molecular studies have reported that up to 15% of sporadic colorectal cancers and approximately 90% of syndrome of hereditarynon - polyposis colorectal cancer ( hnpcc ) , which accounts for 1 to 3 percent of all colorectal cancers , were caused by inactivation of mismatch repair ( mmr ) genes , such as msh2 , mlh1 , pms1 , pms2 , hmsh6/gtbp , and hmsh3 [ 1 - 3 ] . inactivation of one of these genes results in dna microsatellite instability ( msi ) , characterized by alterations in the length of simple repetitive microsatellite sequences found throughout the genome.while germline mutations of dna mmr genes have been identified as a causative event in hereditary nonpolyposis , development of sporadic colorectal cancer with msi - high ( msi - h ) is most commonly a result of promoter methylation , leading to epigenetic silencing of the mlh1 gene . some studies have suggested that colorectal carcinomas with msi - h tend to have an improved survival rate , and may respond differently to adjuvant chemotherapy than non - msi tumors . msi tests may be used routinely as a first - line screening tool for identification of msi - h colorectal carcinomas for diagnosis of suspected hnpcc and for determination of the clinical implications of msi - h status in sporadic colorectal cancer . however , to date , msi tests are complex , time - consuming , and expensive , and are not widely accepted as a screening test . clinical data , including family history of cancer , including amsterdam criteria   and/or bethesda guidelines , have been used for selection of suspected hnpcc for msi testing . in addition to clinical data , a number of studies have suggested that msi - h colorectal carcinomas may have morphologic characteristics that differ from those of non - msi - h tumors and therefore may be used as a first - line screening tool for identification of tumors for further molecular testing . however , the utility of histology as a screening tool for msi - h colorectal carcinomas has yet to be defined . immunohistochemistry ( ihc ) for mlh1 and msh2 protein may increase accuracy of prediction and aid in selection of patients who may have msi , and it can be used as a screening tool for msi testing in colorectal cancers . however , these guidelines or histopathologic parameters are not adequate for selection of colorectal cancer patients because their results were studied independently and a false negative result may be obtained . in this study , we attempted to identify the combination of parameters from clinical data , histopathology , including ihc for mlh1 and msh2 protein that may be helpful in selection of patients with msi - h . between january 2004 and june 2006 , the 120 patients who underwent surgery for treatment of colorectal cancers at daegu catholic university hospital , daegu , korea were retrospectively enrolled in the present study . using medical records or telecommunication , the questionnaire included cancer history in first- and second - degree relatives and contained questions regarding their age at diagnosis , type of cancer , hospital at which the diagnosis was made , current age , and current status . patients with hnpcc meeting the amsterdam criteria , patients with familial adenomatous polyposis , and patients with a vague family history were excluded . of the 120 enrolled cases of primary colorectal cancer , 65 were male and 55 were female , with a mean age at the time of surgery of 62.411.1 years ( range , 36 to 83 years).twenty patients had a family history of cancer , except hepatocellular carcinoma or cervical cancer , within a second degree pedigree ( table 1 ) . among them , 10 patients had a family history of colorectal cancer , and only one patient had met the full requirement for diagnosis of amsterdam , and five were included according to bethesda guidelines . the numbers according to tumor location , which were classified as rt . sided colon ( from ileocecal valve to splenic flexure ) , lt . sided colon ( from descending colon to sigmoid colon ) , and rectum ( from recto - sigmoid colon to distal rectum ) and multiple colon ( two or more synchronous colon cancers at diagnosis ) were 27 , 23 , 69 , and 1 , respectively ( table 2 ) . tumors in which less than 10% of the cells formed glands were classified as high grade ( poorly differentiated ) , while those containing more than 50% extracellular mucin were classified as mucinous type . the numbers of well / moderately differentiated adenocarcinoma ( wd / md ) , poorly differentiated adenocarcinoma ( pd ) , and mucinous