University of Groningen

Fluorescence molecular endoscopyHartmans, Elmire

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C H A P T e R 1

General introduction

and outline of the thesis

C H A P T e R 1

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General introduction and outline of the thesis

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1GeNeRAL INTRODUCTION

Globally, colorectal cancer (CRC) and esophageal cancer (EC) are respectively the second and eighth most commonly diagnosed types of cancer.1 Endoscopic screening is used to prevent gastrointestinal cancer development through detection and removal of premalig-nant lesions, or decrease mortality via early cancer diagnosis and subsequent treatment. For inspection of the gastrointestinal tract in day-to-day clinic, conventional white-light (WL) endoscopy is the current standard. However, WL-endoscopy has substantial detection miss rates when considering early lesions in both the upper2 and lower digestive tract.3,4 To enhance the diagnostic yield of surveillance WL-endoscopy, and to improve the detection of small and flat lesions, the demand for new endoscopic techniques arose. Hence, a wide variety of imaging modalities have been developed over the past few decades.

The newly developed endoscopic technologies can grossly be divided into two different categories (Figure 1): the first category comprises techniques that detect areas of interest based on improved image quality, such as high-definition (HD) WL-endoscopy and magni-fication endoscopy. The second category includes techniques that aim to perform ‘real-time’ characterization of mucosal lesions based on enhancement of tissue contrast. Examples of such techniques are conventional chromoendoscopy (using methylene blue, indigo carmine or acetic acid application), narrow band imaging (NBI) and autofluorescence imaging (AFI), which delineates cancer-associated alterations by the use of endogenous fluorescence. The second category also includes another subcategory of techniques that are able to visualize the tissue of interest at a microscopic level, such as optical coherence tomography (OCT) or volumetric laser endomicroscopy (VLE) and confocal laser endomicroscopy (CLE).8,9

To date, HD WL-endoscopy and NBI, and to a lesser extent AFI and CLE, are commercially available methods that are used in day-to-day clinical practice. However, the majority of the characterization techniques are still constricted to their application in research setting only. The overall marginal evidence of superiority, and in some cases false-positive outcomes, resulted in an overall negative stand towards the use of characterization techniques instead of conventional HD WL-endoscopy. Moreover, some of the promising techniques like CLE are less attractive for clinical application due to their small field of view, their dependence on expensive equipment and the necessity of highly specialized interpretation. Noteworthy is the fact that most of the above-described characterization techniques require highly experi-enced operators, which make them mainly suitable for use in tertiary care centers. As a result, easy-to-use and easy to interpret techniques with the ability to combine both lesion detection and lesion characterization have been of special interest; thus, techniques that are able to

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Chapter 1

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delineate superficial mucosal changes at a macroscopic level and at the same time identify changes associated with neoplastic progression on a more biological level.5 Consequently, optical molecular imaging made its appearance in the field of gastroenterology. Whereas most endoscopic techniques differentiate aberrant lesions based on morphological aspects and architectural changes only, molecular imaging enables minimally invasive visualization based on specific molecular alterations.6,7 These specific molecular ‘fingerprints’ - e.g. genetic mutations, copy number variations or deletions - can lead to phenotypic cellular changes. Since these molecular changes often occur prior to visible morphologic features, these cell-specific ‘fingerprints’ provide a window of opportunity for early lesion detection. When combining molecular imaging with endoscopy, biological insights can be obtained and clinical problems may be solved. This novel technique is known as Fluorescence Molecular Endoscopy (FME).

