hereditary non - polyposis colorectal cancer ( hnpcc ) or lynch syndrome ( ls ) is a chronic disease in which there is familial aggregation of colorectal cancer ( crc ) and other associated tumors .
this disease now is described as a cancer susceptibility syndrome secondary to a germ - line mutation in at - least one of the dna mismatch repair genes ( mmrs ) including mlh1 , msh2 , msh6 , and pms2 or some large deletion mutations in last exon of epcam , a gene located next to msh2 , or epcam - msh2 locus [ 2 , 3 ] .
it leads to accumulate of mutations in other genes responsible to apoptosis and cell cycle control , accelerating an adenoma - to - carcinoma transition event [ 3 , 4 ] .
moreover , mlh1 promoter hypermethylation , an epigenetic phenomenon , can inactivate the gene , mostly in sporadic crcs [ 5 , 6 ] . screening for molecular detection
is both time - consuming and expensive due to the heterogeneity of so variable mutations in mmrs . among two molecular screening tools which are being used commonly to detect ls , microsatellite instability ( msi ) testing and immunohistochemical ( ihc ) staining of mmr proteins .
msi is sensitive but not specific for ls , as only 20 - 25% of all msi - high ( msi - h ) tumors are associated with germline mutations in a mmr gene .
although msi has enough sensitivity to identify ls , it will not be detected in about 5% of all ls tumors . 

mmr defects can be identified by ihc staining in tumor tissue sections in which expression of mmr genes is lost ( ihc - absent ) , while their expression in healthy adjacent tissue sections is intact ( ihc - present ) .
ihc has about 77 - 100% sensitivity and 98 - 100% specificity to detect mmr defects compared to msi - testing [ 4 , 7 - 9 ] .
though , ihc has been reproduced as complement of msi - testing in many studies [ 1 , 10 ] .
it has been suggested as the choice screening method prior to genetic testing by some authors .
they believe this technique has some advantages compared to msi - testing and should be replaced as the first molecular screening tool .
ihc is more available than msi as part of the routine service in the general pathology laboratories .
also , ihc is regarded more inexpensive than msi , so earlier analysis indicated that ihc was about threefold less expensive than msi - testing .
ihc , additionally , may consider as genetic testing because it can reveal which particular mmr gene may be defective , and as such it enables efficient mutation analysis on the target gene [ 8 , 14 ] . meanwhile , there are some limitations in ihc - mmrs such as uncertainty in interpretation due to variable tissue fixation and other technical issues which can result in weak or equivocal staining patterns .
it has also a low sensitivity to detect mutation of mlh1 with mlh1 anti body alone , because it is possible that some missense mutations in this gene will not result in the absence of a detectable protein product .
although the incidence of crc has increased among iranian population within recent decades [ 16 , 17 ] , it has not been yet established any systematic screening program to identify ls affected families among iranian population .
so , we designed a study to set up a molecular screening program in central iran for the first time .
we discuss in this article the results of ihc - mmrs concluded from the study .
this study was a descriptive retrospective in order to screen crc patients , in collaboration between two provinces : isfahan and charmahal va bakhtiari , to identify hnpcc families .
we screened 1659 crc patients registered in poursina hakim research center ( phrc ) , a famous referral gastroenterology clinic in isfahan , center of iran , during about 14 years from 2000 to the end of 2013 . at first , we selected all patients with age of fifty years or less , as early - onset patients
. then using amsterdam ii criteria , at risk families for lynch syndrome were included for next molecular analyses .
our study included all individuals participating in counseling sessions with history of bowel resection during the past decade whose paraffin - embedded blocks were available . during genetic counseling ,
the participants were interviewed about cancer related family history at - least up to three generations .
the drawn pedigrees were reconfirmed by at - least two other members of every family .
moreover , the reported malignancies within families were possibly verified by searching for their medical documents , if available .
otherwise , we could trust them . because according to strong familial relationship among iranian people , they have usually awareness of serious diseases such as cancer in their relatives . 