adenocarcinoma ( mu ) were 112 , 3 , and 5 , and two were diagnosed as lymphoma . the depth of invasion to mucosa / submucosa , muscularis layer , pericolic ( or perirectal area ) , or invasion to adjacent other organs were 11 , 19 , 82 , and 6 , respectively . according to international union against cancer ( uicc ) classification , 19 of these tumors were tumor - node - metastasis ( tnm ) stage i , 55 were stage ii , 37 were stage iii , and nine were stage iv . dna from blood and tissue were amplified for the five mononucleotide markers of nr21 , nr22 , nr24 , bat25 , and bat26 . amplification of nr21 , 22 , and 24 was performed using the home brew pcr system and denaturing high performance liquid chromatography ( transgenomic inc . , omaha , ne ) was used for analysis of the pcr products ( fig . a hybridization probe melting point analysis was performed   using light cycler and dna master hybridization probe reagents ( roche diagnostics , mannheim , germany ) for detection of bat25 and 26 instability ( fig . one block of formalin fixed paraffin embedded colorectal cancer tissue was selected for ihc . in all cases , the block was comprised of an area of normal colonic mucosa adjacent to the tumor . sections ( 4 m ) were affixed , dried , de - waxed , and rehydrated , followed by inhibition of endogenous peroxidase activity ; they were then subjected to heat antigen retrieval . the mlh1 antibody ( g168 - 15 , pharmingen , san diego , ca ) and the msh2 antibody ( g219 - 1129 , pharmingen ) were used for ihc stain using the streptavidin biotin universal detection system ( immunotech , marseille , france ) . finally , the sections were counterstained in mayer 's haematoxylin . among 120 cases , ihc could not be performed for three microsatellite stable ( mss ) cases , and , for 117 cases , one pathologist ( h.k . oh ) , without knowledge of msi status , performed scoring of ihc staining expression . for statistical analysis , the  or fisher 's exact test was used as an appropriate univariate test for selection of optimal clinico - pathologic factors between groups . we then performed multiple logistic regression for identification of clinico - pathologic factors among all selected optimal factors described above as a univariate test . for selection of clinico - pathologic factors , we used the forward selection method in multiple logistic regression . using clinico - pathologic parameters identified in multiple logistic regression , we evaluated the prediction of msi - h as the sensitivity and specificity for the single and all combinations of identified clinico - pathologic parameters . in addition , we used receiver operatring characteristic curve and area under curve to obtain the most appropriate sensitivity and specificity . between january 2004 and june 2006 , the 120 patients who underwent surgery for treatment of colorectal cancers at daegu catholic university hospital , daegu , korea were retrospectively enrolled in the present study . using medical records or telecommunication , the questionnaire included cancer history in first- and second - degree relatives and contained questions regarding their age at diagnosis , type of cancer , hospital at which the diagnosis was made , current age , and current status . patients with hnpcc meeting the amsterdam criteria , patients with familial adenomatous polyposis , and patients with a vague family history were excluded . of the 120 enrolled cases of primary colorectal cancer , 65 were male and 55 were female , with a mean age at the time of surgery of 62.411.1 years ( range , 36 to 83 years).twenty patients had a family history of cancer , except hepatocellular carcinoma or cervical cancer , within a second degree pedigree ( table 1 ) . among them , 10 patients had a family history of colorectal cancer , and only one patient had met the full requirement for diagnosis of amsterdam , and five were included according to bethesda guidelines . the numbers according to tumor location , which were classified as rt . sided colon ( from ileocecal valve to splenic flexure ) , lt . sided colon ( from descending colon to sigmoid colon ) , and rectum ( from recto - sigmoid colon to distal rectum ) and multiple colon ( two or more synchronous colon cancers at diagnosis ) were 27 , 23 , 69 , and 1 , respectively ( table 2 ) . tumors in which less than 10% of the cells formed glands were classified as high grade ( poorly differentiated ) , while those containing more than 50% extracellular mucin were classified as mucinous type . the numbers of