The purpose of this study was to determine the safety and feasibility of fluorescence molecular imaging using a commercially available system and intravenous injection of fluorescently labeled bevacizumab (bevacizumab-800CW) to visualize bevacizumab distribution in patients with neovascular age-related macular degeneration (nAMD)s. Methods: Twelve patients with active nAMD aged 60 y or older, who were either on anti-vascular endothelial growth factor (VEGF) therapy or were anti-VEGF therapy naïve, received an intravenous injection of 4.5 mg (n = 3) or 15 mg (n = 9) of bevacizumab-800CW. This clinical trial was divided into 2 parts. An interim analysis was performed after the inclusion of 3 patients per dose group (study part 1) to determine the more optimal dose. Then 6 additional patients were included with the optimal dose in study part 2. All patients underwent standard clinical imaging, including fundus photography, optical coherence tomography, optical coherence tomography angiography, and fluorescein angiography. Fluorescence imaging was performed 1 min, 60 min, and 3-4 d after bevacizumab-800CW injection. Results: Bevacizumab-800CW injections were safe and well tolerated. One minute after injection, only the 15-mg group demonstrated a significantly higher contrast-to-noise ratio (median, 6.32) in vessels compared with baseline (median, -4.44; P = 0.0342). This, combined with the clear visual uptake after 3-4 d, supported 15 mg as the preferred dose. Contrast-to-noise ratio values inside the macula of patients receiving 15 mg of bevacizumab-800CW (n = 8) were significantly higher after 3-4 d (median, 4.45) compared with baseline (median, 0.19; P = 0.0078). Conclusion: Targeted fluorescent tracers such as bevacizumab-800CW can visualize VEGF expression, providing important insights into nAMD, and can pave the way toward personalized treatments and targeted drug development.

Representativeness of Study Participants

Overview of the absorption and scattering parameters used for tissue characterisation

INTRODUCTION:Achieving gross total resection and endocrine remission in pituitary neuroendocrine tumours (PitNET) can be challenging, especially in PitNETs with cavernous sinus (CS) invasion, defined as a Knosp grade of 3 or 4. A potential target to identify PitNET tissue is vascular endothelial growth factor A (VEGF-A), which expression is known to be significantly higher in PitNETs with CS invasion. METHODS AND ANALYSIS:The aim of this non-randomised, non-blinded, single centre, feasibility and dose-finding phase 1 trial is to determine the feasibility of intraoperative fluorescence imaging detection of PitNET tissue during endoscopic transsphenoidal surgery using the VEGF-A targeting optical agent bevacizumab-800CW (4, 5, 10 or 25 mg). Nine to fifteen patients with a PitNET with a Knosp grade of 3 or 4 will be included. Secondary objectives are: (1) To identify the optimal tracer dose for imaging of PitNET tissue during transsphenoidal surgery for further development in a phase 2 fluorescence molecular endoscopy trial. (2) To quantify fluorescence intensity in vivo and ex vivo with multidiameter single-fibre reflectance, single-fibre fluorescence (MDSFR/SFF) spectroscopy. (3) To correlate and validate both the in vivo and ex vivo measured fluorescence signals with histopathological analysis and immunohistochemical staining. (4) To assess the (sub)cellular location of bevacizumab-800CW by ex vivo fluorescence microscopy. Intraoperative, three imaging moments are defined to detect the fluorescent signal. The tumour-to-background ratios are defined by intraoperative fluorescence in vivo measurements including MDSFR/SFF spectroscopy data and by ex vivo back-table fluorescence imaging. After inclusion of three patients in each dose group, an interim analysis will be performed to define the optimal dose. ETHICS AND DISSEMINATION:Approval was obtained from the Medical Ethics Review Board of the University Medical Centre Groningen. Results will be disseminated through national and international journals. The participants and relevant patient support groups will be informed about the results. TRIAL REGISTRATION NUMBER:NCT04212793.

