Performance Assessment and Quality Control of Fluorescence Molecular Endoscopy with a Multi-Parametric Rigid Standard.

Fluorescence molecular endoscopy (FME) is emerging as a "red-flag" technique with potential to deliver earlier, faster, and more personalized detection of disease in the gastrointestinal tract, including cancer, and to gain insights into novel drug distribution, dose finding, and response prediction. However, to date, the performance of FME systems is assessed mainly by endoscopists during a procedure, leading to arbitrary, potentially biased, and heavily subjective assessment. This approach significantly affects the repeatability of the procedures and the interpretation or comparison of the acquired data, representing a major bottleneck towards the clinical translation of the technology. Herein, we propose a robust methodology for FME performance assessment and quality control that is based on a novel multi-parametric rigid standard. This standard enables the characterization of an FME system's sensitivity through a single acquisition, performance comparison of multiple systems, and, for the first time, quality control of a system as a function of time and number of usages. We show the photostability of the standard experimentally and demonstrate how it can be used to characterize the performance of an FME system. Moreover, we showcase how the standard can be employed for quality control of a system. In this study, we find that the use of composite fluorescence standards before endoscopic procedures can ensure that an FME system meets the performance criteria and that components prone to performance degradation are replaced in time, avoiding disruption of clinical endoscopy logistics. This will help overcome a major barrier for the translation of FME into the clinics.

Fluorescently labelled vedolizumab to visualise drug distribution and mucosal target cells in inflammatory bowel disease.

OBJECTIVE: Improving patient selection and development of biological therapies such as vedolizumab in IBD requires a thorough understanding of the mechanism of action and target binding, thereby providing individualised treatment strategies. We aimed to visualise the macroscopic and microscopic distribution of intravenous injected fluorescently labelled vedolizumab, vedo-800CW, and identify its target cells using fluorescence molecular imaging (FMI). DESIGN: Forty three FMI procedures were performed, which consisted of macroscopic in vivo assessment during endoscopy, followed by macroscopic and microscopic ex vivo imaging. In phase A, patients received an intravenous dose of 4.5 mg, 15 mg vedo-800CW or no tracer prior to endoscopy. In phase B, patients received 15 mg vedo-800CW preceded by an unlabelled (sub)therapeutic dose of vedolizumab. RESULTS: FMI quantification showed a dose-dependent increase in vedo-800CW fluorescence intensity in inflamed tissues, with 15 mg (153.7 au (132.3-163.7)) as the most suitable tracer dose compared with 4.5 mg (55.3 au (33.6-78.2)) (p=0.0002). Moreover, the fluorescence signal decreased by 61% when vedo-800CW was administered after a therapeutic dose of unlabelled vedolizumab, suggesting target saturation in the inflamed tissue. Fluorescence microscopy and immunostaining showed that vedolizumab penetrated the inflamed mucosa and was associated with several immune cell types, most prominently with plasma cells. CONCLUSION: These results indicate the potential of FMI to determine the local distribution of drugs in the inflamed target tissue and identify drug target cells, providing new insights into targeted agents for their use in IBD. TRIAL REGISTRATION NUMBER: NCT04112212.

Detection of Early Esophageal Neoplastic Barrett Lesions with Quantified Fluorescence Molecular Endoscopy Using Cetuximab-800CW.

Esophageal adenocarcinoma causes 6% of cancer-related deaths worldwide. Near-infrared fluorescence molecular endoscopy (NIR-FME) uses a tracer that targets overexpressed proteins. In this study, we aimed to investigate the feasibility of an epidermal growth factor receptor (EGFR)-targeted tracer, cetuximab-800CW, to improve detection of early-stage esophageal adenocarcinoma. Methods: We validated EGFR expression in 73 esophageal tissue sections. Subsequently, we topically administered cetuximab-800CW and performed high-definition white-light endoscopy (HD-WLE), narrow-band imaging, and NIR-FME in 15 patients with Barrett esophagus (BE). Intrinsic fluorescence values were quantified using multidiameter single-fiber reflectance and single-fiber fluorescence spectroscopy. Back-table imaging, histopathologic examination, and EGFR immunohistochemistry on biopsy samples collected during NIR-FME procedures were performed and compared with in vivo imaging results. Results: Immunohistochemical preanalysis showed high EGFR expression in 67% of dysplastic tissue sections. NIR-FME visualized all 12 HD-WLE-visible lesions and 5 HD-WLE-invisible dysplastic lesions, with increased fluorescence signal in visible dysplastic BE lesions compared with nondysplastic BE as shown by multidiameter single-fiber reflectance/single-fiber fluorescence, reflecting a target-to-background ratio of 1.5. Invisible dysplastic lesions also showed increased fluorescence, with a target-to-background ratio of 1.67. Immunohistochemistry analysis showed EGFR overexpression in 16 of 17 (94%) dysplastic BE lesions, which all showed fluorescence signal. Conclusion: This study has shown that NIR-FME using cetuximab-800CW can improve detection of dysplastic lesions missed by HD-WLE and narrow-band imaging.

