Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using a Fluorescently Labelled Anti-CEA Nanobody Probe: A Preclinical Study.

Molecular fluorescence-guided surgery using near-infrared light has the potential to improve the rate of complete resection of cancer. Typically, monoclonal antibodies are being used as targeting moieties, however smaller fragments, such as single-domain antibodies (i.e., Nanobodies®) improve tumor specificity and enable tracer injection on the same day as surgery. In this study, the feasibility of a carcinoembryonic antigen-targeting Nanobody (NbCEA5) conjugated to two zwitterionic dyes (ZW800-1 Forte [ZW800F] and ZW800-1) for visualization of pancreatic ductal adenocarcinoma (PDAC) was investigated. After site-specific conjugation of NbCEA5 to the zwitterionic dyes, binding specificity was evaluated on human PDAC cell lines with flow cytometry. A dose escalation study was performed for both NbCEA5-ZW800F and NbCEA5-ZW800-1 in mice with subcutaneously implanted pancreatic tumors. Fluorescence imaging was performed up to 24 h after intravenous injection. Furthermore, the optimal dose for NbCEA5-ZW800-1 was injected in mice with orthotopically implanted pancreatic tumors. A dose-escalation study showed superior mean fluorescence intensities for NbCEA5-ZW800-1 compared to NbCEA5-ZW800F. In the orthotopic tumor models, NbCEA5-ZW800-1 accumulated specifically in pancreatic tumors with a mean in vivo tumor-to-background ratio of 2.4 (SD = 0.23). This study demonstrated the feasibility and potential advantages of using a CEA-targeted Nanobody conjugated to ZW800-1 for intraoperative PDAC imaging.

Side-by-Side Comparison of uPAR-Targeting Optical Imaging Antibodies and Antibody Fragments for Fluorescence-Guided Surgery of Solid Tumors.

PURPOSE: Radical resection is paramount for curative oncological surgery. Fluorescence-guided surgery (FGS) aids in intraoperative identification of tumor-positive resection margins. This study aims to assess the feasibility of urokinase plasminogen activator receptor (uPAR) targeting antibody fragments for FGS in a direct comparison with their parent IgG in various relevant in vivo models. PROCEDURES: Humanized anti-uPAR monoclonal antibody MNPR-101 (uIgG) was proteolytically digested into F(ab')2 and Fab fragments named uFab2 and uFab. Surface plasmon resonance (SPR) and cell assays were used to determine in vitro binding before and after fluorescent labeling with IRDye800CW. Mice bearing subcutaneous HT-29 human colonic cancer cells were imaged serially for up to 120 h after fluorescent tracer administration. Imaging characteristics and ex vivo organ biodistribution were further compared in orthotopic pancreatic ductal adenocarcinoma (BxPc-3-luc2), head-and-neck squamous cell carcinoma (OSC-19-luc2-GFP), and peritoneal carcinomatosis (HT29-luc2) models using the clinical Artemis fluorescence imaging system. RESULTS: Unconjugated and conjugated uIgG, uFab2, and uFab specifically recognized uPAR in the nanomolar range as determined by SPR and cell assays. Subcutaneous tumors were clearly identifiable with tumor-to-background ratios (TBRs) > 2 after 72 h for uIgG-800F and 24 h for uFab2-800F and uFab-800F. For the latter two, mean fluorescence intensities (MFIs) dipped below predetermined threshold after 72 h and 36 h, respectively. Tumors were easily identified in the orthotopic models with uIgG-800F consistently having the highest MFIs and uFab2-800F and uFab-800F having similar values. In biodistribution studies, kidney and liver fluorescence approached tumor fluorescence after uIgG-800F administration and surpassed tumor fluorescence after uFab2-800F or uFab-800F administration, resulting in interference in the abdominal orthotopic mouse models. CONCLUSIONS: In a side-by-side comparison, FGS with uPAR-targeting antibody fragments compared with the parent IgG resulted in earlier tumor visualization at the expense of peak fluorescence intensity.

