MOLECULAR ANALYSIS : MICROSATELLITY INSTABILITY AND LOSS OF HETEROZYGOSITY OF TUMOR SUPPRESSOR GENE IN HEREDITARY NON-POLYPOSIS COLORECTAL CANCERS ( HNPCC )

HNPCC (Hereditary non-polyposis colorectal cancers) development is caused by mutation of genes included in system of mismatch repair genes. Th e mutation exists at  of patients in hMSH gene,  in hMLH and  both in hPMSand hPMS genes. RER+ exists in about  in hereditary non-polyposis colorectal cancer and about - in sporadic


Introduction
HNPCC is one of the most frequent forms of familial colorectal cancer with about - of all colorectal cancers (,).Its heredity is autosomal dominant, and possibility of its transmission on children is : regardless of sex ().If the members of a family are bearers of genetic mutation there is a possibility that cancer will develop during their lives.Th e risk of development of colorectal cancers is about , endometrium cancers  at females, gastric cancers  and ovary cancers  at females ().HNPCC develops before the age of - in regard to sporadic cancers having their own developing periods average about - ages.
There is an opinion () that HNPCC and sporadic RER+ colorectal tumours having different paths for cancer development.These tumours have increased mutation level, so called mutator phenotype, which is a result of MMR gene inactivating.Th ere are two paths for genesis of colorectal tumours: -tumour suppressors path: mutational inactivation both alleles; -mutator phenotype path: microsatellite instability in HNPCC and sporadic RER + colorectal cancers.
RER+ exists in about  in hereditary non-polyposis colorectal cancer and about - in sporadic cancers.Germline mutation in hMLH and hMSH genes commonly have for consequence a loss of heterozygosity which is inactivated by MMR gene (-).Germline mutation in HNPCC exists in each cell and only one following event, commonly loss of heterozygosity, can inactivate MMR gene.Th is happens in earlier stage, before inactivation of APC tumour suppressor gene, and results in a fast progression from adenoma to cancer, so called »the adenoma-carcinoma sequence».Loss of functions of APC gene leads towards the other mutations, usually to mutation of "ras" oncogene.Next is the third stage of mutation in tumour suppressor gene, which leads to late adenoma development.Only when the both copies p of tumour suppressor gene become defective, will the cells become malignant.At least seven "hits"are necessary for further cancer: two per each tumour suppressor gene (APC, DCC and p) and one on "ras" oncogene.In  in Amsterdam, an international collaboration group established the Amsterdam Criteria () used in diagnostics of HNPCC families, as follows: -at least three relatives with CRC; -one should be fi rst degree relative of other two; -at least two successive generations aff ected; -at least one CRC diagnosed before the age of ; -FAP excluded; -diagnosis confi rmed by histology.
Th e Bethesda criteria were used in the study to assist the decision of which colorectal cancers should be tested for microsatellite instability ().A microsatellite panel must have mononucleotide markers: Bat  and Bat , and obligatory DS  from dinucleotide markers.According to these criteria, there is a cancers classification on microsatellite instability as following: MSI -L -microsatellite instability-low (MSI in locus less than - ; MSI-H -microsatellite instability-high (MSI in locus more than  and replication positive error (RER+) and MSS cancers-microsatellite stable

Materials and Methods
Th rough the study we involved  patients with clinical diagnosed sporadic colorectal cancer.Th e Amsterdam Criteria () and Bethesda Criteria () were used for genetic test and HNPCC was diagnosed in the patients with previously diagnosed sporadic colorectal cancer Therefore, complete study made according to Bethesda and Amsterdam Criteria and  of  patients are belong to HNPCC group.Our study was based on samples of tumor and surrounding healthy tissue of patients with colorectal cancer.Samples were collected from Gastroenterological and Surgical Clinic of University Clinical Center in Tuzla (Bosnia and Herzegovina).Both tumor and healthy surroundings tissue was formalin fi xed and thereafter that embedded in paraffi n blocks.Methods of genomic DNA isolation are made on de-paraffinization of tissue sections as on cell proteolyses with proteinase K Fluoroscent chain synthesis of DNA is a method which has very broad application in tumour detection, and it is especially important in determination of microsatellite instability (MSI) and loss of heterozygosity (LOH) of tumour suppressor gene.We used mononucleotide and dinucleotide microsatellite markers in detection of microsatellite instability.In the group of mononucleotide markers the following were used: BAT, BAT and BAT, but in the group of dinucleotide markers DS and TP . for detection of LOH were used, we used intragene markers for following tumour suppressor genes: NM, p, APC, RB, DCC and DCC ()