*High-definition white-light(~850,000 pixels)

**Magnification endoscopy (~150x)

*Conventional chromoendoscopy**Narrow band imaging

***Autofluorescence imaging

Conventional white-light endoscopy

Lesioncharacterisation

Macroscopiccontrast enhancement

Microscopic imagingreal-time histology

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CLE (~1000x)

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* ** * ** ***

Lesiondetection

Figure 1. Schematic overview of novel endoscopic techniques. Schematic overview of the different endoscopic techniques used in the field of gastroenterology. Roughly, these techniques can be divided into two categories: 1) enhanced image quality to improve detection and surveillance, and 2) contrast enhancement or real-time microscopy, with which lesion characterisation can be accomplished. OCT, optical coherence tomography; VLE, volumetric laser endomicroscopy; CLE, confocal laser endomicroscopy.8,9

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General introduction and outline of the thesis

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1OUTLINe OF THe THeSIS

By specifically targeting upregulated proteins and therefore fluorescently ‘highlighting’ pre-malignant and malignant cells, fluorescence molecular endoscopy is able to differentiate between normal and aberrant gastrointestinal tissue. By creating a wide-field ‘red-flag’ technique by which tissue differentiation is feasible in ‘real-time’, fluorescence molecular endoscopy may optimize the detection of neoplasia. Therefore, the main aim of this thesis is to translate the application of near-infrared fluorescence molecular endoscopy (NIR-FME) towards the clinic.

Part I of this thesis describes the ex vivo evaluation and clinical validation of the custom-made VEGFA-guided NIR-FME technique. To apply molecular-guided strategies, target proteins that are specifically upregulated in the cells of interest need to be identified. When considering optical molecular imaging as a strategy to visualize and distinguish gastrointestinal neoplasia, proteins that are upregulated in the development of cancer could function as a target. Several proteins are known to play a prominent role in the development and spreading of cancer, such as growth receptors (e.g. endothelial growth factor receptor - EGFR) and angiogenic proteins (e.g. vascular endothelial growth factor A - VEGFA).10-13

Very few clinical FME trials have been executed over the past few years, which is mostly because clinical translation of the targeted tracers is logistically challenging. Since therapeu-tic agents are far more interesting from a commercial point of view, translation of targeted imaging agents to the clinic is scarce. A multidisciplinary team is required to translate a tracer to the clinic; integrated knowledge from clinicians, physicists, chemists and pharmacists is required to find the right target and develop high quality fluorescent tracer products. For our initial preclinical and clinical studies, which are included in this thesis, we have selected a VEGFA-targeting tracer. VEGFA is a protein that is a dominant inducer of blood vessel growth and an important driver of tumor angiogenesis.14,15 It is known from literature that VEGFA is overexpressed in various solid tumor types and that the protein plays a critical role in the development of gastrointestinal carcinomas.16-20 To speed up the translation of optical molecular imaging towards clinical practice, we fluorescently labelled the therapeutic agent bevacizumab, which is a commercially available anti-cancer drug. To do so, we made use of dye molecule that fluoresces near-infrared light (665 – 900 nm). NIR fluorophores are considered to be optimal as they create minimal tissue autofluorescence and maximal tissue penetration. We produce this NIR-VEGFA tracer in-house under GMP-conditions, which enabled rapid translation and application in preclinical and clinical NIR-FME trials.

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CHAPTERS 2, 3 and 4 describe the validation of VEGFA as a target for NIR-FME for col-orectal adenomas and dysplastic Barrett lesions in the esophagus. Based on immuno-stainings performed on various human colorectal adenomas (CHAPTER 2) and esophageal samples - including dysplastic Barrett lesions and esophageal adenocarcinoma (CHAPTER 4) - we demonstrate that VEGFA is indeed a discriminating target protein, suitable for early detection of lesions by molecular imaging. Moreover, CHAPTER 2 describes the potential of VEGFA and EGFR-targeting NIR-fluorescent antibodies for visualizing small colorectal lesions. This validation was performed by simulating a NIR-FME procedure in an ex vivo model, with the use of a segment of freshly resected human colon with stitched-in, mice-de-rived tumor xenografts. Since no commercial video endoscopes were available that are able to excite and detect signals of NIR wavelengths, we have developed a NIR-FME system in coop-eration with the Technical University of Munich and its spin-off company SurgVision. The NIR-FME system makes use of an external ultrasensitive NIR fluorescence camera, to which an endoscope or fiber-bundle (“mother-daughter technique”) can be attached (Figure 2). We first started imaging with an old Olympus fiberscope,21 which we subsequently changed to a miniature fiber-bundle with limited resolution (SpyGlass, Boston scientific; 6000 pixels), and eventually optimized our system with a custom-made and easily applicable fiber-bundle containing 30,000 miniature fibers. CHAPTER 2 of this thesis describes the ex vivo eval-uation and validation of this custom-made NIR-FME approach.