Single fiber reflectance spectroscopy for pancreatic cancer detection during endoscopic ultrasound guided fine needle biopsy: a prospective cohort study.

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.

Resection of the Portal-Superior Mesenteric Vein in Pancreatic Cancer: Pathological Assessment and Recurrence Patterns.

OBJECTIVES: The portal vein (PV)-superior mesenteric vein (SMV) margin is the most affected margin in pancreatic cancer. This study investigates the association between venous resection, tumor invasion in the resected PV-SMV, recurrence patterns, and overall survival (OS). METHODS: This multicenter cohort study included patients who underwent pancreatoduodenectomy for pancreatic cancer (2010-2017). In addition, a systematic literature search was performed. RESULTS: In total, 531 patients were included, of which 149 (28%) underwent venous resection of whom 53% had tumor invasion in the resected PV-SMV. Patients with venous resection had a significant higher rate of R1 margins (69% vs 37%) and had more often multiple R1 margins (43% vs 16%). Patient with venous resection had a significant shorter time to locoregional recurrence and a shorter OS (15 vs 19 months). At multivariable analyses, venous resection and tumor invasion in the resected PV-SMV were not predictive for time to recurrence and OS. The literature overview showed that pathological assessment of the resected PV-SMV is not adequately standardized. CONCLUSIONS: Only half of patients with venous resection had pathology confirmed tumor invasion in the resected PV-SMV, and both are not independently associated with time to recurrence and OS. The pathological assessment of the resected PV-SMV needs to be standardized.

Detection of cutaneous oxygen saturation using a novel snapshot hyperspectral camera: a feasibility study.

BACKGROUND: Tissue necrosis, a consequence of inadequate tissue oxygenation, is a common post-operative complication. As current surgical assessments are often limited to visual and tactile feedback, additional techniques that can aid in the interrogation of tissue viability are needed to improve patient outcomes. In this bi-institutional pilot study, the performance of a novel snapshot hyperspectral imaging camera to detect superficial cutaneous oxygen saturation (StO2) was evaluated. METHODS: Healthy human volunteers were recruited at two participating centers. Cutaneous StO2 of the forearm was determined by a snapshot hyperspectral camera on two separate study days during occlusion-reperfusion of the brachial artery and after induction of local vasodilation. To calculate the blood StO2 at each pixel in the multispectral image, spectra were selected, and fitting was performed over wavelengths ranging from 470 to 950 nm. RESULTS: Quantitative detection of physiological changes in cutaneous StO2 levels was feasible in all sixteen volunteers. A significant (P<0.001) decrease in cutaneous StO2 levels from 78.3% (SD: 15.3) at baseline to 60.6% (SD: 19.8) at the end of occlusion phase was observed, although StO2 levels returned to baseline after five minutes. Mean cutaneous StO2 values were similar in the same subjects on separate study days (Pearson R2: 0.92 and 0.77, respectively) at both centers. Local vasodilation did not yield significant changes in cutaneous StO2 values. CONCLUSIONS: This pilot study demonstrated the feasibility of a snapshot hyperspectral camera for detecting quantitative physiological changes in cutaneous StO2 in normal human volunteers, and serves as a precursor for further validation in perioperative studies.

Quantitative dynamic near-infrared fluorescence imaging using indocyanine green for analysis of bowel perfusion after mesenteric resection.

