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 .
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 .
regard , crc is considered as the fourth most common cancer in iranian population [ 4 , 5 ] .
regard , crc is the fourth most common cancer in iranian male and the second in female [ 4 , 69 ] .
two distinct pathways have been identified as main players in crc : the microsatellite instability ( msi ) and the chromosomal instability ( cin)/microsatellite stable ( mss ) pathway .
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 .
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 [ 1012 ] .
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 .
the prognostic significance of msi for patients with colorectal cancer is a subject of controversy .
based on the number of markers displaying msi per tumor , three groups of tumors are defined : those with 3040% of the markers showing instability ( msi - h ) ; those with less than 3040% of the markers showing instability ( msi - l ) , and those showing no instability ( mss ) .
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 .
several studies have demonstrated the prevalence of msi among crc patients in iran [ 2023 ] .
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 .
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 .
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 .
serial sections ( 5  m ) from formalin - fixed 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 .
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 .
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 .
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 .
statistical analysis was performed using the spss software program for windows , release 13.0.0 ( spss inc . ,
comparison of variables was performed using pearson 's chi - square test , fisher 's exact test , or the mann - whitney u test , depending on the nature of the data .
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 ) , tumor - node - 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 ( log - rank test ) curves were plotted .
we identified 158 consecutive patients with colorectal cancer of whom pathological results and follow - up data were available .
the clinicopathological features of patients enrolled in this study is present at table 1 . of 158 samples analyzed ,
the subjects were subdivided into three groups by msi testing : thirty - five ( 22.2% ) tumors were msi - h , 21 msi - l ( 13.3% ) and 102 mss ( 64.55% ) . among 35 msi - h tumors ,
instability of two markers was detected in 15 tumors ( 42.8% ) , 12 tumors ( 34.2% ) had instability in three markers and 5 tumors ( 14.3% ) had instability in four markers and 3 tumors ( 8.6% ) were instable in all five mononucleotide markers .
nr24 was the most instable marker ( 33 tumor ) among msi - h patients , however in 21 msi - low patients , the most instable marker was nr24 detected in 9 cases followed by bat 25 ( 6 case ) , nr21 ( 3 case ) and bat26 ( 3 case ) .
the mean period of follow - up was 60.2  24.5 months and the median period of follow - up were 53 months ( range 2120 ) .
of 158 patients , 70 cases had family history of crc and 88 did not have any history of crc or other gastrointestinal cancers . according to our findings 29 patients had metastatic in the time of diagnosis and 129 were negative for metastatic status .
patients in the mss group were found to be older than patients in the msi - l and msi - h groups ( mean ages 53.16 years , 49.6 years and 50.32 years resp . ) .
interestingly , the high incidence of msi - h ( 24/35 , 68.6% ) were presented in male gender and there was statistically significant relationship between msi status and gender ( p = 0.019 ) .
we found that msi - h colorectal cancer were located predominantly in proximal colon sites versus rectal sites ( p = 0.030 ) , associated with poorer differentiation ( p = 0.003 ) , have a family history for gi cancer ( p < 0.001 ) , and showed less frequent systemic metastasis ( p = 0.050 ) than msi - l and mss colorectal cancer .
distribution of tumor stages i  iv differed among patients with msi - h , msi - l and mss tumors ( p = 0.028 ) .
msi - h distribution in stage iii , ii and i were 60.0% , 31.4% and 8.6% respectively .
none of the 7 tumors with iv stage were shown to be msi - h .
we found no significant differences among patients with msi - h , msi - l and mss tumors for t status , m status , vital status or adjuvant therapy , however the distribution of lymph node metastasis and age of diagnosis in msi - h crcs was statistically different with msi - l and mss tumors . to assess the effect of msi status on survival
, we used a cox regression model for univariate and multivariate analysis . among the prognostic variables for overall survival entered into univariate and multivariate analysis , all characteristic such as diagnostic age , tumor stage , adjuvant therapy , msi status , tumor stage , and family history
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 on overall survival in all colorectal cancer patients ( p = 0.426 ) .
we also obtained kaplan - meier curves of overall survival in patients according to msi status , stratified based on tumor location and tnm stages ( figures 2 and 3 ) . based on our results , in crc patients with stage i ,
msi status is not considered as a valuable prognostic markers , long rank p = 0.742 .
whereas in crc patients with stage ii , msi - l showed significantly poorer survival compared with patients who had msi - h or mss tumors , long rank p = 0.048 , ( 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 p = 0.430 , ( figure 2(b ) ) .
kaplan - meier curves of overall survival in patients according to msi status stratified based on tumor location including colon and rectum are presented in figures 3(a ) and 3(b ) respectively . according to our findings , patients with ms - l colon tumors , show significantly poorer survival compared with patients who had msi - h or mss tumors , long rank p = 0.028 .
but we did not observe the similar results in rectal tumors , long rank p = 0.180 .
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% [ 2530 ] .
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 .
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 ( p = 0.03 ) and were associated with poorer differentiation ( p = 0.003 ) and tumor , lymph nodes , and metastasis ( tnm ) stage ii / iii of tumors ( p = 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 [ 2023 ] , but it was also higher than other reports from other ethnic groups ( 520% ) [ 1517 ] .
documented a high incidence of msi ( 45% ) in crc patients from african americans population .
the incidence of msi - l reported in this study was higher than another study in iran . in this study
we used 5 mononucleotide markers to evaluate msi status . probably different sample sizes and different threshold markers
were applied to assign msi in each previous study , indicating this variety . in present study
crc patients with stage ii msi - l showed significantly poorer survival compared with patients who had msi - h or mss tumors ( p = 0.04 ) .
this indicates that msi - l was clearly a marker of poorer prognosis in stage ii colorectal cancers .
our results documented distinct clinicopathologic characteristics of msi - h , msi - l , and mss colorectal cancers . in line with other reports ,
most of the tumors in msi - h were located in colon , poorly differentiated and frequent in stage iii .
high incidence of msi - h in proximal located tumors in the colon was reported in many papers [ 32 , 33 ] . however , brim et al .
have documented higher frequency of msi - h tumors in distal colon in omani patients .
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 ( p = 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 ( p = 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 ] .
this observation is in contrast to several other studies that showed the highest frequency of msi in stage ii [ 1517 , 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 , 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 [ 3638 ] and msi - l differ from mss tumors , but the biological background of this feature is unknown [ 19 , 3943 ] .
it was shown that mlh1 and msh2 genes do not seem to be contributed to etiology of msi - l . studies which indicated the k - ras mutations , o - 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 , 3943 ] .
whereas several studies and two current meta - analyses suggested better prognosis and outcome for patients with msi - h tumors [ 4447 ] , 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 ] .
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 .
nevertheless , there is now clear evidence that msi - l tumors are a discrete molecular group on basis of gene expression data .
together , these data suggest that msi - l cancer may arise from a distinct carcinogenic pathway as compared to mss and msi - h crc .
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 .
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 .
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 .
 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 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion