Multiplexed endoscopic imaging of Barrett’s neoplasia using targeted fluorescent heptapeptides in a phase 1 proof-of-concept study

Message

Imaging molecular expression patterns may improve methods for early cancer detection in patients with Barrett’s esophagus (BE). A multiplexed strategy that detects two targets simultaneously is demonstrated. Heptapeptides specific for EGFR and ErbB2 were labeled with Cy5 and IRDye800, respectively. Fluorescence images from n = 22 BE patients were collected in vivo using a multi-modal scanning fiber endoscope. The images were evaluated using support vector machine and logistic regression methods, and revealed 92% sensitivity and 89% specificity. This first-in-human pilot study demonstrates feasibility to detect multiple targets concurrently and potential for early detection of cancers that are molecularly heterogeneous.

BACKGROUND

Esophageal adenocarcinoma (EAC) is a deadly disease that has increased dramatically in incidence.^1,2 Endoscopic screening with white light illumination and random biopsy is limited by sampling error.³ Dysplasia often presents with flat architecture and patchy distribution.⁴ EGFR and ErbB2 are transmembrane tyrosine kinase receptors that stimulate epithelial cell growth, proliferation, and differentiation.⁵ Overexpression of these targets reflects a higher risk for cancer progression.^6–8 Multiplexed imaging methods take advantage of the broad spectrum of light over the visible and near-infrared (NIR) regime. We aim to demonstrate clinical feasibility to visualize EGFR and ErbB2 expression simultaneously in vivo to detect Barrett’s neoplasia.

METHODS

Consecutive patients referred for either evaluation or therapy of Barrett’s neoplasia were recruited for the study (NCT03589443). A multi-modal scanning fiber endoscope (mmSFE) was designed to collect two fluorescence images concurrently. Target/background (T/B) ratios were calculated for each fluorescence image. Details are provided online in Supplementary file.

RESULTS

The peptide QRHKPRE specific for EGFR was labeled with Cy5 via a GGGSK linker, Figure 1A,⁹ and KSPNPRF, specific for ErbB2, was labeled with IRDye800 via a GGGSC linker, Figure 1B.¹⁰ These fluorophores were chosen to minimize overlap between absorbance and emission spectra, Figure 1C. The characteristics and stability of fluorescently labeled peptides are shown, Table S1-S4. The pharmacology/toxicology study showed no acute adverse effects in animals for either peptide, Table S5,6. A Phase 1 safety study performed in n = 25 human showed no abnormalities in the laboratory results, urinalysis, and EKG for either peptide, and no adverse events (AE) were found.

Figure 1. Fluorescently labeled peptides for multiplexed imaging.

Biochemical structures are shown for A) QRH*-Cy5, and B) KSP*-IRDye800. C) Peak absorbance of QRH*-Cy5 and KSP*-IRDye800 occurs at λ_abs = 648 and 776 nm, respectively. Peak fluorescence emits at λ_em = 675 and 812 nm, respectively. D) Schematic diagram for the multi-modal scanning fiber endoscope (mmSFE) is shown. Excitation at λ_ex = 638 and 785 nm is delivered through a single-mode fiber (SMF) that is scanned in a spiral pattern by a piezo tube actuator. The beam is focused onto the tissue surface (illumination plane) by a compact lens assembly. E) Fluorescence is collected by a ring of large core multi-mode fibers (MMF) mounted around the instrument periphery. F) The dimensions of the rigid tip are 9 mm in length and 2.4 mm in diameter. G) This instrument passes forward through the 2.7 mm diameter working channel of a standard medical endoscope (Olympus #GIF-HQ190). H) The system is contained within a portable cart.

The mmSFE was designed to collect multiplexed fluorescence images, Figure 1D-H. Contrast agents were administered, and real time images were collected from n = 22 subjects, Table 1 (Videos S1-23). Representative white light images are shown for squamous (SQ) and non-dysplastic BE (NDBE), Figures 2A,B. Minimal background was seen following peptide administration. Fluorescence images were collected in separate channels, and co-registered reflectance provided anatomic landmarks for image interpretation. A representative set of in vivo images for HGD and EAC is shown, Figures 2C,D. Increased fluorescence intensities were seen from regions of HGD and EAC, and were confirmed by pathology. Immunohistochemistry (IHC) was performed to validate EGFR and ErbB2 expression on excised specimens, Figure S1.

Table 1. Patient demographics.

