Validation of Novel Molecular Imaging Targets Identified by Functional Genomic mRNA Profiling to Detect Dysplasia in Barrett’s Esophagus

SIMPLE SUMMARY: Barrett’s esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE), including low-grade dysplasia (LGD) and high-grade dysplasia (HGD), shows a higher progression risk to EAC compared to non-dysplastic BE (NDBE). If LGD or HGD is detected, more intensive endoscopic surveillance or endoscopic treatment is recommended. This results in a significantly improved prognosis compared to EACs treated by surgery and/or chemoradiotherapy. However, the miss rates for detecting DBE by endoscopy remain high. Fluorescence molecular endoscopy (FME) can fill this gap by targeting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. We identified SPARC, SULF1, PKCι, and DDR1 as promising imaging targets for FME to differentiate DBE from NDBE tissue. We are also the first to develop near-infrared fluorescent tracers, SULF1-800CW and SPARC-800CW, for the endoscopic imaging of DBE tissue. ABSTRACT: Barrett’s esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKCι, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKCι, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett’s esophagus

Background Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett’s esophagus. Methods Six L2-IL1B mice with advanced stage of disease (12–16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12–14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls. Results Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results. Conclusions This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett’s esophagus. Further investigations are needed to assess its use in the clinical setting

Detection of Early Esophageal Neoplastic Barrett Lesions with Quantified Fluorescence Molecular Endoscopy Using Cetuximab-800CW

Esophageal adenocarcinoma causes 6% of cancer-related deaths worldwide. Near-infrared fluorescence molecular endoscopy (NIR-FME) uses a tracer that targets overexpressed proteins. In this study, we aimed to investigate the feasibility of an epidermal growth factor receptor (EGFR)–targeted tracer, cetuximab-800CW, to improve detection of early-stage esophageal adenocarcinoma. Methods: We validated EGFR expression in 73 esophageal tissue sections. Subsequently, we topically administered cetuximab-800CW and performed high-definition white-light endoscopy (HD-WLE), narrow-band imaging, and NIR-FME in 15 patients with Barrett esophagus (BE). Intrinsic fluorescence values were quantified using multidiameter single-fiber reflectance and single-fiber fluorescence spectroscopy. Back-table imaging, histopathologic examination, and EGFR immunohistochemistry on biopsy samples collected during NIR-FME procedures were performed and compared with in vivo imaging results. Results: Immunohistochemical preanalysis showed high EGFR expression in 67% of dysplastic tissue sections. NIR-FME visualized all 12 HD-WLE–visible lesions and 5 HD-WLE–invisible dysplastic lesions, with increased fluorescence signal in visible dysplastic BE lesions compared with nondysplastic BE as shown by multidiameter single-fiber reflectance/single-fiber fluorescence, reflecting a target-to-background ratio of 1.5. Invisible dysplastic lesions also showed increased fluorescence, with a target-to-background ratio of 1.67. Immunohistochemistry analysis showed EGFR overexpression in 16 of 17 (94%) dysplastic BE lesions, which all showed fluorescence signal. Conclusion: This study has shown that NIR-FME using cetuximab-800CW can improve detection of dysplastic lesions missed by HD-WLE and narrow-band imaging.</p

Validation of Novel Molecular Imaging Targets Identified by Functional Genomic mRNA Profiling to Detect Dysplasia in Barrett’s Esophagus

Simple Summary: Barrett's esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE), including low-grade dysplasia (LGD) and high-grade dysplasia (HGD), shows a higher progression risk to EAC compared to non-dysplastic BE (NDBE). If LGD or HGD is detected, more intensive endoscopic surveillance or endoscopic treatment is recommended. This results in a significantly improved prognosis compared to EACs treated by surgery and/or chemoradiotherapy. However, the miss rates for detecting DBE by endoscopy remain high. Fluorescence molecular endoscopy (FME) can fill this gap by targeting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. We identified SPARC, SULF1, PKC iota, and DDR1 as promising imaging targets for FME to differentiate DBE from NDBE tissue. We are also the first to develop near-infrared fluorescent tracers, SULF1-800CW and SPARC-800CW, for the endoscopic imaging of DBE tissue. Abstract: Barrett's esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKC iota, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKC iota, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings