Organoids of Barrett*s esophagus:
a feasibility study and creation of a Biobank

Esophageal cancer is the eighth most prevalent cancer in the world (2) and it
has been on the rise in recent decades. The incidence of esophageal cancer in
the Netherlands almost doubled between 1989 and 2009, as it rose from 4.6 to
8.5 cases per 100,000 citizens. This rise is mostly explained by an increase in
esophageal adenocarcinoma (EAC) in men, whereas the incidence of esophageal
squamous-cell carcinoma (ESCC) has remained relatively stable. (Figure 1) (1)

The etiology of EAC is different from ESCC. While Tobacco use is a risk factor
for both; obesity, reflux disease and Barrett*s esophagus are specific for EAC
and increase the risk notably. (3, 4) Especially the latter two have been
considered the key factors responsible for the observed increase in incidence.
(5) Barrett*s esophagus will be described in more detail further on in this
introduction.

The treatment of advanced esophageal cancer is traditionally surgical. This
surgery, however, has a relatively high morbidity and mortality. (6) Recent
discoveries have proven that neoadjuvant chemoradiotherapy (nCRT) significantly
increased survival as compared to surgery alone in patients with potentially
curable esophageal cancer. Moreover, in the nCRT group of the study, 49% of
patients with ESCC and 23% of patients with EAC had a pathologically complete
response in the resection specimen. (1, 7) This finding led to the start of the
preSANO and SANO trial, of which the first started in 2013. The goal in this
study is to assess if remission after nCRT can be assessed and if so, if
surgery is needed at all in patients that show remission after nCRT. (6) As of
now all patients receive the same kind of chemotherapy. However, there are
multiple effective pharmacological treatments known which are not equally
effective in every patient. (1, 4, 8) In light of these findings, trying to
improve the efficacy of chemotherapy is even more imperative.

We want to try to investigate these esophageal tissues using organoids.
Organoids are defined as miniaturized and simplified versions of an organ,
produced in vitro in three-dimensional culture systems, that are
self-organizing and show realistic micro-anatomy. (9) This is done by placing
adult stem cells, isolated from tissue, in extracellular matrix (i.e. Matrigel®
containing laminin, collagen IV, heparin sulfate proteoglycans,
entactin/nidogen and growth factors) and adding the correct growth factors. The
stem cells will then start to build organoids through mitosis. (10, 11).

The creation of organoids has already been accomplished for healthy esophageal
tissue at our facilities and for Barrett*s esophagus elsewhere. (10, 11) The
purpose of this study is to create organoids of patient-derived Barrett*s
esophagus tissue at our own facility. If this is successful we can use this
technique for further research.

We want to use these tissue models to test different cytotoxic agents or
chemotherapeutics. Hopefully this knowledge will ultimately lead to a validated
model that can assess treatment-efficacy of chemotherapy. By *treating* these
organoids first to see what therapy works the best, we hope that a more
patient-specific and therefore possibly superior treatment can be achieved.

In conclusion, the primary aim of this pilot-study is to test the feasibility
of culturing patient-derived Barrett*s esophagus organoids on a small scale at
our facilities.

Secondary aims will be:
- To create a small Biobank of 10 patients for further study in the near future.
- To use organoids of Barrett*s esophagus tissue to test cytotoxic agents or
chemotherapeutics in vitro.

Primary Objectives:
- To test the feasibility of culturing patient-derived Barrett*s esophagus
organoids
- To test pharmacological and chemotherapeutical agents in vitro on the
cultured organoids

Secondary Objectives:
- To create a small Biobank (of 10 patients) for further study

Patients with a confirmed Barrett*s esophagus diagnosis are added to a
surveillance program whereafter a surveillance endoscopy is recommended to take
place every 3-5 years, dependent on absence or presence of dysplasia. During
the surveillance endoscopy four quadrant-biopsies are taken at every 2
centimeters of the Barrett segment. Endoscopic biopsy of Barrett*s esophagus is
considered a safe procedure; a study of 1.458 procedures found no deaths, no
esophageal perforations and no gastro-intestinal bleeding that was attributable
to endoscopic biopsies alone. The average number of biopsies taken during a
single procedure was 35 with a maximum of 120 (12).

In addition to the standard-care biopsies, a total of 5 extra biopsies of the
Barrett*s esophagus is deemed to be necessary for subsequent cell culture in
the form of organoids. Members of the gastroenterology laboratory will pick the
tissue up from the endoscopy ward and bring it to the laboratory for cell
culture preparation.

After sufficient growth of organoids has been observed, they will be stored in
a Biobank at
-80 ºC. The tissue will not leave the facilities of the Radboud University
Medical Center.

Patients with a confirmed Barrett*s esophagus diagnosis are added to a
surveillance program whereafter a surveillance endoscopy is recommended to take
place every 3-5 years, dependent on absence or presence of dysplasia. During
the surveillance endoscopy four quadrant-biopsies are taken at every 2
centimeters of the Barrett segment. Endoscopic biopsy of Barrett*s esophagus is
considered a safe procedure; a study of 1.458 procedures found no deaths, no
esophageal perforations and no gastro-intestinal bleeding that was attributable
to endoscopic biopsies alone. The average number of biopsies taken during a
single procedure was 35 with a maximum of 120 (12).
In addition to the standard-care biopsies, a total of 5 extra biopsies of the
Barrett*s esophagus is deemed to be necessary for subsequent cell culture in
the form of organoids.

Participation in the study does not cause any additional charge to patients.
The patient does not benefit from participating in the study. On the other
hand, the risk of taking an additional 5 biopsies in these patients is
negligible.

The risk classification is determined as negligible based on the guideline of
the *Nederlandse Federatie van Universitair Medische Centra*. The risks
associated with the participation in the study are similar to the risks of
multiple biopsies in surveillance of Barrett*s esophagus.