The feasibility of preparing untreated esophageal adenocarcinoma endoscopic biopsies towards DNA/RNA samples suitable for next-generation sequencing.

Esophageal carcinoma is the fastest growing malignancy in the gastrointestinal
tract in the Netherlands. In 2014, 2549 patients were diagnosed consisting in
68% of adenocarcinoma and 32% of squamous cell carcinoma. Neoadjuvant
chemoradiation combined with radical surgical resection is the standard of
treatment in the Netherlands and offers the best chance of cure. Chemoradiation
results in a complete tumor response in 25%, partial response in 35% and no
response in 40% of the patients (non-responders). Patients with squamous cell
carcinoma have a higher reponse rate to chemoradiation compared to patients
with adenocarcinoma. Chemoradiation is associated with toxicity, including
leukopenia, thrombocytopenia, fatigue, nausea and anorexia. It is of high
clinical importance to identify the non-responders to further refine the
treatment regimen, and to apply more selective treatment options. This group of
patients suffer from the potential adverse effects without clinical benefit and
have surgical delay of 3 months.
As yet, there are no methods available for response prediction to neoadjuvant
therapy. Preliminary studies from our research group with tissue microarrays
showed an association of the expression of different biomarkers and survival of
patients with esophageal adenocarcinoma. The results indicate that expression
analysis of many more genes in combination is required for accurate
classification.
Next generation sequencing (NGS) can lresult in the identification of
expression profiles for predicting the repsonse to neoadjuvant chemoradiation.
This will ultimately lead to optimal individualized tailored treatment of
patients with esophageal adenocarcinoma. Non-responders will not be exposed to
this toxic, ineffective therapies but can be treated directly by surgical
resection.
In order to be able to apply NGS, good quality DNA or RNA samples are necessary.
The aim of this pilot study will be to determine the feasibility of preparing
DNA/RNA samples from untreated esophageal adenocarcinoma biopsies suitable for
next-generation sequencing..

The feasibility of preparing untreated esophageal adenocarcinoma endoscopic
biopsies towards DNA/RNA samples suitable for next-generation sequencing.

- Patients are selected by the treating physician and informed about the study.
If the patient is interested, information about the study is given.
- After at least 24 hours, informed consent is written during the next
policlinic visit.
- On the day of the standard endoscopic ultrasonography, the gastroenterologist
will take four extra tumor biopsies.
- The PhD candidate will ensure that all biopsies are directly fresh frozen and
stored at -80ºC in the tissue biobank. The laboratory manual sampling
instructions for sequencing is used for the collecting, processing, storing and
shipping of the samples.
- The department of pathology will confirm the diagnosis of esophageal
adenocarcinoma and the tumor percentage and evaluate the representativeness of
the biopsies.
- Only biopsies with >30% tumor percentage will be used for DNA/RNA extraction.
- DNA or RNA will be extracted from every sample using the automatic setup of
QiaSymphony by Hartwig Medical Foundation.
- All biopsy samples with a tumor percentage of at least 30%, a minimum of
500ng DNA, and a minimum of 1 µg RNA can be sequenced under supervision of
prof. Cuppen by Hartwig Medical Foundation. Whole exome sequencing can be
applied to analyze DNA with the Agilent SureSelect enrichment followed by
Illumina NextSequencing. For RNA sequencing, samples can be prepared with the
Illumina Neoprep microfluidics system and sequenced on Illumina Nextsequencing.
- After sequencing, data analysis and interpretation can be performed with the
standard CPCT pipelines using BWA and GATK best practice.
- The clinical, surgical and pathological variables of enrolled patients will
be kept in our electronic database.

Standard endoscopic ultrasonography with taking of biopsies is performed to
determine the diagnosis and to make a treatmentplan. Only in rare cases (<1%),
a tear or hole (perforation) or bleeding in the esophagus wall can be caused.
The taking of additional biopsies is associated with the same risk.