Colorectal cancer is a major cause of mortality and morbidity throughout the world. With adjuvant chemotherapy as standard management following surgery to treat stage III and stage II patients with high risk factors, the 5-year relative survival rate of locally advanced colorectal cancer was still 69.2% compared with 90.1% among patients with localized disease [
Promoter CpG island hypermethylation resulting in the transcriptional silencing of tumor suppressor genes has been widely observed in colorectal cancer and been increasingly recognized to contribute to the pathogenesis of colorectal cancer. The subset of colorectal cancers with exceptionally high frequency of CpG island methylation were referred to as CpG island methylator phenotype (CIMP) [
Our study was designed to investigate the prognostic effect of CIMP epigenotype in stages II and III sporadic colorectal cancer interacting with adjuvant chemotherapy in Chinese population utilizing the five-gene panel. The aim of the study was to provide evidence contributing to risk stratification and individualized management for patients with locally advanced sporadic colorectal cancer.
Between July 2004 and November 2004, 50 patients with stage II/III colorectal cancer who had received curative resection and were pathologically confirmed as adenocarcinoma or mucinous adenocarcinoma at the Colorectal Surgery Department, Fudan University Shanghai Cancer Center, were included for analysis. None of the patients were diagnosed as hereditary colorectal cancer (Lynch syndrome or familial adenomatous polyposis) or malignant tumors of other organs. Patients who underwent preoperative chemoradiation therapy or were treated with local excision were excluded. Eighteen and 32 patients were staged into stages II and III, respectively, according to the current TNM staging system of the 7th edition of the American Joint Committee on Cancer’s (AJCC) Staging Manual [
Clinicopathological characteristics associated with CIMP status.
CIMP− | CIMP+ |
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No. (%) | No. (%) | |
Age (yr) | 0.270 | ||
Mean ± SD |
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Sex | 0.750 | ||
Male | 17 (44.7) | 6 (50.0) | |
Female | 21 (55.3) | 6 (50.0) | |
Sitea | 0.046 | ||
Proximal | 5 (13.2) | 5 (41.7) | |
Distal | 33 (86.8) | 7 (58.3) | |
Stage | 0.022 | ||
II | 17 (44.7) | 1 (8.3) | |
III | 21 (55.3) | 11 (91.7) | |
Histology | 0.059 | ||
Adenocarcinoma | 32 (84.2) | 7 (58.3) | |
Mucinous adenocarcinoma | 6 (15.8) | 5 (41.7) | |
Grade | 0.007 | ||
Well/moderate | 31 (81.6) | 5 (41.7) | |
Poor | 7 (18.4) | 7 (58.3) | |
Lymphovascular invasion | 0.121 | ||
Present | 3 (8.1) | 3 (25.0) | |
Absent | 34 (91.9) | 9 (75.0) | |
Perineural invasion | 0.961 | ||
Present | 3 (7.9) | 1 (8.3) | |
Absent | 35 (92.1) | 11 (91.7) | |
CEA level | 0.802 | ||
Normal | 28 (80.0) | 10 (83.3) | |
Elevated | 7 (20.0) | 2 (16.7) | |
Adjuvant chemotherapy | 0.637 | ||
Not received | 10 (26.3) | 4 (33.3) | |
Received | 28 (73.7) | 8 (66.7) |
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SD: standard deviation, CIMP: CpG island methylator phenotype, CEA: carcinoembryonic antigen.
aProximal location included the cecum, ascending colon, hepatic flexure of colon, and transverse colon while distal location included the splenic flexure of colon, descending colon, sigmoid colon, and rectum.
Fresh tissue samples were collected within half an hour after the removal of tumor from the patient and were stored at −80°C. Fresh frozen tissues of colorectal cancer were reviewed pathologically to ensure that the sample tissue contained more than 80% cancer cell proportion. Genomic DNA was extracted using EZNA Tissue Kit (Omega Bio-Tek, Norcross, GA, USA) according to manufacturer’s instructions. A260 and A280 of the DNA samples were tested to measure the quantity and purity of genomic DNA.
