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.

EGFR-targeted fluorescence molecular imaging for intraoperative margin assessment in oral cancer patients: a phase II trial.

Inadequate surgical margins occur frequently in oral squamous cell carcinoma surgery. Fluorescence molecular imaging (FMI) has been explored for intraoperative margin assessment, but data are limited to phase-I studies. In this single-arm phase-II study (NCT03134846), our primary endpoints were to determine the sensitivity, specificity and positive predictive value of cetuximab-800CW for tumor-positive margins detection. Secondary endpoints were safety, close margin detection rate and intrinsic cetuximab-800CW fluorescence. In 65 patients with 66 tumors, cetuximab-800CW was well-tolerated. Fluorescent spots identified in the surgical margin with signal-to-background ratios (SBR) of ≥2 identify tumor-positive margins with 100% sensitivity, 85.9% specificity, 58.3% positive predictive value, and 100% negative predictive value. An SBR of ≥1.5 identifies close margins with 70.3% sensitivity, 76.1% specificity, 60.5% positive predictive value, and 83.1% negative predictive value. Performing frozen section analysis aimed at the fluorescent spots with an SBR of ≥1.5 enables safe, intraoperative adjustment of surgical margins.

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.

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.

Standardization and implementation of fluorescence molecular endoscopy in the clinic.

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.

Epidermal Growth Factor Receptor-Targeted Fluorescence Molecular Imaging for Postoperative Lymph Node Assessment in Patients with Oral Cancer.

In most oral cancer patients, surgical treatment includes resection of the primary tumor combined with excision of lymph nodes (LNs), either for staging or for treatment. All LNs harvested during surgery require tissue processing and subsequent microscopic histopathologic assessment to determine the nodal stage. In this study, we investigated the use of the fluorescent tracer cetuximab-800CW to discriminate between tumor-positive and tumor-negative LNs before histopathologic examination. Here, we report a retrospective ad hoc analysis of a clinical trial designed to evaluate the resection margin in patients with oral squamous cell carcinoma (NCT02415881). Methods: Two days before surgery, patients were intravenously administered 75 mg of cetuximab followed by 15 mg of cetuximab-800CW, an epidermal growth factor receptor-targeting fluorescent tracer. Fluorescence images of excised, formalin-fixed LNs were obtained and correlated with histopathologic assessment. Results: Fluorescence molecular imaging of 514 LNs (61 pathologically positive nodes) could detect tumor-positive LNs ex vivo with 100% sensitivity and 86.8% specificity (area under the curve, 0.98). In this cohort, the number of LNs that required microscopic assessment was decreased by 77.4%, without missing any metastases. Additionally, in 7.5% of the LNs false-positive on fluorescence imaging, we identified metastases missed by standard histopathologic analysis. Conclusion: Our findings suggest that epidermal growth factor receptor-targeted fluorescence molecular imaging can aid in the detection of LN metastases in the ex vivo setting in oral cancer patients. This image-guided concept can improve the efficacy of postoperative LN examination and identify additional metastases, thus safeguarding appropriate postoperative therapy and potentially improving prognosis.

Identification and Validation of Esophageal Squamous Cell Carcinoma Targets for Fluorescence Molecular Endoscopy.

Dysplasia and intramucosal esophageal squamous cell carcinoma (ESCC) frequently go unnoticed with white-light endoscopy and, therefore, progress to invasive tumors. If suitable targets are available, fluorescence molecular endoscopy might be promising to improve early detection. Microarray expression data of patient-derived normal esophagus (n = 120) and ESCC samples (n = 118) were analyzed by functional genomic mRNA (FGmRNA) profiling to predict target upregulation on protein levels. The predicted top 60 upregulated genes were prioritized based on literature and immunohistochemistry (IHC) validation to select the most promising targets for fluorescent imaging. By IHC, GLUT1 showed significantly higher expression in ESCC tissue (30 patients) compared to the normal esophagus adjacent to the tumor (27 patients) (p < 0.001). Ex vivo imaging of GLUT1 with the 2-DG 800CW tracer showed that the mean fluorescence intensity in ESCC (n = 17) and high-grade dysplasia (HGD, n = 13) is higher (p < 0.05) compared to that in low-grade dysplasia (LGD) (n = 7) and to the normal esophagus adjacent to the tumor (n = 5). The sensitivity and specificity of 2-DG 800CW to detect HGD and ESCC is 80% and 83%, respectively (ROC = 0.85). We identified and validated GLUT1 as a promising molecular imaging target and demonstrated that fluorescent imaging after topical application of 2-DG 800CW can differentiate HGD and ESCC from LGD and normal esophagus.

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity.

The increasing number of patients suffering from chronic obstructive pulmonary disease (COPD) represents a major and increasing health problem. Therefore, novel therapeutic approaches are needed. Class I HDACs 1, 2 and 3 play key roles in the regulation of inflammatory gene expression with a particular pro-inflammatory role for HDAC 3. HDAC 3 has been reported to be an important player in inflammation by deacetylating NF-κB p65, which has been implicated in the pathology of COPD. Here, we applied the pharmacological HDAC 3-selective inhibitor RGFP966, which attenuated pro-inflammatory gene expression in models for inflammatory lung diseases. Consistent with this, a robust decrease of the transcriptional activity of NF-κB p65 was observed. HDAC 3 inhibition affected neither the acetylation status of NF-κB p65 nor histone H3 or histone H4. This indicates that HDAC 3 inhibition does not inhibit NF-κB p65 transcriptional activity by affecting its deacetylation but rather by inhibiting enzymatic activity of HDAC 3. Taken together, our findings indicate that pharmacological HDAC 3-selective inhibition by inhibitors such as RGFP966 may provide a novel and effective approach toward development of therapeutics for inflammatory lung diseases.