First in-human intraoperative optical imaging of ...scripties.umcg.eldoc.ub.rug.nl/FILES/root/... · Intra-operatieve NIRF afbeelding van peritonitis carcinomatosa tijdens cytoreductie

First in-human intraoperative optical imaging of peritoneal carcinomatosis of colorectal origin using the VEGF-targeted near-infrared fluorescent tracer

bevacizumab-IRDye800CW: a feasibility study.

HI-LIGHT study, NL45588-042-13

Research Report of S.J. de Jongh, S1827626 10-09-2015, Groningen

Supervisor Prof. dr. Gooitzen M. van Dam [email protected] Co-supervisors Drs. Niels J. Harlaar Drs. Marjory Koller [email protected] [email protected] Department University Medical Centre Groningen (UMCG) Surgery – Surgical Oncology Image and Fluorescence-guided Surgery Working Group

University of Groningen (RuG) Faculty of Medical Sciences

Research Report S.J. de Jongh

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Preface and acknowledgements This report is part of my research internship at the department of surgical oncology of the University Medical Centre of Groningen (UMCG). Prof. dr. Go van Dam gave me the opportunity to participate in one of his innovative research projects concerning optical fluorescence imaging and its translation into the clinic. This report is the result of the research I performed over the past five months.

First of all, I would like to thank my direct supervisors, Marjory Koller and Niels Harlaar. Without their guidance and advice, my internship would not have been possible. I also would like to thank prof. dr. Go van Dam, who gave me this wonderful opportunity and motivated me to carry on with performing research. I am looking forward to the future collaboration as a PhD-student.

Then I would like to thank Dr. Gursah Kats, who supported me with the histopathological analyses and lend me her microscope. Also Dr. Wouter Nagengast, thank you for your critical notes and comments.

Finally, I want to thank all of my other colleagues during this research project: Elmire Hartmans, Jolien Tjalma, Matthijs Linssen and the people from the medical oncology lab. You were a great help on the work floor.


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Abstract

English abstract Introduction Optimal cytoreduction (CRS) in addition to Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) is essential for the curative treatment of peritoneal carcinomatosis (PC) of colorectal origin. Currently, differentiation between benign and malignant lesions can be very difficult and the diagnostic accuracy profile of tactile and visual inspection remains unknown. Better tumor detection could lead to improved radical cytoreduction. In this study we used near-infrared fluorescence (NIRF) imaging to enhance surgical vision. Methods Patients scheduled for CRS and HIPEC were included in this feasibility study (n=7). The NIRF tracer bevacizumab-IRDye800CW targeting VEGF-A was administered intravenously two days prior to surgery. After exposure of the abdominal cavity, two surgeons independently calculated the peritoneal cancer index (PCI) first by inspection and palpation, subsequently using the NIR intraoperative camera system. Biopsies were taken from fluorescent and non-fluorescent areas for ex-vivo validation and correlation of fluorescence. Results No (serious) adverse events related to tracer administration occurred in any of the seven patients. In total 87 peritoneal lesions were imaged and histologically analyzed. In 31 out of 58 fluorescent lesions cancerous cells were detected during histopathological analysis. All 23 non-fluorescent lesions were cancer negative, indicating a sensitivity of 100%. Additionally, in two patients this method detected cancerous tissue that was otherwise missed by inspection and palpation alone. In one patient this concerned a positive resection margin and in another patient a para-aortal lymph node metastasis was detected. Despite the relatively high false positive rate (47%), the PCI decreased 2.9 points on average per patient. Most false positive lesions appeared to be highly fibrotic and vascularized lesions or foreign inclusion bodies such as suturing material from previous surgery. Fluorescence was strongly correlated with vital tumor tissue ex-vivo at a microscopic level, with a tumor-to-normal ratio of 6,92 ± 2,47 (mean ± sd). Conclusion Intraoperative NIRF-imaging of PC during CRS is technically feasible and safe. This highly sensitive technique can lead to a more optimal cytoreduction by leaving suspicious lesions that are not fluorescent in situ. Moreover, stage migration might occur due to better patient selection, possibly enabling more patients to benefit from the curative CRS and HIPEC procedure. Therefore, this technique has to potential to prevent both over and under treatment. Additionally, NIRF-imaging enables the evaluation of possible positive resection margins, which might be of use in the treatment of locally advanced rectal cancer. We state that these preliminary results are promising for a subsequent phase II study.


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Nederlandse samenvatting Introductie Optimale cytoreductie voorafgaand aan hypertherme intraperitoneale chemotherapie (HIPEC) is van essentieel belang voor de curatieve behandeling van peritonitis carcinomatosa van colorectale origine. Differentiatie tussen benigne en maligne peritoneale laesies kan zeer lastig zijn. Bovendien is de diagnostische betrouwbaarheid van inspectie en palpatie vooralsnog onbekend. Betere intra-operatieve detectie van peritoneale metastasen zou kunnen bijdragen aan een radicale cytoreductie. In de huidige studie hebben wij nabij-infrarode fluorescente (NIRF) beeldvorming gebruikt ter uitbreiding van het zicht van chirurgen. Methode Patiënten waarbij een behandeling middels cytoreductie gevolgd door HIPEC gepland stond, werden geïncludeerd in de studie (n=7). Twee dagen pre-operatief werd de NIRF tracer bevacizumab-IRDye800CW gericht op VEGF-A intraveneus toegediend. Na het openen van het abdomen werd de peritoneale kanker index (PCI) door twee chirurgen onafhankelijk van elkaar gescoord. Allereerst werd dit gedaan middels de conventionele techniek van inspectie en palpatie, vervolgens met behulp van het intra-operatieve camerasysteem. Zowel van fluorescente als niet-fluorescente laesies werden biopten genomen voor ex-vivo validatie en correlatie met fluorescente signalen. Resultaten Er zijn geen tracer-gerelateerde ongewenste reacties of complicaties gezien in de zeven patiënten. In totaal zijn er intra-operatief 87 peritoneale laesies bekeken en geanalyseerd middels histologie. In 31 van de 58 fluorescente laesies werden kankercellen gevonden tijdens histopathologische analyse. Alle 23 niet-fluorescente laesies bleken negatief voor kanker cellen, wat resulteert in een sensitiviteit van 100%. Bovendien is met behulp van deze techniek bij twee patiënten tumorweefsel gedetecteerd, dat initieel door middel van inspectie en palpatie was gemist. In één patiënt betrof dit een positief snijvlak en in een andere patiënt een para-aortale lymfeklier metastase. Ondanks het relatief hoge aantal fout-positieven (47%) bleek de PCI te zakken met gemiddeld 2.9 punten per patiënt bij toepassing van deze techniek. Het merendeel van de fout-positieve laesies bleek zeer fibrotisch en gevasculariseerd weefsel te zijn, of bevatte lichaamsvreemd materiaal afkomstig van eerdere operaties. Fluorescentie was tijdens ex-vivo analyses duidelijk gecorreleerd met vitaal tumor weefsel op een microscopisch niveau. De tumor-tot-normaal ratio was 6,92 ± 2,47 (gem ± sd). Conclusie Intra-operatieve NIRF afbeelding van peritonitis carcinomatosa tijdens cytoreductie is technisch haalbaar en veilig. Deze sensitieve techniek kan bijdragen aan een optimale cytoreductie door verdachte, maar niet fluorescente laesies in situ te laten. Daarnaast kan de stagering van patiënten veranderen door een betere preselectie, waardoor potentieel meer patiënten curatief behandeld kunnen worden middels cytoreductie en HIPEC. Derhalve heeft deze techniek de potentie om zowel over als onder behandeling te voorkomen. Bovendien zou NIRF beeldvorming van toegevoegde waarde kunnen zijn in patiënten met een lokaal uitgebreid rectum carcinoom ter evaluatie van positieve snijvlakken. Wij stellen dat deze resultaten veelbelovend zijn voor een volgende fase-II studie.