SIGNIFICANCE: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has proven to be a feasible application for real-time intraoperative assessment of tissue perfusion, although quantification of NIR fluorescence signals is pivotal for standardized assessment of tissue perfusion. AIM: Four patients are described with possible compromised bowel perfusion after mesenteric resection. Based on these patients we want to emphasize the difficulties in the quantification of NIR fluorescence imaging for perfusion analysis. APPROACH: During image-guided fluorescence assessment, 5 mg of ICG (2.5 mg / ml) was intravenously administered by the anesthesiologist. NIR fluorescence imaging was done with the open camera system of Quest Medical Imaging. Fluorescence data taken from the regions of interest (bowel at risk, transition zone of bowel at risk and adjacent normally perfused bowel, and normally perfused reference bowel) were quantitatively analyzed after surgery for fluorescence intensity-and perfusion time-related parameters. RESULTS: Bowel perfusion, as assessed clinically by independent surgeons based on NIR fluorescence imaging, resulted in different treatment strategies, three with excellent clinical outcome, but one with a perfusion related complication. Post-surgery quantitative analysis of fluorescence dynamics showed different patterns in the affected bowel segment compared to the unaffected reference segments for the four patients. CONCLUSIONS: Similar intraoperative fluorescence results could lead to different surgical treatment strategies, which demonstrated the difficulties in interpretation of uncorrected fluorescence signals. Real-time quantification and standardization of NIR fluorescence perfusion imaging could probably aid surgeons in the nearby future.

Stage-Specific Value of Carbohydrate Antigen 19-9 and Carcinoembryonic Antigen Serum Levels on Survival and Recurrence in Pancreatic Cancer: A Single Center Study and Meta-Analysis.

This study aimed to determine the stage-specific prognostic value of carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) serum levels at diagnosis on overall survival (OS) and time to local recurrence or distant metastases in patients with pancreatic ductal adenocarcinoma (PDAC). Consecutive PDAC patients, discussed at multidisciplinary team meetings from 2013 through 2017, were reviewed. Prognostic factors were stage-specific (resection vs. advanced PDAC) evaluated in Cox proportional hazard models. Additionally, a systematic literature search and meta-analysis was performed, as current literature is lacking a complete overview of used cut-off values and the added value of CEA as prognostic marker. In the retrospective cohort, elevated CA19-9 (>305 kU/L) level was independently associated with poor OS (Hazard ratio (HR): 1.72(1.31-2.26)) and early recurrence (HR: 1.74(1.06-2.86)), whereas CEA was not significantly associated. The meta-analysis showed that both elevated CA19-9 and CEA serum levels were predictors for poor OS (pooled HR: 1.29(1.17-1.42) and HR: 1.51(1.33-1.73), respectively). In the resected cohort, elevated CA19-9 level was significantly associated with early recurrence (pooled HR: 2.41(1.77-3.29)), whereas CEA was not. Elevated CA19-9 serum level appear to be an independent prognostic factor for poor OS and early recurrence in PDAC patients, whereas the prognostic value of CEA is disputable.

Elevated CEA and CA19-9 serum levels independently predict advanced pancreatic cancer at diagnosis.

Purpose: It is suggested that tumour markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) could be used to predict the stage of pancreatic cancer. However, optimal cut-off values for CEA and CA19-9 are disputable. This study aimed to assess the value of CEA and CA19-9 serum levels at diagnosis of pancreatic ductal adenocarcinoma (PDAC) as predictors for the advanced stage of PDAC in patients discussed at pancreatic multidisciplinary team (MDT) meetings.Methods: Patients with suspected PDAC discussed at MDT meetings from 2013 to 2017 were reviewed, in order to determine optimal cut-off values of both CEA and CA19-9.Results: In total, 375 patients were included. Optimal cut-off values for predicting advanced PDAC were 7.0 ng/ml for CEA and 305.0 U/ml for CA19-9, resulting in positive predictive values of 83.3%, 73.6%, and 91.4% for CEA, CA19-9 and combined, respectively. Both tumour markers were independent predictors of advanced PDAC, demonstrated by an odds ratio of 4.21 (95% CI:1.85-9.56; p = 0.001) for CEA and 2.58 for CA19-9 (95% CI:1.30-5.14; p = 0.007).Conclusions: CEA appears to be a more robust predictor of advanced PDAC than CA19-9. Implementing CEA and CA19-9 serum levels during MDT meetings as an additional tool for establishing tumour resectability is worthwhile for tailored diagnostics.