Bisulfite modification of 500 ng genomic DNA was performed using EZ DNA methylation kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions. Methylation specific PCR (MSP) was performed as described previously [
CIMP classification was based on the number of methylated genes of the panel markers. Tumors were classified as CIMP positive if 3 or more markers were methylated or CIMP negative if none or less than 3 methylated markers were observed.
Tumor size and age were categorized into two groups using median as cut-off value. Tumors located from the cecum to transverse colon were classified into proximal colon cancer. Tumors located from the left colonic flexure to rectum were categorized into distal colorectal cancer. Correlations of CIMP epigenotype with categorical clinicopathological variables were assessed using Chi-square test or Fisher’s exact probability tests as appropriate. Comparisons of continuous variables were performed using Mann-Whitney
The primary clinical outcome was disease-free survival (DFS). DFS was defined as the time from surgical resection of colorectal cancer to the date of the local recurrence or first distal metastasis confirmed pathologically or by clinical imaging or to the last follow-up date. Overall survival (OS) was defined as the time from surgery to the date of patient’s death or to the last follow-up date. The impact factors of DFS and OS were analyzed using Kaplan-Meier method (log-rank test) and adjusted Cox proportional hazards models, respectively. Hazard ratios (HRs) and 95% confidence intervals (CIs) for DFS and OS were estimated using Cox regression. Furthermore, stratified survival analysis was performed according to CIMP epigenotype.
All
CIMP positive epigenotype was detected in 24.0% (12/50) of patients. Representative results of the electrophoresis of MSP products can be seen in our previous paper [
The median DFS was 60 months. Three local recurrences were identified. Nineteen cases of distal metastasis were confirmed before the last follow-up date. Liver metastasis was most common, occurring in 9 patients while the lung was the second common metastatic organ involving 6 patients. The 5-year disease-free survival was 58% in all patients.
In the univariate analysis, TNM stage and CIMP epigenotype were the only variables which showed significant impact on DFS. Patients with stage III disease (5-year DFS: 47.1% versus 74.1%,
Univariate and multivariate analysis of the prognostic effect of CIMP status and clinicopathological features in stage
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Univariate analysis | Multivariate analysisb | ||||
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No. | 5 yr DFS |
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HR | 95% CI |
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Age (yr) | 0.182 | |||||
≤55 | 30 | 69.7% | ||||
>55 | 20 | 55.0% | ||||
Sex | 0.990 | |||||
Male | 23 | 64.6% | ||||
Female | 27 | 63.0% | ||||
Stage | 0.049 | 0.242 | ||||
II | 18 | 83.3% | 1 | reference | ||
III | 32 | 52.9% | 3.075 | (0.468–20.205) | ||
Sitea | 0.092 | |||||
Proximal | 10 | 40.0% | ||||
Distal | 40 | 69.6% | ||||
Histology | 0.671 | |||||
Adenocarcinoma | 39 | 66.7% | ||||
Mucinous | 11 | 53.0% | ||||
Grade | 0.681 | |||||
Well/moderate | 36 | 66.7% | ||||
Poor | 14 | 56.3% | ||||
CEA level | 0.304 | |||||
Normal | 38 | 56.3% | ||||
Elevated | 9 | 44.4% | ||||
Adjuvant chemotherapy | 0.849 | |||||
Not received | 14 | 71.4% | ||||
Received | 36 | 61.0% | ||||
CIMP epigenotype | 0.014 | 0.019 | ||||
Negative | 38 | 73.7% | 1 | reference | ||
Positive | 12 | 31.3% |
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2.935 | (1.193–7.220) |
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5-yr DFS: five-year disease-free survival, CIMP: CpG island methylator phenotype, CEA: carcinoembryonic antigen, HR: hazard ratio.
aProximal location included the cecum, ascending colon, hepatic flexure of colon, and transverse colon while distal location included the splenic flexure of colon, descending colon, sigmoid colon, and rectum.
bOnly factors which showed significant impact on DFS in the univariate analysis were included in the Cox regression analysis.
