the database of the korea university anam hospital , seoul , korea was searched for all crc patients who underwent surgical resection between 2002 and 2013 . a cohort of patients who underwent hepatic resection or pulmonary resection for crc metastases was also identified . sections were incubated for 15 minutes with antibodies against mlh1 protein ( 1:200 , es05 , leica , newcastle upon tyne , uk ) , msh2 protein ( 1:100 , g219 - 1129 , cell marque , rocklin , ca , usa ) , msh6 protein ( 1:50 , 44 , cell marque ) , and pms2 ( 1:50 , mrq-28 , cell marque ) . tumors were considered deficient in mlh1 , msh2 , msh6 , and pms2 expression when there was a complete absence of detectable nuclear staining in neoplastic cells . intact nuclear staining of the adjacent non - neoplastic epithelium , stromal cells , or lymphocytes served as an internal positive control ( appendix 1 ) . whole sections were stained and reviewed for cases that showed a loss of expression on a microarray . clinicopathological features , including age , size , gender , site of primary crc , lymph node status , lymphovascular invasion , perineural invasion , carcinoembryonic antigen ( cea ) levels at diagnosis , t category , tumor differentiation , and metastatic site , were compared between dmmr and proficient mmr ( pmmr ) patients used fisher exact ,  tests and mann - whitney test . this case was reviewed by the institutional review board of korea university medical center ( an15349 - 00 ) . the database of the korea university anam hospital , seoul , korea was searched for all crc patients who underwent surgical resection between 2002 and 2013 . a cohort of patients who underwent hepatic resection or pulmonary resection for crc metastases was also identified . to characterize the mmr system , ihc was performed using 4-m - thick paraffin tissue sections . sections were incubated for 15 minutes with antibodies against mlh1 protein ( 1:200 , es05 , leica , newcastle upon tyne , uk ) , msh2 protein ( 1:100 , g219 - 1129 , cell marque , rocklin , ca , usa ) , msh6 protein ( 1:50 , 44 , cell marque ) , and pms2 ( 1:50 , mrq-28 , cell marque ) . tumors were considered deficient in mlh1 , msh2 , msh6 , and pms2 expression when there was a complete absence of detectable nuclear staining in neoplastic cells . intact nuclear staining of the adjacent non - neoplastic epithelium , stromal cells , or lymphocytes served as an internal positive control ( appendix 1 ) . whole sections were stained and reviewed for cases that showed a loss of expression on a microarray . clinicopathological features , including age , size , gender , site of primary crc , lymph node status , lymphovascular invasion , perineural invasion , carcinoembryonic antigen ( cea ) levels at diagnosis , t category , tumor differentiation , and metastatic site , were compared between dmmr and proficient mmr ( pmmr ) patients used fisher exact ,  tests and mann - whitney test . this case was reviewed by the institutional review board of korea university medical center ( an15349 - 00 ) . the clinicopathological features of 61 patients included in the study are detailed in table 1 . the mean patient age was 58 years ( range , 31 to 78 years ) ; 26% of the patients were female and 73% were male . the mean tumor size was 5.4 cm ( range , 1.8 to 15 cm ) . the anatomical location of the primary tumor was classified as left ( rectum , rectosigmoid colon , splenic flexure , and descending colon ) or right side ( ascending colon , hepatic flexure , and cecum ) . in total , 55/61 ( 90.1% ) were t3 cancers that invade through the muscularis propria into pericolorectal tissues . neither patient had a family history of cancer . using fisher exact ,  tests and mann - whitney tests to analyze the relationship between mmr status and clinicopathological features , significant relationships were not detected for age ( p=.58 ) , size ( p=.14 ) , sex ( p=.34 ) , site ( p=.96 ) , t stage ( p=.36 ) , tumor differentiation ( p=.32 ) lymph node metastasis ( p=.13 ) , lymphovascular invasion ( p=.75 ) , perineural invasion ( p=.30 ) , or cea level ( p=.49 ) . of the primary tumors , 7/61 ( 11.4% ) showed a dmmr system based on ihc , while secondary tumors showed a deficiency in 13/61 cases ( 21.3% ) . in total , three cases showed deficiencies in both the primary and the paired metastatic lesions . in 10 cases , proficient expression was found in the primary lesions , but not in the corresponding metastatic lesions . in four cases , proficient expression was detected in the secondary tumor , but not in the primary tumor . in total , 14 patients died of cancer , and the median survival was 33 months from the date of initial diagnosis . a survival analysis was performed assuming that the primary and metastatic tumors showing intact expression using ihc group are pmmr and the loss of expression in either tumor indicates dmmr ( fig . this result was not statistically significant ( log - rank test , p=.22 ) , but the pmmr group showed a more favorable prognosis compared to the dmmr group . the clinicopathological features of 61 patients included in the study are detailed in table 1 . the mean patient age was 58 years ( range , 31 to 78 years ) ; 26% of the patients were female and 73% were male . the mean tumor size was 5.4 cm ( range , 1.8 to 15 cm ) . the anatomical location of the primary tumor was classified as left ( rectum , rectosigmoid colon , splenic flexure , and descending colon ) or right side ( ascending colon , hepatic flexure , and cecum ) . in total , 55/61 ( 90.1% ) were t3 cancers that invade through the muscularis propria into pericolorectal tissues . using fisher exact ,  tests and mann - whitney tests to analyze the relationship between mmr status and clinicopathological features , significant relationships were not detected for age ( p=.58 ) , size ( p=.14 ) , sex ( p=.34 ) , site ( p=.96 ) , t stage ( p=.36 ) , tumor differentiation ( p=.32 ) lymph node metastasis ( p=.13 ) , lymphovascular invasion ( p=.75 ) , perineural invasion ( p=.30 ) , or cea level ( p=.49 ) . 