Search

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 cetuximab800CW, 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.

Purpose: To provide proof of principle of safety, breast tumor–specific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab–IRDye800CW targeting VEGF-A in patients with breast cancer.Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab–IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and coregistration of tumor tissue and healthy tissue.Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared with those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level.Conclusions: Our study shows that systemic administration of the bevacizumab–IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake as evaluated by a systematic validation methodology. The findings are a step toward a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in the tumor tissue. Clin Cancer Res; 23(11); 2730–41. ©2016 AACR.

Supplemental Materials and Methods; Suppl. Fig. 1. Comparison of tumor vs. skin fluorescence obtained from human breast tissue specimen; Suppl. Fig 2: Magnified comparison between fluorescence images obtained from 3mm thick paraffin slices and 4microm paraffin slices.

Near-Infrared Optical Imaging of Positive Tumor Margin.

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623–31. ©2016 AACR.

Co-localization of Bevacizumab-IRDye800CW, hematoxylin / eosin (H/E), VEGF-A, Collagen and CD34 in Ductal Carcinoma In Situ (DCIS).

Co-localization of Bevacizumab-IRDye800CW, hematoxylin/eosin (H/E), VEGF-A, Collagen and CD34 in Breast Cancer and a Satellite Lesion.

Supplementary Material and Methods / Legends of Supplemental Figures

Multiplex pathology imaging of white-light microscopy, bevacizumab-IRDye800CW fluorescence imaging (green pseudocolor), VEGF-A (red pseudocolor), collagen (red pseudocolor) and CD34 (blue pseudocolor) in breast cancer, including overlay images is shown in a stepwise manner for reasons of clarity.

Intraoperative and Ex Vivo Optical Imaging of Axillary Lymph Nodes.

Microscopic Biodistribution of Bevacizumab-IRDye800CW

Co-localization of Hematoxylin / Eosin (H/E), VEGF-A, and Bevacizumab-IRDye800CW in Tumor-negative Lymph Nodes.

Representative Fluorescence Flatbed Scans of Paraffin Blocks of all Patients (N=20) and Macroscopic Segmentation of Fluorescence Tissue Localization and Target-to-Background Ratios (TBR).

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 &lt, 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 &lt, 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.

Rationale: Resection of soft tissue sarcomas (STS) is accompanied by a high rate of tumor-positive surgical margins (14-34%), which potentially leads to decreased disease-free survival. Vascular Endothelial Growth Factor-A (VEGF-A) is overexpressed in malignant tumors, including STS, and can be targeted with bevacizumab-800CW during fluorescence-guided surgery for real-time tumor detection. In this phase 1 clinical trial, we determined the feasibility, safety and optimal dose of bevacizumab-800CW for fluorescence-guided surgery (FGS) in STS for in- and ex vivo tumor detection. Methods: Patients with a histopathologically diagnosis of STS were included. In the dose-escalation phase, patients received bevacizumab-800CW intravenously 3 days prior to surgery (10, 25 and 50 mg, n = 8). In the subsequent dose-expansion phase, 7 additional patients received bevacizumab-800CW at the optimal dose. Fluorescence images in- and ex vivo were obtained during all stages of standard of care. The optimal dose was determined by calculating in- and ex vivo Tumor-to-Background ratios (TBR) and correlating these results with histopathology. Results: Fifteen patients with STS completed this study. All tumors could be visualized during in- and ex vivo imaging. The optimal bevacizumab-800CW dose proved to be 10 mg with a median in vivo TBR of 2.0 (±0.58) and an ex vivo TBR of 2.67 (±1.6). All seven tumor-positive margins could be observed real-time after surgical resection. Conclusion: FGS using 10 mg bevacizumab-800CW is feasible and safe for intra-operative imaging of STS, potentially allowing tumor detection and margin assessment during surgery. An additional follow-up phase II study is needed to confirm the diagnostic accuracy.

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques (n = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p p

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.