well / moderately differentiated adenocarcinoma ( wd / md ) , poorly differentiated adenocarcinoma ( pd ) , and mucinous adenocarcinoma ( mu ) were 112 , 3 , and 5 , and two were diagnosed as lymphoma . the depth of invasion to mucosa / submucosa , muscularis layer , pericolic ( or perirectal area ) , or invasion to adjacent other organs were 11 , 19 , 82 , and 6 , respectively . according to international union against cancer ( uicc ) classification , 19 of these tumors were tumor - node - metastasis ( tnm ) stage i , 55 were stage ii , 37 were stage iii , and nine were stage iv . dna from blood and tissue were amplified for the five mononucleotide markers of nr21 , nr22 , nr24 , bat25 , and bat26 . amplification of nr21 , 22 , and 24 was performed using the home brew pcr system and denaturing high performance liquid chromatography ( transgenomic inc . , omaha , ne ) was used for analysis of the pcr products ( fig . a hybridization probe melting point analysis was performed   using light cycler and dna master hybridization probe reagents ( roche diagnostics , mannheim , germany ) for detection of bat25 and 26 instability ( fig . one block of formalin fixed paraffin embedded colorectal cancer tissue was selected for ihc . in all cases , the block was comprised of an area of normal colonic mucosa adjacent to the tumor . sections ( 4 m ) were affixed , dried , de - waxed , and rehydrated , followed by inhibition of endogenous peroxidase activity ; they were then subjected to heat antigen retrieval . the mlh1 antibody ( g168 - 15 , pharmingen , san diego , ca ) and the msh2 antibody ( g219 - 1129 , pharmingen ) were used for ihc stain using the streptavidin biotin universal detection system ( immunotech , marseille , france ) . ihc could not be performed for three microsatellite stable ( mss ) cases , and , for 117 cases , one pathologist ( h.k . oh ) , without knowledge of msi status , performed scoring of ihc staining expression . for statistical analysis , the  or fisher 's exact test was used as an appropriate univariate test for selection of optimal clinico - pathologic factors between groups . we then performed multiple logistic regression for identification of clinico - pathologic factors among all selected optimal factors described above as a univariate test . for selection of clinico - pathologic factors , we used the forward selection method in multiple logistic regression . using clinico - pathologic parameters identified in multiple logistic regression , we evaluated the prediction of msi - h as the sensitivity and specificity for the single and all combinations of identified clinico - pathologic parameters . in addition , we used receiver operatring characteristic curve and area under curve to obtain the most appropriate sensitivity and specificity . of the 120 tumors , 11 ( 9.2% ) were msi - h , four ( 3.3% ) were msi - low ( msi - l ) , and 105 ( 87.5% ) were mss ( table 1 ) . among the 11 patients with msi - h , there were seven females , which appeared to be higher than that of males , without statistical significance . ages ( meanstandard deviation , years ) of msi - h and mss patients were 529.6 and 63.310.9 , respectively , which was significantly different ( p=0.004 ) . of patients with a family history of cancer other than hepatocellular carcinoma or cervical cancer , the ratio of msi - h was 30% ( 6/20 ) , which was significantly higher , compared to those who had no family history of cancer ( p=0.001 ) . among patients with a family history of colorectal cancer , the ratio of msi - h was 40% ( 4/10 ) , which was significantly higher than that of those who had no family history of colorectal cancer ( p=0.002 ) . the only patient who met amsterdam criteria showed msi - h , and , among five patients who were included according to bethesda guidelines , three ( 60% ) were msi - h . the ratio of increased preoperative serum cea level was 9% ( 1/11 ) in the msi - h group and 33.3% ( 33/99 ) in the msi - l or mss group when the cut off value of cea was regarded as 5 ng / dl ( p=0.038 ) . the ratios of msi - h in tumors from the right colon and synchronous multiple colon cancers were 29.6% ( 8/27 ) and 100% ( 1/1 ) , which was higher than those of the left colon or rectum ( p=0.001 ) ( table 2 ) . among five tumors of mu , three ( 60% ) showed msi - h , which was higher than those of wd / md , or pd or lymphoma ( p=0.001 ) . except for one patient , all tumors with msi - h invaded to the pericolic ( or perirectal ) area , and no difference for status of regional lymph node