgeon intraoperative modification of the resection. Intraoperative frozen section is widely used but has significant limitations.(2) There is therefore a need for novel intraoperative imaging tools that can rapidly assess margin status by comprehensive examination of the excised surgical specimen in the operating room. Objective: Our team has explored the applications of PARPi-FL for molecular imaging of several cancers including OSCC.(3) PARPi-FL is a small molecule that targets poly-ADP ribose polymerase 1 (PARP1), an enzyme involved in DNA damage. We hypothesize that PARPi-FL is able to differentiate tumor cells from adjoining benign tissue in surgical specimens of OSCC and therefore, would be a rapid and reliable method of intra-operative margin assessment during surgery. In this pilot study, our aim was to analyze the fluorescence signal of PARPi-FL in tumor versus marginal normal tissue after topical application in OSCC specimens immediately after surgical resection. Methods: Previously untreated patients undergoing definitive surgery as their primary therapy for OSCC were eligible for the study (IRB 20-422). Immediately after surgical resection, the fresh surgical specimen (tumor and surrounding tissue) was submerged en bloc in 100mL of a solution containing 1,000nM of PARPi-FL in 30% PEG/PBS. Mucosal margins were imaged using a Quest Spectrum imaging device with an endoscopic camera and a PARPi-FL optimized LED-filter system, testing three different exposure times (30ms, 60ms, and 80ms). The specimen was then sectioned by the study pathologist per standard of care, and the sectioned specimen was imaged again after submersion in PARPi-FL to study depth of infiltration. Biopsies of the transition between margins and tumor were performed to compare the fluorescence imaging results with the standard H&E, and PARP1 expression on immunohistochemistry (IHC), and fluorescence imaging. All fluorescent images were analyzed in ImageJ by quantifying the average radiant efficiency of the PARPi-FL signal in regions of interest placed in tumor and normal tissue. For the statistical analysis, a GEE model was used to determine the association between uptake values and tissue, exposure time, and section (surface vs depth). Results: In the preliminary analysis of 8 patients of this ongoing study we observed a tumor mucosa fluorescence in average 2.85 times higher than in normal, adjusting for section and exposure time (p<0.001). Analyzing the IHC and H&E slides on tumor sections, we could confirm higher levels of PARP1 expression in the tumor areas, aligning with the higher fluorescent signals observed on the QUEST images (Figure 1). A major advantage of our method is that we were able to study wide areas of interest on the ex vivo specimens, including the evident malignant tissue and transition to normal tissue. Conclusion: PARPi-FL based ex vivo molecular imaging of surgically resected specimens holds promise for rapid and comprehensive intraoperative assessment of surgical margins in OSCC. [ABSTRACT FROM AUTHOR]