Trial Information

Confocal Laser Endomicroscopy as Diagnostic Tool for Gastrointestinal MALT-lymphoma

Introduction

Extranodal marginal zone B-cell lymphoma (MZBL) of the mucosa associated lymphoid tissue
(MALT-lymphoma) is the most common form of extranodal lymphoma [1, 2]. Although
MALT-lymphoma may involve different organs, the stomach represents the site of predilection
of this malignant disease. About 90% of gastric MALT-lymphomas are associated with infection
with Helicobacter pylori (HP) [3] and eradication of the pathogen leads to tumor regression
in a high percentage of patients at early tumor stages [4, 5]. Relapses after HP-eradication
and primary advanced tumor stages require chemotherapeutic treatment, radiation therapy and,
in some cases, surgery [6]. Following complete remission lifelong follow-up examinations
should be performed in order to detect relapses [7].

Histologically, MALT-lymphoma is characterized by an infiltration of the lamina propria by
lymphomatous centrocyte-like cells that spread into the surrounding mucosal layers [8]. In
immunohistochemistry they show CD 20 positive staining properties. When they spread to lymph
nodes or spleen they accumulate typically within the marginal zone (for this reason they are
classified as "marginal zone lymphoma") [2]. Dissemination to other organs, especially to
other mucosal sites may occur frequently wherefore detailed diagnostics are required during
MALT-lymphoma staging and follow-up. Spreading within the gastrointestinal tract (GI-tract),
to the small bowel, for example, is rare, but can be assessed very easily with standard
endoscopic procedures as recently reported at our department [9].

For gastric MALT-lymphoma gastroscopy with biopsy assessment is the current follow-up
examination of choice. Multiple biopsies should be taken randomly from all gastric sites and
specifically from all suspicious lesions. Recent developments for the enhancement of the
tissue contrast during the endoscopic examinations (chromoendoscopy or Narrow Band Imaging,
for example) helped to detect MALT-lymphoma lesions. Nevertheless, detection rates hardly
changed, since all these techniques rely on the presence of superficial features[10].

An endoscopic technique that allows an evaluation of deeper wall layers is endoscopic
ultrasonography. During the last years it has become an important amending tool in the
diagnostic management of gastric MALT-lymphoma. Although it provides essential information
about tumor infiltration depth and lymph node involvement in the context of staging
procedures, it cannot replace histological assessment during follow-up, since
endosonographic findings do not show a good correlation to histological results over time
[11, 12].

The newest development that has been introduced 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 [13].

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 an 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) [13].

The in vivo visualisation of different GI-pathologies led to the first publications on the
clinical use of CLE. Kiesslich et al., as one of the pioneers of the technique, published a
case on live imaging of Helicobacter pylori by CLE in the stomach [14]. At our department we
recently reported the endomicroscopic findings of a patient with Whipple's disease.
Detecting foamy macrophages within the lamina propria which were clearly distinguishable
from goblet cells in the surrounding CLE could provide in vivo histology at the single cell
level [15].

Assessing GI-pathologies more systematically there have been many publications dealing with
the use of CLE in the context of follow-up examinations of conditions that predispose to the
development of malignancies. For Barrett's oesophagus, for example, Kiesslich et al. found
high sensitivity and specificity rates for the detection of Barrett's metaplasia as well as
for the prediction of Barrett's associated neoplastic changes - for both results CLE derived
pictures were compared to conventional histology [16]. In a first prospective, randomized,
double-blind, controlled, crossover trial Dunbar et al. proved these findings, as
CLE-targeted biopsies had a higher diagnostic yield for Barrett's oesophagus associated
neoplasia than standard endoscopy with 4-quadrant random biopsy [17]. Similar results have
already been published for patients with ulcerative colitis that were routinely screened for
colon cancer. It could be shown that CLE is a useful tool to improve the diagnostic yield.
At the same time, it helped to take conventional biopsies more accurately, which can spare
patients unnecessary biopsies [18].

Combining the potential of CLE to improve the diagnostic yield during surveillance with its
properties of scanning the upper 250µm of the GI-wall (which equates nearly the whole depth
of the mucosa) makes it the ideal tool for the diagnostic management of gastric
MALT-lymphoma, where pathologies typically arise from the lamina propria, a deeper layer of
the mucosa [8].

Study Aims

In this clinical pilot study the investigators aim to examine patients with gastric
MALT-lymphoma by confocal laser endomicroscopy (CLE) in the context of staging or follow-up
endoscopies. To our knowledge no systematic study on the use of CLE in MALT-lymphoma
patients has yet been performed.

- The investigators aim to determine whether the diagnosis of MALT-lymphoma can be
achieved by evaluating endomicroscopic features of suspicious lesions of both the
stomach and the small bowel, as these GI-parts are easily accessible by upper
endoscopy. Conventional biopsies from the GI-parts observed should serve as comparable
gold standard.

- Including endomicroscopic results of patients with chronic gastritis, gastric
adenocarcinoma and healthy subjects as controls the investigators aim to establish
MALT-lymphoma specific markers that can be used for a "biopsy-free" in vivo diagnosis
of the disease.

Study Design

Prospective clinical trial without randomisation or blinding

Study Population

Patients referred to our department for upper endoscopy in the context of staging or
follow-up of gastric MALT-lymphoma.

Exclusion criteria:

- 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 Division of Gastroenterology and Hepatology,
Department of Internal Medicine III., in close cooperation with the Division of Oncology,
Department of Internal Medicine I. and the Clinical Institute of Pathology at the Medical
University of Vienna.

As this study has been planned as a pilot trial, it mainly focuses on the qualitative
endomicroscopic features drawn from our examinations 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 MALT-lymphoma diagnosis. Based on current incidence and relapse
rates of the disease the investigators estimated to include 50 patients in order to get a
meaningful patient sample for qualitative and quantitative analysis. Given the current
number of MALT-lymphoma patients who undergo staging or follow-up endoscopies at the
investigational site, this patient sample can be included within a period of two 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 practised at our unit. If the patient agrees to
participate in the study he will be prepared for endoscopy with our confocal laser
endomicroscope (Pentax EC3870K with the ISC-1000 confocal endomicroscopy processor - Pentax,
Tokyo, Japan and Optiscan Pty Ltd, Notting Hill, Victoria, Australia) 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.

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 small bowel. 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 during the endoscopic procedure together with a
board certified GI-pathologist, who will establish an initial diagnosis.

Conventional biopsies will be processed and evaluated according to the criteria as outlined
in the recent WHO-classification for MALT-lymphoma. In all patients, immunologic phenotyping
on paraffin sections will be done for demonstration of light chain restriction and for the
phenotype CD20+CD5-CD10-cyclinD1- which, in context with the microscopic appearance, is
consistent with MALT-lymphoma. In addition, all patients will be assessed for MALT-lymphoma
specific translocations, especially t(11;18)(q21;q21).

Endomicroscopic controls (patients with chronic gastritis, gastric adenocarcinoma 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.

Ethical implications

Confocal laser endomicroscopy is a safe new technique that has already been studied in
clinical trials [16, 17]. 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 [13].

This study protocol had been already 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 MALT-lymphoma specific markers that can be used for a
"biopsy-free" in vivo diagnosis, this could lead to another reduction of invasive procedures
for the affected patients which may have an important clinical impact, as biopsy assessment
is often limited by the rather bad coagulation status of oncologic patients.

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 diagnostic yield will be performed.