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Table of contents 1 – PREFACE AND ACKNOWLEDGEMENTS 2

2 – ABSTRACT 3 2.1 – English abstract 3 2.2 – Nederlandse samenvatting 4

3 – TABLE OF CONTENTS 5

4 – LIST OF ABBREVIATIONS 6

5 – LIST OF TABLES AND FIGURES 6

6 – INTRODUCTION 7 6.1 – Background 7 6.2 – CRS and HIPEC procedure 7

6.2.1 –Peritoneal Cancer Index 8 6.3 – Need for real-time tumor-specific imaging technique 9

6.3.1 – Near-infrared fluorescence (NIRF) optical imaging 9 6.3.2 – Multispectral Fluorescence Reflectance Imaging (MFRI) camera system 10

6.4 – VEGF-A targeted optical fluorescent tracer: bevacizumab-IRDye800CW 11

7 – RESEARCH QUESTION 12 7.1 – Aim of the research project 12

8 – MATERIALS AND METHODS 13 8.1 – Trial design 13 8.2 – Injection of bevacizumab-IRDye800CW 13 8.3 – Surgical procedure and specimen handling 13

8.3.1. – Follow-up 14 8.4 – Ex-vivo analysis of tumor samples 14

8.4.1. – Imaging surgical specimen 14 8.4.2. – Bevacizumab-IRDye800CW quantification 14 8.4.4. – Immunohistochemical staining 16

8.5 – Statistical analysis 16

9 – RESULTS 17 9.1 – Patient characteristics 17 9.2 – (Serious) Adverse Events 17 9.3 – Intraoperative tumor-specific fluorescence imaging 17

9.3.1. – Peritoneal Cancer Index 18 9.4 – Postoperative histopathological analyses 19

10 – DISCUSSION 22

11 – CONCLUSION 25 11.1 – Future perspectives 25

12 – APPENDICES 26 12.1 – Immunohistochemical staining protocol for VEGF 26

13 – REFERENCES 28


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List of abbreviations CRC Colorectal Cancer CCD Charge-Coupled Device CT-scan Computed Tomography-scan DLS Diagnostic Laparoscopy EGFR Endothelial Growth Factor Receptor FFPE Formalin Fixated Paraffin Embedded FR-α Folate Receptor-α Folate-FITC Folate Fluorescein Isothiocynate H/E Hematoxylin and Eosin HIPEC Hyperthermic Intraperitoneal Chemotherapy PC Peritoneal Carcinomatosis PCI Peritoneal Cancer Index mAb Monoclonal Antibody MFI Mean Fluorescent Intensity MFR Multispectral Fluorescence Reflectance MFRI Multispectral Fluorescence Reflectance Imaging MMC Mytomicin-C NIR Near-infrared NIRF Near-infrared Fluorescence ROI Region Of Interest (S)AE (Serious) Adverse Event SOP Standard Operating Procedure SUSAR Suspected Unexpected Serious Adverse Reaction TBR Tumor-to-Background Ratio UMCG University Medical Centre of Groningen VEGF-A Vascular Endothelial Growth Factor – A VEGFR1 Vascular Endothelial Growth Factor Recepor-1 5-ALA 5-aminolevulinic acid

List of tables and figures Figure 1 Open ‘coliseum’ technique for HIPEC Figure 2 Peritoneal Cancer Index (PCI) Figure 3 Absorption coefficient of the light spectrum Figure 4 Image and schematic of the MFRI camera system Figure 5 Workflow ex-vivo analyses Figure 6 Intraoperative images of the MFR camera system Figure 7 Results Peritoneal Cancer Index (PCI) Figure 8 Mean Fluorescent Intensity (MFI) per group Figure 9 Mean Fluorescent Intensity (MFI) per patient Figure 10 Correlation of fluorescence, tumor margins and VEGF Figure 11 Representative samples Table 1 Scan-settings for the Odyssey scanner. Table 2 Demographics and individual data of the patients.


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Introduction

Background Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide (1). The majority of patients is diagnosed in a relatively advanced stage of disease, due to the fact that presentation is often asymptomatic and non-specific. The advanced stage is characterized by distant metastases that occur mainly in lungs, liver and peritoneum. Distant metastases are present in one fifth of patients at first presentation (2), thereby reflecting a poor prognosis with a five year survival of 5-6 months (3).

Peritoneal carcinomatosis (PC), defined as metastases limited to the peritoneal surface, occurs in 3-28% of all patients with CRC (4). Data on the exact incidence of PC varies widely, possibly due to differences in methods used to detect or define tumor deposits. In approximately 25% of patients with PC of colorectal origin, the metastases are limited to the peritoneal surface. This suggests that the tumor biology of PC is different from systemic metastasized disease and might even imply that PC can be regarded as a first step of local tumor dissemination (5).

Currently, the three pillars of CRC treatment are surgery, chemotherapy and radiotherapy. For metastatic CRC, palliative chemotherapy used to be the only remaining treatment option, with a median survival of 12.6 months (4). With the suggestion that PC should not be regarded and treated as generalized metastatic disease, the treatment strategy has changed considerably. The cornerstone of this change was the introduction of cytoreductive surgery (CRS) followed by intraoperative flushing of the abdominal cavity with heated intraperitoneal chemotherapy (HIPEC) (6). This potentially curative therapy for PC of colorectal origin significantly improved survival to 22.2 months (7,8).

Completeness of cytoreduction and the extent of the disease are important prognostic factors that influence the benefit of the procedure. Irradical resected tumor tissue or local recurrence of disease negatively affect survival and may require additional invasive treatments. This emphasizes that complete removal of all macroscopic visible tumor nodules is essential for treatment success (5) as disease-free resection margins increase the 5-year survival to 45% (7). However, peritoneal metastases are difficult to detect and can be sometimes not be differentiated from normal healthy tissue.

Therefore, to improve the success rate of cytoreduction, an intraoperative ‘red-flag’ technique is needed to assist in optimal tumor detection. For that purpose, we aim to use targeted near-infrared fluorescence (NIRF) optical imaging of tumor lesions, which will be elucidated further in this report.

CRS and HIPEC procedure The fundamentals for peritonectomy procedures and subsequent preparation techniques of the abdomen for perioperative HIPEC were laid by Sugarbaker in the 1980’s (6). In short, an incision from xyphoid to pubis is made to perform CRS. After estimating the extent of peritoneal disease, surgeons aim to remove all macroscopic visible tumor load.

Figure 1: Open ‘coliseum’ technique for HIPEC. The abdominal wall is elevated, creating a funnel in which the chemotherapy circulates through inflow and outflow lines, that are attached to a pump and heating unit.


