Bishnu P. Joshi

Targeted Imaging of Esophageal Neoplasia with a Fluorescently Labeled Peptide: First-in-Human Results

A fluorescently labeled peptide enables first-in-human targeted endoscopic imaging of esophageal neoplasia. Fluorescent Peptide Probe for Esophageal Cancer Detecting cancerous tissue isn’t always easy—and it can be particularly difficult for the early stages of esophageal cancer because the new lesions are often flat (versus a bulky tumor, for example) and thus invisible to the naked eye. To confidently detect esophageal adenocarcinoma (EAC), Sturm and colleagues designed a fluorescently labeled synthetic peptide, named ASY*-FITC, that recognizes cancer tissue and allows for in vivo imaging with a clinical endoscope. The cancer-targeting ASY*-FITC peptide was discovered using phage display technology and was found to bind tightly to human EAC cells and tissues ex vivo, but not to normal (squamous) tissue or metaplastic tissue, such as Barrett’s esophagus (BE). The tissues identified as cancerous were confirmed via histology. The authors then took this peptide into 25 patients. The fluorescent peptide was administered as would be expected during clinical exam: sprayed on the suspect area and then imaged with an endoscope. No ASY*-FITC bound to the squamous areas, and only minimal amounts of peptide bound to BE. However, areas of EAC as well as a high-grade dysplasia (HGD) were brightly illuminated and easily detected. The peptide was found to be safe and well tolerated in both humans and animals, and was synthesized according to good manufacturing practices (GMPs), suggesting that translation to a clinical setting will be possible in the near future. Further testing is needed to address optical limitations, such as imaging depth. Nevertheless, this first-in-human study paves the way for detection of HGD and EAC and other neoplasias, potentially without invasive biopsy. 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.

Exogenous Molecular Probes for Targeted Imaging in Cancer: Focus on Multi-modal Imaging

Cancer is one of the major causes of mortality and morbidity in our healthcare system. Molecular imaging is an emerging methodology for the early detection of cancer, guidance of therapy, and monitoring of response. The development of new instruments and exogenous molecular probes that can be labeled for multi-modality imaging is critical to this process. Today, molecular imaging is at a crossroad, and new targeted imaging agents are expected to broadly expand our ability to detect and manage cancer. This integrated imaging strategy will permit clinicians to not only localize lesions within the body but also to manage their therapy by visualizing the expression and activity of specific molecules. This information is expected to have a major impact on drug development and understanding of basic cancer biology. At this time, a number of molecular probes have been developed by conjugating various labels to affinity ligands for targeting in different imaging modalities. This review will describe the current status of exogenous molecular probes for optical, scintigraphic, MRI and ultrasound imaging platforms. Furthermore, we will also shed light on how these techniques can be used synergistically in multi-modal platforms and how these techniques are being employed in current research.

In Vivo Fluorescence-Based Endoscopic Detection of Colon Dysplasia in the Mouse Using a Novel Peptide Probe

Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli) gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61) binds ∼2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15), p<0.01. The peptide did not bind to the non-dysplastic (hyperplastic) epithelium of the Kras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs.

Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

Gastrointestinal cancers are heterogeneous and can overexpress several protein targets that can be imaged simultaneously on endoscopy using multiple molecular probes. We aim to demonstrate a multispectral scanning fiber endoscope for wide-field fluorescence detection of colonic dysplasia. Excitation at 440, 532, and 635 nm is delivered into a single spiral scanning fiber, and fluorescence is collected by a ring of light-collecting optical fibers placed around the instrument periphery. Specific-binding peptides are selected with phage display technology using the CPC;Apc mouse model of spontaneous colonic dysplasia. Validation of peptide specificity is performed on flow cytometry and in vivo endoscopy. The peptides KCCFPAQ, AKPGYLS, and LTTHYKL are selected and labeled with 7-diethylaminocoumarin-3-carboxylic acid (DEAC), 5-carboxytetramethylrhodamine (TAMRA), and CF633, respectively. Separate droplets of KCCFPAQ-DEAC, AKPGYLS-TAMRA, and LTTHYKL-CF633 are distinguished at concentrations of 100 and 1 μM. Separate application of the fluorescent-labeled peptides demonstrate specific binding to colonic adenomas. The average target/background ratios are 1.71 ± 0.19 and 1.67 ± 0.12 for KCCFPAQ-DEAC and AKPGYLS-TAMRA, respectively. Administration of these two peptides together results in distinct binding patterns in the blue and green channels. Specific binding of two or more peptides can be distinguished in vivo using a novel multispectral endoscope to localize colonic dysplasia on real-time wide-field imaging.

In vivo targeting of colonic dysplasia on fluorescence endoscopy with near-infrared octapeptide

Objective To demonstrate a near-infrared (NIR) peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo. Design A 3 mm diameter endoscope was adapted to deliver 671 nm illumination and collect NIR fluorescence (696–736 nm). Target (QPIHPNNM) and control (YTTNKH) peptides were labelled with Cy5.5, a NIR dye, and characterised by mass spectra. The peptides were topically administered separately (100 μM) through the endoscopes instrument channel into the distal colon of CPC;Apc mice, genetically engineered to spontaneously develop adenomas. After 5 min for incubation, the unbound peptides were rinsed off, and images were collected at a rate of 10 frames/s. Regions of interest were identified around the adenoma and adjacent normal-appearing mucosa on white light. Intensity measurements were made from these same regions on fluorescence, and the target-to-background ratio (TBR) was calculated. Results An image resolution of 9.8 μm and field of view of 3.6 mm was achieved at a distance of 2.5 mm between the distal end of the instrument and the tissue surface. On mass spectra, the experimental mass-to-charge ratio for the Cy5.5-labelled target and control peptides agreed with expected values. The NIR fluorescence images of adenomas revealed individual dysplastic crypts with distorted morphology. By comparison, only amorphous surface features could be visualised from reflected NIR light. The average TBR for adenomas was found to be 3.42±1.30 and 1.88±0.38 for the target and control peptides, respectively, p=0.007. Conclusion A NIR peptide was shown to be highly specific for colonic adenomas on fluorescence endoscopy in vivo and to achieve sub-cellular resolution images.