metastasis , lymphovascular invasion , perineural invasion , distant metastasis , or tnm stage was observed between msi and msi - l or mss groups . the numbers for expression of mlh1 were 94 ( 92.2% ) , 3 ( 75.0% ) , and 5 ( 45.5% ) , while 8 ( 7.8% ) , 1 ( 25.0% ) , and 6 ( 54.5% ) were not expressed in mss , msi - l , and msi - h , in order , and the sensitivity and specificity of mlh1 for msi - h were 54.5% and 92.2% , respectively ( table 4 ) . in msh2 , the numbers for expression were 101 ( 99.0% ) , 4 ( 100% ) , and 10 ( 90.9% ) , while 1 ( 1% ) , 0 ( 0% ) , and 1 ( 9.1% ) were not expressed in mss , msi - l , and msi - h , respectively , and the sensitivity and specificity for msi - h were 9.1% and 99.0% . among factors that were proved to be statistically significant on a univariate test , multiple logistic regression analysis was performed for identification of parameters that were significantly affected or related to msi - h . immunohistochemical staining for mlh1 , tumor location , bethesda guidelines , and cell types showed a significant association with msi - h . msi - h tumors had at least one or more parameters among negative expression of mlh1 protein , right sided colon and/or synchronous multiple colorectal cancer , one of the bethesda guidelines , or poorly differentiated or mucinous adenocarcinima . sensitivity and specificity were calculated for all combinations of immunohistochemical staining for mlh1 , tumor location , bethesda guideline , and cell types ( table 5 ) . according to the results , three clinico - pathologic parameters with mlh1 , tumor location , and bethesda guidelines were appropriate and important factors with high sensitivity of 100% and specificity of 72.5% . of the 120 tumors , 11 ( 9.2% ) were msi - h , four ( 3.3% ) were msi - low ( msi - l ) , and 105 ( 87.5% ) were mss ( table 1 ) . among the 11 patients with msi - h , there were seven females , which appeared to be higher than that of males , without statistical significance . ages ( meanstandard deviation , years ) of msi - h and mss patients were 529.6 and 63.310.9 , respectively , which was significantly different ( p=0.004 ) . of patients with a family history of cancer other than hepatocellular carcinoma or cervical cancer , the ratio of msi - h was 30% ( 6/20 ) , which was significantly higher , compared to those who had no family history of cancer ( p=0.001 ) . among patients with a family history of colorectal cancer , the ratio of msi - h was 40% ( 4/10 ) , which was significantly higher than that of those who had no family history of colorectal cancer ( p=0.002 ) . the only patient who met amsterdam criteria showed msi - h , and , among five patients who were included according to bethesda guidelines , three ( 60% ) were msi - h . the ratio of increased preoperative serum cea level was 9% ( 1/11 ) in the msi - h group and 33.3% ( 33/99 ) in the msi - l or mss group when the cut off value of cea was regarded as 5 ng / dl ( p=0.038 ) . the ratios of msi - h in tumors from the right colon and synchronous multiple colon cancers were 29.6% ( 8/27 ) and 100% ( 1/1 ) , which was higher than those of the left colon or rectum ( p=0.001 ) ( table 2 ) . among five tumors of mu , three ( 60% ) showed msi - h , which was higher than those of wd / md , or pd or lymphoma ( p=0.001 ) . except for one patient , all tumors with msi - h invaded to the pericolic ( or perirectal ) area , and no difference for status of regional lymph node metastasis , lymphovascular invasion , perineural invasion , distant metastasis , or tnm stage was observed between msi and msi - l or mss groups . the numbers for expression of mlh1 were 94 ( 92.2% ) , 3 ( 75.0% ) , and 5 ( 45.5% ) , while 8 ( 7.8% ) , 1 ( 25.0% ) , and 6 ( 54.5% ) were not expressed in mss , msi - l , and msi - h , in order , and the sensitivity and specificity of mlh1 for msi - h were 54.5% and 92.2% , respectively ( table 4 ) . in msh2 , the numbers for expression were 101 ( 99.0% ) , 4 ( 100% ) , and 10 ( 90.9% ) , while 1 ( 1% ) , 0 ( 0% ) , and 1 ( 9.1% ) were not expressed in mss , msi - l , and msi - h , respectively , and the sensitivity and specificity for msi - h were 9.1% and 99.0% . among factors that were proved to be statistically significant on a univariate test , multiple logistic regression analysis was performed for identification of parameters that were significantly affected or related to msi - h . immunohistochemical staining for mlh1 , tumor location , bethesda guidelines , and cell types showed a significant association