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). Patients with LARC receive nCRT followed by surgery to achieve local disease control. Interestingly, 15%–27% of patients have a pathological complete response, that is, no residual cancer cells are found in the surgical specimen.1–3 There is an increasing interest in identifying patients with a clinical complete response before surgery, as non-operative management for these patients is associated with high survival rates, reduced morbidity and improved functional outcomes.4–8 However, assessing tumour response after nCRT is challenging. White-light endoscopy provides only morphological information, while MRI cannot always distinguish viable tumour from fibrosis.9–11 QFE is a novel endoscopy technique that visualises and quantitatively measures the presence of targeted fluorescence tracers in tissue. We hypothesised that VEGFA-targeted QFE can be of additional value in restaging patients with LARC. In untreated patients, QFE showed clearly enhanced fluorescence in all rectal tumours compared with normal rectal tissue (figure 1A). The tumour-to-normal ratio of 3.1 (figure 1B) signifies QFE can be used to localise rectal cancer. Figure 1 (A) Representative fluorescence images of the quantitative fluorescence endoscopy (QFE) procedure in untreated rectal cancer. From left to right: a high-definition white-light video endoscope image; a white-light image from the QFE fibreoptic, followed by the corresponding near-infrared …

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.

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. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( < 3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas.

Esophageal carcinoma (EC) is a global health problem, with disappointing 5-year survival rates of only 15-25%. Near-infrared targeted photodynamic therapy (NIR-tPDT) is a novel strategy in which cancer-targeted phototoxicity is able to selectively treat malignant cells. In this in vitro report we demonstrate the applicability of antibody-based NIR-tPDT in esophageal adenocarcinoma (EAC), using the phototoxic compounds cetuximab-IRDye700DX and trastuzumab-IRDye700DX, targeting respectively epidermal growth factor receptor 1 (EGFR) and 2 (HER2). Furthermore, we demonstrate that NIR-tPDT can be made more effective by tyrosine kinase inhibitor (TKI) induced growth receptor upregulation. Together, these results unveil a novel strategy for non-invasive EAC treatment, and by pretreatment-induced receptor upregulation its future clinical application may be optimized.

The incidence of oesophageal adenocarcinoma (EAC) is steeply rising. Early lesion detection is a critical factor for improving disease prognosis. We developed and investigated wide-field near-infrared fluorescence molecular endoscopy (NIR-FME), using systemic and topical administration of a fluorescence-labelled antibody against vascular endothelial growth factor (VEGFA). Fourteen patients with Barrett’s oesophagus (BE) underwent endoscopic mucosal resection (EMR) combined with NIR-MFE. From a total of 20 confirmed aberrant lesions identified with NIR-FME, 4 lesions were missed by high-definition (HD) narrowband imaging (NBI) and white-light endoscopy (WLE). This overall 25% detection enhancement advocates NIR-FME as a promising ‘red-flag’ technique for improving early oesophageal lesion detection. ClinicalTrials.gov ID NCT02129933. Worldwide, >450 000 people are diagnosed with oesophageal cancer each year. In the western world, at least 80% of these cancers concern EAC.1–4 Late-stage disease detection challenges the efficacy of therapies, resulting in 400 000 deaths each year. Therefore, there is a pressing need for new endoscopic techniques that enable early EAC detection. Endoscopic imaging of targeted fluorescent agents directed against cancer-specific cellular and subcellular moieties, that is, fluorescence molecular endoscopy (FME), has been heralded as a novel method for oesophageal dysplasia and cancer detection.5 FME enables surface and subsurface visualisation of cancer-specific pathophysiology, going beyond the surface-only morphology and tissue discolourations visible with HD-WLE.6–10 In this study, we investigated for the first time real-time NIR-FME of EAC and dysplasia. A pilot study in patients with BE was based on a novel custom-built NIR fluorescence endoscope, paired to a HD-WL clinical scope (online supplementary figure 1) and employed the anti-VEGFA antibody bevacizumab, labelled with the NIR-fluorescent 800CW. The potential of VEGFA as an early discriminating target in BE was first confirmed by immunohistochemistry, demonstrating that all dysplastic tissues expressed VEGFA (online supplementary figure 2). ### Supplementary file 1 [SP1.pdf] The study furthermore compared systemic versus topical tracer administration during clinical inspection of …