Near-infrared-labeled peptide multimer functions as phage mimic for high affinity, specific targeting of colonic adenomas in vivo (with videos)

BACKGROUND Fluorescent-labeled peptides are being developed to improve the endoscopic detection of colonic dysplasia. OBJECTIVE To demonstrate a near-infrared peptide multimer that functions as a phage mimic for in vivo detection of colonic adenomas. DESIGN A peptide multimer was synthesized by using trilysine as a dendritic wedge to mimic the presentation of peptides on phage, and all peptides, including the multimer, were fluorescent-labeled with Cy5.5. SETTING Small-animal imaging facility. ANIMAL SUBJECTS: Genetically engineered CPC;Apc mice that spontaneously develop colonic adenomas. INTERVENTION Near-infrared-labeled AKPGYLS peptide multimer was administered topically into the distal colons of the mice, and endoscopic images of adenomas were captured. Fluorescence intensities were quantified by target-to-background (T/B) ratios, and adenoma dimensions were measured with calipers after imaging. Validation of specific peptide binding was performed on cryosectioned specimens and cells by using confocal microscopy and flow cytometry. MAIN OUTCOME MEASUREMENTS Fluorescence T/B ratios from colonic adenomas and adjacent normal-appearing mucosa. RESULTS AKP-multimer, monomer, trilysine core, and Cy5.5 resulted in mean (± SD) T/B ratios of 3.85 ± 0.25, 2.21 ± 0.13, 1.56 ± 0.12, and 1.19 ± 0.11, respectively, P < .01 on in vivo imaging. Peptide multimer showed higher contrast and greater specificity for dysplastic crypts as compared with other probes. Peptide multimer demonstrated significantly greater binding to HT29 cells on flow cytometry and fluorescence microscopy in comparison to monomer and trilysine core. A binding affinity of 6.4 nm/L and time constant of 0.1136 minutes(-1) (8.8 minutes) was measured for multimer. LIMITATIONS Only distal colonic adenomas were imaged. CONCLUSION Peptide multimers combine strengths of multiple individual peptides to enhance binding interactions and demonstrate significantly higher specificity and affinity for tumor targets.

Multispectral endoscopic imaging of colorectal dysplasia in vivo

Light provides a broad range of colors in the visible and near-infrared (400–900 nm) spectrum that can be used to transmit information about molecular expression in normal and diseased tissues. The absorbance spectrum of hemoglobin, the primary chromophore in tissue, is shown in Fig. 1A. Genetic changes that occur in cancer transformation can be heterogeneous, and several signaling pathways may be activated concurrently.1 Moreover, molecular activity levels may vary between individual patients, at different time points, and within the tumor. Thus, a single genetic mutation is unlikely to characterize most disease processes over a general population. Unfortunately, most endoscopic imaging methods are sensitive to only one molecular parameter.2 Thus, a methodology that can image multiple molecular targets simultaneously is better suited to address the heterogeneity of cancer and achieve disease detection with high sensitivity and specificity desired for efficient surveillance. In addition, these multiplexed strategies can potentially visualize the interactions between signaling pathways and allow for better understanding of disease pathogenesis on a systems level. Figure 1 A) The visible and near-infrared spectrum of light provides a broad range of colors to generate molecular images from digestive tract mucosa that can be collected with a B) 1.6 mm diameter multispectral scanning fiber endoscope. C) Excitation is delivered ... Recently, significant progress has been made in advancing new methods of endoscopic imaging to target disease in the digestive tract, including the development of novel instruments and specific contrast agents.3 In this video journal, we aim to demonstrate the application of a novel, multispectral endoscope that uses a scanning fiber to image multiple targets at the same time. Fluorescence imaging can be achieved with high target-to-background ratios, and allow us to see the different target expression patterns in an Apc-mutation dependent mouse model of spontaneous colorectal adenomas using multiple peptides as targeting ligands.4

Multimodal endoscope can quantify wide-field fluorescence detection of Barrett's neoplasia.

BACKGROUND AND STUDY AIMS To demonstrate the clinical use of a multimodal endoscope with a targeted fluorescently labeled peptide for quantitative detection of Barretts neoplasia. PATIENTS AND METHODS We studied 50 patients with Barretts esophagus using a prototype multimodal endoscope with a fluorescently labeled peptide. Co-registered fluorescence and reflectance images were converted to ratios to correct for differences in distance and geometry over the image field of view. The ratio images were segmented using a unique threshold that maximized the variance between high and low intensities to localize regions of high grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). RESULTS Early neoplasia (HGD and EAC) was identified with 94 % specificity and 96 % positive predictive value at a threshold of 1.49. The mean results for HGD and EAC were significantly greater than those for squamous/Barretts esophagus and low grade dysplasia by one-way analysis of variance (ANOVA). The receiver operator characteristic curve for detection of early neoplasia had an area under the curve of 0.884. No adverse events associated with the endoscope or peptide were found. CONCLUSION A multimodal endoscope can quantify fluorescence images from targeted peptides to localize early Barretts neoplasia. (ClinicalTrials.gov number NCT01630798.).

EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

Objectives:Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears “invisible” on conventional wide-field endoscopy.Aims:We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging.Methods:We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence.Results:After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.Conclusions:We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0–400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.