Endomicroscopy and Graft-versus-host Disease
Endomicroscopy and Graft-versus-host Disease
Early diagnosis of acute Gastrointestinal Graft-versus-Host disease (aGI-GvHD) has a strong
 impact on morbidity and mortality of patients who underwent haematopoietic stem cell
 transplantation (HSCT). Recent results at the investigators department showed that mucosal
 biopsies from the small intestine have a high diagnostic yield for aGI-GvHD specific changes.
 By performing an enteroscopic examination, aGI-GvHD suspected patients can be prevented from
 colonoscopy and prior bowel preparation which is clinically important, considering the rather
 bad general condition of this patient group. To further reduce invasive procedures the
 investigators want to evaluate the in vivo histological features of aGI-GvHD in the small
 bowel. Therefore aGI-GvHD suspected patients will undergo confocal laser endomicroscopy of
 the upper GI-tract, including duodenum and jejunum, in the context of a prospective clinical
 pilot trial. The histological evaluation of biopsy samples taken from these sites will be
 used as comparable gold standard. Endomicroscopic aspects of patients with celiac disease,
 infectious enteritis, inflammatory bowel disease and healthy subjects should serve as
 controls. If it is possible to diagnose aGI-GvHD from endomicroscopic features of the small
 bowel alone, this could be another important step to improve the diagnostic management of
 post HSCT patients, especially when taking of biopsy samples is difficult because of a bad
 coagulation status. Additionally, an accurate diagnosis in vivo could lead to immediate
 treatment to prevent progression and site spreading of the disease.
Graft-versus-host disease (GvHD) represents a major cause for morbidity and mortality in
 patients who underwent haematopoietic stem cell transplantation (HSCT) [1]. In the
 gastrointestinal tract as one of the main target organs of GvHD diagnosis may be difficult
 because of many similar presenting differential diagnoses, most notable, infections by
 cytomegalovirus [2]. Experts disagree about the best diagnostic approach for patients
 suspected of acute gastrointestinal GvHD (aGI-GvHD). There have been many studies supporting
 the endoscopic evaluation of either the lower [3, 4, 5], the upper [6, 7] or both parts of
 the GI-tract [8, 9]. In either case, the histological evaluation of biopsy samples serves as
 standard tool to establish the final diagnosis [10].

 Considering the rather bad general condition of patients after HSCT as a limiting factor for
 endoscopic and histological evaluation, some new approaches have been introduced into the
 diagnostic manual of aGI-GvHD in order to reduce the amount of invasive examinations.
 Conventional imaging techniques like magnetic resonance imaging [11], computer tomography
 [12] and high-resolution ultrasonography with Doppler Imaging [13] reported bowel wall
 thickening in GvHD patients. But as such findings are only unspecific signs of
 gastrointestinal pathology, conventional imaging techniques cannot replace endoscopy with
 biopsies [14]. The same holds for video capsule endoscopy which was indeed found to have a
 high negative predictive value for the detection of aGI-GvHD, but shares the important
 shortcoming of missing biopsy samples for histological evaluation [15, 16, 17].

 At least this method shifted the focus of interest onto the small bowel. Biopsies deriving
 from this former neglected GI part had shown a high sensitivity for GvHD-related changes in
 previous studies [18]. As similar results were seen at the study site, the investigators
 prospectively compared enteroscopy with biopsies to the present gold standard in the
 diagnostic aGI-GvHD management, colonoscopy with biopsies. In the context of this
 investigation the large intestine showed much stronger affection by aGI-GvHD than all other
 locations, although there was no pair of examinations, in which colonoscopy revealed aGI-GvHD
 without the presence of such findings at enteroscopy. Quite the opposite, enteroscopy even
 revealed GvHD related pathologies in one case where the respective colonoscopy remained
 inconspicuous. Consequently, they postulated that enteroscopic findings may replace the need
 for colonoscopy [19], wherefore post-HSCT patients could remain without stressful bowel
 preparation, often limited by their rather bad general condition.

 One of the latest developments in the endoscopic management of the gastrointestinal tract is
 the so called confocal laser endomicroscope (CLE). Confocal microscopy was developed by
 Marvin Minsky in the late 1950s. Its principle is the microscopic scanning of focal points
 below the surface of an object. In comparison to conventional light microscopy it uses a
 special filter system to avoid image overlapping by surrounding tissue. In detail, a light
 source (normally a laser) is focused by a microscope objective lens to a diffraction limited
 spot on or inside the object. Light that is scattered, or fluorescence excited (achieved
 through fluorescein staining, for example) and emitted, at the focus in the sample will
 partially return back through the optics along the path from which it arrived. A
 beam-splitter placed into the path reflects the return light towards a detector. The optics
 will focus the light from the focal point in the specimen to its conjugate focus near the
 detector (hence the technology is termed "con-focal"). Here a spatial filter ("pinhole") is
 used to extinguish all light deriving from areas outside the focal point. Light reflections
 from the focal point itself will be forwarded to the detector which is connected to a
 computer system that digitalises the optical signal and creates the in vivo histological
 image [20].