BACKGROUND: Optimum cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is essential for the curative treatment of peritoneal carcinomatosis of colorectal origin. At present, surgeons depend on visual inspection and palpation for tumour detection. Improved detection of tumour tissue using molecular fluorescence-guided surgery could not only help attain a complete cytoreduction of metastatic lesions, but might also prevent overtreatment by avoiding resection of benign lesions.METHODS: For this non-randomised, single-centre feasibility study, we enrolled patients with colorectal peritoneal metastases scheduled for cytoreductive surgery and HIPEC. 2 days before surgery, 4·5 mg of the near-infrared fluorescent tracer bevacizumab-IRDye800CW was administered intravenously. The primary objectives were to determine the safety and feasibility of molecular fluorescence-guided surgery using bevacizumab-IRDye800CW. Molecular fluorescence-guided surgery was deemed safe if no allergic or anaphylactic reactions were recorded and no serious adverse events were attributed to bevacizumab-IRDye800CW. The technique was deemed feasible if bevacizumab-IRDye800CW enabled detection of fluorescence signals intraoperatively. Secondary objectives were correlation of fluorescence with histopathology by back-table imaging of the fresh surgical specimen and semi-quantitative ex-vivo analyses of formalin-fixed paraffin embedded (FFPE) tissue on all peritoneal lesions. Additionally, VEGF-α staining and fluorescence microscopy was done. This study is registered with the Netherlands Trial Registry, number NTR4632.FINDINGS: Between July 3, 2014, and March 2, 2015, seven patients were enrolled in the study. One patient developed an abdominal sepsis 5 days postoperatively and another died from an asystole 4 days postoperatively, most probably due to a cardiovascular thromboembolic event. However, both serious adverse events were attributed to the surgical cytoreductive surgery and HIPEC procedure. No serious adverse events related to bevacizumab-IRDye800CW occurred in any of the patients. Intraoperatively, fluorescence was seen in all patients. In two patients, additional tumour tissue was detected by molecular fluorescence-guided surgery that was initially missed by the surgeons. During back-table imaging of fresh surgical specimens, a total of 80 areas were imaged, marked, and analysed. All of the 29 non-fluorescent areas were found to contain only benign tissue, whereas tumour tissue was detected in 27 of 51 fluorescent areas (53%). Ex-vivo semi-quantification of 79 FFPE peritoneal lesions showed a tumour-to-normal ratio of 6·92 (SD 2·47).INTERPRETATION: Molecular fluorescence-guided surgery using the near-infrared fluorescent tracer bevacizumab-IRDye800CW is safe and feasible. This technique might be of added value for the treatment of patients with colorectal peritoneal metastases through improved patient selection and optimisation of cytoreductive surgery. A subsequent multicentre phase 2 trial is needed to make a definitive assessment of the diagnostic accuracy and the effect on clinical decision making of molecular fluorescence-guided surgery.FUNDING: FP-7 Framework Programme BetaCure and SurgVision BV.

There is a dire need for better visualization of cancer and analysis of specific targets in vivo. Molecular imaging with fluorescence is gaining more and more attention, as it allows detection of these targets and has advantages over radioactivity, such as no radiation dose, and lower costs. A key challenge in optical imaging however, is translation of the newly developed tracers from pre-clinical phase to clinical application. We describe the development and safety testing of clinical grade bevacizumab-800CW, an antibody-based targeted agent for non-invasive imaging of vascular endothelial growth factor A (VEGF-A).Development included implementing the manufacturing process and analytical methods according to current Good Manufacturing Practice (cGMP), formulation studies, extended characterization and stability testing. For safety pharmacology an extended single dose toxicity study in mice was performed.Bevacizumab-800CW was formulated in isotonic phosphate buffered sodium chloride solution at pH 7. The production was robust and showed a reproducible labeling efficiency, and no impurities. The binding affinity to VEGF-A remained intact. The optimized product meets all release specifications, is stable up to at least 3 months and its characteristics did not significantly differ from the unlabeled bevacizumab. Toxicity testing in mice showed no remarkable findings.In conclusion, sterile bevacizumab-800CW (6 mg = 6 ml) can be produced in stock according to current Good Manufacturing Practice. It is ready for first-in-human use. (C) 2016 Elsevier B.V. All rights reserved.

