Low Level of Microsatellite Instability Correlates with Poor Clinical Prognosis in Stage II Colorectal Cancer Patients

The influence of microsatellite instability (MSI) on the prognosis of colorectal cancer (CRC) requires more investigation. We assessed the role of MSI status in survival of individuals diagnosed with primary colorectal cancer. In this retrospective cross-sectional study the MSI status was determined in 158 formalin-fixed paraffin-embedded tumors and their matched normal tissues from patients who underwent curative surgery. Cox proportional hazard modeling was performed to assess the clinical prognostic significance. In this study we found that MSI-H tumors were predominantly located in the colon versus rectum (p = 0.03), associated with poorer differentiation (p = 0.003) and TNM stage II/III of tumors (p = 0.02). In CRC patients with stage II, MSI-L cases showed significantly poorer survival compared with patients who had MSI-H or MSS tumors (p = 0.04). This study indicates that MSI-L tumors correlate with poorer clinical outcome in patients with stage II tumors (p = 0.04) or in tumors located in the colon (p = 0.02). MSI-L characterizes a distinct subgroup of CRC patients who have a poorer outcome. This study suggests that MSI status in CRC, as a clinical prognostic marker, is dependent on other factors, such as tumor stage and location.


Introduction
Colorectal cancer (CRC) develops either sporadically (85% of the cases), as part of a hereditary cancer syndrome (≤10%), or as a background of inflammatory bowel disease [1]. The incidence of CRC in Iran has been significantly increased since 1970 [2,3]. According to Iran National Cancer Registry (INCR) report, approximately 51,000 new cases of cancers with mortality rate of 35,000 are identified every year. This makes cancer the third most common cause of mortality in Iran. In this regard, CRC is considered as the fourth most common cancer in Iranian population [4,5]. The prevalence however is different in Iranian male and female gender. In this regard, CRC is the fourth most common cancer in Iranian male and the second in female [4,[6][7][8][9].
Two distinct pathways have been identified as main players in CRC: the microsatellite instability (MSI) and the chromosomal instability (CIN)/microsatellite stable (MSS) pathway [10]. Findings of these two pathways have led to the paradigm of CRC as a genetically heterogeneous disease. MSI pathway in hereditary and sporadic colorectal cancer occurs through two different mechanisms. In hereditary nonpolyposis colorectal cancer (HNPCC) the cause is a germline mutation in one of the DNA mismatch repair genes (MLH1, MSH2, MSH6, and PMS2), while MSI in sporadic colorectal cancer is predominantly due to hypermethylation of the MLH1 promoter and sometimes sporadic mutations [11]. The DNA mismatch repair system works as a spell checker that identifies and then corrects mismatched base pairs in the DNA. Defects in the mismatch repair mechanisms lead to MSI status [1,11,12]. Currently, tumor stage is the most important predictor of prognosis for CRC patients. Implications of MSI in colorectal cancer continue to increase and many studies have evaluated the role of MSI test in clinical management [10][11][12]. Investigation of MSI status in CRC is warranted for three reasons: (a) as a potential screening tool for HNPCC, (b) as a potential predictive factor of chemotherapy response, and finally (c) as a prognostic marker [13]. The prognostic significance of MSI for patients with colorectal cancer is a subject of controversy. The mechanism by which MSI possibly influences clinical outcome is unknown. Based on the number of markers displaying MSI per tumor, three groups of tumors are defined: those with 30-40% of the markers showing instability (MSI-H); those with less than 30-40% of the markers showing instability (MSI-L), and those showing no instability (MSS) [14]. Some investigations reported that patients with MSI-H tumors have a better prognosis than those with MSI-L or MSS tumors [15,16], whereas others reported that MSI in colorectal cancer was not an independent prognostic factor or had no prognostic significance in colorectal cancer [17,18]. The biologic defect producing the low MSI (MSI-L) phenotype in colorectal cancer is poorly recognized; since there is no obvious biologic differentiation between MSI-L and microsatellite stable (MSS) colorectal cancer, these two phenotypes are generally merged when analyzed against the well-defined high MSI (MSI-H) phenotype. Recently, there is controversy in the definition of MSI-L because most tumors could be classified as MSI-L if an adequate number of markers are studied [19]. Several studies have demonstrated the prevalence of MSI among CRC patients in Iran [20][21][22][23]. However, information about the survival or mortality rate is unknown. In the present study, we evaluated the possible prognostic significance of MSI in colorectal cancer patients by determining the relationship between MSI status (MSI-H, MSI-L, and MSS) and prognosis in 158 patients who underwent resection for primary colorectal cancer.

