Choline Kinase α (ChoKα) is overexpressed in many human tumors and is associated with poor clinical outcome in breast, prostate and lung cancer. In non-small cell lung carcinoma (NSCLC), ChoKα is overexpressed in 60% of human lung tumors (1).  Local recurrence occurs in up to 40% of patients after tumor resection (2). We hypothesize that the use of fluorophores for optical surgical navigation can improve margin detection, identify micro metastases, and ultimately reduce local recurrence. A novel near-infrared (NIR) fluorescent competitive ChoKα inhibitor, JAS239 (l_ex,l_em: 745, 775 nm), has been developed for intraoperative fluorescence imaging. The initiation of a clinical trial in patient dogs with spontaneous NSCLC will establish the proof-of-concept that JAS239 can provide contrast to lung tumors in a clinical setting. Twenty canine patients with operable NSCLCs, 18 adenocarcinomas, 1 squamous cell carcinoma, and 1 renal carcinoma metastasis, were recruited into an informed consent clinical trial. Patients received 0.25, 0.5, or 1 mg/kg JAS239 17-25 hours prior to intraoperative imaging with the EleVision (Medtronic) system. The trial included both male neutered and female spayed dogs. The mean weight of the dogs was 19.2 ± 11.0 kg and the mean age was 10.3 ± 1.9 years old. Dogs had 1-3 tumors with a mean primary tumor size of 186.8 ± 279.4 cm³ originating in left or right lung lobes. After resection, tumors were imaged on the back table with the EleVision and tumor and normal lung sections were imaged on the LI-COR Pearl and submitted to the Comparative Pathology Core (CPC) for H&E and ChoKα immunohistochemistry (IHC). Tumor to background ratios (TBRs) were calculated and used to determine the optimal imaging dose of JAS239. The average TBR for the 0.5 mg/kg dose, the dose of choice, was 5.0±2.0. IHC staining intensity was compared to fluorescence intensity. JAS239 was able to identify 16/20 tumors, including the squamous cell carcinoma. Two of the nonfluorescent tumor tissues were necrotic, where JAS239 was unable to accumulate. The third nonfluorescent tumor was a renal lung metastasis and the fourth was removed from the study due to the patient receiving repeat doses of JAS239 due to surgery delay. Clinically significant events (CSE) were classified as an event in which the imaging agent was able to identify a primary tumor, tumor margin, or synchronous lesion that the surgeon could not. A CSE was identified in one patient in which the surgeon resected a piece of normal lung for back bench imaging. Fluorescence imaging showed a fluorescent ROI with a TBR of 2.7. Pathology confirmed that this ROI was in fact malignant. Another CSE identified a close margin, prompting the surgeon to further investigate the cavity and margin, though no further disease was found. This veterinary trial provides the proof of concept that JAS239 can successfully identify NSCLCs in a clinical intraoperative setting. Additionally, it showed that JAS239 can identify synchronous lesions and close margins which can impact the surgical plan and further care. The ultimate goal of this study is translation of JAS239 for optical surgical navigation in human NSCLC patients. 

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