During the last decade, the emerging field of molecular fluorescence imaging has led to the development of tumor-specific fluorescent tracers and an increase in early-phase clinical trials without having consensus on a standard methodology for evaluating an optical tracer. By combining multiple complementary state-of-the-art clinical optical imaging techniques, we propose a novel analytical framework for the clinical translation and evaluation of tumor-targeted fluorescent tracers for molecular fluorescence imaging which can be used for a range of tumor types and with different optical tracers. Here we report the implementation of this analytical framework and demonstrate the tumor-specific targeting of escalating doses of the near-infrared fluorescent tracer bevacizumab-800CW on a macroscopic and microscopic level. We subsequently demonstrate an 88% increase in the intraoperative detection rate of tumor-involved margins in primary breast cancer patients, indicating the clinical feasibility and support of future studies to evaluate the definitive clinical impact of fluorescence-guided surgery.

Optical molecular imaging using fluorescently labeled monoclonal antibodies is of significant added value in guiding surgical or endoscopic procedures. However, development of tracers for clinical trials is complex, and implementation in the clinic is therefore slow. We present a roadmap for development and translation of monoclonal antibody tracers into a drug product compliant with current good manufacturing processes (cGMPs). Methods: The production process for cetuximab-800CW and trastuzumab-800CW was optimized with regard to dye-to-protein ratio and formulation buffer. Promising formulations were produced under cGMP conditions and advanced to a full-scale stability study. Tracers were analyzed for stability by size-exclusion high-pressure liquid chromatography, pH measurement, osmolality, visual inspection, and sterility, as required by the European Pharmacopeia and cGMP guidelines. Results: Seven formulations were investigated for cetuximab-800CW and 10 for trastuzumab-800CW. On the basis of the formulation study results, we chose 2 formulations per antibody for investigation during the full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product. Conclusion: We designed a roadmap to standardize the development, formulation, and cGMP translation of molecular fluorescent tracers. Using our standardized approach, we developed 2 stable antibody-based tracers for clinical use. The proposed roadmap can be used to efficiently develop a cGMP-compliant formulation and improve the translation of newly developed optical tracers to first-in-human use.

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

BackgroundThe tissue factor (TF)- Factor VIIa (FVIIa) complex has a pivotal role in inflammatory and coagulation responses in patients with systemic inflammatory response syndrome (SIRS) and sepsis. Because zymogen FVII (FVII) and FVIIa compete for binding to TF, their plasma levels determine if a catalytically active TF-FVIIa complex will be formed.ObjectiveTo study mortality in SIRS patients as a function of FVIIa and FVII levels in plasma.MethodsThis was a cohort study of 275 patients presenting with SIRS, aged 18 years or older and with an anticipated Intensive Care Unit (ICU) stay of at least 24h. FVIIa was measured using a novel, quantitative assay that recognizes FVIIa, but not FVII. All-cause hospital mortality was followed over a period of 60days.ResultsThe percentage of FVII measured as FVIIa was higher in non-survivors than survivors (2.8%, IQR=1-5.5% vs. 1.5%, IQR=0.6-3.3%; P=0.034). High levels of FVIIa were associated with decreased 60-day cumulative survival (62% vs. 81%, P=0.030); the opposite was observed for FVII (84% vs. 76%, P=0.039). Patients with high-FVIIa and low-FVII levels had a three-fold increased hazard ratio (HR) compared with the patients that had low-FVIIa and high-FVII levels (HR=3.24, 95% confidence interval [CI]=1.41-7.36). This association persisted after adjusting for the APACHE IV score (adjusted HR=2.75, 95% CI=1.2-6.27).ConclusionsSIRS patients with high-FVIIa and low-FVII on admission have an increased mortality risk, an association that is independent from the parameters included in the APACHE IV score.