Methods

Patient eligibility

The target number of recruited patients with CRC was 320, and more than 30%

should be EOCRC. Patients who met the following inclusion criteria were eligible for

this study: (1) age ≥ 20 y/o, (2) cytologically or pathologically proven CRC and

adequate staging (American Joint Cancer Committee on Cancer, 7^th edition) by

computed tomography, and (3) undergoing a colectomy surgery. The exclusion criteria

were that they received systemic chemotherapy or radiotherapy before colectomy.

Written informed consent was obtained from all patients before collecting the tumor

specimens. We also collected the patients’ clinical and pathological characteristics,

including age, sex, histology and grade of tumors, tumor location, the risk factors of

CRC, and BMI. The definition of these clinicopathologic variables is described below.

For age, the patients were categorized as age < 50 y/o and age ≥ 50 y/o. The histology

of tumors included the presence or absence of mucinous components, signet-ring cells,

and medullary histology, which were observed by microscopic examination. Mucinous

carcinoma was designated if more than 50% of the tumor volume was a mucinous

component. The tumor grade was classified into low and high grades. Tumors were

graded as a low grade if ≥ 95% of the tumor has glandular formation or MSI-high.

Otherwise, tumors were graded as a high grade. Tumor location was grouped into

right-sided and left-right-sided. Right-right-sided tumors were designated if the primary tumors were

located from the cecum to the splenic flexure of the transverse colon, and left-sided

tumors were located from descending colon to the rectum. The Institute Review Board

of National Taiwan University Hospital (NTUH) approved this study.

Risk factors

In this study, risk factors include first-degree family history for CRC, history of

colorectal polyp, DM, hyperlipidemia, BMI at diagnosis, pre-diagnosis use of NSAIDs,

and hormonal replacement therapy (HRT). These data were obtained from interviews

with the patients and the survey of the electronic chart of NTUH. The definitions of

these risk factors are described below. Patients with more than one first-degree family

member diagnosed with CRC were grouped into a group with a family history of CRC.

BMI at diagnosis was retrieved from a medical record. We categorized BMI into three

groups based on WHO criteria of 18–24.9, 25–29.9, and ≧30 kg/m². We also

categorized BMI into two groups (< 27.5 and ≧ 27.5 kg/m²) according to the experts’

opinion for appropriate BMI in Asian populations from WHO expert consultation⁶⁷. In

that report, a BMI of ≧ 27.5 kg/m² was regarded as a high risk for public health.

Histories of DM and hyperlipidemia were determined according to self-reports and/or

the identification of the use of medication for these diseases. Pre-diagnosis NSAIDs use

was defined if two or more times per week in one month or longer within one year of

NSAIDs use was reported. Pre-diagnosis of HRT use was determined if six months or

longer of HRT use within one year was reported.

CIMP analysis

We used a QIAamp DNA formalin-fixed paraffin-embedded (FFPE) tissue kit to

extract genomic DNA from tumor specimens (Qiagen, Santa Clarita, CA, USA). Then

the genomic DNA was treated with sodium bisulfide according to the EZ DNA

Methylation Kit protocol (Zymo Research, Irvine, CA, USA). We evaluated the

methylation status of five loci: MINT1, MINT2, MINT31, p16, and MLH1 (classical

panel) using MethyLight assay, which was a probe-based, methylation-specific

real-time polymerase chain reaction technology. The primers used for the MethyLight study

are listed in Table 1. The methylation-independent Alu repeat was measured for

normalization control reaction. The methylation levels of five loci in tumor samples and

a constant reference sample were measured, and quantification analysis of DNA

methylation was performed. The percentage of methylated reference (PMR) of each

locus was calculated based on the equation proposed in a previous study as the

following: PMR = 100 × (methylated reaction / control reaction)sample / (methylated

reaction / control reaction)M.SssI-Reference. A PMR of > 10 was regarded as

hypermethylated⁴⁹. Finally, CIMP-high tumors were determined if there were three or

more loci with a PMR of >10 identified. Otherwise, the tumors were determined as

CIMP-low/negative.

RAS/BRAF mutation analysis

We evaluated RAS (KRAS and NRAS) and BRAF mutations by PCR coupled with

Sanger’s sequencing method. The primers in this study covered exons 2, 3, and 4,

including codons 12, 13, and 61 of KRAS and NRAS, and covered exon 15 of BRAF.

After PCR, we used an automated ABI 3730 sequencer (Applied Biosystems, Foster

City, CA, USA) for direct sequencing.

MSI analysis

We used the MSI Analysis System (Promega Corp., Madison, WI, USA), a

PCR-based assay for detecting MSI, in five mononucleotide loci (NR-21, NR-24, MONO-27,

BAT-25, and BAT-26). Tumors were designated MSI-high if abnormal allele length

was identified in two or more loci. Otherwise, tumors were MSS.

Statistical analysis

The patient’s clinicopathological characteristics were presented as percentages and

frequencies. The association between CIMP-high CRC and clinicopathological

variables was estimated by logistic regression (Table 2), Fisher’s exact test, and

Chi-square test (Table 3 and Table 6). The association between risk factors and CIMP-high

CRC was also evaluated by logistic regression (Table 4 and Table 7). Finally, we used a

multivariate logistic regression model to evaluate the odds ratio of each variable for

CIMP-high tumors. Only a 2-sided p-value of ≤ 0.05 was considered statistically

significant. Only the variables with a p-value less than 0.1 in univariate analysis (Table

3 and Table 4) were put into a multivariate logistic regression model. All the statistical

analyses were performed using SAS statistical software (version 9.4, SAS Institute,

Cary, NC, USA).