Proteomics in Early Neoplasia in Barrett*s Esophagus: Biomarkers for Early Detection.

The incidence of esophageal adenocarcinoma (EAC) has increased almost fourfold
in the last three decades. At a symptomatic stage, EAC carries a poor
prognosis. At this stage the tumor can only be cured by extensive surgical
treatment, which has a mortality rate of 3-5% and a morbidity rate of 30-50%.
Furthermore, at the time of diagnosis 50% of patients are found to have local
irresectable disease, distant metastases or poor general condition, making them
not eligible for curative treatment. Presence of Barrett*s esophagus (BE) is
the most important risk factor for developing EAC. In BE, the epithelium of the
distal esophagus has been replaced by columnar epithelium containing
specialized intestinal metaplasia, due to chronic gastro-esophageal reflux.
Malignant transformation of BE into EAC is a gradual progress, which may take
up to several decades. Progression into cancer is through the histopathological
stages classified as no dysplasia, low-grade dysplasia and high-grade
dysplasia. In order to detect malignant progression of BE at an early and
curable stage, regular endoscopic surveillance is indicated in patients with
BE. Studies have shown that patients with a BE in whom the cancer is diagnosed
while under surveillance have an excellent prognosis.

In the USA the costs of Barrett*s surveillance are estimated at 290 million
dollars annually, the annual Dutch costs are estimated at approximately
¤4,000,000 with approximately 8,000 gastroscopies per year. Beside the high
annual costs of endoscopic surveillance, endoscopy is uncomfortable for the
patient. A less invasive, less costly diagnostic test would be of great value.
Analysis of protein profiles for example in serum or tissue of patients using
mass spectrometry, potentially has the ability to detect specific profiles that
can be used as a high-throughput diagnostic test, which could be an alternative
for endoscopic surveillance and potentially even for Barrett's screening.

To obtain more information on which proteins should be looked for in the serum,
the tissue protein profile of Barrett*s mucosa with and without early neoplasia
(HGD and carcinoma tissue with or without early neoplasia. Mass spectrometry (MS) coupled with
Liquid Chromatography (LC) of epithelial has proven a valuable tool in proteome
exploration of tissue. A drawback of the technique is that lower abundant
proteins often remain undetected in complex protein samples. One approach to
look more specific and deeper into tissue proteomes is by selective sampling of
distinct cell types by Laser Captured Microdissection (LCM). A pilot study was
performed on non-dysplastic biopsies, to assess the feasibility of the current
study. This approach resulted in high numbers of protein and peptide
identifications in biopsies, with good reproducibility of the detected
differences between epithelial and stromal cells.

To compare the protein profile of Barrett*s mucosa with and without early
neoplasia in ER specimens in epithelial and stroma aiming to identify a
biomarker indicating presence of early neoplasia in BE.

To be able to investigate a larger amount of neoplastic cells in the current
study, we will use endoscopic resection (ER) specimens instead of biopsy
specimens, for the following reasons: 1) an ER specimen will contain more early
neoplastic cells for research than biopsies, 2) ER specimens are larger than
biopsies, therefore orientation of the frozen sample before cutting is
possible; orientation enables better histopathological assessment. This is
important for frozen samples in particular because histopathological assessment
of frozen tissue is complicated by freeze artifacts.

This non-randomized study includes two patient groups, each with 10 consecutive
patients. Protein profiles of Barrett*s mucosa of patients with early neoplasia
and without any dysplasia are compared on the epithelial and stromal level
using mass spectrometry. Patient recruitment and sample collection will be
performed at the AMC in Amsterdam. Samples will be processed and analyzed in
the EMC Rotterdam at the Netherlands Proteomics Center.

In the non-dysplastic group, a regular follow-up endoscopy will be performed,
with standard biopsy sampling. General risks associated with a gastroscopy are:
mild irritation of the throat due to introduction of the endoscope,
difficulties swallowing and retrosternal pain. Additionally, an endoscopic
resection will be performed by using the cap-technique, minor bleeding may
occur in 6% of the cases, usually easily managed with endoscopic hemostatic
techniques.

Participation of patients in the dysplastic group does not contain any
additional risks, given the fact that these patients undergo an endoscopic
resection as a regular therapy for carcinoma or high-grade dysplasia.