1. The study aimed to identify DNA methylation markers in normal colon tissue that are associated with increased risk of metachronous polyp formation after colorectal cancer (CRC) resection. 2. DNA methylation analysis of normal colon samples from 41 CRC patients identified 5 differentially methylated loci between those with and without subsequent polyps. These mapped to genes implicated in cancer development. 3. Characterizing a pre-malignant field through aberrant DNA methylation could help identify high-risk individuals for CRC prevention and lend insight into CRC pathogenesis.

1. Discovery and Validation of Potential Field Cancerization Molecular Markers that Associate with Metachronous Polyp Formation
1. Division of Gastroenterology, University of Washington, Seattle, WA, United States 2. Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, United States. 3. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, United States.
Barahimi, Mitra1,2; Maden, Sean2; Carter, Kelly2; Yu, Ming2; Vickers, Kathy2; Li, Christopher3; Grady, William M.1,2
INTRODUCTION
METHODS
There are a number of factors that increase the risk of colorectal cancer
(CRC) development. These include genetic susceptibility, behavioral factors and
environmental exposures. These factors appear to influence colon polyp
formation and/or progression of the polyp to CRC.
Field carcinogenesis, the concept that an organ may be predisposed to
cancer development due to molecular changes in the normal appearing tissue,
has been observed in the colon and associated with an increased risk of CRC. A
variety of putative field cancerization alterations have been shown, including
chromosomal losses and gains, aberrantly methylated genes and loci (e.g. EVL,
LINEs).
Importantly, these alterations have been implicated in field cancerization
by their association with concurrent colorectal cancer. None of these alterations
have been shown to associate with the risk for forming polyps or CRC in the
future.
The purpose of this study was to identify and characterize a pre-malignant
field in the form of aberrant DNA methylation in CRC patients who have
metachronous polyps after CRC resection.
To accomplish this aim, we quantified aberrant DNA methylation at CpG
dinucleotides (CpGs) in normal colon biopsies obtained in patients at the time of
CRC resection and compared these findings in patients with and without
metachronous polyps after CRC resection.
1. DNA methylation analysis: DNA from the normal colon at the edges of
resected colon segments was extracted and subjected to bisulfate treatment.
We then assayed the DNA samples using DNA methylation arrays (HM450 or HM
EPIC arrays, Illumina). We analyzed genome-wide methylation at over 450,000
CpG loci in normal colon tissue from 41 patients. The samples were divided into
a discovery set of samples (N=20) and a validation set of samples (N=21).
2. Tissue samples: Samples were collected within 30 cm of the CRC site from
patients at the University of Washington Medical Center between 2008-2016
(Table 1). The study subjects are enrolled in the ColoCare CRC cohort study.
Patients were classified according to whether they had 0 (N = 18) or >1 (N
= 23) metachronous polyps.
3. Data Analysis: These sample groups were used to identify significant (F-test,
q-value < 0.01) differentially methylated CpG probes (DMPs) in the HM450K
discovery dataset (N = 20; 8 with and 12 without metachronous polyps).
We then validated a subset of significant DMPs using a validation dataset
generated with EPIC arrays (N = 21; 15 with and 6 without metachronous
polyps).
RESULTS
CONCLUSIONS
ACKNOWLEDGEMENTS
1. In this comparative analysis, we identified five aberrantly methylated CpG loci
in patients with metachronous polyps on one or more follow up colonoscopies
after CRC resection.
2. These DMPs map to several genes implicated in cancer development and
therefore may represent functionally relevant pre-malignant field markers.
3. Characterizing a pre-malignant field in the colon could shed more light on the
biology of CRC formation and lend itself to the development of biomarkers that
could identify individuals at higher risk of CRC formation.
4. Furthermore, aberrant DNA methylation is a promising target for future CRC
prevention strategies.
RESULTS
Figure 1. Heatmap demonstrating differentially methylated CpG Probes in
normal colon samples from patients with and without metachronous polyps
on surveillance colonoscopy
MAIN FINDINGS
1. Identification of differentially methylated DNA loci or genes (DMPs) in patients with
metachronous colon polyps:
We identified 5 DMPs that showed significant and substantial (>|0.1| β-value difference, q-value
< 0.01, F-test with FDR adj.) differences in mean methylation values between patients with and without
metachronous polyps across both validation and discovery datasets (Figure 1).
2. Location of several candidate field cancerization methylated CpGs is in genes, which
suggests that the methylated genes may have a functional role in CRC formation.
These DMPs mapped to several gene regions which have previously been implicated in
gastrointestinal and non-gastrointestinal cancer development. The methylation of CpGs in gene
promoters can cause gene silencing.
These include ANXA1, implicated in glioblastoma development, as well as GRM8 and PFKP,
which are molecularly altered in gastric and breast cancer, respectively.
Table 1. Baseline and other patient demographics in discovery and validation data sets.
DM = distal margin, PM = proximal margin, DA = distal adjacent, NA = not available, CRC = colorectal cancer
Funding provided by the following: NIH grants (P30CA15704, UO1CA152756, R01CA194663, RO1CA220004,
RO1189184, U54CA143862, P01CA077852), R.A.C.E. Charities, Cottrell Family Fund, Listwin Family
Foundation, and Seattle Translational Tumor Research program. We wish to also to thank the staff and
patients of the ColoCare Study, GICaRes biorepository, and CHTN.
HM450K Discovery Data Set (N = 20) EPIC Validation Data Set (N = 21)
No Polyps (N = 12) >1 Polyp (N = 8) No Polyps (N = 6) >1 Polyp (N = 15)
Age, mean  s.d. 59.0  7.2 61.3  6.1 55.2  10.1 51.8  13.4
Female, n (%) 5 (41.7) 3 (37.5) 4 (66.7) 9 (60)
Male, n (%) 7 (58.3) 5 (62.5) 2 (33.3) 6 (40)
Location of Normal Colon
Biopsy
DM, n (%) 1 (8.3) 2 (25) 1 (16.7) 3
PM, n (%) 9 (75.0) 4 (50) 5 (83.3) 12
DA, n (%) 1 (8.3) 0 0 0
NA, n (%) 1 (8.3) 2 (25) 0 0
CRC Location
Right Colon, n (%) 5 (41.7) 3 (37.5) 0 0
Left Colon, n (%) 7 (58.3) 5 (62.5) 6 (100) 15 (100)
BMI (kg/m2), mean  s.d. 25.5  6.4 34.1  14.1 25.7  3.9 29.7  6.8
Tobacco use
Yes, n (%) 4 (33.3) 4 (50) 2 (33.3) 6 (40)
No, n (%) 8 (66.7) 4 (50) 4 (66.7) 9 (60)
NSAID use
Yes, n (%) 6 (50) 2 (25) 1 (16.7) 3 (20)
No, n (%) 6 (50) 6 (75) 5 (83.3) 12 (80)
Lynch Syndrome, n (%) 0 1 (12.5) 0 1 (6.7)