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During HIPEC, the abdominal cavity is flushed with a high concentration of heated chemotherapeutics to eradicate all microscopic disease (Fig. 1). Hyperthermia shows a synergism with certain cytostatic drugs and is therefore applied to enhance tumor penetration. (9,10). Using a direct and local way of delivering the chemotherapy, high regional concentrations can be achieved, while keeping systemic drug levels low (7). After removal of the chemotherapy, surgeons restore the bowel continuity by making anastomosis, whenever necessary.

The CRS and HIPEC procedure is considered a high-risk procedure. Reported mortality and grades III to IV morbidity rates at 30 days were 3% and 31% respectively (11). Other studies reported even higher mortality and morbidity rates (12). Therefore, patients usually stay for several days at the intensive care unit for observation after surgery.

Peritoneal Cancer Index CRS followed by HIPEC is a curative treatment strategy when implemented on well-selected patients. Careful preoperative assessment of distant metastases, technical resectability and the extent of peritoneal disease is therefore needed.

The imaging modality of first choice for staging and localizing the primary tumor is a Computed Tomography scan (CT-scan) of the thorax and abdomen, on which distant metastases and invasive tumor-growth can be detected. However, its accuracy for detecting peritoneal metastases is low, due to limited specificity and resolution. Predominantly, the small size of tumor deposits (typically less than 1 cm) negatively affects sensitivity. Other imaging modalities, such as the Positron Emission Tomography (PET) scan and Magnetic Resonance Imaging (MRI) scan, also lack these properties (13,14).

Therefore, a diagnostic laparoscopy (DLS) is performed prior to surgery to estimate the peritoneal cancer index (PCI). The PCI represents the extent of the disease and is scored by estimating the number and size of all suspicious lesions (Fig. 2). A PCI greater than 20 is regarded as a contraindication for surgery, as the risks of surgery then outweigh the benefits (i.e. prognosis) (15). Preoperative evaluation of the PCI during DLS is known to improve patient selection for the CRS and HIPEC procedure (16).

Figure 2: Peritoneal Cancer Index (PCI). The abdomen is divided into 13 regions. Each region is carefully inspected and scored by the surgeon. A score of 0 to 3 is given, depending on the number and size of the peritoneal lesions, with a maximum score of 39.


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However, in the presence of adhesions due to previous surgery, a limited view of the abdominal cavity can cause technical difficulties for reliable PCI assessment. Therefore, the PCI-score is always confirmed directly after laparotomy, as this is considered to be the golden standard.

Need for real-time tumor-specific imaging technique Surgeons use imaging modalities (e.g. CT/MRI/PET scans) for preoperative planning, tumor detection and guidance during surgery. Current cancer diagnostics are mainly based on the visualization of tumors, and to a significantly lesser extent on their heterogeneity and molecular composition. However, cancer classification is still largely based on the morphology of tumor cells.

Most imaging modalities are not practical for intraoperative use, nor tumor-specific and lack sensitivity and specificity for detection of PC. Visual and tactile inspection is currently the only tool available for intraoperative detection of PC, but its diagnostic accuracy remains unknown. Molecular changes cannot be detected if the colors or physical properties of lesions are similar, making it very difficult to distinguish tumor lesions from for example benign scar tissue.

Complete cytoreduction and tumor-free resection margins are associated with an increase of survival rates (15,17). Considering that tumor recurrence rates after the CRS and HIPEC procedure are reported of up to 46% (18), novel molecular and tumor-targeted diagnostic and therapeutic tools for tumor detection are urgently needed.

In recent years, there is increased interest in the clinical application of intraoperative optical imaging techniques. Significant progress is made in the development of real-time fluorescence imaging systems and optical contrast agents (19). We aim to use intraoperative optical fluorescence imaging using a fluorescent and tumor-targeted tracer, to detect and remove tumor deposits on a more microscopic level, so both staging and CRS will be more optimal.

Near-infrared fluorescence (NIRF) optical imaging The principle of fluorescence imaging is based on the phenomenon where a fluorescence dye absorbs light, then emits light with a different wavelength. When an excitation source sends out a bundle of high-energy light with a specific wavelength, a fluorophore can absorb this energy, thus reaching an excited state. After releasing its stored energy, the fluorophore than emits a low-energy photon with a longer wavelength. This fluorophore can either be an intrinsic tissue component (auto-fluorescence) or an injected external fluorescent agent (targeted-fluorescence) (20).

Fluorescence imaging is influenced by multiple factors, including light scattering (i.e. changes in photon direction), light absorption and fluorescence emission. Light absorption and scattering are mainly dependent on tissue properties and the wavelength of the excitation source. All sorts of tissue components can absorb fluorophores, but water, lipids and hemoglobin are the most relevant photon absorbers. Less absorption and scattering occurs

Figure 3: Absorption coefficient of the light spectrum. The absorption coefficient of light in tissue is dependent on wavelength, and results from absorbers such as hemoglobin, lipids and water. Visible light (400 to 650 nm) displays significantly higher tissue absorption than light from the near-infrared spectrum (650 nm to 900 nm).


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in light with higher wavelengths (Fig. 3). Therefore, in the near-infrared (NIR) light spectrum penetration depth increases to more than a centimeter, depending on the type of tissue (20,21).

The near-infrared (NIR) light spectrum (650 – 900 nm) thus provides favorable properties for fluorescence imaging: low tissue absorption, high penetration depth and in addition, relatively low autofluorescence. This results in higher signal-to-background ratios (SBR). Moreover, NIR optical imaging forms a low risk to the patient compared to other molecular imaging techniques, due to the use of non-ionizing radiation. It is cost-efficient in terms of preparation of molecular probes and the detection hardware is relatively simple to operate (22). Compared to other imaging modalities, fluorescence imaging techniques fit well in an operation room and enables real-time feedback to the surgeon.

Multispectral Fluorescence Reflectance Imaging (MFRI) camera system Because the human eye is not sensitive for light in the NIR range, a special camera system is required to detect fluorescence signals. In collaboration with the Technical University of Munich, a real-time optical imaging system has been developed. Themelis et al have previously described this Multispectral Fluorescence Reflectance Imaging (MFRI) system extensively (23) (Fig 4).

Briefly, this camera system uses video-rate concurrent multi-spectral NIRF imaging (21,24). After intravenous injection of an optical contrast agent, an external light source with a defined wavelength is used to illuminate a subject. As light propagates through the tissue, it will excite both surface and subsurface localized fluorescent contrast agents. Immediately after absorption of excited fluorophores, the contrast agent responds by releasing low-energy light of a longer wavelength, which can subsequently be detected by higly sensitive charge-coupled device (CCD) cameras (Fig. 4) (25).

This MFRI system has safely been used intraoperatively for real-time tumor detection, margin assessment and lymph node mapping in multiple studies. Van Dam et al showed the potential application for intraoperative tumor-specific fluorescence imaging in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery

Figure 4: Image and schematic of the MFR camera system. Reproduction of the intraoperative MFR camera system, currently wrapped in sterile transparent drapes (a). Schematic of a MFRI camera system capable of capturing three imaging channels in real time simultaneously: color reflectance, fluorescence and intrinsic excitation (b). A halogen light source is used for white light illumination and a diode laser for fluorescence excitation. MFR(I): multispectral fluorescence reflectance (imaging), CCD: charge-coupled device.


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(26). The same imaging technique has also been used for breast cancer surgery (27) and for sentinel lymph node mapping in cervical (28) and vulvar (29) cancer.