 Focusing on its clinical impact, confocal microscopy is the first technique to allow in vivo
 evaluation of tissue structures beneath their surface. Because of many breakthroughs in
 miniaturisation (mostly in the 1990s) this technology could be applied for intraluminal use
 in gastroenterology, integrated into a otherwise standard endoscope. It allows the in vivo
 histological visualisation of the upper 250 micrometers of all walls within the
 gastrointestinal tract, additionally to the normal function of white light endoscopy
 (provided by two separate screens on top of the workstation) [20].

 The first clinical use of CLE in a prospective pilot trial including 35 patients after HSCT
 was reported by Bojarski et al. in 2009. CLE of the colonic mucosa could predict the positive
 diagnosis of aGI-GvHD in 14 of 19 patients, later confirmed by histological evaluation. The
 in vivo findings of aGI-GvHD, varying from single crypt apoptosis to complete crypt loss,
 were clearly distinguishable from pathologies of other origin like infectious colitis or
 ulcerative colitis, both used as comparative control [21].

 Study Aims

 In this clinical investigation the investigators intend to continue the diagnostic approach
 for acute Gastrointestinal Graft-versus-Host Disease (aGI-GvHD). Based on the recent results
 at the study institution (explained above, [19]) that showed a remarkable diagnostic yield
 for aGI-GvHD associated pathologies in the small intestine the investigators intend to
 evaluate confocal laser endomicroscopy (CLE) of the small intestine as primary diagnostic
 procedure in patients with suspected aGI-GvHD after haematopoietic stem cell transplantation
 (HSCT).

 -  The investigators aim to examine whether early diagnosis of aGI-GvHD in the duodenum and
 jejunum can be achieved by evaluating endomicroscopic features.

 -  In comparison with conventional biopsies from the mentioned GI-parts as gold standard
 the investigators want to test whether the markers described by Bojarski et al. for the
 colon also hold true for the small intestine.

 -  Including endomicroscopic results of patients with celiac disease, enteral infections,
 inflammatory bowel disease and healthy subjects as controls the investigators aim to
 establish aGI-GvHD specific markers that can be used for a "biopsy-free" in vivo
 diagnosis of aGI-GvHD.

 Study Design

 Prospective clinical pilot trial without randomisation or blinding

 Study Population

 Patients after haematopoietic stem cell transplantation (HSCT) who are referred to the study
 site for clarification of one of the following symptoms:

 -  anorexia

 -  nausea

 -  vomiting

 -  abdominal pain

 -  diarrhea

 -  intestinal bleeding

 To exclude possible side effects of the conditioning therapy, GI symptoms must have occurred
 or persisted 20 days after the respective transplantation date [22].

 Exclusion criteria:

 -  infection with CMV or HSV

 -  bacterial infection of the GI tract

 -  medication related symptoms

 -  patients allergic to one of the drug components (including drugs used for conscious
 sedation like propofol or midazolam as well as fluorescein, the fluorescent dye used for
 CLE )

 -  refusal to participate in the study

 -  patient's age below 18 years.

 Methods

 This investigation will be performed at the Medical University of Vienna, Department of
 Internal Medicine III, Division of Gastroenterology and Hepatology. Patient recruitment will
 be achieved in close cooperation with the Department of Internal Medicine I, Division of
 Oncology, Bone Marrow Transplantation Unit.

 As this study has been planned as a pilot trial, it mainly focuses on the qualitative
 endomicroscopic features of the small intestine in GvHD-patients to yield reference
 pathologies for accurate diagnosis in the future. Nevertheless, a quantitative analysis will
 be performed by comparing CLE-derived diagnoses with histological results to calculate
 sensitivity and specificity of CLE-based aGI-GvHD diagnosis in the small intestine.
 Considering the current diagnostic yield of 62% for the detection of aGI-GvHD in symptomatic
 post-HSCT-patients by conventional histology [19], the investigators estimated to include 40
 patients with suspected aGI-GvHD in order to discover a meaningful sample of patients
 affected by aGI-GvHD for qualitative and quantitative analysis. In comparison, Bojarski et al
 included 35 patients of whom 19 finally turned out to suffer from aGI-GvHD by conventional
 histology. They could then calculate a sensitivity of 74% and specificity of 100% for the
 prediction of the histological diagnosis by CLE [21]. On the basis of the current frequency
 of post-HSCT-patients at the study site the investigators estimate to include the aimed
 amount of 40 patients within a time period of 2 years.

 All patients who fulfill the listed inclusion criteria, will receive the patient information
 form of this study together with the usual informed consent form of the respective endoscopic
 examination they are about to undergo. Patient information will be done at least 24 hours
 before the intervention, as practiced at our unit. If the patient agrees to participate in
 the study he/she will be prepared for endoscopy by administration of intravenous propofol
 and/or midazolam as routinely used for conscious sedation during endoscopic procedures at our
 department. Additionally, 5-10 ml of a 10% solution of fluorescein sodium will be
 administered intravenously to enhance tissue fluorescence during endomicroscopy. As so far no
 specific CLE-enteroscope is available, we will use the CLE-colonoscope (Pentax EC-3870 CIFK
 with the ISC-1000 confocal endomicroscopy processor - Pentax, Tokyo, Japan and Optiscan Pty
 Ltd, Notting Hill, Victoria, Australia) to reach the small intestine, comparable to an
 enteroscopic procedure (oral access).

