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Detection by fluorescence of pituitary neuroendocrine tumour (PitNET) tissue during endoscopic transsphenoidal surgery using bevacizumab-800CW (DEPARTURE trial): study protocol for a non-randomised, non-blinded, single centre, feasibility and dose-finding trial

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

ABSTRACT 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 dosefinding 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 singlefibre 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 tumourto-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.

INTRODUCTION
Pituitary neuroendocrine tumours (PitNETs) are usually benign tumours arising from adenohypophyseal cells. They are a Strengths and limitations of this study ► The DEPARTURE trial is the first study on detection of pituitary neuroendocrine tumour (PitNET) tissue during endoscopic transsphenoidal surgery using the fluorescent tracer molecule bevacizumab-800CW. ► An intraoperative near infrared camera system will be used that has shown successful tumour detection using bevacizumab-800CW in tumours with high vascular endothelial growth factor A (VEGF-A) expression in multiple clinical trials. ► Multidiameter single-fibre reflectance, single-fibre fluorescence spectroscopy will be used to allow quantification of fluorescent intensities both in vivo and ex vivo. ► This pilot study is a phase 1 exploratory trial, which means that data recorded within this study cannot be directly used for the current endoscopic transsphenoidal treatment of patients with PitNET. ► A heterogeneous group of PitNETs with possible different VEGF-A expression is included in this study.
Open access heterogeneous group of tumours in their origin, growth patterns and biological behaviours. PitNETs account for 15% of all intracranial neoplasms and are the third most frequent tumour type after meningiomas and gliomas. They generally present either due to hypersecretion of pituitary hormones and/or due to local space occupying effects (optico-chiasmatic compression/cavernous sinus invasion) and/or hyposecretion of some or all of the pituitary hormones. PitNETs can be divided histopathologically into six main categories (somatotroph, lactotroph, thyrotroph, corticotroph, gonadotroph and plurihormonal tumours) 1 and are associated with considerably mortality 2 and morbidity. 3

METHODS AND ANALYSIS
Near-infrared fluorescence imaging for endoscopy Near-infrared (NIR) fluorescence imaging combines optical imaging with a NIR (wavelength: 650-900 nm) targeted fluorescence tracer binding to a specific molecule. By choosing a fluorescence tracer binding to a tumour specific molecule, it is possible to visualise the presence of tumour tissue in vivo. NIR optical imaging in the area of the sella has several advantages over current methods for visualising tumour characteristics: it does not use ionising radiation, it provides real-time molecular information, it is affordable and it can be used as part of endoscopic proceduresallowing parallel biopsy acquisition of relevant tumour areas for optimal ex vivo analyses. The NIR window allows for both improved tissue penetration by reduced absorption of haemoglobin and reduced autofluorescence resulting in lower background signal and higher signalto-background ratio. These advantages make NIR fluorescence imaging appropriate for in vivo imaging.
Multidiameter single-fibre reflectance, single-fibre fluorescence spectroscopy To allow quantification of fluorescent intensities in vivo, multidiameter single fibre reflectance (MDSFR) and single-fibre fluorescence (SFF) spectroscopy has been developed by the Erasmus Medical Centre Rotterdam. MDSFR spectroscopy determines the tissue absorption coefficient and reduced scattering coefficient of the tissue, while SFF spectroscopy measures the tissue fluorescence. Afterwards, the fluorescence is corrected for the optical properties resulting in the intrinsic fluorescence which is a quantitative value. 9-11 MDSFR-SFF spectroscopy has been successfully used previously in vivo and ex vivo in multiple ongoing clinical studies by our group (NCT02113202, NCT01972373, NCT02583568 and NCT03205501).

Clinical molecular imaging of VEGF-A
A potential target to be used to identify PitNET tissue is vascular endothelial growth factor A (VEGF-A). VEGF-A is a potent, endothelial-cell specific activator of angiogenesis and its expression is known to be significantly higher in PitNETs with CS invasion. 12 Bevacizumab binds and neutralises all isoforms of human VEGF-A. VEGF-A is endothelial cell-specific and is one of the key regulators of angiogenesis in general, including the promotion of tumour progression and metastasis. 13 In PitNETs, it is known to correlate with disease progression and haemorrhage. 14 15 Overexpression of VEGF has been observed in lactotroph PitNETs, especially in men, and in pituitary carcinomas. [16][17][18][19] Bevacizumab can be labelled (ratio 1:2) with the NIR organic fluorophore IRDye800CW, developed by LI-COR Biosciences (Lincoln, Nebraska, USA) and can be used for NIR imaging. Labelling of bevacizumab to IRDye800CW is performed at the Department of Clinical Pharmacy and Pharmacology of the University Medical Centre Groningen under Good Manufacturing Practice (GMP) conditions. Therefore, it is expected that the tracer bevacizumab-800CW accumulates in the pituitary microenvironment. The aim of this study is to visualise the fluorescence in the NIR spectrum using the fluorescence endoscopy system during pituitary surgery. In addition, this technique may be able to differentiate the poorly vascularised microcorticotroph tumour from the well vascularised normal surrounding pituitary.