Materials and Methods
In this study 158 consecutive CRC patients referred to Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, from 2004 to 2010. The patients, who underwent surgical resection of adenocarcinoma of the colon or rectum and their characteristic and clinical data were available, were retrospectively included in this study. Informed consent was obtained from all patients or their relatives. Clinical information was recorded prospectively and registered in a database; this information included (i) age, sex, and personal and family medical history and (ii) tumor location, tumor differentiation, and TNM stage. Ethical approval for the study was obtained from the institutional review boards of the relevant centers.

MSI Analysis.
Serial sections (5 m) from formalinfixed paraffin-embedded matched normal and tumor tissues were routinely stained, and representative normal and tumor regions were identified by microscopic examination. Genomic DNA from each tumor and from corresponding normal tissue was purified using the QIAamp Tissue Kit (QIAGEN GmbH, Germany). Yield and purity of products were determined by electrophoresis on 0.8% agarose gel and spectrophotometry absorbance at 260 nm. Determination of MSI status was carried out using five mononucleotide repeat microsatellite targets (BAT25, BAT26, NR21, NR24, and NR27) using standard PCR techniques [24]. PCR products were denatured by electrophoresis on 5% denaturing polyacrylamide gels and were analyzed by an ABI 3130xl automated sequencer (Applied Biosystems, USA). Tumor samples that exhibited different allele peaks than the corresponding normal sample(s) were classified as MSI for that particular marker. MSI-H is defined when at least two of the five standard markers show instability in tumor DNA. MSI-L is defined when one MSI marker shows instability and others were microsatellite stable (MSS) when there was no instability detected on tumors. Analysis was performed twice if the results were ambiguous. The TNM (tumor, lymph nodes, and metastasis) staging system was applied to determine the severity of disease and the local or distant extent of disease spread. The TNM staging system of the American Joint Committee on Cancer (AJCC) is the preferred and standard staging system for CRC.

Statistical Analysis.
Since the significance of MSI-L in CRC is poorly understood, phenotype of MSI was originally grouped into three levels including MSI-High, MSI-Low, and MS-Stable. Analysis was performed twice if the results were ambiguous. Statistical analysis was performed using the SPSS software program for Windows, Release 13.0.0 (SPSS Inc., Chicago, IL). Comparison of variables was performed using Pearson's Chi-square test, Fisher's exact test, or the Mann-Whitney test, depending on the nature of the data. Two-tailed < 0.05 was considered statistically significant. Colorectal cancer (CRC) overall survival was computed since the date of cancer diagnosis up to the date of death or end of follow-up (February, 2014). Patients who died due to causes which were unrelated to colorectal cancer were censored at the time of death and were excluded from the analyses. For survival analyses, the following variables were assessed: age, sex, location of the tumor (colon versus rectum), tumornode-metastasis stage, and grade of differentiation (well/ moderate versus poor), use of adjuvant therapy, age of diagnosis, family history and MSI. Overall survival analyses were done through a Cox proportional hazard function for both univariate and multivariate analyses and Kaplan-Meier (logrank test) curves were plotted. Significance for all statistics were recorded if < 0.05.

Patients Descriptive.
We identified 158 consecutive patients with colorectal cancer of whom pathological results and follow-up data were available. The clinicopathological   (Table 4).

Survival.
Overall survival curves relative to MSI status was obtained for all colorectal cancer patients presented in Figure 1. We did not observe clear influence of MSI status  (Figure 2(a)). This indicates that MSI-L was clearly a marker of poorer prognosis in stage II colorectal cancers. However, we did not observe any significant association between overall survival of patients in advanced stages III/IV with MSI status, Long Rank = 0.430, (Figure 2