VEGF-A targeted optical fluorescent tracer: bevacizumab-IRDye800CW Depending on which receptors are overexpressed in a tumor, several targeted optical contrast agents have been identified. CRC tissues are characterized by the up-regulation of angiogenesis-related molecules (30). A potent angiogenic growth factor commonly involved in tumor-induced angiogenesis is the Vascular Endothelial Growth Factor-A (VEGF-A). By activating a down-stream signaling pathway it enhances tumor growth and tumor-induced neovascularization. VEGF-A is up-regulated in colorectal cancer cells (31) as well as in metastasis of CRC, such as PC (32), suggesting this might be a suitable candidate for targeted-based therapies

The registered monoclonal antibody (mAb) bevacizumab that targets VEGF, is widely used in the clinic. This mAb is known to neutralize all VEGF-A isoforms and could therefore be used as an imaging agent (33). Successful and specific radiolabeled VEGF-imaging with 111Indium and 89Zirconium has been performed in patients with stage III/IV melanoma (34), liver metastases (35), renal cell cancer (36,37), neuroendocrine tumors (38) and breast cancer (39). However, despite showing good results, fluorescent-labeled tracers are preferable in an intraoperative image-guided surgery setting due to the use of non-ionizing radiation.

The NIR fluorophore IRDye800CW (wavelength 800 nm) is a very promising signal-generating molecule. Previously, single administration of IRDye800CW did not cause any toxicity in doses up to 20 mg/kg in a preclinical mouse toxicity study (40). Terwisscha van Scheltinga et al already applied antibody-based tumor detection using intraoperative optical imaging in preclinical in-vivo mouse models (41). They labeled the monoclonal antibody bevacizumab with the NIR fluorophore IRDye800CW to the VEGF-targeted fluorescence tracer bevacizumab-IRDye800CW. Using real-time intraoperative imaging they were able to detect tumor lesions at even the sub-millimeter level in mouse models with peritoneal tumor dissemination.

These preclinical findings using bevacizumab-IRDye800CW as a VEGF-targeted optical fluorescent tracer encouraged future clinical studies regarding intraoperative image- guided surgery in cancer patients. Bevacizumab-IRDye800CW has already been used clinically at the University Medical Centre Groningen (UMCG) in breast cancer surgery for margin assessment, evaluation of lymph node metastasis and primary tumor detection (NCT01508572). Other ongoing in-human endoscopic studies make use of this tracer for MFR optical imaging of patients with rectal cancer (NCT01972373), familial adenomatous polyposis (NCT02113202) and premalignant esophageal lesions (NCT02129933). Preliminary results of all of these studies show that the application of bevacizumab-IRDye800CW is safe and feasible, providing a solid base for the intraoperative detection of PC of colorectal origin.


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Research question

Aim of the research project Targeted NIRF optical imaging has already widely been applied for intraoperative real-time tumor detection and margin assessment. Preliminary results from ongoing trials using bevacizumab-IRDye800CW and the MFR camera system reported sufficient tumor-to-normal ratios and highly sensitive tumor detection. Therefore we hypothesize that this technique may be of additional value when applied during CRS in patients with PC of colorectal origin, as optimal tumor detection and removal is crucial for treatment outcome. In this research project, the primary objective was to demonstrate the safety and feasibility of intraoperative NIRF imaging using the fluorescent tracer bevacizumab-IRDye800CW targeting VEGF-A. Therefore, we aimed to determine ex-vivo:

• The specific tumor-uptake of becavizumab-IRDye800CW in peritoneal metastases of colorectal origin;

• The correlation of fluorescence signals with vital tumor tissue; • The mean fluorescent intensity (MFI), subsequently the tumor-to-normal ratio of

signals from bevacizumab-IRDye800CW in peritoneal metastases and normal tissue; • The diagnostic accuracy in terms of sensitivity and specificity of the MFRI camera

system for tumor detection using bevacizumab-IRDye800CW compared to standard histopathological examination.

We hypothesize to find a high tumor-specific uptake in peritoneal metastases, whereas non-tumorous lesions will show a negligible level of fluorescence. The sensitivity is expected to be high, indicating that non-fluorescent lesions correspond with non-tumorous or normal tissue. Specificity might be influenced by the fact that VEGF is an angiogenic growth factor and therefore also present in for example blood vessels. As VEGF-A is upregulated in tumor tissue, we hypothesize to find a significant difference in mean fluorescent intensity between tumor and normal tissue. Since bevacizumab-IRDye800CW uptake can be quantified ex vivo, the level of fluorescence in tumor and normal tissue can be determined.


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Material and methods

Trial design The Hi-Light study is a first-in-human, non-blinded, prospective, single center feasibility study. Bevacizumab-IRDye800CW was administered intravenously to a total of seven patients scheduled for the CRS and HIPEC procedure for treatment of PC of colorectal origin at the UMCG. For all patients injected with the tracer, intraoperative MFR imaging was performed. Intermediate evaluation was carried out after five patients. The study was to be suspended if no accumulation of bevacizumab-IRDye800CW in the tumor tissue could be demonstrated in these patients (5/5). The study was also to be suspended immediately if any suspected unexpected serious adverse reaction (SUSAR) related to the administration of the tracer occurred in any of the patients. All SUSAR and serious adverse event (SAE) were reported to the Medical Research Ethics Committee (METc, in Dutch: medisch etische toetsing commissie) and to the Data Safety Monitoring Board (DSMB) of the UMCG. The full trial aimed at evaluation of 10 patients for the accumulation of bevacizumab-IRDye800CW in tumor tissue. The final result was considered successful if seven out of ten patients did have targeting of tumor tissue. Patients were eligible if they were ≥ 18 years of age and had a WHO performance score of 0-2. They had to be admitted to the UMCG to undergo CRS for PC of colorectal origin. Signed written informed consent had to be obtained. Exclusion criteria were the presence of distant metastases, other malignancies (except for adequately treated basocellular carcinoma of the skin or in situ carcinoma of the cervix uteri), other concurrent uncontrolled medical conditions, clinically significant cardiac disease or myocardial infarction within the past 12 months and pregnancy or lactation. Patients with prior malignancies had to be disease-free for at least five years. The METc of the UMCG approved the study.

Injection of bevacizumab-IRDye800CW Clinical grade bevacizumab-IRDye800CW was produced in the Good Manufacturing Practice (GMP) facility of the UMCG. Bevacizumab (Roche AG) and IRDye800CW-NHS (LI-COR Biosciences Inc.) were labeled under regulated conditions.

It is known from studies using radiolabeled bevacizumab that the uptake of the tracer increases in time (34). In the present study, MFR imaging was performed two days after administration of bevacizumab-IRDye800CW. Patients received 4.5 mg bevacizumab-IRDye800CW intravenously in a bolus injection 2 days prior to surgery. After injection, the infusion line was flushed with 0.9% sodium chloride. The total dose of 4.5 mg bevacizumab is considered a microdose, compared to the therapeutic dose of 5-10 mg/kg every two weeks. Before and up to one hour after tracer administration patients were monitored for the occurrence of any adverse events related to the tracer.

