Elmire Hartmans

Molecular Fluorescence Endoscopy Targeting Vascular Endothelial Growth Factor A for Improved Colorectal Polyp Detection

Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular fluorescence endoscopy with targeted near-infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR)–targeted fluorescent tracers during ex vivo colonoscopy with an NIR endoscopy platform. Methods: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples—48 sessile serrated adenomas/polyps, 70 sporadic high-grade dysplastic adenomas, and 19 hyperplastic polyps—and tissue derived from patients with Lynch syndrome—78 low-grade dysplastic adenomas, 57 high-grade dysplastic adenomas, and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116luc tumors received intravenous bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW, or sodium chloride. Three days later, 8 resected HCT116luc tumors (2–5 mm) were stitched into 1 freshly resected human colon specimen and followed by an ex vivo molecular fluorescence colonoscopy procedure. Results: Immunohistochemistry showed high VEGF-A expression in 79%–96% and high EGFR expression in 51%–69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions, compared with the adjacent normal colon crypts. During ex vivo molecular fluorescence endoscopy, all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing, respectively, stromal and cell membrane fluorescence. Conclusion: VEGF-A is a promising target for molecular fluorescence endoscopy because it showed a high protein expression, especially in sessile serrated adenomas/polyps and Lynch syndrome. We demonstrated the feasibility to visualize small tumors in real time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field red-flag technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach.

Towards clinically translatable NIR fluorescence molecular guidance for colonoscopy

White-light surveillance colonoscopy is the standard of care for the detection and removal of premalignant lesions to prevent colorectal cancer, and the main screening recommendation following treatment for recurrence detection. However, it lacks sufficient diagnostic yield, exhibits unacceptable adenoma miss-rates and is not capable of revealing functional and morphological information of the detected lesions. Fluorescence molecular guidance in the near-infrared (NIR) is expected to have outstanding relevance regarding early lesion detection and heterogeneity characterization within and among lesions in these interventional procedures. Thereby, superficial and sub-surface tissue biomarkers can be optimally visualized due to a minimization of tissue attenuation and autofluorescence by comparison with the visible, which simultaneously enhance tissue penetration and assure minimal background. At present, this potential is challenged by the difficulty associated with the clinical propagation of disease-specific contrast agents and the absence of a commercially available endoscope that is capable of acquiring wide-field, NIR fluorescence at video-rates. We propose two alternative flexible endoscopic fluorescence imaging methods, each based on a CE certified commercial, clinical grade endoscope, and the employment of an approved monoclonal antibody labeled with a clinically applicable NIR fluorophore. Pre-clinical validation of these two strategies that aim at bridging NIR fluorescence molecular guidance to clinical translation is demonstrated in this study.

A Standardized Light-Emitting Diode Device for Photoimmunotherapy

Antibody-based photodynamic therapy—photoimmunotherapy (PIT)—is an ideal modality to improve cancer treatment because of its selective and tumor-specific mode of therapy. Because the use of PIT for cancer treatment is continuing to be described, there is great need to characterize a standardized light source for PIT application. In this work, we designed and manufactured a light-emitting diode (LED)/PIT device and validated the technical feasibility, applicability, safety, and consistency of the system for cancer treatment. Methods: To outline the characteristics and photobiologic safety of the LED device, multiple optical measurements were performed in accordance with a photobiologic safety standard. A luciferase-transfected breast cancer cell line (2LMP-Luc) in combination with panitumumab-IRDye 700DX (pan-IR700) was used to validate the in vitro and in vivo performance of our LED device. Results: Testing revealed the light source to be safe, easy to use, and independent of illumination and power output (mW cm−2) variations over time. For in vitro studies, an LED dose (2, 4, 6 J cm−2)–dependent cytotoxicity was observed using propidium iodide exclusion and annexin V staining. Dose-dependent blebbing was also observed during microscopic analysis. Bioluminescence signals of tumors treated with 0.3 mg of pan-IR700 and 50 J cm−2 decreased significantly (>80%) compared with signals of contralateral nontreated sites at 4 h and at 1 d after PIT. Conclusion: To our knowledge, a normalized and standardized LED device has not been explicitly described or developed. In this article, we introduce a standardized light source and validate its usability for PIT applications.

Potential Red-Flag Identification of Colorectal Adenomas with Wide-Field Fluorescence Molecular Endoscopy

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas (<3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas.