Multiplexed images were collected in vivo from the distal esophagus of n = 22 patients with a mean (±SD) age of 70.0±10.8 years. Squamous (SQ), non-dysplastic Barrett’s esophagus (NDBE), and low-grade dysplasia (LGD) were identified in a total of n = 2, 3, and 4 subjects, respectively. High-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) were found in n = 7 and 6 subjects, respectively. Modified Prague classification includes length in centimeters of circumferential Barrett’s esophagus (C), maximal tongue (M), and any proximal island (I). These findings were confirmed by histopathology from either endoscopic mucosal resection (EMR) or biopsy.

Age	Gender	Prague/Stage	Tissue Sampling	Pathology
68	M	C0M0I0	EMR/biopsy	SQ
57	M	C0M0I0	biopsy	SQ
84	F	C0M0I9	biopsy	NDBE
60	M	C0M1	EMR/biopsy	NDBE
56	M	C1M3	EMR/biopsy	NDBE
57	M	C7M9	biopsya	LGD
56	F	C0M1	biopsya	LGD
80	F	C0M0I0	biopsya	LGD
67	M	C0M0I7	EMR/biopsy	LGD
79	F	C0M2	biopsy	HGD
88	M	C0M3	EMR/biopsy	HGD
79	M	C0M1I1.5	EMR/biopsy	HGD
85	M	C12M13	biopsy	HGD
79	M	C4M5	biopsy	HGD
66	M	C0M0	biopsy	HGD
60	M	C9M10	biopsy	HGD
75	M	T3N1	biopsy	EAC
73	F	C0M0I2	EMR/biopsy	EAC
81	M	C10M10	biospyb	EAC
71	M	C9M12I13	EMR/biopsy	EAC
55	F	T1a	biopsy	EAC
64	F	C0M1	EMR/biopsy	EAC

^aNo tissue sampling performed at time of fluorescence imaging. Pathology based on findings from the most recent pathological reports before and after the imaging procedure.

^bA mass was found in the thoracic esophagus and the tissue diagnosis was obtained prior to this exam.

Figure 2. Barrett’s esophagus.

Representative in vivo images collected endoscopically are shown from patients with (A) squamous (SQ), (B) non-dysplastic Barrett’s esophagus (NDBE), (C) high grade dysplasia (HGD), and (D) esophageal adenocarcinoma (EAC). The presence of NDBE is identified by salmon red patches (arrows) in the white light images. Fluorescence images are collected after topical administration of QRH*-Cy5 and KSP*-IRDye800 separately. The merged images show high contrast regions-of-interest (ROIs) where EGFR and ErbB2 (orange) are co-expressed. Co-registered reflectance images provide anatomical landmarks to help interpret the location of the ROIs.

The T/B ratios using QRH*-Cy5 and KSP*-IRDye800 were measured from individual patients, and are shown, Figure 3A,B. For SQ (n = 2) or NDBE (n = 3), a mean (±SD) T/B ratio of 1.28±0.07 for QRH*-Cy5 and 1.33±0.15 for KSP*-IRDye800, respectively, was calculated. The T/B for (n = 4) LGD was 1.23±0.05 and 1.18±0.10, respectively. For HGD (n=7) and EAC (n = 6), a mean (±SD) T/B ratio of 1.61±0.21 and 1.68±0.24, respectively, was found. Leave-one-out cross-validation (LOOCV) was used to classify results, Table S7. Support vector machine (SVM) and logistic regression (LR) provided the highest classification accuracy of 91%. The imaging results revealed n = 12, 1, 8, and 1 true positives, false positives, true negatives, and false negatives, respectively, resulting in 92% sensitivity and 89% specificity. The decision boundaries using SVM and LR are shown. The ROC curves using these methods with LOOCV are displayed, Figure 3C. A higher AUC was achieved with multiplexed detection versus either target alone from bootstrap, Figure 3D.

Figure 3. In vivo imaging performance.

Scatter plots show T/B ratios measured for EGFR and ErbB2 expression in the fluorescence images collected in vivo from the distal esophagus of n = 22 patients. Decision boundaries show regions classified as either negative (blue) or positive (brown) for neoplasia using A) support vector machine (SVM) and B) logistic regression (LR) trained on all data. C) ROC curves for classifying HGD/EAC from SQ/NDBE/LGD are shown using SVM and LR algorithms with leave-one-out cross-validation (LOOCV). D) Average ROC curves from bootstrap using SVM (AUC = 0.97) model trained on all data show that multiplexed detection provides improved performance than using either EGFR (AUC = 0.95) or ErbB2 alone (AUC = 0.94).