Surgical procedure and specimen handling Two surgeons performed cytoreductive surgery for patients with PC of colorectal origin according to standard procedures. First, the abdomen was prepared to obtain a maximum field-of-view for estimation of the PCI. Second, both surgeons scored the PCI independently. Therefore one surgeon left the operating room. In the meanwhile the other surgeon estimated the PCI by visual and tactile inspection alone. Next, the PCI was determined when guided by the MFRI camera system. This system was operated at a distance suitable for detection of the fluorescence signal (on average 15 cm above the operating field). The presence of a


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fluorescence signal was determined in tumor and peritoneal lesions. Both surgeons switched places and this procedure was repeated.

Subsequently, cytoreductive surgery was performed using visual inspection and palpation. Thereafter, the abdominal cavity was inspected using the MFRI camera system. In case initially missed fluorescent lesions were detected, these were removed additionally if technically safe as judged by the surgeons. Real-time fluorescence images and videos of the operative field were made.

After cytoreduction, the abdominal cavity was flushed with heated intraperitoneal chemotherapy (Mitomycin C at 41°C) conform the open ‘coliseum’ technique for 90 minutes. Patients were monitored on the intensive care unit as a standard procedure after a HIPEC procedure. Next, ex-vivo imaging with the MFRI camera system was performed on the surgical specimen.

Follow-up Adverse events occurring within 2 weeks after surgery were reported to the METc and DSMB of the UMCG, according to standard procedure as described in the case report form.

Ex-vivo analysis of tumor samples

Imaging surgical specimen After surgical removal, the fresh surgical specimen was imaged directly after surgery. Fluorescence signals were determined in tumor and surrounding healthy tissue using the MFRI camera system. Both non-fluorescent lesions suspect for tumor based on visual inspection and palpation and fluorescent lesions were marked with colored pins, to determine the accuracy of the MFRI camera system.

The surgical specimen was fixated in formalin and cut into bread-loaf slices. The pathologist was blinded for fluorescence signals during histopathological analysis. Subsequently, these slices were processed to formalin fixed and paraffin embedded (FFPE) tissue blocks as a routine procedure. All tissue was handled and stored in the dark as much as possible to prevent photo bleaching.

Bevacizumab-IRDye800CW quantification A standard operating procedure (SOP) was developed to measure fluorescence intensities in a reproducible and non-biased way, as illustrated in Figure 6. Odyssey scanning All available FFPE blocks of peritoneal lesions were selected for further analysis. These FFPE blocks were scanned using the Odyssey® CLx fluorescence flatbed scanner (LI-COR Biosciences Inc.) for assessment of intensity and localization of fluorescence signals (Fig. 6 panel A and B). This scanner is specifically designed for detection of the IRDye800CW. A pre-scan was made to check for oversaturation in images, as this negatively influences results. Scan settings were used according to Table 1. Cutting of paraffin sections FFPE tissue blocks of peritoneal lesions were cut into multiple paraffin tissue sections of 4

Table 1: Scan-settings for the Odyssey scanner.


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and 10 micrometer (µm). These sections were mounted on silane-coated slides and dried overnight at 37°C. Tissue slides were deparaffinized with xylene for 10 minutes and air-dried for 15 minutes. Subsequently, the 10µm tissue slides were scanned on the Odyssey (Fig. 6 panel C and D, settings in table 1). Hematoxylin and Eosin (H/E) staining The 4µm slides were used to identify tumor tissue and define regions of interest (ROI). Therefore, 4µm slides and the previously scanned corresponding 10µm slides were H/E stained manually (Fig. 6 panel E to F). Hematoxylin staining was performed for 10 minutes and after a washing step with water, eosin staining was performed for 2 minutes. Thereafter, two-times washing steps with 96% and 100% alcohol were performed. Thin glass coverslips were mounted on the slides using the Eukitt® quick-hardening mounting medium and air-dried overnight. H/E stained slides were digitalized using a digital slide scanner (Hamamatsu®). Defining regions of interest Tumor lesions were identified and highlighted at a microscopic level on the 4µm H/E stained slides by an experienced pathologist specialized in gastroenterology. Subsequently, the ROIs defined on the 4µm H/E stained slides were matched digitally with the corresponding 10µm H/E stained slides in an exact overlay. On the fluorescence scans of the 10µm slides, MFIs of these predefined ROIs could be measured (Fig. 6 panel H to J).

Microscopic evaluation of all 4μm slides; tumor localization by pathologist

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4μm slideNIRF image(Odyssey)

FFPE-blockColor image

FFPE-blockNIRF image(Odyssey)

10μm slideNIRF image(Odyssey)

10μm slideH/E stained

4μm slideH/E stained

ROI: tumor

ROI: no tumor

ROI: tumor(ImageJ)

ROI: no tumor(ImageJ)

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E

F

H

I!

GC

B

D

J!

K

Figure 5: Workflow ex-vivo analyses. A formalin fixed paraffin embedded (FFPE) tissue block (A) was scanned for fluorescence (B) on the Odyssey CLx NIR fluorescence scanner, 800nm. This FFPE-block was cut into paraffin sections from 4µm and 10µm, which were subsequently scanned using the Odyssey scanner (C, D) and H/E stained afterwards (E, F). A pathologist evaluated all 4µm slides for tumor localization with a microscope (G). Regions-of-interest (ROIs) were defined on the 10µm H/E stained slide and colored black (H, I). This predefined selection was then transformed to ImageJ for quantification of the fluorescence signal using these standard ROI’s (J, K).


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Quantifying fluorescence intensity The MFI is among other factors, such as tissue scattering and absorption properties, dependent on the thickness of a tissue part, as multiple cell layers imply more tracer uptake. Comparable measurements could not be obtained from FFPE-blocks, as these vary in thickness. Due to the limited layer of cells, there was relatively low fluorescence signal in the 4µm slides. Intensity settings from the Odyssey scanner had to be increased for optimal scanning properties of the 4µm slides. As a consequence, this also resulted in higher background signals, which created too much background noise for measurements. Therefore, to quantify fluorescence and compare the MFI of both tumor lesions and non-tumorous lesions, Odyssey scanned 10µm tissue slides were used for quantification.

Fluorescence images from the Odyssey scanned 10µm slides and correlating regions-of-interest (ROIs) were imported in ImageJ (1.48v) for measurement of the MFI. Using auto-threshold the black-colored ROIs were selected and could be overlaid on the fluorescence image (Fig. 6 panel J to K). This was applied for tumor ROIs as well as non-tumorous ROIs. Afterwards, MFIs of tumor slides were statistically analyzed with MFIs of non-tumorous slides.

Immunohistochemical staining Next to standard H/E staining, additional immunohistochemical (IHC) staining was performed for VEGF, to correlate the fluorescence signal with VEGF expression. The immunohistochemical staining protocol was added supplementary in appendix 9.2. For VEGF staining, the rabbit polyclonal antibody RB-9031 was used.

Statistical analysis Data-analysis of the outcome measures was exploratory in nature. The secondary outcome measures included:

• Measurement of the fluorescent intensity of signals from bevacizumab-IRDye800CW ex-vivo, thereby determining the sensitivity and specificity.

• Assessment of tumor-to-background ratios by comparing the presence of the mean fluorescent intensity of peritoneal cancer tissue and normal tissue.