Early detection of (pre)malignant esophageal lesions is critical to improve esophageal cancer morbidity and mortality rates. In patients with advanced esophageal adenocarcinoma (EAC) who undergo neoadjuvant chemoradiation therapy, the efficacy of therapy could be optimized and unnecessary surgery prevented by the reliable assessment of residual tumors after therapy. Optical coherence tomography (OCT) provides structural images at a (sub)-cellular level and has the potential to visualize morphological changes in tissue. However, OCT lacks molecular imaging contrast, a feature that enables the study of biological processes at a cellular level and can enhance esophageal cancer diagnostic accuracy. We combined OCT with near-infrared fluorescence molecular imaging using fluorescently labelled antibodies (immuno-OCT). The main goal of this proof of principle study is to investigate the feasibility of immuno-OCT for esophageal cancer imaging. We aim to assess whether the sensitivity of our immuno-OCT device is sufficient to detect the tracer uptake using an imaging dose (similar to 100 times smaller than a dose with therapeutic effects) of a targeted fluorescent agent. The feasibility of immuno-OCT was demonstrated ex-vivo on dysplastic lesions resected from Barrett's patients and on esophageal specimens resected from patients with advanced EAC, who were respectively topically and intravenously administrated with the tracer bevacizumab-800CW. The detection sensitivity of our system (0.3 nM) is sufficient to detect increased tracer uptake with micrometer resolution using an imaging dose of labelled antibodies. Moreover, the absence of layered structures that are typical of normal esophageal tissue observed in OCT images of dysplastic/malignant esophageal lesions may further aid their detection. Based on our preliminary results, immuno-OCT could improve the detection of dysplastic esophageal lesions.

AbstractPurpose: The ability to identify residual tumor tissues in patients with locally advanced esophageal cancer following neoadjuvant chemoradiotherapy (nCRT) is essential for monitoring the treatment response. Using the fluorescent tracer bevacizumab-800CW, we evaluated whether ultrasound-guided quantitative fluorescent molecular endoscopy (US-qFME), which combines quantitative fluorescence molecular endoscopy (qFME) with ultrasound-guided needle biopsy/single-fiber fluorescence (USNB/SFF), can be used to identify residual tumor tissues in patients following nCRT. Experimental Design: Twenty patients received an additional endoscopy procedure the day before surgery. qFME was performed at the primary tumor site (PTS) and in healthy tissue to first establish the optimal tracer dose. USNB/SFF was then used to measure intrinsic fluorescence in the deeper PTS layers and lymph nodes (LN) suspected for metastasis. Finally, the intrinsic fluorescence and the tissue optical properties—specifically, the absorption and reduced scattering coefficients—were combined into a new parameter called omega. Results: First, a 25-mg bevacizumab-800CW dose allowed for clear differentiation between the PTS and healthy tissue, with a target-to-background ratio (TBR) of 2.98 (IQR, 1.86–3.03). Moreover, we found a clear difference between the deeper esophageal PTS layers and suspected LN compared to healthy tissues, with TBR values of 2.18 and 2.17, respectively. Finally, our new parameter, omega, further improved the ability to differentiate between the PTS and healthy tissue. Conclusions: Combining bevacizumab-800CW with US-qFME may serve as a viable strategy for monitoring the response to nCRT in esophageal cancer and may help stratify patients regarding active surveillance versus surgery.