immunohistochemistry 
  we tried to select one paraffin - embedded tissue block for each case from resected bowel specimen containing tumoral and preferably adjacent normal mucosa . about 1 - 2 m .
thick tissue sections were cut after that deparaffinized in xylene and rehydrated it through graded alcohols . then slides were washed in running tap water , immersed in tris - edta buffer at ph 9.0 in a pressure chamber at microwave for antigen retrieval for 20 minutes ( 5  in high and 15  in m - high degree ) .
after washing the slides in deionized water , we used peroxidase block reagent to neutralize endogenous peroxidase for at - least 5 minutes .
then we washed the slides twice in tbs ( tris - buffered saline ) , each time for 5 minutes .
the slides were incubated with protein block reagent for 5 minutes and washed again in tbs twice as mentioned .
the next step was incubation the slides overnight with optimally diluted mouse monoclonal primary antibodies as following : msh2 ( leica biosystems : novocastra , uk , lyophilized , product code ( pc ) : ncl - msh2 ) at 1/80 dilution , mlh1 ( leica biosystems : novocastra , uk , liquid , pc : ncl - l - mlh1 ) at 1/100 dilution , msh6 ( leica biosystems : novocastra , uk , liquid , pc : ncl - l - msh6 ) at 1/100 dilution , and pms2 ( leica biosystems : novocastra , uk , liquid , pc : ncl - l - pms2 ) at 1/100 dilution .
the next morning , after twice washing the slides in tbs , they were incubated with post primary block reagent for 30 minutes .
washing again twice in tbs , we incubated the slides with novolink polymer for 30 minutes
. once again , twice washing in tbs with gentle rocking followed with developing peroxidase activity with dab working solution for 5 minutes .
after rewashing the slides in water for 5 minutes , we finally dehydrated , cleared and mounted sections .
overall , of 1659 crc patients registered in phrc , 413 patients ( 24.9% ) were 50 years at diagnosis .
219/413 successful calls , 45 hnpcc families were screened using amsterdam ii criteria of which 14 affected families were excluded from molecular testing stage . of excluded families , 10 individuals were omitted because of being unavailable their tumor tissues , and 4 others were excluded due to being unwilling for incorporation .
ihc - mmr staining was absent in 7.31 of the individuals ( 22.6% ) ( ihc - a ( absent ) versus ihc - p ( present ) families ) , of which 4 cases determined as ihc - a for both msh2 and msh6 antibodies ( 57.1% ) , in 2 cases both mlh1 and pms2 antibodies showed ihc - a ( 28.6% ) , and just in one case msh6 was defective ( 14.3% ) .
there were 187 cancer patients in all 31 hnpcc families of which 40 affected members ( ~21% ) were related to 7 ihc - a families .
the mean of affected members in ihc - a families was 5.7 while it was 6.1 in ihc - p families ( p value=0.513 ) .
the most frequent cancers among ihc - a families were : crc ( 67.5% ) , stomach ( 10% ) , small bowel ( 5% ) , and prostate ( 5% ) ; while in ihc - p families : crc ( 27.9% ) , stomach ( 18.4% ) , lung ( 10.9% ) , and breast ( 7.5% ) were the most common cancers ( table 1 ) .
mean age of ihc - a individuals at diagnosis was 38.0 years ( range 31 - 50 ) , while ihc - p individuals had averagely 45.3 years at diagnosis ( range 24 - 69 ) ( p value=0.146 ) .
on the other hand , the mean age of tumor diagnosis in 147 affected members within 24 ihc - p families was nearly the same as 40 cancer patients within 7 ihc - a families ( ~51 years : range 2 - 82 years ) .
the most frequent colorectal cancer tumor sites among ihc - p individuals were : rectum ( 41.7% ) , sigmoid colon ( 33.3% ) , cecum ( 12.5% ) , and ascending colon ( 8.3% ) ; while in ihc - a individuals : ascending colon and descending colon ( 28.6% ) , and transverse colon , sigmoid colon and cecum ( each one 14.3% ) were the most common involved sites .
meanwhile , there was no case with rectum involvement among ihc - a individuals ( table 2 ) .
just 1 of 7 ihc - a individuals ( ~14% ) was diagnosed at i or ii colorectal cancer tnm stage , while 8 of 24 ihc - p individuals ( ~33% ) were found at these early stages ( p value=0.345 ) ( table 3 ) .
although 11/24 of ihc - p individuals ( ~46% ) had been deceased at the screening time , 6.7 of ihc - a individuals ( ~86% ) were alive at this time ( p=0.382 ) .
in this study we evaluated expression of mmr genes containing mlh1 , msh2 , pms2 , and msh6 using ihc staining in 31 index early - onset crc patients among identified hnpcc families within isfahan , iran for the first time .
the gene expression is normal if nuclear staining in crc cells would be observed intact as in normal adjacent epithelial cells . if nuclear staining is absent in cancer cells versus positive staining in nuclei of normal colon epithelial cells , it would be indicated defective expression of a gene . 