Analysis of genetic instability of microsatellite loci and RER phenotype of hereditary non-polyposis colorectal cancers
In total sample of  patients,  or , belong to hereditary non-polyposis colorectal cancer, according to Amsterdam Criteria and Bethesda Criteria.Analysis of microsatellite instability showed that mono-nucleotide marker Bat  was presented in / (,) of tumour samples, than Bat  and Bat  had in / (,) (Figures  and ).From dinucleotide markers, microsatellite instability showed TP  in / (,) and DS  in / (,) of tumour samples (Table ).
The research showed that / (,) of tumour samples belong to RER positive phenotype, and / (,) belong to RER negative phenotype.From RER positive phenotype / (,) of tumours showed instability in three loci, and / (,) in four loci.There was a significant difference between RER+ and RER-tumour phenotype in occurrence of number of microsatellite instability loci (p<,).
In the tumour group of RER+ phenotype, it was detected that mononucleotide marker Bat  showed microsatellite instability in / (,).
In the tumour group of RER-phenotype, it was detected that mononucleotide markers Bat  and Bat  showed microsatellite instability in / (,) tumor tissues.
There was no significant difference between mononucleotide and dinucleotide markers in regard to RER status (p>,).Analysis of seperate clinico-pathological parameters (sex, age, tumour localization, histopathological type) showed that there were more males / (,); furthermore that the age category was over  in / (,); then that cancers were located on left side (region of rectum and sigma) / (,) and that according to histopathological finding they were in adenocarcinomas / (,).Analysis of microsatelite instability of separate markers and clinicopathological characteristics showed that there was no significant difference (p>,).Analysis RER phenotype and clinicopathological characteristics showed that RER + phenotype was presented at males in / (, ).In the age group over , there was in / (, ) and in tumour localization RER+ phenotype was presented at left side tumours in / (, ) and adenocarcinomas were in / (,).RER-phenotype was most presented at females in ½ (); in age group below  in / (,); and belong to the group of right side tumours  / () and adenocarcinomas in / (,) (Table )

Discussion
Th e heterogeneous pattern of tumor mutation suggest that multiple alternative genetic pathway to colorectal cancer exist and accepted genetic model of cancer development is not representative of major tumors.().Microsatellite instability was detected in the group of  tumours HNPCC with high frequency of  () .RER+ phenotype showed  tumours ().Authors of the study concluded that RER+ tumour can be found in earlier stages of carcinogenesis in HNPCC, except nonhereditary for which we can suppose that HNPCC tumours can be developed through diff erent genetic changes in the frame of own adenoma-carcinoma sequences .
Results of our study showed that RER phenotype was presented in , of tumour tissues, and RER-phenotype in , of samples.Th ere is a signifi cant diff erence between RER+ and RER-phenotype of tumours in appearance of the number of microsatellite locus (p<,).However, other study found () that MSI was detected in  of tumour HNPCC,  from that belongs to the group of MSI-L level, but only  belongs to MSI-H level.Both tumour groups did not show simple clinico-pathological forms and they showed a significant connection between MSI and mucinous histological tumour type.Furlan et al. () con-cluded that microsatellite screening could be an the most efficient strategy for HNPCC identification.At Slovakia patients, report () showed that MSI-H tumour status was noted only at patients younger than  and MSI-H was not fi nd at patients older than , alhough they had a positive familiar history for colorectal cancer.Analyses confi rmed that  patients had instability in  locus and  had only one instability locus.Between markers used for MSI tumor status, the biggest frequency of microsatellite instability showed mononucleotide marker Bat  with frequency of .
According to the results of study, Fridrichova et al. () concluded hat some patients which with MSI-H tumor status at HNPCC group of ''suspicious'' patients had better expressed clinico-pathological characteristics than patients with positive familial illness history.. Some study () found that RER+ phenotype had all patients / in HNPCC group, in the group of patients with CRC family, but with uncompleted data for HNPCC diagnosis only ..