Today's Mistakes and Tomorrow's Wisdom in Endoscopic Imaging of Barrett's Esophagus.

Background: Esophageal adenocarcinoma (EAC) is one of the main causes of cancer-related deaths worldwide and its incidence is rising. Barrett's esophagus (BE) can develop low- and high-grade dysplasia which can progress to EAC overtime. The golden standard to detect dysplastic BE (DBE) or EAC is surveillance with high-definition white-light endoscopy (HD-WLE) and random biopsies according to the Seattle protocol. However, this method is time-consuming and associated with a remarkable miss rate. Therefore, there is great need for the development of novel reliable techniques to optimize surveillance strategies and improve detection rates. Summary: Optical chromoendoscopy (OC) techniques like narrow-band imaging have shown improved detection of DBE and EAC compared to HD-WLE and random biopsies. Most recent OC techniques, including the iSCAN optical enhancement system and linked color imaging, showed improved characterization of DBE and EAC retrospectively. Fluorescence molecular endoscopy (FME) presented promising results to highlight DBE and EAC. Moreover, with the establishment of well-performing delineation computer-aided detection (CAD) algorithms and the first real-time CAD system for EAC, we expect clinical application of CAD in the near future. Key Messages: Despite impressive progress made in the development of advanced endoscopic techniques, combined HD-WLE/OC followed by random biopsies remains the golden standard for BE surveillance. Surveillance depends on appropriate mucosal cleansing, sufficient inspection time, and competence of the performing gastroenterologist to improve detection of EAC. In addition, to facilitate the clinical implementation of advanced endoscopic techniques, multicenter prospective clinical studies are demanded for OC and FME. Meanwhile, further optimization of CAD algorithms, the education of gastroenterologists, and analysis of the interaction between the clinician and the computer should be performed.

Mucosal Eosinophil Abundance in Non-Inflamed Colonic Tissue Is Associated with Response to Vedolizumab Induction Therapy in Inflammatory Bowel Disease.

Vedolizumab is used as a treatment for patients with inflammatory bowel disease (IBD), but induction therapy leads to clinical response and remission in approximately 55% and 30% of patients with IBD, respectively. In this study, we aimed to explore the predictive value of mucosal eosinophils and serum eotaxin-1 regarding response to vedolizumab induction therapy. Eighty-four (84) patients with IBD (37 Crohn's disease [CD], 47 ulcerative colitis [UC]) were included. For 24 patients with IBD, histopathology was assessed for eosinophil counts in non-inflamed colonic tissue prior to vedolizumab treatment. For 64 patients with IBD, serum eotaxin-1 levels were quantified prior to (baseline) and during vedolizumab treatment. Serum samples of 100 patients with IBD (34 CD, 66 UC) from the GEMINI 1 and 2 trials were used for external validation. Baseline mucosal eosinophil numbers in non-inflamed colonic tissue were significantly higher in responders to vedolizumab induction therapy when compared to primary non-responders (69 [34−138] vs. 24 [18−28] eosinophils/high-power field, respectively, p < 0.01). Baseline serum eotaxin-1 levels in the discovery cohort were significantly elevated in responders, compared to primary non-responders (0.33 [0.23−0.44] vs. 0.20 [0.16−0.29] ng/mL, p < 0.01). Prediction models based on mucosal eosinophil counts and serum eotaxin-1 showed an area under the curve (AUC) of 0.90 and 0.79, respectively. However, the predictive capacity of baseline serum eotaxin-1 levels could not be validated in the GEMINI cohort. Mucosal eosinophil abundance in non-inflamed colonic tissue was associated with response to vedolizumab induction therapy in patients with IBD. Future studies are warranted to further validate the potential value of mucosal eosinophils and serum eotaxin-1 as biomarkers for response to vedolizumab therapy.

Validation of Novel Molecular Imaging Targets Identified by Functional Genomic mRNA Profiling to Detect Dysplasia in Barrett's Esophagus.

Barrett's esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKCι, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKCι, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings.

Endoscopic imaging in inflammatory bowel disease: current developments and emerging strategies.