Following the development, the ex vivo evaluation and the validation of our imaging system, we gained permission to clinically evaluate our VEGFA-targeted NIR-FME approach for the detection of colorectal polyps and Barrett lesions (CHAPTER 3 and CHAPTER 4). Both chapters describe clinical studies, in which we made use of the GMP-produced tracer tar-geted against VEGFA: bevacizumab-800CW.

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General introduction and outline of the thesis

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Tip of fiber-bundle

working channel

Fiber-bundle inserted through working channel of video endoscope

Endoscopy suite

Single photondetection CCD

camera

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Band pass filter819 nm, ±44 nm

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ITControl and image

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ATTACHED FIBER-BUNDLE

Figure 2. Schematic overview of the custom-made Near-infrared Fluorescence Molecular Endoscopy (NIR-FME) system. Schematic overview of the external NIR-fluorescence camera system, to which the cus-tom-made fiber-bundle can be attached. This fiber-bundle can be inserted through a conventional high-definition white-light video endoscope (bottom images).

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CHAPTER 3 describes a clinical proof-of-principle study for the detection of colorectal adenomas. In this chapter we evaluated the application of VEGFA-guided NIR-FME for ade-noma detection, performed in patients with familial adenomatous polyposis (FAP). This study was designed with a tracer dose escalation set-up to evaluate the best-performing systemic tracer dose for colorectal adenoma detection. To identify the optimal dose, the observed in vivo fluorescent signals need to be quantified and validated. Several factors, such as local tissue optical properties - i.e. light absorption and scattering - and the distance to the target tissue, influence the fluorescent signals observed and therefore impede reliable quantitative measurements based on in vivo NIR-FME images. Multi-Diameter Single Fiber Reflectance and Single Fiber Fluorescence (MDSFR/SFF) spectroscopy can correct “raw” fluorescence signals for these influencing factors.10-13 As such, this chapter describes the use of MDSFR/SFF spectroscopy to validate the in vivo NIR-FME findings and select the best-performing tracer dose. In addition, NIR-fluorescence flatbed scanning, fluorescence microscopy and VEGFA immunohistochemistry (IHC) was performed to visualize tracer distribution at a microscopic level, enabling correlation with histopathology and VEGFA protein expression.

Our other clinical proof-of-principle study is described in CHAPTER 4. This chapter describes the evaluation of two bevacizumab-800CW application routes: topical application (luminal spraying) and systemic administration (intravenous injection). Hence, in this clinical study we evaluate whether VEGFA-guided NIR-FME, following both topical or systemic tracer administration, can be used to detect dysplastic and early esophageal adenocarcinoma (EAC) lesions in Barrett Esophagus (BE) patients.

Part II of this thesis describes the search for novel molecular targets for colorectal adenomas and pancreatic cancer. In general, the cellular localization of proteins determines whether they are attractive as an imaging target. Cell membrane receptors and specific extracellularly secreted proteins are presumed to be the most attractive targets, because proteins on the cell surface or in the extracellular space are easily accessible for targeting tracers. In addition, imaging targets should preferably be restricted to the tissue of interest: for example by anchor-ing to the cell membrane or binding to membrane receptors. This enables ‘highlighting’ of the aberrant area and fluorescent visualization during endoscopy. In the case of VEGFA, where the protein gets excreted into the extracellular matrix, the large VEGFA isoforms will anchor to the cell and accumulate within the tumor.