prevalence of mmr deficiencies 
  we found absent ihc - mmr staining in 22.6% of the early - onset hnpcc individuals ( ihc - a ) who had met amsterdam ii criteria . given high sensitivity of four - antibody ihc - mmr to identify msi - crcs more than 92%
, it seems a significant portion of amsterdam positive families in our population has no mmr mutations .
although we can not find definite data in iranian population according to other studies , about 35 - 70% of hnpcc families meeting amsterdam criteria do not have mmr deficiency and are considered  familial colorectal cancer type x  ( fcc - x ) or  non - syndromic familial colorectal cancer  [ 20 , 21 ] .
it seems more evaluations on larger samples using complementary techniques such as msi - testing and mutation analyses are necessary to estimate a more accurate frequency of x syndrome among iranian population .
the most frequent ( ~57% ) deficiency of gene expression was related to both msh2 and msh6 genes .
msh6 and pms2 proteins are accessory to major mmr proteins : msh2 and mlh1 , respectively .
so the loss of msh2 expression in a tumor tissue leads to loss of msh6 expression in that tissue .
germline mutations , however , in msh6 or pms2 , as minor mmr genes , lead to single loss of expression of their associated proteins .
the prevalence of msh2 defect in our study was near to some large early studies .
about 29% of the ihc - a tumors did nt show any nuclear ihc - staining for both mlh1 and pms2 proteins .
it predicts existence of germline mutation in mlh1 of near to 30% of the ihc - a individuals . although it is similar to some early valid studies , developing the ihc results by msi - testing and mutation analyses , preferably on a larger sample , will be more informative .
we found just one individual with absent ihc - staining in msh6 protein ( 14.3% ) .
interestingly , the genetic pedigree shows the number of affected relatives is fewer on average than msh2 or mlh1 families .
so , there are only two cancer patients among second degree relatives of the individual with more than 65 years old . according to some studies
, we expect the patients with msh6 mutations would be more likely amsterdam negative , so the bethesda guidelines are more sensitive than the amsterdam criteria to identify it .
consequently , we may identify more patients with msh6 defect among all our crc patients using bethesda guidelines . as some studies have presented , pms2 loss in ihc - staining is the rarest event .
clinicopathological features 
  although there was no significant difference between average count of cancer patients among the ihc - a and ihc - p hnpcc families , the frequency of crc among ihc - a families was predominantly more than ihc - p families , nearly 2.5 fold ( 67.5% versus 27.9% ) , while we had asked cancer - related family history up to three generations in both groups . moreover , the mean age at diagnosis in ihc - a individuals was more than 7 years earlier than ihc - p individuals ( 38 versus 45.3 years ) , whereas there was no significant difference between other cancer patients in both groups of families ( about 51 years ) .
studies on fcc - x families have shown that crc risk among their kindred s is lower than hnpcc families .
also , crc diagnosis has occurred averagely 10 - 15 years later in fcc - x families [ 20 , 27 ] . of our index crcs ,
20.8% and 57.1% were localized proximal to the splenic flexure in ihc - p and ihc - a groups , respectively ( p<0.001 ) .
more studies on families with amsterdam criteria , with and without mmr deficiency , have shown a higher proportion of crcs are located proximally of the splenic flexure in patients with mmr deficiency than those with intact mmrs .
for example , mueller - koch and his coworkers found 68% of crcs proximal to the splenic flexure in amsterdam positive families with mmr mutations versus 14% in families without mmr mutations .
although the proportion of our alive ihc - a individuals at screening to the alive ihc - p individuals was about 2 fold , the early - stage diagnosis among ihc - p individuals was more than 2 fold of ihc - a individuals , according to their pathologic documents .