Intraoperative detection of the remnant cystic duct during robot-assisted surgery using near-infrared fluorescence imaging: a case report.

BACKGROUND: Post cholecystectomy syndrome is characterized as recurrence of symptoms as experienced before cholecystectomy. In rare cases, a remnant cystic duct is causing these symptoms and occasionally surgical resection is performed. During surgery, visualization of the biliary ducts could be difficult due to inflammation and dense adhesions. CASE PRESENTATION: In this article, we presented a 36-year old woman with post-cholecystectomy syndrome in which we evaluated the feasibility of near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) for visualization of the remnant cystic and common bile duct during robot-assisted surgery. Intraoperative visualization of the remnant biliary duct and other important structures was feasible, and resection of the remnant cystic duct was successfully performed under fluorescence guidance, without any complications. CONCLUSIONS: NIR fluorescence imaging of the biliary ducts using ICG does not prolong the operating time, and could potentially decrease the operation time in difficult procedures, because of easy and fast detection of the biliary tract. Furthermore, it is a non-hazardous and non-invasive technique, as it does not require use of radiation and cannot cause bile duct injury. This case illustrated that ICG NIR fluorescence imaging during difficult robot-assisted surgical procedures of the bile ducts is effective and therefore highly recommended.

Latest developments in molecular tracers for fluorescence image-guided cancer surgery.

Real-time intraoperative guidance is essential during oncological surgery for complete and safe tumour resection. Fluorescence imaging in the near-infrared spectrum has shown potential for guiding surgeons during complex interventions. Recently, there has been a shift towards the use of fluorescence contrast agents for molecular imaging. The first targeted fluorescent agents, of which most consist of approved therapeutic antibodies conjugated to a fluorescent dye, have been evaluated in several early-phase clinical trials. Moreover, advances in protein engineering and drug design have led to the development of a variety of tracers suitable for molecular fluorescence image-guided surgery. In this Review, we discuss preclinical and clinical evidence, ongoing clinical trials, and the latest developments in the field of molecular near-infrared tracers for fluorescence-guided cancer surgery.

A systematic review of the use of near-infrared fluorescence imaging in patients with peripheral artery disease.

OBJECTIVE: In the diagnosis of peripheral artery disease (PAD), the ankle-brachial index plays an important role. However, results of the ankle-brachial index are unreliable in patients with severe media sclerosis. Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) can provide information about tissue perfusion and has already been studied in oncologic, reconstructive, and cardiac surgery. For patients with PAD, this technique might give insight into skin perfusion and thereby guide treatment. We performed a systematic review of the literature on the use of NIR fluorescence imaging in patients with PAD. METHODS: PubMed, MEDLINE, Embase, and Cochrane were searched for articles and abstracts on the application of NIR fluorescence imaging using ICG as fluorescent dye in patients with PAD. Our search strategy combined the terms "fluorescence," "ICG," or synonyms and "peripheral artery disease" or synonyms. The extracted data included fluorescence parameters and test characteristics for diagnosis of PAD. RESULTS: Twenty-three articles were found eligible for this review using 18 different parameters for evaluation of the fluorescence signal intensity. NIR fluorescence imaging was used for four main indications: diagnosis, quality control in revascularization, guidance in amputation surgery, and visualization of vascular structures. For the diagnosis of PAD, NIR fluorescence imaging yields a sensitivity ranging from 67% to 100% and a specificity varying between 72% and 100%. Significant increases in multiple fluorescence parameters were found in comparing patients before and after revascularization. CONCLUSIONS: NIR fluorescence imaging can be used for several indications in patients with PAD. NIR fluorescence imaging seems promising in diagnosis of PAD and guidance of surgeons in treatment, especially in patients in whom current diagnostic methods are not applicable. Further standardization is needed to reliably use this modality in patients with PAD.