COMMENTS

Here, we demonstrate feasibility to detect Barrett’s neoplasia endoscopically by imaging two targets concurrently in vivo. Fluorescently labeled peptides specific for EGFR and ErbB2 were administered topically in the distal esophagus of n = 22 BE patients. With conventional white light illumination, structural abnormalities associated with Barrett’s neoplasia appeared subtle. By comparison, spatial patterns of target expression were visualized with high contrast using fluorescence. Two laser excitation wavelengths were delivered concurrently through a single flexible optical fiber using a prototype wide-field endoscope accessory. Adequate signal was collected by using large core, high numerical aperture fibers. The regions imaged were compared with histopathology of specimens excised via either EMR or biopsy. Immunohistochemistry of these specimens confirmed heterogeneous expression of EGFR and ErbB2.

To our knowledge, this study is the first to demonstrate clinical application of multiplexed imaging during endoscopy. Many cancers, such as EAC, are molecularly heterogeneous, thus the capacity to detect multiple targets concurrently may result in more accurate clinical diagnosis. Mucosal abnormalities with non-specific features, such as nodularity, ulceration, and irregularity, are not specific for accurate location of Barrett’s neoplasia. Leading medical societies recommend random 4-quadrant biopsies for EAC surveillance, but this sampling method is inefficient and has been poorly adopted by community physicians.¹¹ Molecular biomarkers can be highly specific for disease, and are expressed before neoplastic lesions become grossly apparent. Endoscopic imaging strategies for detecting these targets in vivo can be used to guide and prioritize high risk regions for resection, reduce surveillance frequency, and minimize over diagnosis.

Recently, detection of dysplasia and early EAC in BE patients was demonstrated using an antibody specific for vascular endothelial growth factor A (VEGFA). Bevacizumab was originally developed for cancer therapy, and was repurposed for diagnostic imaging by labeling with IRDye800.¹² Compared with antibodies, peptides are much smaller in size, have faster binding kinetics, and can be mass manufactured at lower costs.^13–15 The peptides were delivered topically to the mucosal surface in the distal esophagus. This method was effective for staining regions of neoplastic BE involvement.¹⁶ Compared with systemic delivery, this approach localizes the distribution of exogenous imaging agents to the target tissues only, minimizes background, and maximizes image contrast. This strategy could be performed because the peptides used have similar binding kinetics.^9,10 Lectins have been investigated for detection of Barrett’s neoplasia ex vivo,¹⁷ but have not been demonstrated clinically. Fluorescently-labeled peptides for targeted detection of pre-malignant lesions in the colon have also been shown clinically.^18,19 Folate has been developed for targeted imaging of ovarian cancer,²⁰ however, small molecules have limited flexibility for fluorescence labeling and would be difficult to arrange for multiplexed imaging.

In this work, light over a broad optical spectrum was collected using a flexible fiber endoscope accessory, and was separated into fluorescence and reflectance channels.²¹ Multi-pixel photon counters with much higher sensitivity than the charge-coupled devices (CCD) found in video endoscopes were used. Multiplexed detection was achieved by exciting Cy5 and IRDye800 at wavelengths with minimal overlap between the absorption and emission bands. Additional targets can be detected by extending this strategy to the full visible and NIR spectrum.²² Fluorescence emission in the NIR regime mitigates the effects of hemoglobin absorption and tissue scattering, and minimizes tissue autofluorescence background.²³ The distal optics and scan strategy used provide much greater spatial resolution than an optical fiber bundle.

The clinical usefulness of this technology can be improved by addressing several study limitations. The peptides were administered separately to minimize potential binding interactions but can be combined to reduce time needed to reconstitute and prepare the peptides for delivery. After inserting the mmSFE accessory through the working channel, the fluorescence and HD-WLE images were not registered. Accurate alignment would allow the fluorescence images to be more effective as a real-time guide for tissue resection. This study was performed at a tertiary referral center that specializes in treatment of patients with advanced BE, thus a cohort highly enriched with neoplasia was studied. Inclusion of more non-neoplastic subjects would better reflect the prevalence of disease seen in the community. In conclusion, we demonstrated proof-of-concept for detecting multiple targets concurrently in patients with Barrett’s neoplasia and promise for this strategy to detect cancers in hollow organs early.

Abbreviations –

BE

Barrett’s esophagus

SQ

squamous

LGD

low grade dysplasia

HGD

high grade dysplasia

EAC

esophageal adenocarcinoma

mmSFE

multi-modal scanning fiber endoscope

NIR

near infrared