Colorectal cancer (CRC) is the third most common cancer in males and the second in female worldwide (1). Chromosomal instability (CIN) and microsatellite instability (MSI) are two main pathways for the development of CRC (2). The CIN pathway involves the accumulation of mutations in oncogenes, such as KRAS and tumour-suppressor genes, especially on chromosomes 5q (the adenomatous polyposis coli gene), 17p (the P53 gene), 18q [the deleted in colorectal carcinoma (DCC) and SMAD4 genes]. Approximately 85% of sporadic CRC cases involve CIN (3). In contrast, the MSI pathway involves a unique molecular alteration that is induced by deficiencies in the DNA mismatch repair (MMR) system, and is characterized by unstable microsatellites. The CpG island methylator phenotype has also recently been reported as a third mechanism of colorectal carcinogenesis (4,5).
The cause of MSI is the inactivation of at least one of the MMR genes, which include MLH1, MSH2, MSH6 or PMS2. Germline mutations in the MMR genes represent a major cause of the hereditary MSI-high CRCs in Lynch syndrome. The most frequent mutations in the MMR system affect the MLH1 gens (6), which has been identified as the most common cause of the development of hereditary nonpolyposis colorectal cancer (HNPCC). The sporadic CRCs with a high level of MSI account for nearly 3–15% of all CRCs (7), which are mainly caused by promoter CpG island hypermethylation, especially on MLH1 (8). Researchers have demonstrated that immunohistochemistry can be used to test for the loss of MMR gene protein expression (9), which offers a highly specific and technically simple alternative to the characterization of MSI tumours.
The correlation between MMR status and the clinicopathological features of CRC have been reported by several researchers. However, few studies have focused on the relationship between the molecular characteristics of CRC and MMR status. Therefore, we investigated the relationship between MMR status and the clinicopathological and molecular features in Chinese patients with sporadic CRC, using a large-scale retrospective analysis. The findings of the present study contribute to our understanding of the relationship between MSI and the clinicopathological and molecular features of CRCs.
This retrospective study evaluated 1,405 patients with primary CRC that was diagnosed between 2012 and 2014 at Fudan University Shanghai Cancer Center. Patients who were suspected of having hereditary or familial CRC were excluded from our analysis. All patients had undergone primary tumour resection.
Clinicopathological and molecular features
The clinicopathological characteristics were summarized using data from the patients’ medical histories and histopathology reports. These data included age, sex, tumour location, TNM stage, tumour type, histological type, differentiation, tumour size, vascular and peripheral nerve invasiveness. The molecular features were acquired from the immunohistochemistry report, and included the expression of the p53, Bcl-2, topoisomerase II (TopoII), CD44, and glutathione S-transferase (GST) proteins. Patients were classified as having MMR deficiency (dMMR) if they exhibited loss of at least one of the MMR genes (MLH1, MSH2, PMS2 or MSH6).
Univariate analysis was performed using the χ2 test, and the t-test was used for continuous variables analysis. Multivariate correlation analysis was performed using the logistic regression test. The statistical analyses were performed using SPSS software (version 20, SPSS Inc., Chicago, IL, USA). Differences with a P value of <0.05 were considered statistically significant.
MMR status and clinicopathological parameters
The present study included 822 (58.5%) men and 583 (41.5%) women, and the average age of the included patients was 59.8 years. We observed dMMR in 125 (8.9%) cases of CRC, and 1,280 (91.1%) cases of CRC exhibited MMR proficiency (pMMR) (Table 1). The number of patients with dMMR decreased with increasing age (P<0.05) (Table 2). The prevalence of dMMR varied according to tumour stage, with the lowest frequency observed in stage III (5.6%) and the highest frequency observed in stage II (13.1%). As in previous studies, the dMMR phenotype was more common in right-side colon cancer (22.35), compared to in left-side colon cancer (7.9%) and rectal cancer (1.9%); this difference was statistically significant (P<0.001). In addition, dMMR was more prevalent in poorly differentiated tumours, mucinous or signet-ring cell tumours, and tumours with lesser lymph node metastasis (Table 1) or a larger tumour diameter (Table 2). We also analysed the number of lymph nodes that had been acquired and the frequency of their metastasis, which revealed that more lymph nodes were examined, and a lower metastatic frequency was observed, in cases of dMMR CRCs (Table 2).
