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-guided 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 a 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 (SSA/P), 70 sporadic high-grade dysplastic (HGD) adenomas, 19 hyperplastic polyps (HP) and tissue derived from patients with Lynch syndrome (LS): 78 low-grade dysplastic (LGD) adenomas, 57 HGD adenomas and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116luc tumors received intravenously 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 one freshly resected human colon specimen and followed by an ex vivo molecular-guided 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 to the adjacent normal colon crypts. During ex vivo molecular-guided 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-guided fluorescence endoscopy as it showed a high protein expression, especially in SSA/P and LS. We demonstrate the feasibility to visualize small tumors 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.

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.

Quantitative fluorescence endoscopy (QFE) is a new technique that can visualise and quantify fluorescently tagged tumour tissue. In 25 patients with locally advanced rectal cancer (LARC), we evaluated QFE targeting vascular endothelial growth factor A (VEGFA) to detect residual tumour after neoadjuvant chemoradiotherapy (nCRT). QFE detected significantly higher fluorescence in tumour compared with normal rectal tissue and fibrosis, and improved prediction of final pathology results in 16% of patients compared with standard MRI and white-light endoscopy. QFE is a promising technique to aid clinical response assessment in patients with LARC and warrants further validation in larger clinical trials. ClinicalTrials.gov (NCT01972373).

Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2–3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence-guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 ± 2.5 (mean ± SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM.

Nearly one third of patients undergoing neoadjuvant chemoradiotherapy (nCRT) for locally advanced esophageal cancer have a pathologic complete response (pCR) of the primary tumor upon histopathological evaluation of the resection specimen. The primary aim of this study is to develop a model that predicts the probability of pCR to nCRT in esophageal cancer, based on diffusion-weighted magnetic resonance imaging (DW-MRI), dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and 18F-fluorodeoxyglucose positron emission tomography with computed tomography (18F-FDG PET-CT). Accurate response prediction could lead to a patient-tailored approach with omission of surgery in the future in case of predicted pCR or additional neoadjuvant treatment in case of non-pCR. The PRIDE study is a prospective, single arm, observational multicenter study designed to develop a multimodal prediction model for histopathological response to nCRT for esophageal cancer. A total of 200 patients with locally advanced esophageal cancer - of which at least 130 patients with adenocarcinoma and at least 61 patients with squamous cell carcinoma - scheduled to receive nCRT followed by esophagectomy will be included. The primary modalities to be incorporated in the prediction model are quantitative parameters derived from MRI and 18F-FDG PET-CT scans, which will be acquired at fixed intervals before, during and after nCRT. Secondary modalities include blood samples for analysis of the presence of circulating tumor DNA (ctDNA) at 3 time-points (before, during and after nCRT), and an endoscopy with (random) bite-on-bite biopsies of the primary tumor site and other suspected lesions in the esophagus as well as an endoscopic ultrasonography (EUS) with fine needle aspiration of suspected lymph nodes after finishing nCRT. The main study endpoint is the performance of the model for pCR prediction. Secondary endpoints include progression-free and overall survival. If the multimodal PRIDE concept provides high predictive performance for pCR, the results of this study will play an important role in accurate identification of esophageal cancer patients with a pCR to nCRT. These patients might benefit from a patient-tailored approach with omission of surgery in the future. Vice versa, patients with non-pCR might benefit from additional neoadjuvant treatment, or ineffective therapy could be stopped. The article reports on a health care intervention on human participants and was prospectively registered on March 22, 2018 under ClinicalTrials.gov Identifier: NCT03474341 .

Endoscopic techniques such as High-definition and optical-chromoendoscopy have had enormous impact on endoscopy practice. Since these techniques allow assessment of most subtle morphological mucosal abnormalities, further improvements in endoscopic practice lay in increasing the detection efficacy of endoscopists. Several new developments could assist in this. First, web based training tools could improve the skills of the endoscopist for enhancing the detection and classification of lesions. Secondly, incorporation of computer aided detection will be the next step to raise endoscopic quality of the captured data. These systems will aid the endoscopist in interpreting the increasing amount of visual information in endoscopic images providing real-time objective second reading. In addition, developments in the field of molecular imaging open opportunities to add functional imaging data, visualizing biological parameters, of the gastrointestinal tract to white-light morphology imaging. For the successful implementation of abovementioned techniques, a true multi-disciplinary approach is of vital importance.