For the determination of Tumor-to-Background ratios (TBRs) and MFIs obtained by fluorescence flatbed scans in regions of tumor, stroma and healthy surrounding tissue, the Mann-Whitney test for unpaired data was used. All MFIs were checked per patient individually for significant differences with the non-parametric Kruskal Wallis test for independent variables in multiple groups. Data were presented as a mean ± standard deviation (s.d.) in case of a normal distribution, whereas median ± s.d. is used in case of a skewed distribution. A two-sided P value of less than 0.05 was considered significant (SPSS, version 19.0; IBM).


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Results

Patient characteristics A total of 7 patients were enrolled in the study. Patient and tumor characteristics are summarized in Table 2. The mean age of all patients was 58 ± 15,7 years (mean ± s.d.). Three patients were admitted with a primary tumor with peritoneal metastasis, four patients with recurrence of disease. All tumors were adenocarcinomas, as confirmed by histopathology. All patients had non-favorable characteristics varying from stage II-IV. The mean PCI as estimated during DLS was 6,4 ± 3,2 (mean ± s.d.). Patient 5 and 7 were treated neoadjuvantly with chemoradiation and chemotherapy respectively. Adjuvant individualized therapy was given to five patients and consisted of a fluoropyrimidin (capecitabin/5-flourouracil/ leucovorin) and/or oxaliplatin, based on recommendations of a multidisciplinary team and patients’ preferences.

Sex Age (y)

ASA Tumor PCI (DLS)

Primary tumor TNM Site Stage Differentiation Adj. Therapy

AE SAE

Patient 1 F 67 2 Recurrence 6 pT4 N0 M0 Asc. colon II Poor Yes + – Patient 2 M 68 2 2nd Recurrence 10 pT4 N1 M0 Cecum II Well Yes + – Patient 3 M 26 1 Primary 9 pT4 N2 M1 Sigmoid IV Poor Yes – – Patient 4 M 70 2 Recurrence 5 pT3 N1 M0 Sigmoid III Poor Yes + – Patient 5 M 61 2 Recurrence 9 pT3 N0 M1 Rectum IV Well No + + Patient 6 F 65 3 Primary 5 pT4 N1 M1 Asc. colon IV Moderate Yes + – Patient 7 F 49 2 Primary 1 pT3 N0 M1 Sigmoid IV Moderate No + +

(Serious) Adverse Events No adverse or serious adverse events related to tracer administration occurred in any of the patients. The METc and DSMB of the UMCG did not attribute the two SAEs to tracer administration. Patient 5 suffered from an abdominal sepsis, requiring a re-laparotomy and antibiotic treatment. This was complicated by an entero-atmospheric fistula and an abdominal fluid collection, which was drained CT-guided. Patient 7 died from an asystole 3 days post-operatively, also not attributable to the tracer.

Intraoperative tumor-specific fluorescence imaging All patients underwent the CRS and HIPEC procedure. Use of the intraoperative imaging system did not interfere with the standard surgical procedure. Real-time image-guided excision of fluorescent tumor deposits smaller than 5 mm was feasible (data not shown). Fluorescence was detectable intraoperatively in all patients injected with bevacizumab-IRDye800CW. An example of an intraoperatively detected peritoneal metastases as displayed by the MFR camera system is illustrated in Figure 6 (panel A to C). Additionally, in two patients this method detected cancerous tissue that was otherwise missed by inspection and palpation alone. In one patient this concerned an additional fluorescent para-aortal lymph node metastasis, as confirmed by histopathology (Fig 6, panel D to F). In another patient a fluorescent signal was detected at the resection plane near the sacral bone. Histopathological analysis of biopsies taken from this fluorescent area confirmed a positive resection margin (Fig. 6, panel G to I).

Table 2: Demographics and individual data of the patients. N = 7 patients. M, Male; F, Female; y, years; ASA, American Society of Anesthesiologists; PCI, PCI, Peritoneal Cancer Index; DLS, Diagnostic Laparoscopy; TNM, Tumor Nodes Metastases; Asc, Ascending; Adj, Adjuvant; (S)AE, (Serious) Adverse Event; +, present; –, absent (see text for further explanation).


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A blinded pathologist used standard histopathology to analyze a total of 87 lesions that were detected while imaging the complete surgical specimen directly after surgery. All of the 23 non-fluorescent lesions that were imaged were cancer negative, indicating a sensitivity of 100%. In 31 out of the 58 fluorescent lesions cancerous cells were found. Due to this relatively high false positive rate, the specificity was 52%. Fluorescence in most of the false positive lesions could be attributed to the presence of foreign inclusion bodies such as suturing material from previous surgery, reactive lymph nodes, fibrosis, highly vascularized or scar tissue.

Peritoneal Cancer Index The PCI detected when guided by tumor-specific fluorescence imaging was significantly lower (P = 0.018, Wilcoxon signed-rank test) in all patients compared to visual observation and palpation alone. The PCI decreased with 2.9 points on average per patient (Fig. 7).

Figure 7: Results Peritoneal Cancer Index (PCI). PCI scored by two surgeons using inspection and palpation alone (dark gray, PCI) compared to the PCI scored when guided using additional Multispectral Fluorescence Reflectance Imaging (light gray, PCI + MFRI).

Figure 6: Intraoperative images of the MFR camera system. Images are shown from a peritoneal metastasis (panel A to C), para-aortal lymph node metastasis (panel D to F) and positive resection margin (G to I), all confirmed by histopathology (indicated with a white arrow). Both the para-aortal lymph node and the positive resection margin were initially missed by visual and tactile inspection, however detected using the MFR camera system. Physiological excretion of bevacizumab-IRDye800CW was seen via the ureter (panel G to I).


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Postoperative histopathological analyses All available tumor positive and negative peritoneal lesions were analyzed ex-vivo for validation and correlation of tumor-specific fluorescence. Tumor deposits could be visualized ex-vivo with a resolution of ≤ 3 mm (data not shown). An experienced pathologist analyzed a total of 79 peritoneal lesions using standard histopathology. Of these lesions, 33% has been issued as peritoneal metastases, whereas 67% did not contain any tumor cells. Additional biopsies from peritoneal lesions obtained from previous surgery, that therefore did not contain the tracer, were evaluated as negative controls (4 peritoneal metastases and 3 tumor negative lesions).

Quantitative analysis of fluorescence is shown in Figure 8 and 9. There was a significant difference in mean fluorescent intensity of tumor lesions (i.e. peritoneal metastases) versus non-tumorous lesions (P < 0.001, Mann Whitney U test). The MFI of peritoneal metastases was 6338 (n=26 lesions), whereas the MFI of non-tumorous lesions was 748 (n=53 lesions). Comparison of the MFI of peritoneal metastases to tumor negative lesions revealed a mean tumor-to-background (TBR) ratio of 6,92 ± 2,47 (mean ± sd).

Peritoneal metastases from patients administered with the tracer showed a significantly higher level of fluorescence compared to negative control tissue without the tracer (P = 0.003, Mann Whitney U test). The MFI of negative control tissue was 1703 for the peritoneal metastases (n=4) and 1129 for the non-tumorous lesions (n=3). The relatively high fluorescence signal from tumor positive negative controls was attributed to fibrosis and necrosis, which displays proportions of relatively high autofluorescence.

However, non-tumorous or healthy tissue also showed fluorescence signal in-vivo and ex-vivo, as illustrated in Figure 8 (outliers and extremes). Seven non-tumorous lesions displayed values outside the interquartile range. Upon detailed analysis, three of these lesions appeared to contain foreign body material, most likely originating from previous surgery (i.e.

