Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: first-in-human results

Abstract

Esophageal adenocarcinoma is rising rapidly in incidence and usually develops from Barrett's esophagus, a precursor condition commonly found in patients with chronic acid reflux. Premalignant lesions are challenging to detect on conventional screening endoscopy because of their flat appearance. Molecular changes can be used to improve detection of early neoplasia. We have developed a peptide that binds specifically to high-grade dysplasia and adenocarcinoma. We first applied the peptide ex vivo to esophageal specimens from 17 patients to validate specific binding. Next, we performed confocal endomicroscopy in vivo in 25 human subjects after topical peptide administration and found 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett's esophagus and squamous epithelium with 75% sensitivity and 97% specificity. No toxicity was attributed to the peptide in either animal or patient studies. Therefore, our first-in-human results show that this targeted imaging agent is safe and may be useful for guiding tissue biopsy and for early detection of esophageal neoplasia and potentially other cancers of epithelial origin, such as bladder, colon, lung, pancreas, and stomach.

Fig 1. Fluorescently labeled peptide specific for Barrett’s neoplasia

(A) Chemical structure of ASYNYDA peptide (black) with a GGGSK linker (blue) and a FITC label (green). (B) Fluorescence emission spectra for ASY*-FITC and FITC with λ_ex = 471 nm shows emission peaks 519 nm. (C) Mass spectrum of ASY*-FITC. (D) Binding affinity (apparent dissociation constant, k_d) for ASY*-FITC to H460 cells. Data are averages from 3 independent experiments. (E) Binding kinetics (apparent association time constant, k) for ASY*-FITC to H460 cells. (F) Relative fluorescence intensity (mean±SD) from binding of ASY*-FITC to human H460 adenocarcinoma cells decreases in a concentration-dependent manner on addition of the unlabeled ASY* peptide. The control was unlabeled GGG* at two different concentrations. P-values determined by unpaired t-test.

Fig 2. Ex vivo validation of peptide specific for Barrett’s neoplasia

(A) White light stereomicroscope image of resected esophageal specimen shows differences in color but not in architecture for squamous (yellow box), HGD (red box), and Barrett’s esophagus (blue box). Histology was cut along dashed lines 1–6. Scale bar, 2 mm. Image is representative of 12 specimens. (B) Fluorescence image collected after staining (A) with ASY*-FITC. Scale bar, 2 mm. (C) Histology (H&E stain) from sections 1–6. Scale bar, 2 mm. (D) Expanded region of red box in (B) section 3 shows features of HGD, including large, stratified nuclei and lack of cytoplasmic mucin (black arrow). (E) Fluorescence intensities from ASY*-FITC and GGG*-FITC (control) binding to human esophageal mucosa ex vivo in 1 mm² intervals for squamous (Squ), BE, HGD, and EAC. Data are means ± SEM. **P<0.01, determined by Dunn’s multiple comparison test.

Fig 3. Clinical validation of specific peptide binding in vivo

(A) Enrollment scheme for 25 patients with Barrett’s esophagus (BE). (B) Representative white light endoscopy image of the distal esophagus shows a typical salmon-pink region of BE (black arrow) and normal-appearing squamous epithelium (white arrow). (C) Peptide was applied endoscopically to distal esophagus via spray catheter (arrow). (D) In vivo imaging with confocal endomicroscope (arrow). (E to H) Representative peptide-based fluorescence image of normal squamous epithelium (E), BE with a benign crypt (arrow) (F), HGD dysplastic crypts (arrows) (G), and EAC-transformed crypts (arrows) (H). (I to L) Histology (H&E). Squamous epithelium alongside BE is shown (I). Surface epithelium in (J) confirms HGD in (G). Magnified view of the epithelium lining the mucosa in (J) reveals large, irregularly shaped nuclei that are fully stratified and have little cytoplasm (K). EAC from (H) shows malignant cells with glandular differentiation (L).

Fig 4. Quantitative analyses of imaging performance

(A) Target-to-background (T/B) ratio for a representative confocal image of HGD was calculated by measuring the mean fluorescence intensity from 4 solid boxes (dimensions 25×25 μm²) around the outer rim of crypts and 4 dashed boxes within the crypt lumen from 3 consecutive images. (B) T/B ratio (mean±SD) on in vivo confocal images collected from autofluorescence, squamous, BE, HGD, and EAC. **P<0.01, with P-values determined by unpaired t-test. (C) Receiver-operator curve (ROC) shows an optimum sensitivity of 75% (95% CI: 43%–95%) and specificity of 97% (95% CI: 85%–100%) for detection of HGD/EAC at T/B ratio of 4.2. (D) The sensitivity and specificity for peptide targeting HGD/EAC varies with T/B ratio as a function of detection threshold. Dashed line shows results at T/B ratio of 4.2. (E) Leukocytosis (average number of white blood cells measured as a ratio of post/pre-EMR) was quantified with and without peptide administration. ** P<0.01, with P-values determined by Mann Whitney test.