Univariate analysis of the prognostic effect of CIMP status and clinicopathological features in stage
Univariate analysisb | |||
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No. | 5-yr OS |
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Age (yr) | 0.078 | ||
≤55 | 30 | 74.2% | |
>55 | 20 | 58.5% | |
Sex | 0.459 | ||
Male | 23 | 60.5% | |
Female | 27 | 74.4% | |
Stage | 0.222 | ||
II | 18 | 78.2% | |
III | 32 | 62.2% | |
Sitea | 0.117 | ||
Proximal | 10 | 50.0% | |
Distal | 40 | 82.0% | |
Histology | 0.559 | ||
Adenocarcinoma | 39 | 64.3% | |
Mucinous | 11 | 81.8% | |
Grade | 0.820 | ||
Well/moderate | 36 | 65.2% | |
Poor | 14 | 56.3% | |
CEA level | 0.255 | ||
Normal | 38 | 71.4% | |
Elevated | 9 | 50.0% | |
Adjuvant chemotherapy | 0.083 | ||
Not received | 14 | 35.7% | |
Received | 36 | 78.0% | |
CIMP epigenotype | 0.354 | ||
Negative | 38 | 69.0% | |
Positive | 12 | 65.6% |
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5-yr OS: five-year overall survival, CIMP: CpG island methylator phenotype, CEA: carcinoembryonic antigen, HR: hazard ratio.
aProximal location included the cecum, ascending colon, hepatic flexure of colon, and transverse colon while distal location included the splenic flexure of colon, descending colon, sigmoid colon, and rectum.
bOnly univariate analysis was performed since all factors considered were not significantly associated with OS.
Disease-free survival analysis according to stage and CIMP status. Patients with CIMP positive tumors had a marginally significant poor DFS than those with CIMP negative tumors with
Among CIMP negative patients, adjuvant chemotherapy had no effect on DFS (
Disease-free survival analysis stratified by CIMP status and treatment jointly. In CIMP negative tumors, adjuvant chemotherapy showed no effect on DFS with
In the present study, we identified a cohort of 50 stage II/III colorectal cancer patients treated with curative surgery alone or curative surgery followed by 5-FU based adjuvant chemotherapy and characterized the patients as CIMP positive or CIMP negative by performing methylation specific PCR (MSP) of 5 genes.
The prevalence of CIMP positive was reported to be 9%–90% in colorectal cancer and ranged widely between studies of populations of different ethnic backgrounds [
Furthermore, the prognostic effect of clinicopathological variables and CIMP status on stage II/III colorectal cancer was investigated. Our data suggested an unfavorable prognosis in patients with CIMP positive tumors and stage III tumors (Figure
In metastatic or recurrent colorectal cancer treated with 5-fluorouracil based chemotherapy, Ogino et al. [
Small sample size within each subgroup categorized by CIMP status, TNM stage, and chemotherapy limited the detection of the difference in prognostic significance. Potential selection bias could not be excluded in this retrospective study. It is noteworthy that the prognostic influence of CIMP status on stage II tumors and potential interactions with lymphovascular invasion, perineural invasion, and other clinical high risk factors of stage II colorectal cancer remains unclear since only 18 stage II cases were include in our study. The clinical decision-making regarding the adjuvant treatment of stage II is on the basis of identifying high-risk individuals by assessing the clinicopathological high risk factors. However, conventional clinicopathological parameters cannot yield satisfying performance to guide the individualized therapy. Integrating molecular biomarkers such as CIMP epigenotype seems to be promising in improving the predictive accuracy. Therefore, a prospective study with abundant case numbers of stage II patients is needed to evaluate the prognostic value of CIMP status in order to improve individualized therapeutic strategies.
To conclude, our data suggested that CIMP positive was an unfavorable independent prognostic factor in stage II/III sporadic colorectal cancer in Chinese population. Stage II/III patients with CIMP positive epigenotype may not benefit from 5-fluorouracil based adjuvant chemotherapy. Further studies are merited to confirm the potential role of CIMP status assessment as a high risk factor improving the stratifying of stage II/III colorectal cancer. If CIMP positive epigenotype was validated as a marker for chemoresistance of 5-fluorouracil and oxaliplatin based standard regimen, the benefit of these patients from irinotecan based chemotherapy should be evaluated.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Yuwei Wang and Yadong Long contributed equally to this work.
This study was supported by the grants of the National Natural Science Foundation of China (no. 81001055), Shanghai Pujiang Program (no. 13PJD008), and China Medical Foundation (no. 201302).