*Deep phenotyping of the gut immune system during Immune Checkpoint Inhibitor therapy* (DEFENCE)

Activation of the immune system by Immune checkpoint inhibitors (ICIs) results
in long lasting anti-tumour activity in many patients with various cancer
types. However, the majority of patients do not benefit from ICI treatment.
There is an unmet need to better understand the determinants of ICI tumour
response and to develop novel therapeutic strategies to enhance this response.
Patients responding to ICIs have been shown to have higher peripheral and
tumour-infiltrating CD8+ T cells than non-responders at baseline and during ICI
therapy. The cytotoxic, tumour-directed effect of CD8+ T cells is affected by
interactions with other immune cells including CD4+ T cells and macrophages.
The gut is the largest barrier between the body and the outside world.
The gut wall is a crucial immune organ, hosting over 70% of our immune cells.
ICI therapy affects the gut, as evidenced by: (1) a per-treatment increase of
faecal calprotectin - indicating intestinal immune activation, (2) shifts in
immune cell subtypes including CD8+ T cells due to ICI therapy. CTLA-4 and
PD-1, the targets of ICI, are expressed by immune cells in the gut wall, and
moreover, have been shown to play an important role in the regulation of the
gut wall immune system. Furthermore, recent gut microbiome studies indicate
that the tumour response to ICI can be modified by gut-directed interventions.
Surprisingly, the gut wall has hardly drawn attention in the context of
ICI therapy. The effects of ICIs on the gut immune system are not fully
understood. We hypothesise that the gut immune system reflects relevant immune
changes taking place in the tumour due to ICI therapy. More specifically, we
hypothesize that in line with what has been reported within the tumour
microenvironment, ICI leads to an increase in CD8+ T cells in the lamina
propria and responders have higher CD8+ T cell counts than non-responders.

To define the immunohistopathological changes induced in the colon lamina
propria by ICI therapy.

This project is an exploratory study aiming to gain insight in gut immune
system phenotypes before and after ICI therapy. After informed consent is
obtained, sigmoidoscopies limited to the sigmoid and rectum will be performed.
Eight biopsies will be taken at both locations at baseline and 6-8 weeks after
initiation of ICI therapy. The immune cell infiltrates in these biopsies will
be analysed by immunohistochemistry (IHC), RNA sequencing and flowcytometry.
The design of the present study is based on ongoing as well as completed
successful studies (e.g. GEID study and VISION study) at the Department of
Gastroenterology and Hepatology of the UMCG. Both sample acquisition as well as
analyses will be done according to standardised procedures of the Department of
Gastroenterology and Hepatology, Medical Oncology and Pathology.

Sigmoidoscopies
Bowel preparation will be limited to 2 x 0.5 L PLEINVUE (oral solution) the day
before the endoscopy. The endoscope will be inserted up to upper border of the
descending colon. After white light endoscopic observation, biopsies will be
taken from the following areas: descending colon, sigmoid colon and rectum. Per
location, five biopsies will be taken. Biopsies will only be taken if judged
safe by the gastroenterologist. In order to minimise discomfort, patients may
undergo the procedure under sedation if desired. In case patients are sedated
with propofol, the vitals will be monitored by an employee of anaesthesiology
department (standard clinical care).

Peripheral Blood
Ten millilitres of peripheral blood will be collected using 368589 - 16x100 mm
10.0 mL BD Vacutainer® Plus Plastic whole blood tube, K2EDTA 18.0 mg.
Peripheral blood will be used to measure gut-specific inflammatory markers
before and during ICI therapy.

By taking part in this trial, patients can help to increase knowledge on the
effects of ICI therapy on the gut wall immune cell composition and to identify
new targets to improve ICI therapy. Patients will not directly benefit from
participating in this study.
Sigmoidoscopies with gut wall biopsies will be performed at baseline
and at 6-8 weeks after starting ICI therapy. In addition to the sigmoidoscopy,
blood will be drawn at both timepoints. Thus, patients will be required to make
2 visits outside of their routine care to the hospital. The sigmoidoscopies are
minimally invasive and take about 15 minutes.
Patients may experience some degree of discomfort. No anaesthesia nor
sedation will be required. However, patients may be sedated during the
procedure if desired. The risks are limited and include temporary complaints
such as mild abdominal pain and bloating. The risk of severe complications such
as intestinal perforation or haemorrhage is <1%. The preparation for the
sigmoidoscopy is limited to bowel preparation with PLEINVUE. This study causes
no delay of starting ICI therapy.