Abstract
Aim: To evaluate the contribution of ERCC1 rs11615 and rs3212986 genotypes regarding the risk of colorectal cancer (CRC) in Taiwan. Materials and Methods: In this case–control study, ERCC1 rs11615 and rs3212986 genotypes and their interaction with consumption of cigarettes and alcohol in determining CRC risk were investigated among 362 CRC patients and 362 age- and gender-matched healthy controls. Results: The percentages of CC, CT and TT for ERCC1 rs11615 genotype were 44.2%, 36.2% and 19.6% in the CRC group and 49.7%, 38.4% and 11.9% in the control group, respectively (p for trend=0.0158). The allelic frequency distribution analysis showed that the variant T allele of ERCC1 rs11615 conferred increased CRC susceptibility to the wild-type C allele (odds ratio (OR)=1.34, 95% confidence interval (CI)=1.08-1.67, p=0.0079). As for the gene-lifestyle interaction, there were obvious joint effects of ERCC1 rs11615 genotype on the risk of CRC among ever smokers and alcohol drinkers, but not non-smokers or non-drinkers. There is a positive correlation of ERCC1 rs11615 genotype with lymph node metastasis, but not other CRC prognosis, including tumor size and location. Conclusion: ERCC1 rs11615 T allele serves as a predictive marker for CRC risk and future studies with larger samples and functional evaluation are warranted to validate these findings.
Statistically, nearly one million cases of colorectal cancer (CRC) diagnoses worldwide each year and the incidence, as well as age-adjusted mortality of CRC, keep on increasing in recent years (1). In Taiwan, the incidence and mortality of CRC has occupied the first and third places among the common types of cancer for many years and the high incidence has been proposed to be closely associated with dietary changes to Western food style and a decreased consumption of dietary fiber or grain-made foods. Etiological studies have attributed more than 85% of CRC to risk environmental factors (1-3), particularly meat consumption, cigarette smoking, exposure to carcinogenic aromatic amines, such as arylamines and heterocyclic amines (4, 5). About 15-20% of CRC cases are with strong familial history of cancer that have interested the epidemiologists to figure out additional inherited susceptibility factors (6-8). In Taiwan, although specific biomarkers for CRC prediction and detection have keeping on being reported in recent years (9-16), the genomic susceptibility of CRC and the interactions among the genomic and environmental risk factors are mostly unknown.
Our genome is regularly and frequently damaged by various kinds of endogenous and exogenous mutagens and the DNA repair systems play a vital role in protecting our genome from irreversible mutations leading to carcinogenesis, among which the nucleotide excision repair (NER) pathway is one of the nuclear DNA repair systems used in correcting subtle DNA lesions and bulky DNA damage (17, 18). Among the DNA repair proteins involved in NER, excision repair cross-complementing group 1 (ERCC1) is located on chromosome 19q13.3 and participates as the central rate-limiting enzyme in the multistep NER process. For instance, Shirota and his colleagues have suggested that down-regulation of ERCC1 expression is associated with increased chemotherapeutic sensitivity and, thus, considered a predictive marker for CRC patients receiving combination of oxaliplatin and fluorouracil chemotherapy (19). In addition, Huang and his colleagues reported that A2251C variants of ERCC2 were associated with increased risk of early relapse in CRC (20). In the literature, several single-nucleotide polymorphisms (SNPs) of ERCC1 have been well identified, of which ERCC1 rs11615 and rs3212986 SNPs (Asn118Asn and C8092A) both have transcriptional moderating effects on their mRNA expression (21). Given the role of ERCC1 in genomic stability maintenance and carcinogenesis progression, we hypothesized that genomic variations in the ERCC1 gene may determine the individual susceptibility of Taiwanese to CRC. Therefore, we conducted a hospital-based case-control study to investigate the genotypes of ERCC1 firstly among Taiwanese and examine the association of ERCC1 genotypes with the risk of CRC in a Taiwanese population.