Though, for colorectal adenomas, where the lesions are fairly small and the area of interest is rather limited, it could well be that other target proteins would accumulate more effectively. As a result, the required amount of tracer necessary to accomplish ‘real-time’ imaging may be reduced. In CHAPTER 5 we describe the search for new target proteins for the identification

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1of colorectal adenomas. In order to do so, we applied in silico Functional Genomic mRNA (FGmRNA) profiling on publicly available genetic expression datasets; FGmRNA profiling is a recently developed method that results in an enhanced view on the downstream effects of genomic alterations on gene expression levels in premalignant and neoplastic lesions.22 FGmRNA profiles of sporadic adenomas were compared to normal colon tissue to estimate biologically relevant overexpression of target antigens at the protein level. In this chapter we evaluate the protein expression of the top identified genes, select the most relevant target protein and perform molecular-targeted imaging experiments in ApcMin/+ mice.

In CHAPTER 6 we describe the identification of target proteins for molecular based imaging and treatment strategies in a totally different (gastrointestinal) area of interest: pan-creatic ductal adenocarcinoma (PDA). Molecular targeted therapies may improve the poor disease prognosis by specifically targeting aberrant signaling-pathways in PDA tumor cells. In contrast to the traditional working mechanism of chemotherapy, which has a cytotoxic effect on all rapidly dividing cells, molecular targeted treatments are able to block specific biological signaling pathways that are needed for carcinogenesis and tumor growth. Molecular targeted treatment is therefore expected to be a more effective therapeutic strategy and is assumed to cause fewer side effects. Besides molecularly targeted therapy, molecular guided imaging can have additional benefits in PDA management. In the context of PDA, molecular-guided imaging could be used to better detect micrometastases and therefore improve the selection of patients that are suitable for curative surgical resection. Another suitable application of molecular guided imaging in PDA could be to accomplish complete resections by enabling fluorescence guided molecular surgery. To identify relevant PDA-targeting proteins, the same in silico FGmRNA-profiling method, as described in chapter 5, was applied. Subsequently, we describe a new hybrid reviewing strategy, with which we prioritized the identified FGm-RNA-overexpressing genes based on an extensive literature search for both therapeutic and imaging purposes.

Part III of this thesis describes promising therapeutic possibilities of molecular targeted imaging agents: their so-called theranostic application. The aim of CHAPTER 7 is to evaluate whether this application can be used as a non-invasive treatment strategy in esophageal cancer.

When EAC is in its limited disease-stage, superficial and still restricted to the mucosa, endoscopic resection is the treatment of choice.23-26 However, for optimal treatment of early stage EAC with positive margins after endoscopic resection, additive treatment approaches are needed. A possible ablative approach is photodynamic therapy (PDT). PDT is able to generate cell death via non-ionizing light energy that interacts with photosensitizers, leading to chemical destruction of cells. Although PDT ablation has shown to decrease the development

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of EAC, considerable side effects such as photo cutaneous reactions and esophageal stric-tures have also been described.27-29 Thus, to create a more cancer-selective PDT approach, a new modality known as targeted photodynamic therapy (tPDT) has recently been devel-oped.30 In tPDT cancer-targeting agents, such as monoclonal antibodies, are functionalized with a photosensitizer group to form a phototoxic compound. The targeted photosensitizer accumulates in tissue where the marker is expressed, thereby reducing the PDT exposure of the surrounding healthy tissue to a minimum. As such, tPDT is a novel strategy in which can-cer-targeted phototoxicity is able to selectively treat malignant cells. CHAPTER 7 describes an in vitro report where we, as a first, demonstrate the applicability of antibody-based NIR-tPDT in EAC. Furthermore, we demonstrate that NIR-tPDT can be made more effective by tyrosine kinase inhibitor (TKI) induced growth receptor upregulation.