Dose selection for bevacizumab-800CW
It is important that the tracer only binds selectively to the target tissue with a sufficient amount of fluorophore to detect a signal while any therapeutic effect of the bevacizumab moiety should be prevented.
Based on results of previous studies (table 1), doses of 4.5, 10 and 25 mg can be used safely without observed increase in bleeding risk. Tumour-to-background ratios (TBR) are calculated by fluorescence endoscopy in vivo measurements including MDSFR/SFF spectroscopy data. If background tissue, either normal pituitary or tissue of the medial wall of the cavernous sinus is available, TBR of the ex vivo tissue can be calculated. After inclusion of the first three patients in each dose group (nine patients in total), an interim analysis is performed. After this analysis, three scenarios are possible. If one dose group allows for differentiation between tumour area and surrounding normal tissue, only this dose is expanded. If two dose groups differentiate, these are expanded to a total of six patients. If none of the doses show a sufficient contrast between tumour and normal tissue, the study is terminated after the interim analysis. In case of the first scenario, expansion of two cohorts, a final analysis is performed to define the optimal dose, which will represent a balance between the lowest dose and a clinically usable TBR, sufficient to discriminate between tumorous and non-tumorous tissue. The dose protocol for this study is shown in table 2.

Objectives
Primary objective is to determine the feasibility of bevacizumab-800CW mediated fluorescence for PitNET by comparing intraoperative both in vivo and ex vivo fluorescence measurements including MDSFR/SFF spectroscopy data with ex vivo histological examination in tumours with a Knosp grade of 3 or 4.
Secondary objectives are: ► To identify the optimal tracer dose for imaging of PitNET tissue during TSS for further development in a phase 2 fluorescence molecular endoscopy trial. ► To quantify fluorescence intensity in vivo and ex vivo with MDSFR/SFF spectroscopy. Exclusion criteria ► Medical or psychiatric conditions that compromise the patient's ability to give informed consent. ► Pregnant or lactating women. Documentation of a negative pregnancy test must be available for woman of childbearing potential. We define women of childbearing potential as premenopausal women with intact reproductive organs and women less than 2 years after menopause. ► History of infusion reactions to bevacizumab or other monoclonal antibody therapies. ► Inadequately controlled hypertension with or without current antihypertensive medications. ► Within 6 months prior to inclusion: myocardial infarction, transient ischemic attack, cerebrovascular accident, pulmonary embolism, uncontrolled chronic hepatic failure or unstable angina pectoris.

Sample size calculation
This is a feasibility study to investigate if accumulation of the fluorescent tracer bevacizumab-800CW can be detected with an intraoperative NIR camera system, whether this can be used to identify PitNET tissue during TSS and to define the optimal dose of bevacizumab-800CW. With the dose protocol as described in table 2 we expect to be able to determine whether this approach is feasible to continue to a phase 2 study.

Investigational treatment
Investigational product Bevacizumab (Avastin) is a recombinant, humanised, IgG1 monoclonal antibody with specific affinity for human VEGF. IRDye800CW is a NIR organic fluorophore, developed and produced by LI-COR Biosciences and produced under GMP conditions. For more information regarding IRDye800CW to the toxicity studies of Marshall et al 20 and Ter Weele et al. 21 Conjugation of the fluorescent dye to bevacizumab, purification and formulation is performed at the department of Clinical Pharmacy and Pharmacology of the University Medical Centre Groningen. Bevacizumab-800CW is sterile filled into injection vials as a ready-to-use solution. The product is compounded by drawing the required volume of tracer into a syringe of sufficient size. No dilution of the product is performed.

Dosages, dosage modifications and method of administration
The initial dose level of 4.5 mg flat dose is based on 'FDA Guidance for Industry, Investigators, and Reviewers -Exploratory IND Studies'. This document specifies that a micro dose for proteins, in view of the higher molecular weight of protein molecules compared with conventional drugs, is defined as no more than 30 nanomoles of dust. Bevacizumab-800CW has a molecular weight of 150 000 g/mol, which brings an amount of 30 nanomoles to a dosage of 4.5 mg. This level has been chosen as the starting dose because it is the highest dose level at which a pharmacological effect of the tracer is almost certainly completely absent. Although the higher dose levels from the studies do not fall under microdosing, in terms of safety, the effect and side effects are expected to be very limited, since bevacizumab is therapeutically dosed in schedules up to 10 mg/kg body weight every 2 weeks. The higher doses are thus also far below the therapeutic doses of the antibody. From an imaging point of view, the doses of 10 and 25 mg are based on previous experiences with bevacizumab-800CW, in which other oncological indications showed that 10-25 mg gave a sufficiently strong signal that could be distinguished from background noise. Since these doses cannot be translated directly into other indications, this study has chosen to search for a workable dose for imaging in the same range.

Study procedures
Study flow chart (figure 1).