it may refer to the better survival of msi - crcs compared to mss ( microsatellite stable ) crcs , a fact that has been considered in some studies .
for example , in one study malesci and his coworkers showed that msi was significantly related to a reduced chance of lymph node and distant organ metastases at diagnosis .
for example , mlh1 mutations are related to higher risk of early onset crc cancer and more prevalent crc cancer than extracolonic cancers , while in msh2 mutation carriers there was a higher risk of multiple extracolonic cancers , and the mean age of diagnosis is more than mlh1 mutation carriers [ 30 , 31 ] . in our study
, the patients with mlh1 defect were identified averagely 8 years earlier than the patients with msh2 defect ( ~42 versus ~50 years old ) .
in addition , there were more extracolonic cancer types among families with msh2 defect in comparison to the families with mlh1 or msh6 defects ( 7 types versus 4 and 2 types , respectively ) .
the phenotype of msh6 mutations is somewhat different than mlh1 and msh2 mutations , and this condition has been described as " msh6 syndrome " .
the mean age at cancer diagnosis in msh6 mutation carriers is at least one decade more than msh2 or mlh1 mutation carriers .
in addition , the risk of crc affection in families with msh6 defect is more likely less than hnpcc families with msh2 or mlh1 defects . in our single family with msh6 defect ,
the mean age of three cancer affected members at diagnosis was about 67 years , averagely two decades more than age of the patients in families with mlh1 or msh2 defects .
moreover , the proportion of crc patients was significantly lower than hnpcc families with msh2 or mlh1 defects ( 33% versus 72 and 66 percent respectively ) .
since there is no enough information about molecular aspects of hereditary cancer syndromes like hnpcc in iran , we have not still a definite known plan for molecular screening of the disease . given the limitation of our study , we suggest more evaluations on larger samples by complementary techniques such as msi - testing and mutation analyses to reach more trustworthy results .
since there is no enough information about molecular aspects of hereditary cancer syndromes like hnpcc in iran , we have not still a definite known plan for molecular screening of the disease . given the limitation of our study , we suggest more evaluations on larger samples by complementary techniques such as msi - testing and mutation analyses to reach more trustworthy results .
 backgroundhereditary non - polyposis colorectal cancer ( hnpcc ) is a common hereditary cancer predisposing syndrome has molecular and clinicopathological features still have remained ambiguous within iranian populations . 
 we discuss in this article some molecular and clinicopathological features of the condition.methodsthe study was a descriptive retrospective and designed on 1659 colorectal cancer ( crc ) patients were screened based on early - onset disease and amsterdam ii criteria during 14 years ( 2000 - 2013 ) . 
 immunohistochemistry ( ihc ) staining was set up to detect expression of mismatch repair ( mmr ) genes on paraffin - embedded tissue sections of 31 hnpcc - crc tumors . 
 spss 19 software was used to analyze the data.resultsihc-mmr staining was absent in 7/31 individuals ( 22.6% ) of which 4 cases showed ihc - absent ( ihc - a ) in both msh2 and msh6 ( 57.1% ) , in 2 cases both mlh1 and pms2 had negative staining ( 28.6% ) , and just in one case , msh6 was defective ( 14.3% ) . 
 the frequency of crc among ihc - a and ihc - present ( ihc - p ) families was 67.5% and 27.9% , respectively . also the most frequent extracolonic cancers in ihc - a group were : stomach ( 10% ) , small bowel ( 5% ) , and prostate ( 5% ) ; and in ihc - p group : stomach ( 18.4% ) , lung ( 10.9% ) , and breast ( 7.5% ) . 
 average age of ihc - a individuals at diagnosis was 38.0 versus 45.3 years in ihc - p individuals . 
 overall , 20.8% and 57.1% of our index crcs were localized proximal to the splenic flexure in ihc - p and ihc - a groups , respectively.conclusiongiven the lack of enough information about molecular aspects of hereditary cancer syndromes like hnpcc in iran , more evaluations are necessary on larger samples using complementary techniques such as msi - testing and mutation analyses . 
Introduction
Materials and Methods
Results
Discussion
Conclusion