Pre-clinical evaluation of fluorescent deoxyglucose as a topical contrast agent for the detection of Barrett's-associated neoplasia during confocal imaging

Abstract

The availability of confocal endomicroscopy motivates the development of optical contrast agents that can delineate the morphologic and metabolic features of gastrointestinal neoplasia. This study evaluates 2-NBDG, a fluorescent deoxyglucose, the uptake of which is associated with increased metabolic activity, in the identification of Barrett's-associated neoplasia. Surveillance biopsies from patients with varying pathologic grades of Barrett's esophagus were incubated ex vivo at 37°C with 2-NBDG and imaged with a fluorescence confocal microscope. Images were categorized as neoplastic (high grade dysplasia, esophageal adenocarcinoma) or metaplastic (intestinal metaplasia, low grade dysplasia) based on the degree of glandular 2-NBDG uptake. Classification accuracy was assessed using histopathology as the gold standard. Forty-four biopsies were obtained from twenty-six patients; 206 sites were imaged. The glandular mean fluorescence intensity of neoplastic sites was significantly higher than that of metaplastic sites (p<0.001). Chronic inflammation was associated with increased 2-NBDG uptake in the lamina propria but not in glandular epithelium. Sites could be classified as neoplastic or not with 96% sensitivity and 90% specificity based on glandular mean fluorescence intensity. Classification accuracy was not affected by the presence of inflammation. By delineating the metabolic and morphologic features of neoplasia, 2-NBDG shows promise as a topical contrast agent for confocal imaging. Further in vivo testing is needed to determine its performance in identifying neoplasia during confocal endomicroscopic imaging.

Figure 1:

Representative endoscopic image (A), confocal fluorescence images (B, D) and histologic images (C) of samples diagnosed as IM/LGD are shown. Relevant features such as goblet cells and nuclei are indicated.

Figure 2:

Representative endoscopic image (A), confocal fluorescence images (B, D) and histologic images (C) of samples diagnosed as HGD with intact mucosal surface are shown. Relevant features such nuclei and incomplete glands are indicated. There is no apparent lesion or ulceration indicating neoplasia in endoscopy image; however, biopsy-confirmed neoplasia is present and neoplastic features are visible in the confocal image.

Figure 3:

Representative endoscopic image (A), confocal fluorescence images (B, D) and histologic images (C) of samples diagnosed as EAC with ulcerated mucosal surface are shown. Relevant features such as tumor cells, blood vessels, and the ulcerated surface are indicated. Apparent tumor and ulcerated surface visible during surveillance endoscopy is verified with biopsy-confirmed invasive cancer with surface ulceration.

Figure 4:

Representative confocal fluorescence images of samples diagnosed as (A) IM/LGD with zero to mild levels of chronic inflammation, (B) IM/LGD with moderate to marked levels of chronic inflammation, (C) HGD/EAC with moderate to marked levels of chronic inflammation, and corresponding histopathology (D-F). The presence of moderate-marked inflammation is associated with increased fluorescence in the lamina propria, while the presence of neoplasia is associated with increased glandular fluorescence.

Figure 5:

Demonstration of gland selection for mean glandular intensity (MGI) calculation on a confocal image of Barrett’s metaplasia stained with 2-NBDG. MGI is the average of x, where x is the average intensity of each gland.

Figure 6:

(A) Plot showing mean glandular fluorescence intensity ± one standard deviation, separated according to histologic diagnosis and presence/grade of inflammation: IM/LGD with zero-mild chronic inflammation; IM/LGD with moderated-marked chronic inflammation; and HGD/EAC with zero-marked chronic inflammation. (B) Scatter-plot showing mean glandular fluorescence intensity for each site according to histologic diagnosis and presence/grade of inflammation. Samples with acute inflammation present are indicated by “+” symbols; samples with no acute inflammation present are indicated by “o” symbols. n = the number of images evaluated per category.

Figure 7:

ROC curve for algorithm discriminating samples with HGD/EAC from samples with IM/LGD based on mean glandular fluorescence intensity. Results are shown for a per-site and a per-biopsy analysis using histology as the gold standard.