INTRODUCTION: Developments in enhanced and magnified endoscopy have signified major advances in endoscopic imaging of ileocolonic pathology in inflammatory bowel disease (IBD). Artificial intelligence is increasingly being used to augment the benefits of these advanced techniques. Nevertheless, treatment of IBD patients is frustrated by high rates of non-response to therapy, while delayed detection and failures to detect neoplastic lesions impede successful surveillance. A possible solution is offered by molecular imaging, which adds functional imaging data to mucosal morphology assessment through visualizing biological parameters. Other label-free modalities enable visualization beyond the mucosal surface without the need of tracers. AREAS COVERED: A literature search up to May 2020 was conducted in PubMed/MEDLINE in order to find relevant articles that involve the (pre-)clinical application of high-definition white light endoscopy, chromoendoscopy, artificial intelligence, confocal laser endomicroscopy, endocytoscopy, molecular imaging, optical coherence tomography, and Raman spectroscopy in IBD. EXPERT OPINION: Enhanced and magnified endoscopy have enabled an improved assessment of the ileocolonic mucosa. Implementing molecular imaging in endoscopy could overcome the remaining clinical challenges by giving practitioners a real-time in vivo view of targeted biomarkers. Label-free modalities could help optimize the endoscopic assessment of mucosal healing and dysplasia detection in IBD patients.

A comparative analysis of tioguanine versus low-dose thiopurines combined with allopurinol in inflammatory bowel disease patients.

BACKGROUND: Both tioguanine and low-dose thiopurines combined with allopurinol (LDTA) can be considered for the treatment of inflammatory bowel disease (IBD) when conventional thiopurines fail due to adverse events. AIM: To compare the safety of tioguanine and LDTA in IBD patients. METHODS: Inflammatory bowel disease patients who failed conventional thiopurines due to adverse events and initiated LDTA in standard care were identified in the prospective ICC Registry. IBD patients who failed conventional thiopurines due to adverse events and initiated tioguanine were enrolled in three university hospitals. Patients on concomitant biologicals were excluded. The primary outcome was discontinuation of therapy due to adverse events. Secondary outcomes included: safety outcomes and surgery-, biological- and corticosteroid-free clinical remission (physician global assessment = 0) after 104 weeks. Both multiple logistic regression and propensity score matching were used to correct for confounders. RESULTS: In total, 182 IBD patients treated with tioguanine (n = 94) or LDTA (n = 88) were included with a median follow-up of 104 weeks (IQR 91-104). Of these, 19% (tioguanine: 20%, LDTA: 18%) of patients discontinued therapy due to adverse events. After adjusting for confounders, there were no differences in terms of discontinuation rate due to adverse events (OR 0.50, 95% CI 0.15-1.68, P = 0.26), adverse events (OR 0.89, 95% CI 0.44-1.81, P = 0.75), infections (OR 1.05, 95% CI 0.40-2.73, P = 0.93), hospitalisations (OR 2.00, 95% CI 0.64-6.23, P = 0.23) or clinical remission (OR 0.74, 95%CI 0.33-1.68, P = 0.48). All results are comparable with the propensity score matched cohort. CONCLUSION: Nineteen percent of IBD patients with prior failure to conventional thiopurines due to adverse events discontinued therapy with tioguanine or LDTA due to adverse events. Either therapy may be considered before escalating to biological therapy.

Riboflavin Supplementation in Patients with Crohn's Disease [the RISE-UP study].

BACKGROUND AND AIMS: Crohn's disease [CD] is characterised by chronic intestinal inflammation and dysbiosis in the gut. Riboflavin [vitamin B2] has anti-inflammatory, antioxidant and microbiome-modulatory properties. Here, we analysed the effect of riboflavin on oxidative stress, markers of inflammation, clinical symptoms, and faecal microbiome in patients with CD. METHODS: In this prospective clinical intervention study, patients received 100 mg riboflavin [DSM, Nutritional Products Ltd] daily for 3 weeks. Clinical disease activity [Harvey-Bradshaw Index: HBI], serum biomarkers of inflammation and redox status [plasma free thiols], and faecal microbiome taxonomical composition and functionality [fluorescent in situ hybridisation: FISH; and metagenomic shotgun sequencing: MGS], were analysed before and after riboflavin intervention. RESULTS: In total, 70 patients with CD with varying disease activity were included. Riboflavin supplementation significantly decreased serum levels of inflammatory markers. In patients with low faecal calprotectin [FC] levels, IL-2 decreased, and in patients with high FC levels, C-reactive protein [CRP] was reduced and free thiols significantly increased after supplementation. Moreover, HBI was significantly decreased by riboflavin supplementation. Riboflavin supplementation led to decreased Enterobacteriaceae in patients with low FC levels as determined by FISH; however, MGS analysis showed no effects on diversity, taxonomy, or metabolic pathways of the faecal microbiome. CONCLUSIONS: Three weeks of riboflavin supplementation resulted in a reduction in systemic oxidative stress, mixed anti-inflammatory effects, and a reduction in clinical symptoms [HBI]. FISH analysis showed decreased Enterobacteriaceae in patients with CD with low FC levels, though this was not observed in MGS analysis. Our data demonstrate that riboflavin supplementation has a number of anti-inflammatory and anti-oxidant effects in CD.