Distribution of semi-quantitative VEGF expression

Aims/Purpose: With this phase 1 study, we aim to gain insight into the feasibility of using bevacizumab‐800CW to visualize the upregulation of VEGF‐A in AMD patients for treatment optimization. Methods: First, six wet AMD patients who are currently undergoing treatment with anti‐VEGF agents were included to evaluate the optimal tracer dose and received either 4.5 or 15 mg bevacizumab‐800CW. For the ongoing analysis of targeted tracer accumulation, three additional on‐therapy and three naïve patients will be included with the optimal dose of 15 mg. Fluorescence images (Heidelberg Spectralis) were taken before, up to 60 min and 3–4 days after intravenous injection of bevacizumab‐800CW. The target‐to‐background ratios (TBR) were calculated and compared between dose groups to evaluate the optimal dose. To investigate targeted tracer uptake, images of different time points are normalized and aligned, and a fluorescence intensity line is plotted over the active lesion to locate interesting areas for further quantification. Results: The preliminary results showed a clear inflow of the tracer into the eye vasculature after administration of 15 mg bevacizumab‐800CW. The fluorescence remains visible up to 1 h after tracer administration. Visual image comparison and TBR calculation in the first six patients showed that 15 mg bevacizumab‐800CW was the optimal tracer dose. In patients who received 15 mg bevacizumab‐800CW, accumulation of fluorescence signal in a smaller area within the active lesion was seen after 3–4 days. Conclusions: For the first time, we show that bevacizumab‐800CW (4.5 mg/15 mg) can be imaged in AMD patients using a commercially available system. An increase in signal intensity 3–4 days after bevacizumab‐800CW injection predicts tracer accumulation in the diseased area indicating ongoing active disease. VEGF‐A imaging can act as a proof of concept and pave the way for a broad application to track the performance of drugs by fluorescence molecular imaging.

(1) Introduction: Near-infrared fluorescence (NIRF) combined with tumour-targeted tracers, such as bevacizumab-800CW, could aid surgical decision-making. This study explored the use of IRDye800CW, conjugated to bevacizumab, with four commercially available NIRF laparoscopes optimised for indocyanine green (ICG). (2) Methods: A (lymph node) phantom was made from a calibration device for NIRF and tissue-mimicking material. Serial dilutions of bevacizumab-800CW were made and ICG functioned as a reference. System settings, working distance, and thickness of tissue-mimicking material were varied to assess visibility of the fluorescence signal and tissue penetration. Tests were performed with four laparoscopes: VISERA ELITE II, Olympus; IMAGE1 S™ 4U Rubina, KARL STORZ; ENDOCAM Logic 4K platform, Richard Wolf; da Vinci Xi, Intuitive Surgical. (3) Results: The lowest visible bevacizumab-800CW concentration ranged between 13–850 nM (8–512 times diluted stock solution) for all laparoscopes, but the tracer was not visible through 0.8 cm of tissue in all systems. In contrast, ICG was still visible at a concentration of 0.4 nM (16,384 times diluted) and through 1.6–2.4 cm of tissue. Visibility and tissue penetration generally improved with a reduced working distance and manually adjusted system settings. (4) Conclusion: Depending on the application, bevacizumab-800CW might be sufficiently visible with current laparoscopes, but optimisation would widen applicability of tumour-targeted IRDye800CW tracers.

Fluorescence molecular endoscopy has the potential to improve lesion identification, evaluate therapy response and image biological distribution of medications. The majority of research is performed in gastroenterology to identify polyps and (pre-) cancerous lesions or to evaluate therapy response after neoadjuvant chemoradiotherapy.1–8 However, for extensive use of this technique within the clinical field, standardization and quantification of the fluorescence signal is essential. Quantitative fluorescence molecular endoscopy (qFME) combines the best of two worlds. Wide-field fluorescence imaging allows for a general overview of the fluorescence signal and identifies regions of interest. This thesis investigates the optimalization and implementation of quantitative fluorescence molecular endoscopy into standard clinical care, with a focus on the three aforementioned clinical challenges. The first part of this thesis focusses on standardization of both wide-field fluorescence endoscopy and reflectance spectroscopy system, whereas the second part discusses the clinical application of quantitative fluorescence molecular endoscopy in multiple clinical fields.

Universitair Medisch Centrum Groningen, Netherlands; Erasmus MC, Netherlands.