Data-Driven Prioritization and Review of Targets for Molecular-Based Theranostic Approaches in Pancreatic Cancer

Molecularly targeted therapeutic and imaging strategies directed at aberrant signaling pathways in pancreatic tumor cells may improve the poor outcome of pancreatic ductal adenocarcinoma (PDA). Therefore, relevant molecular targets need to be identified. Methods: We collected publicly available expression profiles of patient-derived normal pancreatic tissue (n = 77) and PDA samples (n = 103). Functional genomic messenger RNA profiling was applied to predict target upregulation on the protein level. We prioritized these targets based on current status of preclinical therapeutic and imaging evaluation in PDA. Results: We identified 213 significantly upregulated proteins in PDA compared with normal pancreatic tissue. We prioritized mucin-1, mesothelin, γ-glutamyltransferase 5, and cathepsin-E as the most interesting targets, because studies already demonstrated their potential for both therapeutic and imaging strategies in literature. Conclusion: This study can assist clinicians and drug developers in deciding which theranostic targets should be taken for further clinical evaluation in PDA.

653 Towards Instant Detection of (Pre)Malignant Lesions by Ultrasensitive Near-Infrared Fluorescence Endoscopy, Using Molecular Targeted Fluorescent Antibodies

Introduction and aim: Colonoscopy is considered to be the most sensitive tool to detect colorectal neoplasms, including small and flat lesions. Nevertheless, detection of such lesions is still incomplete, giving rise to interval cancers, particularly in patients with Lynch syndrome and other hereditary disorders. To improve the detection of (pre)malignant lesions nearinfrared endoscopy, guided bymolecular targeted fluorescent tracers, may hold great promise. Therefore, we developed a combined white-light and near-infrared (WLNI) endoscopic system, as well as two clinically approved fluorescent labeled antibodies, targeting vascular endothelial growth factor-A (VEGF-A) and epithelial growth factor receptor (EGFR). In the present study we investigated VEGF-A and EGFR expression in adenomas and CRC of Lynch patients, as potential targets for (pre)malignant detection. Subsequently we validated the newly developed WLNI endoscopic system, together with intravenously injected fluorescent anti-VEGF-A and anti-EGFR antibodies, in human CRC mouse models. Materials and Methods: VEGF-A and EGFR expression was analyzed by immunohistochemical staining of 34 CRC, 64 high-grade dysplastic (HGD) adenomas, and adjacent normal colon crypts of Lynch patients. The endoscopic system consisted of a custom made clinically approved WLNI camera, comprising a color camera and an ultra-sensitive camera for concurrent white-light and NIR fluorescence acquisition, attached to either a clinical fiberscope or a multi-modal fiber-optic bundle. The WLNI system was evaluated in vivo in CRC subcutaneous (sc) and intraperitoneal (ip) mouse models of bioluminescent CRC cell lines (HCT116-luc, HT29luc2), using bevacizumab-800CW (anti-VEGF-A) and cetuximab-800CW (anti-EGFR). Results: VEGF-A and EGFR were significantly overexpressed in 95% and 74% of HGD adenomas and in 100% and 85% of CRC, compared to adjacent normal crypts (resp. P,0.001/P,0.05 and P,0.05/P,0.001). Tumor-to-background ratio was high (3.2±0.9 for bevacizumab-800CW and 5.7±2.9 for cetuximab-800CW in the HCT116-luc sc model). WLNI endoscopy demonstrated excellent instant visualization of the sc and ip tumors (diameter ≥1 mm), with clear tumor boundaries and a low background fluorescence, demonstrating very high sensitivity and specificity of our WLNI endoscopic system. Conclusion: VEGF-A and EGFR are attractive targets for molecular targeted fluorescence endoscopy in Lynch patients based on their expression patterns. The newly developed WLNI endoscopic system using clinically approved molecular targeted fluorescent antibodies, enables instant visualization of very small tumor lesions in CRC mice models, with an excellent sensitivity and specificity. These results support clinical evaluation of WLNI endoscopy, in order to enhance early detection of colorectal (pre)malignancies and improve potentially outcome in patients.

Tyrosine kinase inhibitor induced growth factor receptor upregulation enhances the efficacy of near-infrared targeted photodynamic therapy in esophageal adenocarcinoma cell lines

Esophageal carcinoma (EC) is a global health problem, with disappointing 5-year survival rates of only 15–25%. Near-infrared targeted photodynamic therapy (NIR-tPDT) is a novel strategy in which cancer-targeted phototoxicity is able to selectively treat malignant cells. In this in vitro report we demonstrate the applicability of antibody-based NIR-tPDT in esophageal adenocarcinoma (EAC), using the phototoxic compounds cetuximab-IRDye700DX and trastuzumab-IRDye700DX, targeting respectively epidermal growth factor receptor 1 (EGFR) and 2 (HER2). Furthermore, we demonstrate that NIR-tPDT can be made more effective by tyrosine kinase inhibitor (TKI) induced growth receptor upregulation. Together, these results unveil a novel strategy for non-invasive EAC treatment, and by pretreatment-induced receptor upregulation its future clinical application may be optimized.