Finally, CHAPTER 8 summarizes the findings of this thesis, and in addition addresses the future research possibilities and developmental perspectives regarding optical molecular imaging in the field of gastroenterology.

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1ReFeReNCeS

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2008;8:880–7. 13. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37 Suppl 4:S9–15. 14. Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–76. 15. Kerbel RS. Tumor angiogenesis. N Engl J Med 2008;358:2039–49. 16. Schneider BP, Miller KD. Angiogenesis of breast cancer. Journal of Clinical Oncology 2005;23:1782–90. 17. Ferroni P, Palmirotta R, Spila A, Martini F, Formica V, Portarena I, et al. Prognostic value of carcinoembry-

onic antigen and vascular endothelial growth factor tumor tissue content in colorectal cancer. Oncology 2006;71:176–84.

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19. Uehara M, Sano K, Ikeda H, Sekine J, Irie A, Yokota T, et al. Expression of vascular endothelial growth factor and prognosis of oral squamous cell carcinoma. Oral Oncol 2004;40:321–5.

20. Xu X-L, Ling Z-Q, Chen W, Xu Y-P, Mao W-M. The overexpression of VEGF in esophageal cancer is associated with a more advanced TMN stage: a meta-analysis. Cancer Biomark 2013;13:105–13.

21. Garcia-Allende PB, Glatz J, Koch M, Tjalma JJ, Hartmans E, Terwisscha van Scheltinga AGT, et al. Towards clinically translatable NIR fluorescence molecular guidance for colonoscopy. Biomed Opt Express 2013;5:78–92.

22. Fehrmann RSN, Karjalainen JM, Krajewska M, Westra H-J, Maloney D, Simeonov A, et al. Gene expres-sion analysis identifies global gene dosage sensitivity in cancer. Nat Genet 2015;47:115–25.

23. Buttar NS, Wang KK, Lutzke LS, Krishnadath KK, Anderson MA. Combined endoscopic mucosal resection and photodynamic therapy for esophageal neoplasia within Barrett’s esophagus. Gastrointest Endosc 2001;54:682–8.

24. May A, Gossner L, Pech O, Fritz A, Günter E, Mayer G, et al. Local endoscopic therapy for intraepithelial high-grade neoplasia and early adenocarcinoma in Barrett’s oesophagus: acute-phase and intermediate

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results of a new treatment approach. Eur J Gastroenterol Hepatol 2002;14:1085–91. 25. Prasad GA, Wu TT, Wigle DA, Buttar NS, Wongkeesong L-M, Dunagan KT, et al. Endoscopic and sur-

gical treatment of mucosal (T1a) esophageal adenocarcinoma in Barrett’s esophagus. Gastroenterology 2009;137:815–23.

26. Ell C, May A, Pech O, Gossner L, Guenter E, Behrens A, et al. Curative endoscopic resection of early esophageal adenocarcinomas (Barrett’s cancer). Gastrointest Endosc 2007;65:3–10.

27. Overholt BF, Panjehpour M, Haydek JM. Photodynamic therapy for Barrett’s esophagus: follow-up in 100 patients. Gastrointest Endosc 1999;49:1–7.

28. Overholt BF, Wang KK, Burdick JS, Lightdale CJ, Kimmey M, Nava HR, et al. Five-year efficacy and safety of photodynamic therapy with Photofrin in Barrett’s high-grade dysplasia. Gastrointest Endosc 2007;66:460–8.

29. Foroulis CN, Thorpe JAC. Photodynamic therapy (PDT) in Barrett’s esophagus with dysplasia or early cancer. Eur J Cardiothorac Surg 2006;29:30–4.

30. Kobayashi H, Choyke PL. Super enhanced permeability and retention (SUPR) effects in tumors following near infrared photoimmunotherapy. Nanoscale 2016;8:12504–9.

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Pa r t 1

Pa r t 1precliNical & CLINICAL VALIDATION

OF VeGFA-GUIDeD NIR-FMe