Detection of Sessile Serrated Adenomas in the Proximal Colon Using Wide-Field Fluorescence Endoscopy

Abstract

Background & aims: Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients.

Methods: We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 μmol/L and 22 patients were given 76.4 μmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hours after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy to identify suspicious regions. The fluorescently labeled peptide (76.4 μmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan.

Results: We identified the peptide sequence KCCFPAQ and measured an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174 min-1 (5.76 minutes). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps.

Conclusions: We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of premalignant serrated lesions during routine colonoscopies. ClinicalTrials.gov ID: NCT02156557.

Keywords: Adenoma; Colorectal Cancer; Peptide; Serrated.

Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Fig. 1. Fluorescently-labeled peptide

A) Chemical structure of KCCFPAQ peptide (black) attached via GGGSK linker (blue) to FITC fluorophore (green), hereafter KCC*-FITC. B) Fluorescence spectra of KCC*-FITC show peak absorbance and fluorescence emission at 490 and 519 nm, respectively. We measured C) an apparent dissociation constant of kd = 72 nM, R2 = 0.925, that reflects binding affinity, and D) an apparent association time constant of k = 0.174 min−1, R2 = 0.989, for binding of KCC*-FITC to HT29 cells, resulting in a time scale of 5.76 min. Results are an average of 5 independent experiments for kd and two independent experiments for k.

Fig. 2. KCC* peptide binding to human colon cells in vitro

On confocal fluorescence microscopy, A) intense staining was observed for binding of KCC*-FITC to the surface (arrows) of HT29 cells with a V600E point mutation in BRAF, but not to B) Hs738St/Int (control) cells. C,D) No binding was observed for the PFA*-FITC control peptide to either cell. Labeling the KCC* peptide with either E) biotin or F) Cy5.5 also resulted in strong cell surface binding. Experiments were performed in triplicate.

Fig. 3. Targeted in vivo images of flat SSA in proximal colon

A) Endoscopic image with white light illumination (Visualization1) shows limited visualization of SSA (arrow) with flat morphology. B) Fluorescence image (Visualization2) shows increased intensity and high contrast from lesion (arrow) while normal colonic mucosa shows minimal background. C) Reflectance (Visualization3) and co-registered fluorescence images are combined in a ratio to correct for differences in distance over the image field-of-view. D) Ratio image shown in pseudo-color enhances signal from SSA (arrow). E) Intensities from fluorescence, reflectance, and ratio images along horizontal dashed line in B–D) show a peak at location of SSA (arrow). F) Corresponding histology shows pathological features of SSA (arrow).

Fig. 4. Endoscopic images from proximal colon

Representative fluorescence images of A) hyperplastic polyp (HP, Visualization4), B) adenoma (Visualization5), and C) adenocarcinoma (ACA, Visualization6) are shown along with corresponding D–F) white light images (Visualization7–9).

Fig. 5. In vivo imaging performance

A) The mean T/B ratios from individual patients are calculated from the “red flag” contours drawn from a ratio of the fluorescence and reflectance images and are classified by histology: hyperplastic (HP), sessile serrated adenoma (SSA), adenoma, adenocarcinoma (ACA), and normal colonic mucosa (Nml). The mean (±std) values were found to be 0.94 for HP (n = 1), 2.24±0.87 for SSA (n = 19), 0.94±0.47 for adenoma (n = 23), 1.26 for ACA (n = 1), and 0.92±0.17 for Nml (n = 24). Fluorescence intensities were measured from adjacent normal colonic mucosa in only n = 24 cases because of the large size of some of the lesions. B) Based on the estimated fold-change, we found the mean fluorescence intensities for SSA to be significantly greater than that for normal and adenoma using Tukey’s single step multiple comparison test, C) For distinguishing SSA from Nml, 89% sensitivity and 92% specificity was achieved at a T/B ratio of 1.16 (dashed line). D) The corresponding receiver operator characteristic (ROC) curve has an AUC of 0.93 with 95% confidence interval: 82–100%. E) For distinguishing SSA from adenoma, 84% sensitivity and 91% specificity was achieved at a T/B ratio of 1.66 (dashed line). F) The corresponding ROC curve has an AUC of 0.90 with 95% confidence interval: 79–100%.

Fig. 6. Ex vivo peptide binding to human colon specimens

A–D) On confocal microscopy, KCC*-FITC (green) binds brightly to surface (arrow) of colonocytes from sessile serrated adenoma (SSA), while minimal signal is seen for hyperplasia (HP), adenoma, and normal colonic mucosa. E) The mean (±std) fluorescence intensities for HP (n = 10), SSA (n = 17), adenoma (n = 31), and normal (n = 26) were found to be 4.04±1.12, 22.93±3.07, 8.81±1.63, and, 3.56±0.64 respectively. The mean fluorescence intensity for SSA was found to be significantly higher than that for HP, adenoma, and normal, *P=<0.01, by unpaired t-test.