Into the light: guided visualization of high-risk cancer precursor lesions

In the Netherlands 14,000 new cases of colorectal cancer (CRC) are diagnosed
annually. Introduction of the screening program has improved early detection of
CRC, however, a significant proportion (~17%) of right-sided CRC precursor
lesions are still missed during colonoscopy because they are often flat and
non-ulcerated (so called sessile serrated lesions (SSLs)). Currently, the
majority of interval cancers (i.e. cancers that occur in between screening
tests) is identified in the right-sided colon and shares the molecular
background of SSLs. The relative high miss rate of SSLs in combination with
their distinct and rapid progression to highly aggressive CRC requires
significant improvements in detection.
To increase detection of CRC precursor lesions several strategies have been
applied. This includes 1) advancing visualization techniques, (e.g. behind
folds visualization), 2) addition of computer aided detection and 3) assisted
viewing (second observer). Only marginal improvements in detection rates have
been achieved so far with these technical and logistical strategies.
The addition of fluorescent labels targeting molecules within the tumor during
colonoscopy has shown promise by focusing on disease based pathologic
characteristics. However, topical administration of these labels has shown
difficulties because mucus barriers need to be passed to reach their targets.

Research direction
Until now only the tumor has been part of the search for new molecular imaging
targets. The associated microbial biofilm that is present on 89% of right-sided
CRCs and conventional advanced adenomas (CAAs) has so far not been incorporated
in imaging optimization, while it has in our view great potential. 1) No mucus
barriers need to be passed for topical administration as the biofilm is part of
the mucus layer, 2) The microbiome in biofilms is very specific for colorectal
tumors and distinct from stool and normal mucosa, and 3) endoscopic
administration of fluorescent tracers against specific microbiome proteins
within the biofilm can be easy and fast and visualized with existing imaging
devices.
Molecular imaging using fluorescence endoscopy of the biofilm could aid the
endoscopist with real-time biological supplementary information complementary
to morphology imaging.

Aim
to potentiate endoscopic detection of high-risk precursor lesions using
targetable proteins within the biofilm

Objectives
1. Assess whether biofilms are reliable biomarkers for all types of right-sided
precursor lesions (conventional advanced adenomas (CAAs) and SSLs).
2. Identify extracellular target peptides specific for precursor lesions using
a discovery-based metagenomics and proteomics approach

Plan of investigation
1. Prevalence and relative abundance of biofilms on right-sided precursor
lesions
Previously we showed that biofilms are associated with right-sided CRCs in
sporadic cases, and are universal on CAAs throughout the colon of patients with
familial adenomatous polyposis. Currently, no information is available about
SSLs in correlation with biofilms. The prevalence of biofilms on all types of
neoplastic precursor lesions (CAAs and SSLs) should at least be 70% and more
abundant (based on thickness and density) than on corresponding normal tissue
to improve detection. When true, our strategy could in theory decrease miss
rates from 17% to 5%. Tissue blocks from 150 CAAs and 150 SSLs larger than 10
mm in size diagnosed between 2010-2018 will be collected from the archives.
First the histological subtype and grade will be re-evaluated by an expert
pathologist to verify correct classification. Next, biofilm prevalence on
adenomas and corresponding normal tissue from the same case will be visualized
with our optimized fluorescent in situ hybridization (FISH) protocol.
2. Identification of targetable extracellular proteins within biofilms
To identify targetable extracellular protein domains we will apply a
discovery-based approach. Prospective fresh frozen material will be collected
from a total of 40 precursor lesions larger than 10 mm in size from the
right-sided colon harboring biofilms and matching normal control biopsies (>10
cm apart from the neoplasia). After pathological classification and diagnosis,
the remaining tissue will be used for biofilm assessment and processed for
analysis by shotgun metagenomics. First, outermembrane proteins, cell-wall
proteins, extracellular proteins and proteins that are known to be shed and
attached to the cell surface will be identified from the assembeled and
annotated metagenomes. Next, precursor lesions will be compared to their
corresponding normal tissue to select extracellular protein domains specific
for the biofilm (using supervised clustering methods). Next, negative selection
will be performed removing proteins that are present in our own and publicly
available metagenomic data of healthy tissue. The remaining top-hits of
extracellular proteins will be selected for the development of single chain
antibodies using phage display libraries in collaboration with Modiquest
Research. The best single chain antibodies will be selected using
immunohistochemistry on clinically diagnosed right-sided precursor lesions by
calculating their positive predictive value.

Expected output
The results of this unique high-risk project will provide a proof-of-concept to
use biofilms for visualization of precursor lesions. The most successful
single-chain antibodies targeting the biofilm on precursor lesions will be
validated in an implementation grant.

For this study, patients will be asked to participate once during one of their
regularly planned colonoscopies with EMR-procedure. During colonoscopy two
extra biopsies will be taken from the mucosa and the participants will fill out
a short questionnaire. the extra risk of taking biopsies during a planned
colonoscopy is estimated as negligible.

The tissue collected for diagnostics will follow an alternative processing
route without any risk for the participant. Instead of placing the tissue
directly in formalin at the endoscopy unit, the tissue will be placed partly in
formalin and cryo-preserved at the pathology suite. Both cryo-preservation and
formalin fixation can be used to set a diagnosis, however, usually formalin is
the preferred choice because it is quicker, cheaper and nice histology. The
tissue will be available for diagnostics at all times. After diagnosis, the
tissue will be made available for the PRELEDE-study.