Discussion
Several epidemiological studies in Iran demonstrated that in comparison to western populations, the incidence of CRC is significantly higher in young Iranian patients and it has been reported that approximately 20% of all CRC cases occur in patients less than 40 years, whereas, this rate is extremely lower in western countries with 2 to 8% [25][26][27][28][29][30].
In addition to this high distribution of CRC prevalence in younger population of Iran in previous studies, in one study Azadeh et al. even reported a considerably higher proportion of 43% CRC incidence in patients less than 50 years old in Iranian population [27]. Although genetic factors have been proposed as one of the main risk factors contributed to this early onset of CRC in Iran, However, some studies indicated that this might be due to the high proportion of   young population in Iran [6,25]. In the present study we identified 35 MSI-H (22%), 21 MSI-L (13%), and 102 MSS (64%) tumors. MSI-H tumors were predominantly located in the colon than rectum ( = 0.03) and were associated with poorer differentiation ( = 0.003) and tumor, lymph nodes, and metastasis (TNM) stage II/III of tumors ( = 0.02) while MSI-L tumors were more frequent in patients aged younger than 50 years when diagnosed with colorectal cancer. The incidence of MSI observed in 158 colorectal cancer patients in the present study was similar to some previous reports from Iran [20][21][22][23], but it was also higher than other reports from other ethnic groups (5-20%) [15][16][17]. Ashktorab et al. documented a high incidence of MSI (45%) in CRC patients from African Americans population [31]. The incidence of MSI-L reported in this study was higher than another study in Iran [23].  [34]. The high incidence of MSI-H (24/35, 68.6%) was presented in male gender and there was statistically significant relationship between MSI status and gender ( = 0.01). In CRC patients with stage II, MSI-L cases showed significantly poorer survival compared with patients who had MSI-H or MSS tumors ( = 0.04). In contrast to several papers showing MSI is more common in women than in men [16,17,35]; we found a significant association between MSI status and male gender. The high prevalence of MSI-H in male was reported previously, but they did not find any significant association [15,20]. The prevalence of MSI varied with tumor stage and was highest in stage III. This observation is in contrast to several other studies that showed the highest frequency of MSI in stage II [15][16][17]20]. Similar to our finding, many other studies showed that the presence of distant metastases at the time of diagnosis (stage IV) is rare in the MSI subgroup of CRC [15,20]. In addition, a relatively high frequency of patients diagnosed in age less than 50 was noted; we identified MSI-L tumors predominantly in this group age. The frequency rate of MSI-L in patients with age less than 40 was reported in a previous study in Iran [23], suggesting that this genetic pathway may play an important role in CRC development in Iranian patients between 40 and 50 years old. It has been reported that MSI-H colorectal tumors differ from MSI-L or MSS tumors in several pathological features [36][37][38] and MSI-L differ from MSS tumors, but the biological background of this feature is unknown [19,[39][40][41][42][43].
It was shown that MLH1 and MSH2 genes do not seem to be contributed to etiology of MSI-L [38]. Studies which indicated the K-ras mutations, O 6 -methylguanine DNA methyltransferase (MGMT) promoter methylation, loss of MGMT gene expression, germline mutation of hMSH6, and a low level of allele loss near APC are associated with the MSI-L phenotype [19,[39][40][41][42][43]. Whereas several studies and two current meta-analyses suggested better prognosis and outcome for patients with MSI-H tumors [44][45][46][47], other studies could not confirm these findings [48,49]. In line with the latter studies we observed no significant difference in overall survival between patients with MSS, MSI-L, and MSI-H tumors. In many prognostic studies, the MSI-L phenotype has not been considered as a separate category; this might be due to a lack of clear single marker for identification of this group of tumors. Our study has shown that MSI-L is associated with poorer survival in colorectal patients with stage II tumors. Many papers suggested that MSI status influences the prognosis of CRC only in specific stages [16,19,33,35,45]. Two recent studies documented poor prognosis of MSI-L in stage III colon cancer [19,50]. Kohonen-Corish et al. showed that MSI-L characterizes a distinct subgroup of stage III colon cancer patients including the MSI-L subset of proximal colon cancer who have a poorer clinical outcome [19]. Nevertheless, there is now clear evidence that MSI-L tumors are a discrete molecular group on basis of gene expression data [41]. Together, these data suggest that MSI-L cancer may arise from a distinct carcinogenic pathway as compared to MSS and MSI-H CRC [51]. Other studies suggest that MSI-L and MSS have a common molecular background [52]. Though the biological basis of the good prognosis of patients with MSI-H is somewhat determined, the poor prognosis effect of MSI-L in colon cancer is unknown. It has been reported that the high mutational load in MSI-H tumors elicited a robust host immune response [53]. MSI-L and MSS have a high frequency of mutation of the p53 suppressor gene, explaining aggressive biological characteristics [37,54]. Few studies have evaluated the effect of MSI status on rectal cancer survival [44,55]. We assessed MSI status in relation to survival of individuals diagnosed with primary rectal cancer and primary colon cancer. We found poorer prognosis for MSI-L patients who had colon tumors and also poorer prognosis for MSI-H patients with rectum tumors, though this latter finding was not statically significant.

Conclusion
In conclusion, this is the first study in Iran to demonstrate the prognostic role of MSI in CRC patients. It seems that the clinical prognostic role of MSI status is dependent on stage and location of the tumor. Further studies with larger sample sizes are required to assess more precisely the impact of MSI-L on clinical outcome.