7/61 ( 11.4% ) showed a dmmr system based on ihc , while secondary tumors showed a deficiency in 13/61 cases ( 21.3% ) . in total , three cases showed deficiencies in both the primary and the paired metastatic lesions . in 10 cases , proficient expression was found in the primary lesions , but not in the corresponding metastatic lesions . in four cases , proficient expression was detected in the secondary tumor , but not in the primary tumor . in total , 14 patients died of cancer , and the median survival was 33 months from the date of initial diagnosis . a survival analysis was performed assuming that the primary and metastatic tumors showing intact expression using ihc group are pmmr and the loss of expression in either tumor indicates dmmr ( fig . this result was not statistically significant ( log - rank test , p=.22 ) , but the pmmr group showed a more favorable prognosis compared to the dmmr group . in this study , mmr proteins were evaluated by ihc for the following reasons : ( 1 ) ihc is less time consuming than pcr , ( 2 ) is easier to implement , and ( 3 ) enables the simultaneous identification of affected mmr genes . first , it is well known that the ihc staining patterns for msh6 ihc are variable . for instance , tissue hypoxia and delayed fixation reduce the sensitivity of detection of mmr gene expression or suppress mmr . third , some mlh1 mutation - positive cases or even cases with mlh1 promoter methylation show false - positive nuclear staining . a missense mutation or an in - frame insertion / deletion mutation in mlh1 does not affect mlh1-pms2 interactions , and thus the protein reacts with the antibody used for ihc . lastly , interpretation of mmr ihc results requires consideration that the mmr proteins act as heterodimers . for example , crcs that show loss of mlh1 and pms2 expression , but intact msh2 and msh6 expression indicate an mlh1 deficiency . the loss of pms2 expression results from mlh1 mutations . in this study , our major interest was investigating the concordance in mmr deficiencies between the primary crcs and its corresponding metastases . reported matching ihc results between the primary tumor and metastatic tissue for all cases examined . however , agoston et al . found discordance in the mmr status between primary tumors and metastases in 20.2% of cases . haddad et al .   found a very low frequency of msi using pcr in resected crc hepatic metastases in 190 patients . unlike in primary crcs , the rate of msi in resectable crc hepatic metastases is approximately 2.5% . first , high - frequency msi primary crcs do not frequently metastasize to the liver . second , high - frequency msi primary crcs spread to the liver extensively and therefore are considered unresectable . however , the incidence was somewhat different between the primary ( 11.5% ) and the metastatic ( 21.3% ) cancer groups . in this study , among the 36 hepatic metastatic cancers , five cases showed dmmr in primary crcs . this frequency of approximately 14% was not as low as that reported in the previous study . the clinicopathological features and each mmr protein expressions of the cases with either primary or metastatic dmmr are summarized in tables 4 and 5 . except one case , based on ihc , primary and metastatic lesions of all the cases showed intact mlh1 expression . in 10 cases , proficient expression was found in the primary tumor , but not in the corresponding metastatic lesions . in four cases , proficient expression was detected in the secondary tumor , but not in the primary tumor . furthermore , the precise mmr proteins that showed a loss of expression were not exactly matched in the primary and the metastatic tumors . to further examine the data , we divided the dmmr cases into five groups . during heterodimeric complex formation , the loss of pms2 is followed by the loss of mlh1 expression owing to functional dimerization . similarly , the loss of msh2 is accompanied by the loss of msh6 ( fig . 2 ) . however , the loss of isolated msh6 or pms2 is not accompanied by mlh1 or msh2 , reflecting mutations in msh6 or pms2 . although three cases were classified as a combined group that do not belong to the above four groups , most of the dmmr cases fit the classification , indicating that the ihc expression is consistent . mlh1 mutations are the most common type among the mmr genes . however , the detected loss of the mlh1 protein was significantly less frequent than the loss of the msh2 protein in this study . as stated above , we attributed this difference to the ihc technique . most mutations in msh2 result in truncated proteins , which consequently showed a loss of expression in ihc analyses . mlh1 mutations are non - functional missense mutations , and mutated - mlh1 cases also show proficient expression . a meta - analysis showed that using ihc , only 74% of mlh1 losses were detected in the mlh1 mutation - positive cases , compared to the 91% detected using msi testing . however , msh2 mutations could be detected in up to 94% of the msh2 mutation - positive cases by ihc . we infer that there may be false - positive mlh1 cases , and additional pcr studies are needed to identify the precise mlh1 mutations . in our study , cases were considered deficient if any target locus ( e.g. , mlh1 , msh2 , msh6 , or pms2 ) was not expressed . this is a limitation of our study , and variable patterns could lead to erroneous interpretations . in one case , we detected the losses of mlh1 , msh6 , and pms2 expression , suggesting a similar scenario . we attributed this discordance in ihc results to the variable reactivity and subjective data interpretations . all cases received adjuvant chemotherapy after resection of the primary tumor , because all cases included in this study were stage iv . most of the patients ( 56 cases ) were treated with folfox regimens ( combination of folic acid , 5-fluorouracil [ 5-fu ] and oxaliplatin ) . three cases were treated only with 5-fu  based chemotherapy . among the 61 cases , 50 cases received chemotherapy before metastatectomy , where s 11 cases received chemotherapy after metastatectomy . of the 50 cases that received chemotherapy before metastatectomy , 11 cases ( 22% ) showed discordance in ihc . in seven cases , proficient expression was found in the primary lesions , but not in the corresponding metastatic lesions . we assume that these losses of expression could be associated with chemotherapy - related effect . since all cases were stage iv tumors , the low survival rates were expected . despite these limitations , we detected a correlation between the mmr status of primary and metastatic crcs using only ihc . further research should focus on the modifications in the biological and biochemical properties of dna mmr proteins during metastatic processes .