Figure 8: Mean Fluorescent Intensity (MFI) per group. Quantification of fluorescence for peritoneal metastases and tumor negative lesions compared to negative control tissue. MFIs of tumor (+) lesions were significantly higher than MFIs of non-tumor (-) lesions and negative controls (P < 0.001 and 0,003 resp.). Outliers/extremes from non-tumorous lesions were mainly due to foreign body material (old suturing material) or highly fibrotic and vascularized lesions. MFI is displayed as median values. Outliers: values > 1,5 interquartile range (IQR), extremes: values > 3 IQR.

Figure 9: Mean Fluorescent Intensity (MFI) per patient. Patient 2, 3, 4 and 6 presented with histopathological confirmed peritoneal carcinomatosis. Patient 1 and 7 presented without any detected peritoneal metastases (no PC), whereas in patient 5 no tumor lesions at all could be detected (no tumor). MFIs for tumor positive versus tumor negative lesions are significantly different for patient 2,3 and 4 (P = 0.037, 0.020, < 0.001 resp.). Tumor (+) lesions were mutually non-significantly different (P = 0.057.)


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old suturing material). These finding were confirmed intraoperative, where suturing material appeared to be highly fluorescent as well. The relatively high fluorescence from the remaining lesions could be attributed to fibrosis and high vascularization. As mentioned previously, this finding was also observed in the tumor positive lesions that were evaluated as negative control.

Differences in MFIs for tumor positive versus tumor negative lesions per patient were significant for patient 2, 3 and 4 (Fig. 9, P = 0.037, P = 0.020, P < 0.001 respectively, Mann-Whitney U test). Although in patient 6 this difference was not significant (P = 0.083, Mann-Whitney U test), the MFIs of all peritoneal metastases alone did not vary significantly either (P = 0.057, Kruskal Wallis test). Two patients presented without peritoneal metastasis, despite the positive PCI estimated during diagnostic laparoscopy. However, in the first patient positive lymph node metastases were detected. In one patient no malignancy was detected during histopathological analysis. This indicates that differentiation between positive and negative peritoneal lesions using only inspection and palpation can be very difficult.

The relationship between fluorescence signals and tumor localization could be illustrated at a microscopic level, as determined by an experienced pathologist using H/E staining. Fluorescence signals correlated perfectly with vital tumor tissue of peritoneal metastases. Normal gastrointestinal mucosa displayed proportions of relatively low fluorescence, which also correlated with VEGF expression in this lesion (Fig. 10). Representative samples of peritoneal metastases, tumor negative peritoneal lesions and negative controls (tumor positive tissue not containing tracer) are illustrated in Figure 11. A perfect correlation of fluorescence, tumor margins and VEGF expression was noted in these lesions. However, additional VEGF staining has to be performed for exact correlation with vital tumor tissue and fluorescence on all peritoneal lesions, as this has not been done yet.

Figure 10: Correlation of fluorescence, tumor margins and VEGF. A perfect correlation is shown between tumor margins, fluorescence signals and VEGF expression on an H/E-stained image (A), NIRF image (B) and VEGF stained image (C) of a 4µm slide from a peritoneal metastasis. Yellow outline represents tumor tissue (T, tumor) as confirmed by histopathology. Normal intestinal mucosa (N, normal) is visible, clearly showing no fluorescence. H/E, haematoxylin and eosin; NIRF, near-infrared fluorescence; VEGF, vascular endothelial growth factor.


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Figure 11: Representative samples. Representative samples of 4 peritoneal metastases, 2 negative peritoneal lesions and 2 negative controls (tumor negative without tracer) are shown. From left to right: a color image of FFPE-blocks, which is scanned for NIRF signal using the Odyssey scanner and subsequently cut to 10µm slides. These 10µm slides were Odyssey scanned again and subsequently H/E-stained and VEGF-stained according to the workflow of the ex-vivo analysis shown in Figure 5. Tumor borders are outlined in yellow. All NIRF images are scaled for brightness and contrast (min: 20, max: 40000). FFPE: formalin fixated paraffin embedded, NIRF: near-infrared fluorescence, H/E: haematoxylin and eosin, VEGF: vascular endothelial growth factor.


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Discussion Colorectal cancer is one of the most common and deadly cancers worldwide. Complete cytoreduction and tumor-free resection margins are associated with an increase of survival. (15,17). To improve cytoreduction, an intraoperative ‘red-flag’ technique is urgently needed, to assist in optimal tumor detection. In this first in-human proof-of-concept study, we demonstrate the feasibility of intraoperative optical fluorescence imaging in patients with PC of colorectal origin to improve tumor detection and enhance surgical view. The intravenously administered tracer bevacizumab-IRDye800CW targeting VEGF was found to be safe and tracer uptake was shown intraoperatively in PC of colorectal origin with a high sensitivity. Ex-vivo analysis of excised specimens, FFPE tissue blocks and tissue slides confirmed tumor-specific uptake with a TBR of 6,92 ± 2,47 (mean ± s.d.).

Fluorescence-guided surgery is emerging as a sensitive intraoperative imaging technique to aid surgeons in achieving a complete cytoreduction. Despite the fact that this phase I feasibility study was not designed to change the standard of care, opportunities were noted to improve surgical decision-making based on fluorescence-guided imaging. A positive resection margin and additional lymph node metastasis were detected using fluorescence, which were initially missed by the surgeons (Fig. 6). Moreover, ex-vivo validation of fluorescence signals correlated seamlessly with tumor areas (Fig. 11). The calculated sensitivity of the MFR camera system for detecting peritoneal metastases using the VEGF targeted tracer bevacizumab-IRDye800CW was 100%. This indicates all non-fluorescent peritoneal biopsies were confirmed as tumor negative by histopathology. Sensitive detection of tumor negative peritoneal lesions may guide surgeons in debulking efforts, thereby resulting in less extensive surgical procedures. Therefore, fluorescence imaging has the potential to prevent overtreatment and contribute to a more effective cytoreduction. Since the majority of the complications result from extensive surgical procedures, prevention of overtreatment would be crucial for reduction of morbidity and mortality. Ultimately, this may permit more patient tailored cancer treatment and improve survival.

The PCI detected when guided by tumor-specific fluorescence imaging decreased significantly compared to visual inspection and palpation alone. Currently, the cut-off point for surgery is situated at a PCI greater than 20 points. This accounts for almost 14% of all patients with PC of colorectal origin (11). By reclassifying patients using fluorescence imaging, stage migration might occur due to more accurate PCI assessment. This might imply that more patients could potentially benefit from the curative CRS and HIPEC procedure, thus potentially preventing under treatment. The application of fluorescence laparoscopy in diagnostic laparoscopy could provide further information on the clinical impact of the decrease in PCI.

Specificity on the other hand was relatively low (54%), due to false positive lesions. Fluorescence in these lesions could be explained after detailed analysis, by either the presence of foreign body material (i.e. autofluorescence from old suturing material from previous surgery), highly vascularized lesions, fibrotic or scar tissue or reactive lymph nodes. Since VEGF is an inducer of angiogenesis and lymphangiogenesis, this implies more bevacizumab-IRDye800CW uptake in highly vascularized lesions and lymph nodes.