Materials and Methods
Investigated population. The investigated population included 724 subjects (362 CRC patients and 362 healthy controls). Patients diagnosed with CRC were recruited at the outpatient clinics of general surgery between 2002 and 2008 at the China Medical University Hospital, Taichung, Taiwan, Republic of China, by the team of Drs. Jeng LB and Yang MD. The clinical characteristics of patients, including histological details, were all graded and defined by expert surgeons (9, 10, 13, 16). All participants have completed a self-administered questionnaire and provided a 5-ml sample of peripheral blood for genotyping work. An equal number of non-cancer healthy volunteers (n=362) were selected as controls by matching for age, gender and some indulgences after initial random sampling from the Health Examination Cohort of the Hospital with the help of colleagues in the Department of Family Medicine. The exclusion criteria of the control group included previous malignancy, metastasized cancer from other or unknown origin and any familial or genetic diseases. This study was approved by the Institutional Review Board of the China Medical University Hospital (IRB project identification coding number: DMR99-IRB-108) and written informed consent was obtained from all participants with the help of Tissue Bank of China Medical University Hospital. The selected patients' characteristics, extracted from personal questionnaires, are summarized in Table I.
Genotyping conditions. Genomic DNA from peripheral blood leucocytes of each investigated subject was prepared applying the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and stored at −80°C until processed as per our recent publications (9, 22, 23). The methodology for ERCC1 rs11615 and rs3212986 genotyping, including the designing of the specific primers and the selection of restriction enzymes, were firstly designed in our lab. Briefly, the sequences for forward and reverse primer pairs for ERCC1 rs11615 were 5’-TTAGGAGGAGAGAGAAGCTG-3’ and 5’-GGCTTCTCATAGAACAGTCC-3’, respectively. The sequences for forward and reverse primer pairs for ERCC1 rs3212986 were 5’-AGGCTGTTTGATGTCCTGCA-3’ and 5’-AGAGGAAGAAGCAGAGTCAG-3’, respectively. The polymerase chain reaction (PCR) cycling conditions were set as one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 30 s; and a final extension step at 72°C for 10 min. After PCR amplification, the PCR products were subject to the digestion by BsrD I and MboI I restriction endonucleases, respectively, for 2 h at 37°C and separation via 3% agarose gel electrophoresis for 25 min. The ERCC1 rs11615 genotypes were identified as homozygous C/C with 393-bp product, heterozygous C/T with 393-, 228- and 165-bp products, as well as homozygous T/T with 228- and 165-bp products, respectively. The ERCC1 rs3212986 genotypes were identified as homozygous G/G with 367-bp product, heterozygous G/T with 367-, 233- and 134-bp products, as well as homozygous T/T with 233- and 134-bp products, respectively. All the genotypic processing was repeated by two researchers independently and blindly; all the genotyping results were 100% concordant.
Summary of selected data of the 362 patients with colorectal cancer and 362 matched non-cancer healthy controls.
Statistical analyses. The Student's t-test was applied for the comparison of ages between the CRC cases and the control groups. Pearson's Chi-square test was applied to compare the distribution of the ERCC1 genotypes among the subgroups. The associations between ERCC1 genotypes and CRC risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from logistic regression analysis. Statistically, any difference at p<0.05 was taken as significant between the two groups compared.
Excision repair cross-complementing group 1 (ERCC1) genotypes among the 362 patients with colorectal cancer and 362 matched healthy controls.
Results
The frequency distributions of selected characters, including age and gender for the 362 CRC patients in the case group and 362 non-cancer healthy subjects in the control group, are summarized and compared in Table I. In addition, tumor size, location,and lymph node metastasis status are also summarized in Table I. Since we applied frequency matching to recruit non-cancer healthy subjects as controls, there was no difference in the distributions of age and gender between the control and case groups (p=0.8654 and 0.6525, respectively) (Table I). The patients with tumor size <5 cm and ≥5 cm were 195 and 167, respectively. The patients with tumor location at colon and rectum were 257 and 105, respectively. The patients with and without lymph node metastasis were 152 and 210, respectively (Table I).