MMR status and molecular features
The molecular markers that we examined were p21, p53, CD44, Her2/neu, E-cadherin, epidermal growth factor receptor (EGFR), Bcl-2, P-glycoprotein (MDR), TopoII, cyclooxygenase 2 (COX2) and GST. Patients with dMMR exhibited a higher frequency of positive p53 and Bcl-2 expression, compared to patients with pMMR (P<0.01). In addition, patients with dMMR exhibited a higher frequency of negative CD44, TopoII and GST expression, compared to patients with pMMR (P<0.05) (Table 1).
Multivariate analysis of factors that were associated with MMR status
Binary logistic regression was performed using age, TNM stage, tumour size, histologic type, differentiation, peripheral nerve invasiveness, p53, Bcl-2, TopoII, CD44 and GST. The independent factors that increased the risk of dMMR-related CRC included younger age (OR: 0.979, 95% CI: 0.960–0.998, P=0.032, <0.05), larger tumour diameter (OR: 1.313, 95% CI: 1.162–1.484, P<0.001), poor differentiation, no peripheral nerve invasiveness (OR: 3.018, 95% CI: 1.258–7.239, P=0.013, <0.05), right-side colon cancer (OR: 10.821, 95% CI: 4.895–23.922, P<0.001), Bcl-2 positivity (OR: 0.209, 95% CI: 0.095–0.458, P<0.001), TopoII negativity (OR: 3.333, 95% CI: 1.563–7.103, P=0.013, <0.01) and GST negativity (OR: 1.748, 95% CI: 1.009–3.027, P=0.046, <0.05) (Table 3).
In the present study, our multivariate analysis revealed that patients with younger age, larger tumour diameter, poor differentiation, no peripheral nerve invasiveness and right-side colon cancer were more likely to have dMMR tumours. Few studies have reported a significant relationship between age and MMR status, although Park et al. (10) have reported that MSI-high colorectal adenoma was more common among younger patients; we also found that dMMR was more common among younger patients. These findings suggest that, when CRC is diagnosed at a young age, clinicians should pay close attention to the patient’s dMMR status, especially in cases with stage II CRC.
Regarding the various tumour characteristics, our findings revealed that dMMR tumours had larger diameters. Similarly, Yoon et al. (11) evaluated 2,028 tumour samples, and found that MSI-high tumours were typically larger. However, both Sun et al. (12) and Faghani et al. (13) have reported that there was no significant relationship between tumour size and MMR status. Interestingly, the MMR system can activate cell-cycle checkpoints or apoptosis, which might partially explain the occurrence of over-proliferation in tumours with defective MMR. We also found that poor differentiation and no perineural invasion were characteristics of dMMR tumours, and previous studies have reported similar findings (14-16). However, the specific mechanism(s) for these characteristics require further exploration. Ye et al. (17) have reported that dMMR tumours were significantly more common in the right colon (20.5%), compared to tumours in the left colon (9.2%) and rectum (5.1%, P<0.001). We also observed a similar trend. In addition, patients with dMMR tumours had a greater number of examined lymph nodes and less lymph nodes metastasis, although these differences were not statistically significant in the multivariate analysis. Interestingly, Takemoto and Smyrk (18,19) have reported that MSI is characterized by tumour-infiltrating lymphocytes, which may explain our findings regarding lymph node involvement. Thus, lymph nodes that are examined due to dMMR tumours may be associated with colitis. Finally, although dMMR in sporadic CRC is associated with a better prognosis (20), we were not able to evaluate this association, as the patients were not followed-up for a long enough period to conduct the relevant analysis.
