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 .
 backgroundcolorectal cancer ( crc ) is one of the most common malignancies worldwide . 
 approximately 10%15% of the crc cases have defective dna mismatch repair ( mmr ) genes . 
 although the high level of microsatellite instability status is a predictor of favorable outcome in primary crc , little is known about its frequency and importance in secondary crc . 
 immunohistochemical staining ( ihc ) for mmr proteins ( e.g. , mlh1 , msh2 , msh6 , and pms2 ) has emerged as a useful technique to complement polymerase chain reaction ( pcr ) analyses . 
 methodsin this study , comparison between the mmr system of primary crcs and paired liver and lung metastatic lesions was done using ihc and the correlation with clinical outcomes was also examined . 
 resultsbased on ihc , 7/61 primary tumors ( 11.4% ) showed deficient mmr systems , while 13/61 secondary tumors ( 21.3% ) showed deficiencies . in total , 
 44 cases showed proficient expression in both the primary and metastatic lesions . 
 three cases showed deficiencies in both the primary and paired metastatic lesions . in 10 cases , 
 proficient expression was found only in the primary lesions , and not in the corresponding metastatic lesions . in four cases , 
 proficient expression was detected in the secondary tumor , but not in the primary tumor . 
 conclusionsalthough each ihc result and the likely defective genes were not exactly matched between the primary and the metastatic tumors , identical results for primary and metastatic lesions were obtained in 77% of the cases ( 47/61 ) . 
 these data are in agreement with the previous microsatellite detection studies that used pcr and ihc . 
MATERIALS AND METHODS
Patient population
Immunohistochemistry
Statistical analysis
RESULTS
Clinicopathological features
Relationship between MMR status and clinicopathological features
Association between primary and metastatic tumors
Survival analysis
DISCUSSION