Identification of novel DNA methylation

biomarkers in colorectal cancer

Hannah Demond1, Javier Retamal1, Jaime Lopez1, Juan Castillo-Fernandez2, Santiago Sepulveda3,
Priscilla Brebi1, Carmen Ili1
1Millennium Institute on Immunology and Immunotherapy, Laboratory of Integrative Biology (LIBi), CEMT-BIOREN, Universidad de
La Frontera, Temuco, Chile. | 2Epigenetics Programme, Babraham Institute, Cambridge, UK. | 3Pathology Department, Faculty of
Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile.

Contact: Hannah.Demond@ufrontera.cl

DNA methylation biomarkers in cancer Pipeline to identify diagnostic biomarkers

Analysis Selection criteria

Logistic regression analysis of Differentially methylated (>30%

unmethylated CGIs difference) between NAT and
5 NAT vs. 5 PT samples PT 152 CGIs

DNA methylation analysis of Average methylation difference

candidates in TCGA database of >35% in both methyl-seq and
74 NAT vs. 74 PT samples TCGA data 28 CGIs

Figure from Luo et al. 2021

Cut-off determined by Youden’s
The tumour sheds DNA and other molecules into its environment. This cell-free or J index:
Analysis of ROC curves using TCGA
circulating tumour DNA can be detected in liquid biopsies of colorectal cancer (CRC) samples • Youden’s J >0.82
• AUC > 0.91 11 CGIs
patients and can be used to identify cancer-specific DNA methylation changes. 74 NAT vs. 374 PT samples • Sensitivity > 82%
• Specificity NAT >94%

Aim: Identification of novel DNA methylation Analysis of ROC curves using whole
Using same cut-off as above:
biomarkers for colorectal cancer to develop a non- blood samples
Specificity >99% 11 CGIs
invasive assay in blood for early diagnosis 656 blood vs. 374 PT samples

Datasets
DNA methylation changes in 5 CRC Normal adjacent 11 diagnostic DNA methylation CRC biomarkers
patients were analysed using whole- tissue were identified with an average sensitivity
genome methyl-sequencing (Ili et al. 5 CRC patients of 85% at an average specificity of 99%
2020). From each patient normal Primary
adjacent tissue (NAT) and primary tumour
tumour (PT) samples were analysed. Novel DNA methylation biomarker candidates
Candidate biomarkers were validated in larger cohorts, including 374 PT samples of
which 74 had patient-matched NAT samples from the cancer genome atlas (TCGA), as Specificity%
Gene* Youden’s J AUC Cut-off Sensitivity% Specificity%
Blood
well as 656 whole-blood samples from healthy controls (GSE40279).
NXPH2 87.92 0.94 > 0.35 87.92 100 99.85
LINC01158 85.57 0.94 > 0.25 85.57 100 100
AC013733.3 85.57 0.93 > 0.27 85.57 100 100
DNA methylation patterns of diagnostic biomarkers ADHFE1 85.38 0.93 > 0.36 85.38 100 100
DNA methylation biomarkers are significantly hypermethylated in PT samples of all GDF6 85.23 0.94 > 0.23 85.23 100 99.85
tumour stages (TNM I-IV) and consensus molecular subtypes (CMS) compared to NAT
OPCML 82.89 0.91 > 0.23 82.89 100 100
samples and are specific for gastro-intestinal adenocarcinoma.
TRBC2 82.85 0.94 > 0.14 88.26 94.59 100
A TCF24 82.85 0.94 > 0.11 88.26 94.59 99.85
SLC35F3 82.83 0.93 > 0.33 82.83 100 100
GALNT13 82.21 0.91 > 0.23 82.21 100 99.09
BRINP2 82.20 0.93 > 0.23 82.20 100 99.7

*Gene overlapping or within 2000bp proximity of differentially methylated CGI

Novel biomarkers: not mentioned as CRC methylation biomarkers in NCBI PubMed

Outlook: Establishing a non-invasive detection assay

In the next part of the project, we will establish and validate methylation-specific qPCR
B C D
(qMSP) assays for the candidate biomarkers using CRC tissue samples (NAT and PT).
NXPH2 - TNM NXPH2 - CMS Finally, the qMSP assays will be tested in blood plasma samples of CRC patients and
1.0 1.0
healthy controls to establish a non-invasive assay to detect CRC in blood.
DNA methylation

DNA methylation

qMSP analysis for two candidate

0.5 0.5
NXPH2 ADHFE1 biomarkers of NAT (n=6) and PT
1000 1000 (n=7) tissue samples from 8 CRC
0.0 patients. Concentration of methylated
Log10 Norm. Conc.

0.0
100 100
T

II

IV
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III
ge
A

L
S1

S2

S3

S4

DNA was measured using a standard

e

e
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ag

B
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CM

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St

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10 10 curve and normalized against ACTB

A) Clustering analysis of DNA methylation values of the biomarkers in 329 PT samples from the TCGA methylation. Each dot represents one

database. B, C) Examples of DNA methylation levels in different cancer stages (B) and subtypes (C) for 1 1 sample and line represents median.

biomarker candidate NXPH2. Dashed line indicates biomarker cut-off. D) Clustering analysis showing Mann-Whitney Test: NXPH2 P͏ =
0.1 0.1
DNA methylation levels of biomarkers in adenocarcinoma of different organs (data: TCGA) NAT PT NAT PT 0.0012** and ADHFE1 P͏= 0.0022**.

Ili et al. Cancers (Basel). 2020. 12:2710. Fondecyt Postdoctorado

Luo H et al. Trends Mol Med. 2021. 27:482–500. Nº 3200676