243 “Optical PET Scan” for the Detection of Barrett's Dysplasia: In Vitro Studies Using Cell Lines and EMR Tissue

“Optical PET Scan” for the Detection of Barrett’s Dysplasia: In Vitro Studies Using Cell Lines and EMR Tissue Emmanuel C. Gorospe*, Cadman Leggett, Joshua D. Penfield, Gang Sun, Marlys Anderson, Lori S. Lutzke, Kenneth K. Wang Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, MN Background: Positron emission tomography (PET) using FDG tracer in cancer imaging relies on the increased glucose metabolism of tumor cells. We examined a fluorescent glucose, 2-NBDG, as an optical analog of FDG-PET for Barrett’s Esophagus (BE)-related neoplasia. We performed a series of experiments using BE cell lines and a unique model, endoscopic mucosal resection (EMR) tissue, to assess the mechanism and feasibility of 2-NBDG as a probe for esophageal dysplasia. Aims: Our fourfold objective include: (a) comparison of 2-NBDG uptake and (b) demonstration of GLUT 1 transporter expression between dysplastic and non-dysplastic BE (ND-BE) cells, (c) demonstration of 2-NBDG uptake by GLUT 1, and (d) feasibility of 2-NBDG imaging in freshly resected esophageal mucosa. Methods: We used hTERT immortalized cell lines, CP-A and CP-B, from ND-BE and high grade dysplasia (HGD), respectively (ATCC). The cells were plated in 35mm dishes and allowed to re-equilibrate for 48 hrs. We used a standardized protocol of 0.5mm 2-NBDG with incubation for 30 min. at 37° C. GLUT 1 antibody (Abcam) was used for immunohistochemical staining per protocol. Fluorescence was recorded for 2-NBDG and GLUT 1 assays separately using an Axiovert (Zeiss) microscope with uniform exposure time at 540 msec. Mean fluorescence intensity (MFI) was recorded in all experiments using ImageJ (NIH). We conducted a competition assay with increasing concentration of D-glucose (1mM, 10mM, 100mM) added to a fixed 2-NBDG concentration of 0.5 mM on CP-B, to show 2-NBDG uptake by glucose transport mechanism. To assess esophageal tissue uptake, we applied 2-NBDG in EMR tissue obtained from 4 patients with history of HGD during their surveillance endoscopy. EMR tissue was obtained for clinically-indicated, suspicious mucosal lesions. Fresh ( 5 min.) ex-vivo EMR tissue was incubated with 0.5mM 2-NBDG for 30 min. We used FIVE1 (Optiscan) endomicroscope to record images in 4quadrants, followed by confirmatory histopathology. Results: There was parallel increase in 2-NBDG uptake and GLUT 1 expression in CP-B, compared to CP-A cell lines (Table 1). The competition assay showed decreasing uptake of 2NBDG with increasing D-glucose concentration (MFI: 75.3 13.1, 69.7 9.2, 51.5 7.6 at 1, 10, and 100mM glucose, respectively). This trend was indicative of 2-NBDG delivery via glucose transport. Final EMR histopathology (2 HGD; 2 ND-BE) correlated with 2-NBDG-enhanced endomicroscopy findings. In our EMR samples, images from areas of HGD (190.6 16.9 MFI) compared to ND-BE (80.2 18.8 MFI) had significantly increased fluorescence (p 0.0001) (Fig. 1). Conclusion: Our study validates 2-NBDG as a marker of BE dysplasia with an uptake similar to FDG in PET. It is a potential agent for “optical PET” imaging that could improve effectiveness of surveillance in patients with BE-associated neoplasia.