Our most important finding was that several molecular biomarkers were related to MMR status in patients with CRC. For example, positive expression of the p53 protein was associated with dMMR status (P<0.05), although this association was only significant in the univariate analysis. Similarly, Christine et al. have evaluated the distinctive patterns of p53 protein expression and MSI in human CRC, and reported that p53 overexpression was significantly more frequent among MSI-high CRCs. In contrast, Park et al. (10) reported that the loss of hMLH1 or hMSH2 expression was correlated with low p53 expression (P<0.001). The reason for this discrepancy may be related to the relationship between P53 gene mutation and its protein expression, which has been extensively evaluated. Therefore, because there are numerous types of P53 gene mutations, it is difficult to use p53 expression to predict P53 mutation(s). The p53 protein acts as a tumour suppressor by triggering cell cycle arrest and then correcting the damage via DNA repair. Interestingly, it has been reported that MMR proteins and p53 have complementary effects on the other’s activity (21). For example, MMR and p53 can cooperate to control the cytotoxic effects of cisplatin and limit its mutagenic potential in colon cancer cells. This mechanism may explain why CRC patients with dMMR are resistant to chemotherapy.
Another molecular biomarker that was significantly different related to MMR status was Bcl-2, which is frequently overexpressed in various types of cancer. This protein enables dysplastic and metaplastic cells to survive, and promotes adenoma development, which can eventually progress to an invasive carcinoma. The relationship between Bcl-2 and MMR was first demonstrated by Youn et al. (22), who reported that Bcl-2 expression could suppress MMR activity via hyperphosphorylation of retinoblastoma protein (pRb), which enhanced the E2F-pRb complex and decreased hMSH2 expression. In addition, the role of MMR is to protect against the accumulation of deleterious mutations and maintain genomic stability. Therefore, decreased MMR activity via Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis, as we found that Bcl-2 expression was more common among dMMR cases, compared to among MMR proficient cases. Similarly, Bendardaf et al. (23) have reported that Bcl-2 expression was closely correlated with hMLH1 and hMSH2 expression (P<0.01). Furthermore, their data also suggested that MSI patients with low Bcl-2/MMR exhibited a shorter disease-free survival.
We also found that other molecular biomarkers, such as TopoII and GST, were correlated with MMR status. For example, the absence of TopoII and GST expression was more common in dMMR patients, and these factors were found the independently influence the dMMR phenotype after our multivariate analysis. Interestingly, the expression of these two proteins is related to chemotherapy resistance, as Kaplan et al. (24) have reported that decreasing TopoIIA, MSH2 and MLH1 expression may help reduce breast cancer resistance to etoposide chemotherapy. In addition, Tsavaris et al. have concluded that levels of TopoII expression were higher in tumours from recurrent CRC (25). Furthermore, Shin et al. have evaluated GST genotypes in Korean patients with HNPCC, and reported that the GSTM1 genotype was related to cancer occurrence in family members with hMLH1/hMSH2 mutations (26). However, few studies have reported the specific relationship between those two proteins and MMR status in sporadic CRC, and further studies are needed to clarify this relationship.
In the present study, we found that younger age, poorly differentiated tumours, negative peripheral nerve invasiveness and right-side tumours were significantly associated with dMMR in Chinese patients with sporadic CRC. Moreover, dMMR status was closely related to the positive expression of Bcl-2 and the absence of TopoII and GST expression. Therefore, future research should evaluate how the TopoII and GST proteins interact with MMR proteins.
We thank all the patients whose data were used in this study.
Funding: This work was partially supported by the Natural Science Foundation of Shanghai (16ZR1406700), the National Natural Science Foundation of China (81472620),and Shanghai Municipal Commission of Health and Family Planning (2014ZYJB0101), the development fund for Shanghai talents No. 2017120.
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the Medical Ethics Committee of Fudan University Shanghai Cancer Center (reference number 050432-4-1212B) and written informed consent was obtained from all patients.
- Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin 2015;65:87-108. [Crossref] [PubMed]
- Jass JR. Molecular heterogeneity of colorectal cancer: Implications for cancer control. Surg Oncol 2007;16 Suppl 1:S7-9. [Crossref] [PubMed]
- Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-67. [Crossref] [PubMed]
- Cunningham D, Atkin W, Lenz HJ, et al. Colorectal cancer. Lancet 2010;375:1030-47. [Crossref] [PubMed]
- Migliore L, Migheli F, Spisni R, et al. Genetics, cytogenetics, and epigenetics of colorectal cancer. J Biomed Biotechnol 2011;2011:792362. [Crossref] [PubMed]
- Marra G, Boland CR. Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives. J Natl Cancer Inst 1995;87:1114-25. [Crossref] [PubMed]
- de la Chapelle A, Hampel H. Clinical relevance of microsatellite instability in colorectal cancer. J Clin Oncol 2010;28:3380-7. [Crossref] [PubMed]
- Imai K, Yamamoto H. Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics. Carcinogenesis 2008;29:673-80. [Crossref] [PubMed]
- Shia J, Ellis NA, Klimstra DS. The utility of immunohistochemical detection of DNA mismatch repair gene proteins. Virchows Arch 2004;445:431-41. [Crossref] [PubMed]
- Park SK, Park DI, Park SH, et al. Microsatellite instability in young patients with sporadic colorectal adenomas. Hepatogastroenterology 2011;58:1531-7. [PubMed]
- Yoon YS, Yu CS, Kim TW, et al. Mismatch repair status in sporadic colorectal cancer: immunohistochemistry and microsatellite instability analyses. J Gastroenterol Hepatol 2011;26:1733-9. [Crossref] [PubMed]
- Sun Z, Yu X, Wang H, et al. Clinical significance of mismatch repair gene expression in sporadic colorectal cancer. Exp Ther Med 2014;8:1416-22. [Crossref] [PubMed]
- Faghani M, Fakhrieh AS, Mansour-Ghanaei F, et al. The Correlation between Microsatellite Instability and the Features of Sporadic Colorectal Cancer in the North Part of Iran. Gastroenterol Res Pract 2012;2012:756263. [Crossref] [PubMed]
- Greenson JK, Bonner JD, Ben-Yzhak O, et al. Phenotype of microsatellite unstable colorectal carcinomas: Well-differentiated and focally mucinous tumors and the absence of dirty necrosis correlate with microsatellite instability. Am J Surg Pathol 2003;27:563-70. [Crossref] [PubMed]
- Ribic CM, Sargent DJ, Moore MJ, et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 2003;349:247-57. [Crossref] [PubMed]
- Samdani T, Schultheis M, Stadler Z, et al. Lymph node yield after colectomy for cancer: is absence of mismatch repair a factor? Dis Colon Rectum 2015;58:288-93. [Crossref] [PubMed]
- Ye JX, Liu Y, Qin Y, et al. KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients. World J Gastroenterol 2015;21:1595-605. [Crossref] [PubMed]
- Takemoto N, Konishi F, Yamashita K, et al. The correlation of microsatellite instability and tumor-infiltrating lymphocytes in hereditary non-polyposis colorectal cancer (HNPCC) and sporadic colorectal cancers: the significance of different types of lymphocyte infiltration. Jpn J Clin Oncol 2004;34:90-8. [Crossref] [PubMed]
- Smyrk TC, Watson P, Kaul K, et al. Tumor-infiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma. Cancer 2001;91:2417-22. [Crossref] [PubMed]
- Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 2005;23:609-18. [Crossref] [PubMed]
- Young LC, Keuling AM, Lai R, et al. The associated contributions of p53 and the DNA mismatch repair protein Msh6 to spontaneous tumorigenesis. Carcinogenesis 2007;28:2131-8. [Crossref] [PubMed]
- Youn CK, Cho HJ, Kim SH, et al. Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity. Nat Cell Biol 2005;7:137-47. [Crossref] [PubMed]
- Bendardaf R, Ristamaki R, Syrjanen K, et al. Bcl-2 expression significantly correlates with thymidylate synthase expression in colorectal cancer patients. World J Gastroenterol 2008;14:6218-23. [Crossref] [PubMed]
- Kaplan E, Gunduz U. Expression analysis of TOP2A, MSH2 and MLH1 genes in MCF7 cells at different levels of etoposide resistance. Biomed Pharmacother 2012;66:29-35. [Crossref] [PubMed]
- Tsavaris N, Lazaris A, Kosmas C, et al. Topoisomerase I and IIalpha protein expression in primary colorectal cancer and recurrences following 5-fluorouracil-based adjuvant chemotherapy. Cancer Chemother Pharmacol 2009;64:391-8. [Crossref] [PubMed]
- Shin JH, Ku JL, Shin KH, et al. Glutathione S-transferase M1 associated with cancer occurrence in Korean HNPCC families carrying the hMLH1/hMSH2 mutation. Oncol Rep 2003;10:483-6. [PubMed]