A Combined Set of Four Serum Inflammatory Biomarkers Reliably Predicts Endoscopic Disease Activity in Inflammatory Bowel Disease.

Introduction: Blood C-reactive protein (CRP) and fecal calprotectin levels are routinely measured as surrogate markers of disease activity in Inflammatory Bowel Disease (IBD), but often do not correlate well with the degree of mucosal inflammation in the intestine as established by endoscopy. Therefore, novel predictive biomarkers are urgently needed that better reflect mucosal disease activity in IBD. The aim of this study was to identify a combination of serum inflammatory biomarkers predictive for endoscopic disease activity. Methods: Serum concentrations of 10 inflammatory biomarkers were analyzed in 118 IBD patients [64 Crohn's disease (CD), 54 ulcerative colitis (UC)] and 20 healthy controls. In a subset of 71 IBD patients, endoscopic disease activity was established. Non-parametric ROC estimation with bootstrap inference was used to establish the best combination of inflammatory biomarkers predicting endoscopic disease activity. Results: Six (6) inflammatory biomarkers (serum amyloid A (SAA), Eotaxin-1, IL-6, IL-8, IL-17A, and TNF-α) showed better prediction of IBD disease activity than routine measures (CRP, fecal calprotectin and HBI/SCCAI scores). The best combination of predictive inflammatory biomarkers consisted of serum SAA, IL-6, IL-8, and Eotaxin-1, showing an optimism-adjusted area under the ROC (AuROC) curve of 0.84 (95% CI: 0.73-0.94, P < 0.0001), which predicted significantly better (P = 0.002) than serum CRP levels with an AuROC of 0.57 (95% CI: 0.43-0.72, P = 0.32). Conclusion: The combination of SAA, IL-6, IL-8, and Eotaxin-1 reliably predicts endoscopic disease activity in IBD and might be valuable for monitoring disease activity and management of the disease.

Serum Free Thiols Are Superior to Fecal Calprotectin in Reflecting Endoscopic Disease Activity in Inflammatory Bowel Disease.

Oxidative stress plays a pivotal role in the pathogenesis of inflammatory bowel diseases (IBD). Serum free thiols (R-SH) reliably reflect systemic oxidative stress, since they are readily oxidized by reactive species. Here, we aimed to establish concentrations of serum free thiols in IBD and assessed their discriminating capacity regarding endoscopic disease activity. Albumin-adjusted serum free thiol concentrations were measured in 78 IBD patients (31 Crohn's disease (CD) and 47 ulcerative colitis (UC) patients) and 50 healthy controls and analyzed for associations with disease parameters and their discriminative value regarding endoscopic disease activity (n = 54) or fecal calprotectin (n = 36) in patients for which those data were available. Mean serum free thiol concentrations were significantly lower in both CD and UC as compared to healthy controls (19.4 ± 3.1 and 17.8 ± 3.4 vs. 21.1 ± 1.9 µmol/g albumin, P < 0.001). Free thiols highly accurately discriminated between mild and moderate-to-severe disease activity, better than fecal calprotectin (FC) levels (AUC = 0.87, P < 0.001 vs. AUC = 0.76, P < 0.05, respectively) and this was maintained after cross-validation (AUC = 0.89, P < 0.001). Serum free thiols are reduced in IBD as compared to healthy controls and strongly correlate with the degree of endoscopic disease activity. Quantifying systemic redox status in IBD may be a promising, minimally invasive strategy to monitor IBD disease activity.

Fitz-Hugh-Curtis syndrome resulting in nutmeg liver on computed tomography.

A 34-year-old woman entered the emergency room with abdominal pain in the right upper quadrant. Computed tomography scan showed a nutmeg liver suspected for increased venous pressure by thrombosis of the liver veins, Budd-Chiari malformation, or right-sided heart failure. Interestingly, the diagnosis was pelvic inflammatory disease complicated by the Fitz-Hugh-Curtis syndrome (FHCS). Pelvic inflammatory disease resulted from an ascended infection by Chlamydia trachomatis. FHCS was caused by perihepatitis defined as inflammation of the peritoneal capsule of the liver. Fast diagnosis and treatment is crucial. Therefore, we report a case of FHCS characterized by a nutmeg liver on computed tomography.