Another hypothesis for the fluorescence detected in reactive lymph nodes is the imaging of a ‘pre-metastatic niche’. Many tumors have a tendency to metastasize towards specific organs, but the molecular and cellular mechanisms that guide tumor cells to specific tissues remain unknown. Recently, Kaplan et al suggested that cellular clusters of bone marrow-derived hematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1) cluster in specific premetastatic locations, previous to metastasizing (42). These progenitor cells mobilize in response to the release of several growth factors produced by the primary tumor. This results in changes of the local


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microenvironment, which is referred to as the premetastatic niche. VEGFR1 acts as a cell surface receptor for VEGF-A, for which it has a high affinity (43). Specific imaging of bevacizumab-IRDye800CW in these premetastatic sites could possibly demonstrate the priming of these cells for metastasis. Focus on the early cellular and molecular events in cancer dissemination and metastasis would likely lead to new approaches for detection and prevention of metastasis at its earliest inception.

Specificity might be improved by advancing the MFRI camera system. Improvements such as implementation of the most advanced CCD chips and correction software for thresholding and quantification of signals might be sufficient for intraoperative tumor differentiation. The development of novel fluorescence tracers for other tumor-targets could also contribute to a higher specificity. Potential novel tumor-targets could for example be the epithelial cell adhesion molecule Ep-CAM or the well-known tumor marker carcinoembryonic antigen (CEA).

Systemic administration of bevacizumab-IRDye800CW showed no toxicity. Both SAE’s that occurred in this trial were not attributed to the tracer administration, as objectively confirmed by the METc and DSMB of the UMCG. Considering the fact that the CRS and HIPEC procedure is a high-risk procedure with a mortality and grade III to IV morbidity rate of 3-8% and 31% respectively (5,8), significant (serious) adverse events could be expected. Remarkably both of the SAE’s occurred in the patients that were treated neoadjuvantly. However, current literature describes that preoperative chemo-radiotherapy does not appear to increase the morbidity rate after cytoreduction.

Although in the sixth patient the MFI of tumor tissue was more than twice as high as non-tumorous lesions, the level of the fluorescence signal compared to background was not significant. However, only two peritoneal metastases were confirmed tumor positive after histopathological analysis in this patient. Possibly this patient presented with a relatively low VEGF-expressing colon cancer. Additional VEGF staining and analysis of the level of VEGF expression of all peritoneal lesions has to be performed for correlation with vital tumor tissue and comparison with other patients. Differences in vital tumor density or tumor lesion size could possibly explain a relatively low tracer uptake and therefore low fluorescence signals, however this still requires further analysis.

As the present study was a phase I feasibility study, the small number of patients precluded randomization. Therefore it was not possible to determine the definitive clinical impact of this imaging technique. As there are no commercially available MFRI camera systems yet, one was developed in collaboration with the Technical University of Munich to perform this study. Since this system has never been used for imaging of PC before, surgeons and researchers had to go through a learning curve. For actual clinical application, an optimized commercially available camera system will have to be developed, also to reduce cost-effectiveness.

Optical fluorescence imaging has previously been applied in humans for resection of malignant gliomas using 5-aminolevulinic acid (5-ALA) (44). In 2011, Van Dam et al applied optical imaging using the folate receptor-α targeted tracer Folate-FITC in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery (26). Recently, the endothelial growth factor receptor (EGFR) targeted fluorescence tracer Cetuximab-IRDye800CW has successfully been applied for surgical navigation in head- and neck cancer (45). Significant advances have been achieved in the clinical translation of intraoperative optical imaging. Although cost-effectiveness and optimal handling still can be improved, fluorescence-guided surgery has clearly demonstrated its potential value to improve treatment outcome.

Here, we demonstrate for the first time that intraoperative NIRF optical imaging of peritoneal carcinomatosis of colorectal origin is feasible. We showed that bevacizumab-


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IRDye800CW can be safely administered intravenously for sensitive tumor detection using the MFR camera system. This technique has the potential to prevent both over and under treatment and may therefore contribute to an optimal cytoreduction. We state that these results are promising and can be used as solid base for a subsequent phase-II study or a sufficiently powered final multicentre diagnostic accuracy study with the study end-point of PCI reduction of for example 3 points.


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Conclusion This study is the first in-human proof-of-principle study to assess the potential application and benefit of optical fluorescence imaging in peritoneal carcinomatosis of colorectal origin. Intraoperative NIRF imaging of PC during the HIPEC procedure using the intravenously administered VEGF-targeted tracer bevacizumab-IRDye800CW is technically feasible and safe. The high sensitivity (100%) gives the surgeon potentially a real-time tool for intraoperative decision-making. Sensitive detection of tumor negative peritoneal lesions may guide surgeons in debulking efforts, thereby resulting in less extensive surgical procedures. Therefore, fluorescence imaging has the potential to prevent overtreatment and contribute to a more effective cytoreduction. By reclassifying patients using fluorescence imaging, stage migration might occur due to more accurate PCI assessment. This might imply that more patients could potentially benefit from the curative CRS and HIPEC procedure, thus potentially preventing under treatment. Additionally, this technique possibly enables the evaluation of positive resection margins. The tumor-to-background ratio found during ex-vivo analysis is considered sufficient for decision-making during surgery. Ex-vivo validation of fluorescence correlated perfectly with tumor areas. We state that these preliminary results are promising and the data will be used to design further studies with regard to intraoperative fluorescent detection of cancer lesions in PC.

Future perspectives Additional immunohistochemical stains and fluorescence microscopy will be performed for CD34 and collagen to determine co-localization of the bevacizumab-IRDye800CW. VEGF expression in tumor tissue still has to be analyzed and correlated with fluorescence and tumor tissue.


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Appendices

Immunohistochemical staining protocol for VEGF Immunohistochemical staining protocol for VEGF (paraffin slides), 698, Thermo sciences RB9-031 Deparaffinization and rehydration

• Dewax or deparaffinize in xylene (2 x 5 minutes) • Washing steps: 100% alcohol, 96% alcohol and 70% alcohol • Rinse with distillated aqua • Wash with phosphate buffered saline (PBS) (5 minutes, refresh 3x)

Antigen retrieval

• Preheat the buffers first in the microwave: 10mM citrate buffer, pH 6.0 • Antigen retrieval: 15 minutes on 400 Watt (4 cups) • Cool down for 15 minutes • Rinse with PBS (5 minutes, refresh 3x)

Staining

• Block endogen peroxidase: 4.5 ml 30% H202 in 45.5 ml PBS, 30 minutes • Rinse with PBS (5 minutes, refresh 3x) • Incubate with first antibody • Thermoscientific, dilution 1:300 in PBS 1% BSA, o/n • Rinse with PBS (5 minutes, refresh 3x) • GARHRP 1:50 + 1% AB-serum 30 min. KT • Rinse with PBS (5 minutes, refresh 3x) • RAGHRP 1:50 + 1% AB-serum 30 min. KT • Rinse with PBS (5 minutes, refresh 3x) • Peroxidase reaction: DAB 10 min., (freezer cup) dilute in 50 ml PBS, than add 50 µl

30% H2O2. Oplossing weggooien in vat III • Rinse with distillated aqua (5 minutes, refresh 3x)

Dehydration and embedding

• Washing steps with 70, 96 and 100% alcohol. • Air-dry for 30 minutes minimal. • Embed with mounting medium.


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