Recruitment
Inclusion of participants started on the 1 October 2020. Identification of potential participants occurs during the multidisciplinary pituitary tumour meeting. Enrolment takes place at the outpatient clinic of the neurosurgery or endocrinology department. Suitability is evaluated based on the inclusion and exclusion criteria. When a patient is identified as a potential participant, Open access the patient information folder in adjusted linguistics is provided during the standard preoperative visit to the neurosurgeon.

Tracer administration
A single dose of bevacizumab-800CW (4.5 mg, 10 mg or 25 mg) is administered intravenously 2-4 days prior to surgery to the patient at the ward of the neurosurgery department. The infusion line is flushed with a saline solution afterwards. During tracer administration, a crash car with necessary equipment is available in case of an adverse reaction. Directly before and after tracer administration, blood pressure, pulse and temperature are measured. The patient is observed for 60 min following tracer injection. After the observation period of 1 hour the intravenous line is removed and the patient is discharged.

Surgical procedure
The surgery is performed according to standard of care. The procedure starts with preparing the endonasal route towards the sella, by the Ear, Nose and Throat (ENT) surgeon When the sellar region is in sight, the anterior wall of the sella is removed and the dura exposed.
Imaging moments 1. After opening of the dura and when the tumour is in sight, in vivo NIR fluorescence imaging is performed using the fluorescence endoscopic system. Additionally, MDSFR/SFF spectroscopy data are obtained by placing the probe in the tumour tissue of interest. 2. After removal of tumour tissue, a second moment of in vivo NIR fluorescence imaging is performed and MDSFR/SFF spectroscopy data are obtained in the sella, including the normal pituitary as background tissue when available. 3. Finally, in vivo NIR fluorescence imaging is performed and MDSFR/SFF spectroscopy data are obtained of the medial wall of the cavernous sinus. If technically possible, both cavernous sinuses are imaged. Within this feasibility and dose finding study, the surgeons do not deviate from the standard surgical procedure based on the findings of the fluorescence endoscopic system, including the number of biopsies taken. At the end of the imaging procedure, the surgery continues as usual. After the surgical procedure, patients are monitored at the recovery care unit as usual, after which they are transported to the neurosurgical ward. The surgical procedure for removal of a PitNET can be classified as a low risk procedure. Mortality rate is below 1%. Median total hospital stay is -5 days.

Specimen related study procedures
Fresh surgical specimen procedures The excised surgical specimen is imaged ex vivo with the Odyssey CLx flatbed scanner (LI-COR Biosciences) for detection of fluorescence. The fresh surgical specimen is transported to the pathology department for standard histopathological analysis including immunohistochemistry. In consultation with the pathologist additional tissue is stored for further analyses.
Procedures after collection of all surgical specimens Additional 4 μm slices are cut from formalin-fixed paraffin-embedded blocks. These slices are scanned for fluorescence on the Odyssey scanner and directly after stained with H&E staining, providing a direct correlation between the fluorescence signal and histology.

Withdrawal of individual subjects
The investigator can decide to withdraw a subject from the study for urgent medical reasons. Withdrawal should always be considered in case of a SAE.

Data management
Clinical record forms are recorded in a web-based data capture system, Research Electronic Data Capture Open access (REDCap), which is hosted by the Trial Coordination Centre of the University Medical Centre Groningen. All imaging data are stored on a secured server of the University Medical Centre Groningen. Ex vivo imaging of surgical specimen ► (Semi-)quantification of fluorescent signal with available fluorescence imaging systems (mean, SD per subject for tumour and surrounding tissue for all subjects together (intra-variability and inter-variability)). ► (Semi-)quantification of fluorescent signal intensity in fresh samples (mean, SD per subject for tumour and surrounding tissue and for all tumour samples together (intra-variability and inter-variability)). ► Ex vivo VEGF levels.

Patient and public involvement
No patients were involved in the design of the study. The participants and relevant patient support groups will be informed about the results of the study.

Ethics and dissemination Regulation statements
The study is conducted according to the principles of the Declaration of Helsinki (Fortaleza 2013 amendment) and in accordance with the medical Research Involving Human Subjects Act (WMO) and other guidelines, regulations and Acts. The protocol has been written and the study is conducted according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Harmonised Tripartite Guideline for Good Clinical Practice.

Consent
An information folder in adjusted linguistics is offered to all participants containing information about the study aims, procedures and risks of potential negative effects. In a conversation with the attending physician, questions regarding the study are answered. After the visit to the neurosurgeon, the patient decides whether he/she wants to participate, for which 7 days may be used. During this period, it is possible to consult with the study coordinator or principal investigator about the study by telephone. After 7 days, the attending physician calls the patient to ask if there are any remaining questions or uncertainties and whether he/she wishes to participate. If the patient wishes to participate, the informed consent form must be signed. The appointment for tracer administration and further study-related procedures are planned after the informed consent form has been signed by both patient and investigator.

Confidentiality
The data of the participant are recorded and analysed without any personal identifiers, by using coded information. The source documents and identification lists are archived in a secured facility. Permission for accessing data are documented per investigator.

Dissemination
Results of this study will be disseminated through national and international papers. The participants and relevant patient support groups will be informed about the results of the study.