A practical guide for the use of indocyanine green and methylene blue in fluorescence-guided abdominal surgery.

Near-infrared (NIR) fluorescence imaging is gaining clinical acceptance over the last years and has been used for detection of lymph nodes, several tumor types, vital structures and tissue perfusion. This review focuses on NIR fluorescence imaging with indocyanine green and methylene blue for different clinical applications in abdominal surgery with an emphasis on oncology, based on a systematic literature search. Furthermore, practical information on doses, injection times, and intraoperative use are provided.

The clinical usefulness of optical coherence tomography during cancer interventions.

INTRODUCTION: Tumor detection and visualization plays a key role in the clinical workflow of a patient with suspected cancer, both in the diagnosis and treatment. Several optical imaging techniques have been evaluated for guidance during oncological interventions. Optical coherence tomography (OCT) is a technique which has been widely evaluated during the past decades. This review aims to determine the clinical usefulness of OCT during cancer interventions focussing on qualitative features, quantitative features and the diagnostic value of OCT. METHODS: A systematic literature search was performed for articles published before May 2018 using OCT in the field of surgical oncology. Based on these articles, an overview of the clinical usefulness of OCT was provided per tumor type. RESULTS: A total of 785 articles were revealed by our search, of which a total of 136 original articles were available for analysis, which formed the basis of this review. OCT is currently utilised for both preoperative diagnosis and intraoperative detection of skin, oral, lung, breast, hepatobiliary, gastrointestinal, urological, and gynaecological malignancies. It showed promising results in tumor detection on a microscopic level, especially using higher resolution imaging techniques, such as high-definition OCT and full-field OCT. CONCLUSION: In the near future, OCT could be used as an additional tool during bronchoscopic or endoscopic interventions and could also be implemented in margin assessment during (laparoscopic) cancer surgery if a laparoscopic or handheld OCT device will be further developed to make routine clinical use possible.

Validation of full-field optical coherence tomography in distinguishing malignant and benign tissue in resected pancreatic cancer specimens.

BACKGROUND: Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States. The minority of patients can undergo curative-intended surgical therapy due to progressive disease stage at time of diagnosis. Nonetheless, tumor involvement of surgical margins is seen in up to 70% of resections, being a strong negative prognostic factor. Real-time intraoperative imaging modalities may aid surgeons to obtain tumor-free resection margins. Full-field optical coherence tomography (FF-OCT) is a promising diagnostic tool using high-resolution white-light interference microscopy without tissue processing. Therefore, we composed an atlas of FF-OCT images of malignant and benign pancreatic tissue, and investigated the accuracy with which the pathologists could distinguish these. MATERIALS AND METHODS: One hundred FF-OCT images were collected from specimens of 29 patients who underwent pancreatic resection for various indications between 2014 and 2016. One experienced gastrointestinal pathologist and one pathologist in training scored independently the FF-OCT images as malignant or benign blinded to the final pathology conclusion. Results were compared to those obtained with standard hematoxylin and eosin (H&E) slides. RESULTS: Overall, combined test characteristics of both pathologists showed a sensitivity of 72%, specificity of 74%, positive predictive value of 69%, negative predictive value of 79% and an overall accuracy of 73%. In the subset of pancreatic ductal adenocarcinoma patients, 97% of the FF-OCT images (n = 35) were interpreted as tumor by at least one pathologist. Moreover, normal pancreatic tissue was recognised in all cases by at least one pathologist. However, atrophy and fibrosis, serous cystadenoma and neuroendocrine tumors were more often wrongly scored, in 63%, 100% and 25% respectively. CONCLUSION: FF-OCT could distinguish normal pancreatic tissue from pathologic pancreatic tissue in both processed as non-processed specimens using architectural features. The accuracy in pancreatic ductal adenocarcinoma is promising and warrants further evaluation using improved assessment criteria.