with msi - h . msi - h tumors had at least one or more parameters among negative expression of mlh1 protein , right sided colon and/or synchronous multiple colorectal cancer , one of the bethesda guidelines , or poorly differentiated or mucinous adenocarcinima . sensitivity and specificity were calculated for all combinations of immunohistochemical staining for mlh1 , tumor location , bethesda guideline , and cell types ( table 5 ) . according to the results , three clinico - pathologic parameters with mlh1 , tumor location , and bethesda guidelines were appropriate and important factors with high sensitivity of 100% and specificity of 72.5% . msi tests can be applied for diagnosis of hnpcc from all colorectal cancers and for detection of msi - h tumors in sporadic colorectal cancers . although a definitive diagnosis of hnpcc could be established by demonstrating a germline mutation , msi tests can be used as a screening test for suspected hnpcc before mutation analysis . as clinical and prognostic implications in sporadic type colorectal cancer with msi - h would predict development of multiple synchronous or metachronous - cancers , responsiveness of adjuvant chemotherapy or to chemoprevention related to cyclo - oxygenase-2 inhibitors as well as prognosis , it is also of great importance for identification of msi - h in sporadic type colorectal cancer in a clinical field [ 13 - 15 ] . for assessment of msi status , an international consensus meeting held in 1997 this included two mononucleotide repeats ( bat25 and bat26 ) and three dinucleotide ( d5s346 , d2s123 , and d17s250 ) repeats . tumors showing instability at two or more of these markers were defined as msi - h , and those showing instability at one repeat or showing no instability were defined as msi - l and mss tumors , respectively . in this study , we used five mononucleotide repeats , including bat25 and bat26 , instead of three dinucleotide ( d5s346 , d2s123 , and d17s250 ) repeats , because each of the dinucleotide repeats in the aforementioned panel generally show instability in only 60 - 80% of msi - h tumors . in addition , when using the bethesda reference panel , misclassifications of msi - h tumors occur if two dinucleotide markers were unstable in the absence of bat26 deletions . as reported by zhou et al . , bat26 identified the msi status in 539 of 542 tumors ( 99.5% ) , and bat26 provided the advantage of being a simple and less expensive method that might be used as a screening procedure prior to performance of mutation analysis . according to our data , sensitivity for bat26 and bat25 for our data also indicated the potential of bat26 for use as a single marker for msi testing ; however , it is not desirable , as there may be the possibility of missing msi - h by bat25 or 26 alone due to polymorphisms . introduction of the msi determination as an initial screening test for hnpcc or msi - h tumors in colorectal cancers has been reported to enable molecular detection of hnpcc in a mass population . salovaara et al . , who examined 535 colorectal cancers , reported that 12% showed msi and 3.4% had germline mutations of msh2 or mlh1.this means that at least more than 85% of colorectal cancers are mss or msi - l and application of msi tests for all colorectal cancers may be ineffective due to low sensitivity and specificity for diagnosis of hnpcc or detection of msi - h in sporadic colorectal cancer . to save time and cost of msi testing , some attempts have been made to increase its sensitivity and specificity ; these include bethesda guidelines , evaluation of clinical and histopathologic parameters , msi tests by one marker or replacement of msi by ihc of mlh1/msh2 protein , or a combination of these trials [ 7 - 10 ] . original bethesda guidelines for testing colorectal tumors for msi were proposed in 1997 to guide clinical use to aid in identification of patients who may have hnpcc , and were then revised in 2004 . bethesda guidelines were established only for diagnosis of hnpcc , not msi - h sporadic colorectal cancers . reported that 29% ( 27/92 ) of bethesda - positive patients showed high msi , compared with 6% ( 4/72 ) of patients who did not meet these criteria . according to our data , five patients were included in the bethesda guidelines and three were msi - h , including one hnpcc . use of bethesda guidelines is a suboptimal method for selection of msi testsin sporadic colorectal cancer and for application to general medical practice outside academic centers , thus , additional clinical or