Significance: The combination of reflectance and fluorescence spectroscopy allows the determination of tissue optical properties and the calculation of the intrinsic fluorescence in vivo. These parameters can discriminate between tissues and may allow the discrimination of malignant from benign tissue. While this approach has significant clinical potential, the lack of standardization and quality assessment prevents the upscaling of research. Aim: Investigate which factors influence device calibration and tissue optical property determination. Improve system quality assessment and allow upscaling of the clinical research using multidiameter single fiber reflectance/singe fiber fluorescence spectroscopy. Approach: Two studies, one phantom based on uniform calibrations and skin measurements and a clinical study including clinical calibrations. The first validates the effect of factors under identical conditions and the effect of calibration quality on the optical property determination of skin. The second shows the effect of different system configurations and the performance of the system and probe over an extended period. Results: Phantom calibrations showed stability over a period of 20 weeks except for a 16-week-old intralipid phantom which showed a significant difference (at least p = 0.0032) for all five probes evaluated. For clinical calibrations, only the fiber tree had a significant influence (probe 4: p < 0.000001 and probe 5: p = 0.00038) on the calibration quality. Interestingly, no degradation of probe performance was detected over a period of 21 months despite the exposure to stress during clinical measurements. Calibration quality affected mu s' and the power law scattering exponent, but the degree of the influence was different per fiber. Conclusions: Intralipid phantom quality and fiber tree performance are the main factors influencing the calibration quality. Probe and user performance did not show any effect, which makes the upscaling of research to multicenter trials easier. A high-quality assessment procedure should be implemented to track changes during clinical trials. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License.

Differentiation of pituitary neuroendocrine tumor (PitNET) tissue from surrounding normal tissue during surgery is challenging. A number of fluorescent agents is available for visualization of tissue discrepancy, with the potential of improving total tumor resection. This review evaluates the availability, clinical and technical applicability of the various fluorescent agents within the field of pituitary surgery. According to PRISMA guidelines, a systematic review was performed to identify reports describing results of in vivo application of fluorescent agents. In this review, 15 publications were included. Sodium Fluorescein (FNa) was considered in two studies. The first study reported noticeable fluorescence in adenoma tissue, the second demonstrated the strongest fluorescence in non-functioning pituitary adenomas. 5-Aminolevulinic acid (5-ALA) was investigated in three studies. One study compared laser-based optical biopsy system (OBS) with photo-diagnostic filter (PD) and found that the OBS was able to detect all microadenomas, even when MRI was negative. The second study retrospectively analyzed twelve pituitary adenomas and found only one positive for fluorescence. The third investigated fifteen pituitary adenomas of which one displayed vague fluorescence. Indocyanine green (ICG) was researched in four studies with variable results. Second-Window ICG yielded no significant difference between functioning and non-functioning adenomas in one study, while a second study displayed 4 times higher fluorescence in tumor tissue than in normal tissue. In three studies, OTL38 showed potential in non-functioning pituitary adenomas. At present, evidence for fluorescent agents to benefit total resection of PitNETs is lacking. OTL38 can potentially serve as a selective fluorescent agent in non-functioning pituitary adenomas in the near future.

This study aimed to determine the ability of single fiber reflectance (SFR) spectroscopy incorporated in endoscopic ultrasound fine needle biopsy (EUS-FNB) procedures in the pancreas to distinguish benign and malignant pancreatic tissue in patient with pancreatic masses suspected for malignancy. Methods: This study was designed as a prospective observational single center study and included consecutive adult patients, who were scheduled for EUS-FNB of a solid pancreatic mass suspected for pancreatic ductal adenocarcinoma (PDAC). In total, seven optical parameters, derived from the absorption acquired spectra, were analyzed: blood volume fraction (BVF), microvascular saturation, average vessel diameter, bilirubin concentration (BIL), Mie amplitude, Mie slope and Rayleigh amplitude. Results: Forty-five patients with a suspicious pancreatic lesion undergoing EUS-FNB were included, of which most of the patients (N=34) were ultimately diagnosed with PDAC. Finally, 27 out of 45 (60.0%) patients were used for the final analysis of the optical parameters. The median (IQR) BVF differed significantly in benign compared to malignant tissue (0.86 [0.30-2.03] and 4.49 [1.28-15.47]; p=0.046). Combining BVF and BIL to a new parameter (θ) improved the discrimination between PDAC and benign pancreatic tissue (p=0.026). The area under the curve of θ was 0.84, resulting in a 92.8%, 75.0%, 97.5%, 50.0% and 91.3% sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy for detection of PDAC. Conclusion: Differentiation between PDAC and benign pancreatic tissue using SFR spectroscopy during EUS-FNB procedures is promising. Future work should focus on comparing the diagnostic performance combining SFR spectroscopy with EUS-FNB and EUS-FNB alone.