 All drugs will be administered by medical specialists, assistant doctors or registered
 nurses, as routinely practiced at our institution.

 CLE picture capturing will start at the deepest point reachable in the jejunum. During
 withdrawal of the scope CLE pictures will be taken every 10 centimetres in the small bowel,
 then from the antrum and body of the stomach and from the esophagus. Furthermore, CLE will be
 applied for distinct looking areas, suspicious of pathologic origin. Wherever CLE pictures
 are obtained, a conventional biopsy will be taken from the same localisation.

 CLE pictures will be captured and stored routinely using our standard data management system.
 They will be evaluated immediately after the endoscopic procedure together with a board
 certified GI-pathologist, who will establish an initial diagnosis.

 Conventional biopsies will be stained with Haematoxylin & Eosin, as well as PAS stain.
 In-situ hybridization for CMV and HSV will be performed to exclude viral infection.
 Classification of GvHD will be done according to McDonald and Sale in 4 grades, appropriately
 modified for the small intestine [23]:

 -  Grade I: individual cell necrosis

 -  Grade II: crypt abscess

 -  Grade III: drop out of one ore more whole crypts in a biopsy

 -  Grade IV: total denudation of epithelium

 Endomicroscopic controls (patients with celiac disease, enteral infections, inflammatory
 bowel disease and healthy subjects) will be collected from present data of endomicroscopic
 procedures already performed within routine examinations.

 At the end of the study all CLE pictures and histological cuts will be evaluated separately
 by two blinded board certified pathologists. In case of discordant diagnoses they will
 analyse the respective cases together to reach an agreement on the final diagnosis. All cases
 where the initial and the final diagnosis are deviating from each other will be re-evaluated
 together by all specialists involved.

 Risk/Benefit assessment

 GvHD represents a major cause of morbidity and mortality after allogeneic HSCT. Approximately
 10-40% of patients who undergo HSCT develop significant GvHD, and about half of these
 patients die from this disease or from therapy resulting complications [1].

 Endoscopic procedures in patients with suspected aGI-GvHD are well established. Until now the
 histological evaluation of GI deriving biopsies is the gold standard tool to set an accurate
 aGI-GvHD diagnosis. Endoscopy of the upper GI-tract is more comfortable than the endoscopic
 assessment of the lower GI-tract as there is no need for bowel preparation. Therefore it is
 the primary technique to assess post HSCT patients at our department. Supported by recent
 results at the study institution [19] these upper GI endoscopies are routinely performed as
 enteroscopies instead of gastroscopies in order to include the small intestine in the
 diagnostic evaluation. The safety of enteroscopy has not been studied systematically but
 reported experience is substantial and complications have been rare [24].

 Confocal laser endomicroscopy is a safe new technique that has already been studied in
 clinical trials [25, 26]. Its safety is being guaranteed by the use of low intensity laser
 light that can at worst cause local bleaching of fluorescein containing cells, which is
 harmless, reversible and even used as diagnostic sign in experimental conditions [20].

 Ethical implications

 This study protocol was approved by the ethics commission of the Medical University of
 Vienna.

 All procedures in the context of this study will be performed in accordance to the
 Declaration of Helsinki as well as to the guidelines for Good Scientific Practice (GSP) of
 the Medical University of Vienna.

 Expected impact and Outlook

 If the investigators find aGI-GvHD specific markers that can be used for a "biopsy-free" in
 vivo diagnosis of aGI-GvHD this could lead to another reduction of invasive procedures for
 patients after HSCT. This would have a big clinical impact, as biopsy assessment is often
 limited by the bad coagulation status of this patient group.

 In case they cannot clearly determine specific markers of aGI-GvHD, CLE should at least
 reduce the amount of conventional biopsies needed for diagnosis by taking them under CLE
 guidance.

 Despite from the expected reduction of invasive procedures, aGI-GvHD diagnosis in vivo could
 lead to immediate initiation of immunosuppressive treatment in order to reduce progression
 and site spreading of the disease.

 In case the study aims will be supported by the results of this pilot trial, a randomized
 controlled trial comparing in vivo diagnosis to conventional histological assessment in
 matters of hospitalisation and survival will be performed.
Inclusion Criteria:

 Patients after haematopoietic stem cell transplantation (HSCT) who are referred to our
 department for clarification of one of the following symptoms:

 -  anorexia

 -  nausea

 -  vomiting

 -  abdominal pain

 -  diarrhoea

 -  intestinal bleeding

 To exclude possible side effects of the conditioning therapy, GI symptoms must have
 occurred or persisted 20 days after the respective transplantation date [22].

 Exclusion criteria:

 -  infection with CMV or HSV

 -  bacterial infection of the GI tract

 -  medication related symptoms

 -  patients allergic to one of the drug components (including drugs used for conscious
 sedation like propofol or midazolam as well as fluorescein, the fluorescent dye used
 for CLE )

 -  refusal to participate in the study

 -  patient's age below 18 years