pathologic data are required for adequate msi - h tumor selection . as reported previously , msi - h tumors are known for having certain clinical and pathological characteristics , including a higher rate of family history of cancer , early age of onset , a tendency for cancers in the proximal colon and excess of poorly differentiated carcinomas or mucinous type , medullary type , contain signet - ring cells , and increased infiltrating t lymphocytes . reported that positive predictabilities of right sided tumor , intraepithelial lymphocytes , poor differentiation , crohn's - like reaction , mucinous tumor , peritumoural lymphocytes , and amsterdam criteria for msi - h in sporadic colorectal cancer were 23% , 35% , 26% , 26% , 20% , 24% and 25% , respectively . our data also showed similar results , indicating that predictabilities of family history of cancer , bethesda guidelines , right sided tumor , and poorly differentiation or mucinous tumor for msi - h in colorectal cancer were 30% , 30% , 28.6% and 14.3% , respectively ( data not shown ) . reported that medullary carcinoma , intraepithelial lymphocytosis , and poor differentiation were the best discriminators between msi - h and microsatellite - stable , and suggested that these histopathological evaluations can be used in prioritization of sporadic colon cancers for msi studies . however , in their study , histopathology alone failed to reliably discriminate msi - h tumors because the minority of specimens showed no major difference in morphology from the usual mss cancer . in addition , approximately 40% of msi - h cancers were not detected , and 6% were never detected by histopathology . these results suggested that these clinical and pathological parameters showed differences between msi - h and mss or msi - l , but are insufficient for clinical application to selection of patients to undergo msi tests independently . also suggested other parameters associated with msi - h , including sex , distant metastasis , and stage , however , these findings were not consistent with those of other reports , and our data also did not show any correlation with msi status . engel et al .   reported that ihc was highly predictive ( 99.1% ) and specific ( 99.6% ) with regard to msi tests . however , their data showed that 14 out of 230 ( 6% ) mutations escaped detection by ihc . in our data , the sensitivity and specificity of mlh1 and msh2 for msi - h were 54.5% , 92.2% and 9.1% , 99.0% , respectively and among 11 cases of msih , seven ( 63.6% ) showed loss of expression of either one of mlh1 or msh2 . , lack of mlh1 nuclear staining was observed much more often than the absence of msh2 nuclear staining . despite a close correlation between ihc and msi , these findings suggested that ihc could not be fully recommended as a substitute for msi tests . in our attempt to find parameters showing significant differences between mss or msi - l and msi tumors by multiple logistic regression tests as a multivariate analysis bethesda guidelines , tumors from the right colon and/or synchronous multiple colon cancers , and negative expression of mlh1 protein in msi - h tumors showed statistical significance . when we applied a selection of patients with one of these three parameters , sensitivity and specificity for msi - h were 100% and 72.5% , respectively . this means that , using these parameters for selection of patients for msi testing , we were able to find all msi - h tumors , with the exclusion of 80 patients who had no msi - h , and to minimize the number of patients for msi testing . using these three parameters , we retrospectively applied 25 patients who underwent surgery before 2003 and were excluded from this study ; all five msi - h patients met at least one of these parameters , and its sensitivity and specificity for msi - h were 100% and 60% , respectively . these findings allowed us to select patients with msi - h tumors who met one of three parameters . except for the bethesda guideline , ihc , tumor location , and cell type showed 100% sensitivity and 70.6% specificity . collection of data for each patient for bethesda guidelines is not difficult ; therefore , careful history taking and routine pathologic data are very important . msi testing should be recommended for positive patients with any of the bethesda guidelines , tumor location , or mlh1 stain . bethesda guidelines , rt . sided colon cancer , and negative expression of mlh1 protein are important parameters for selection of patients with colorectal cancers for msi testing .