Interobserver Agreement and Accuracy in Prediction of Barrett's-Associated Neoplasia Among More-Experienced and Less-Experienced Endomicroscopists

Abstract

Background: Confocal laser endomicroscopy (CLE) provides real time microscopic imaging of the gastrointestinal lining. Fluorescein is the most widely used contrast-enhancing fluorophore for this technique, but fails to provide signal within neoplastic cells that may reflect a more acidic tumor microenvironment. Lysosensor dye DND 189 (LD, Invitrogen) has a fluorescence peak at pH 5.2, compared to fluorescein with a peak at pH 7. The aim of this study was to determine if LD fluorescence might be greater in tumors compared to adjacent normal cells, thereby providing “positive” signals within tumors. Methods: Mice received azoxymethane (AOM, 10 mg/kg body wt) or saline (AOM vehicle) x 6 weeks. Uncontrasted colons were visualized via colonoscopy. Mice were then injected by tail vein with LD (25, 50, or 100 μL). The CLE probe (UHD probe, Mauna Kea Techologies, Paris) was inserted per rectum and images acquired 1-2 cm from the anal verge at 1, 5, 10, 30, 60, 120, and 180 min after injection. To image tumors we used an open laparotomy approach to identify tumors from the serosal side and then position the intraluminal CLE probe directly onto the tumor. After image acquisition, colons were opened and tumors harvested to correlate histological sections with confocal images. Results: Two control mice underwent colonoscopy that revealed endoscopically normal colons. In normal colons, punctate signals were detected 5-10 minutes after injection that were only observed at the highest LD dose. These signals may originate from dye trapped in acidic lysosomes. AOM-treated mice (n=4 mice) were assessed In Vivo for tumors with CLE per rectum assisted by laparotomy. Adequate tumor images were achieved with 50 μL LD. Within 5 min, LD fluorescence was detected in all tumors, but not adjacent normal-appearing mucosa (n=6 tumors, p<0.05). In one mouse without tumors undergoing CLE an inadvertent vascular injury causing ischemia of the colon almost immediately induced marked and diffuse positive signal. Conclusions: Fluorescent dyes, sensitive to low pH, can provide positive signals within tumors in a murine model of colorectal cancer. These metabolic signals could be exploited for enhanced microscopic detection of neoplasia using dynamic contrast CLE. Furthermore, changes in tumor microenvironment pH, induced by chemotherapeutic agents, might prove useful to assess response to therapy.