SIGNIFICANCE:Near-infrared fluorescence molecular endoscopy (NIR-FME) is an innovative technique allowing for in vivo visualization of molecular processes in hollow organs. Despite its potential for clinical translation, NIR-FME still faces challenges, for example, the lack of consensus in performing quality control and standardization of procedures and systems. This may hamper the clinical approval of the technology by authorities and its acceptance by endoscopists. Until now, several clinical trials using NIR-FME have been performed. However, most of these trials had different study designs, making comparison difficult. AIM:We describe the need for standardization in NIR-FME, provide a pathway for setting up a standardized clinical study, and describe future perspectives for NIR-FME. Body: Standardization is challenging due to many parameters. Invariable parameters refer to the hardware specifications. Variable parameters refer to movement or tissue optical properties. Phantoms can be of aid when defining the influence of these variables or when standardizing a procedure. CONCLUSION:There is a need for standardization in NIR-FME and hurdles still need to be overcome before a widespread clinical implementation of NIR-FME can be realized. When these hurdles are overcome, clinical outcomes can be compared and systems can be benchmarked, enabling clinical implementation.

This study investigated the effect of two calibrations on physiological parameters. Pre-calibration significantly influences the scattering parameters. Post-calibration improves the fitting, leading to a better recovery of physiological parameters.

Mutations in USH2A , encoding usherin, are the most common cause of syndromic and non-syndromic retinitis pigmentosa (RP). The two founder mutations in exon 13 (c.2299delG and c.2276G>T) collectively account for ~ 34% of USH2A -associated RP cases. Skipping of exon 13 from the USH2A transcript during pre-mRNA splicing presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in larvae of the previously published ush2a exon 13 zebrafish mutant resulted in the production of usherinΔexon13 and completely restored retinal function. RNA antisense oligonucleotides were investigated for their potential to specifically induce human USH2A exon 13 skipping. Lead candidate QR-421a induced dose-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from a patient homozygous for the USH2A c.2299delG mutation. Intravitreal delivery of QR-421a in non-human primates showed that QR-421a penetrates the retinal outer nuclear layer and induces detectable levels of exon 13 skipping until at least 3 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment for RP caused by mutations in exon 13 of the USH2A gene.

Aims Currently, patients diagnosed with locally advanced esophageal adenocarcinoma receive neoadjuvant chemoradiotherapy (nCRT) followed by esophagectomy independent of the patients’ response to nCRT. Roughly 16-43 % of esophageal cancer patients will achieve a pathological complete response (pCR) after nCRT1. Current imaging devices cannot adequately distinguish pCR from pathological partial response (pPR) before esophagectomy2. There is a need for a reliable pre-operative method which can precisely distinguish pCR from pPR and prevent (possibly) unnecessary surgery.

Esophageal cancer (EC) is affecting more than 450,000 people worldwide and is the 6th leading cause of cancer-related deaths. The poor EC survival is attributed to the insufficient methods for premalignant lesion detection and therefore there is a great need for new endoscopic techniques that can visualize early-stage lesions. In 2017 our group published promising results using near infrared fluorescent molecular endoscopy (NIR-FME) against vascular endothelial growth factor A (VEGF-A) to improve early (pre)malignant lesion detection. Here we show the preliminary results of the phase II study.

Aims Esophageal cancer (EC) is affecting more than 450,000 people worldwide and is the 6th leading cause of cancer-related deaths. The poor EC survival is attributed to the insufficient methods for premalignant lesion detection and therefore there is a great need for new endoscopic techniques that can visualize early stage lesions. In this phase II study we aim to evaluate the sensitivity and specificity of the tracer bevacizumab-800CW in combination with near infrared fluorescent molecular endoscopy (NIR-FME) for detecting (pre)malignant lesions in patients with Barrett’s esophagus (BE).