The distributions of the ERCC1 rs11615 and rs3212986 genotypes among the 326 non-cancer controls and the 326 CRC patients are presented and statistically analyzed in Table II. The results showed that the genotypes of ERCC1 rs11615 were differently distributed between case and control groups (p for trend=0.0158) (Table II). In detail, the ERCC1 rs11615 homozygous TT, but not the heterozygous CT, was associated with CRC risk, compared with wild-type CC genotype (OR=1.86 and 1.06, 95%CI=1.20-2.87 and 0.77-1.46, p=0.0049 and 0.7200, respectively; Table II). In the recessive model, there was a positive association between the TT genotype of ERCC1 rs11615 and CRC risk, compared with CC+CT genotypes (OR=1.81, 95%CI=1.20-2.73, p=0.0043, Table II). The genotypes of ERCC1 rs3212986 were not differently distributed between case and control groups in all models (Table II).
To confirm the results in Table II, analysis of allelic frequency distribution for the ERCC1 rs11615 and rs3212986 was further conducted and the results are presented in Table III. Supporting the findings that genotype of ERCC1 rs11615 was associated with CRC risk, the variant allele T was found at 37.7% in the case group, significantly higher than that of 31.1% in the control group (OR=1.34, 95% CI=1.08-1.67, p=0.0079). At the same time, there was no significant difference in the allelic frequencies of ERCC1 rs3212986 between the case and control groups (Table III).
Since smoking and alcohol drinking habits are well-known risk factors for CRC in Taiwan, we were interested in investigating the interactions between the genotype of ERCC1 rs11615 and personal cigarette smoking and alcohol drinking habits. Among smokers, those with TT genotype at ERCC1 rs11615 were at 4.88-fold odds of having CRC (95% CI=1.90-12.55, p=0.0006) conferring a risky effect, while this was not the case for non-smokers (Table IV). After adjusting for age, gender and alcohol drinking status, statistical significance still existed at a similar level (OR=4.41, 95%CI=1.94-11.53, Table IV). On the other hand, among alcohol drinkers, those with TT genotype at ERCC1 rs11615 were at 5.71-fold odds of having CRC (95% CI=1.72-18.94, p=0.0029) conferring a risky effect, while this was not the case for non-drinkers (Table V). After adjusting for age, gender and smoking status, results were equally significant (OR=5.46, 95%CI=1.68-17.87, Table V).
Distribution of allelic frequencies for excision repair cross-complementing group 1 (ERCC1) among the 362 patients with colorectal cancer and 362 matched healthy controls.
Odds ratios for excision repair cross-complementing group 1 (ERCC1) rs11615 genotype and colorectal cancer after stratification by smoking status.
Odds ratios for excision repair cross-complementing group 1 (ERCC1) rs11615 genotype and colorectal cancer after stratification by alcohol drinking status.
The correlations between genotypes of ERCC1 rs11615 and clinicopathological features of 362 CRC patients were analyzed and summarized in Table VI. No statistically significant correlation was observed between ERCC1 rs11615 genotypic distributions and age, gender, tumor size or location (all p>0.05). The most important finding was that the ERCC1 rs11615 genotype was associated with lymph node metastasis (p=0.0047) (Table VI).
Correlation between excision repair cross-complementing group 1 rs11615 genotypes and clinicopathological properties of 362 colorectal cancer patients.
Discussion
In the literature, genotypes of DNA repair genes may be associated with prognosis of chemotherapy in cancer patients. The DNA repair system plays an important role in maintaining the integrity of human genome that controls the homeostasis of cellular functions via the reversal of all types of DNA damage due to variety of factors, including cancer therapeutic agents. Therefore, overall DNA repair capacity may greatly contribute not only to cancer susceptibility but, also, prognosis (24). ERCC1 plays a central role in NER pathway and is responsible for a major part of routine DNA damage (25). The ERCC1 genotypes of rs11615 and rs3212986 may provide predictive information of platinum-based chemotherapy of advanced gastric cancer (26), advanced non-small cell lung cancer (27), testicular germ cell tumors (28), advanced epithelial ovarian cancer (29) and esophageal cancer (30). Molecular studies showed that ERCC1 rs11615 T allele is associated with diminished mRNA and protein expression levels but represents, however, a controversial predictive marker for cancer therapy (31-35). In the initial steps of carcinogenesis, the defects in DNA repair capacity of cells determined by variant ERCC1 genotypes may also contribute to increased cancer susceptibility. In the current work, we found that ERCC1 rs11615 TT genotype is associated with 1.86-fold enhanced CRC risk (Table II), which is further elevated to 4.88-fold odds of having CRC among smokers and 5.71-fold odds among alcohol drinkers (Tables IV and V). This is the first study to reveal joint effects between ERCC1 rs11615 genotypes with cigarette smoking and alcohol drinking habits on the susceptibility to CRC.
Despite our efforts to conduct an accurate and comprehensive genotyping work and related analysis, there are some limitations that should be noted. Firstly, the lack of recorded follow-ups limited the analysis of the correlation of prognosis indexes, such as survival rates. Table VI only provides evidence for the ERCC1 rs11615 genotype that did not contribute to the prediction of tumor size, location and metastasis. Secondly, lack of tumor and non-tumor samples limited the study of differential expression of ERCC1 mRNA and protein levels among the subjects, in addition to the inter-individual difference of the CRC patients. Further molecular investigations of the genotype-phenotype correlation may help in understanding the contribution of ERCC1 genotypes to not only overall DNA repair capacity but, also, personal susceptibility to CRC and/or other types of cancer. Thirdly, the relatively small sample size, especially in subgroup analysis, such as those in Tables IV and V, may have caused some bias and reduced the statistic power of our estimates.
In conclusion, this study provides evidence that the T allele at ERCC1 rs11615 may interact with smoking and alcohol drinking status to determine personal susceptibility to CRC; however, more research should be conducted to reveal the detailed alteration of DNA repair capacity in relation to CRC susceptibility and prognosis of chemotherapy.
Acknowledgements
The Authors declare no conflict of interest in regard to this study. They also appreciate the Tissue-bank of China Medical University Hospital for their excellent technical assistance and all the subjects, doctors, nurses and colleagues. This study was supported mainly by the Taichung Armed Forces General Hospital 106A14 to Dr. Yueh and partially by research grant from Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW106-TDU-B-212-113004).
Footnotes
↵* These Authors contributed equally to this study.
- Received March 20, 2017.
- Revision received April 4, 2017.
- Accepted April 6, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- Association of Matrix Metalloproteinase-9 rs3918242 Promoter Genotypes With Colorectal Cancer Risk
- Contribution of Caspase-8 Genotypes to Colorectal Cancer Risk in Taiwan
- Contribution of Murine Double Minute 2 Genotypes to Colorectal Cancer Risk in Taiwan
- Association of Matrix Metalloproteinase-7 Genotypes with the Risk of Bladder Cancer
- Association of Matrix Metalloproteinase-8 Genotypes with the Risk of Bladder Cancer
- ERCC1 rs11615 polymorphism increases susceptibility to breast cancer: a meta-analysis of 4547 individuals
- The Contribution of MMP-7 Genotypes to Colorectal Cancer Susceptibility in Taiwan
- The Role of MTHFR Genotype in Colorectal Cancer Susceptibility in Taiwan
- Contribution of Matrix Metalloproteinase-7 Genotypes to the Risk of Non-solid Tumor, Childhood Leukemia