R E R + p h e n o t y p e w a s n o t e d i n f a m ilies showing clinical HNPCC forms and it was commonly varied from - ().
Other researched group () which was classified as HNPCC ''suspicious'' group (because uncompleted data according to Amsterdam Criteria) had RER+ status in  cases.As a consequence, tumours in HNPCC, reveal alterations in the length of simple repetitive genomic sequences like poly A, poly T repeats and least  of cases ().Some report () showed that - families had RER+ phenotype, and analysis confirmed that this group of colorectal patients had a significant difference between RER+ and RER-tumour for each tested marker.
Analysis of microsatellite status of some marker  () showed that mononucleotide marker Bat  was high sensitive marker for screening MLH/ MSH of positive mutation in HNPCC, and Bat  was positive at all  mutations in these studies.
Our study of microsatellite instability showed that mononucleotide marker Bat  had frequency , (Figure ), of tumour samples, than that Bat  and Bat  had the same , (Figure ).
From dinucleotide markers, microsatellite instability was showed at TP  in / or , and DS with , of tumour samples.Some report showed () that there is no signifi cant relation between MSI status and sex, age and adenoma size.Th e biggest number of patients belongs to female sex, and age was about .Th ese analysis' showed that tumors belong to MSI-H were usually located in distal part of colon.. High level of microsatellite instability was detected in patients over  years old and the majority belonged HNPCC group ().
Our study are agreed with some previous cited analysis of clinico-pathological parameters.Analysis of some clinico-pathological parameters (sex, age, tumour localization, histopathological classifi cation) showed that there were more males (,), that age category was over  (,), that cancers were located on left siderectum and sigma region (,) and according histopathological finding it was adenocarcinoma (,).Analyses () showed that there is no significant difference between RER+ and RER-tumours regardless of the age, the average age being ,  at RER-group and ,  at RER+ group.
Further study () showed that RER phenotype showed connection with histopathological variables at patients from Hong Kong.Th ere is no signifi cant diff erence be-tween RER+ phenotype and tumour localization which are usually right-sided.Th ere is a signifi cant diff erence between sex and age.These data showed that RER+ phenotype was more presented at males.Report () showed that young patients' age under  had RER+ tumour phenotype and that there is important difference between RER+ and RER-tumour phenotype.RER+ tumours were found at patients about  years old, while RER-tumours were found at older females about  years old.This study () showed a significant difference between RER+ tumours and tumour localization in proximal colon.There is no significant difference between these two groups of tumours and RER-tumour localization.
Our analysis of RER phenotype and clinico-pathological characteristics showed that RER+ phenotype existed at males , .In age group over , it was in / (,) and at tumour localization RER+ phenotype existed in left side tumours in , and adenocarcinomas were ,.So, RER-phenotype was more present at females in ; in the age group under  in ,, and they belong to the group of right-side cancers in ½ cases or , and adenocarcenomas was in , of tumour samples.Th ere is no signifi cant diff erence between RER tumour status and clinicopathological characteristics (p>,).Alteration of tumour suppressor gene p in HNPCC patients () were in  cases, and alteration of APC gene in  cases.Analyses of loss heterozygosity in APC and p of tumour suppressor genes were signifi cantly less than in HNPCC tumours in regard to sporadic tumours.
Results showed () that  of MSI tumours HNPCC did not show a genetic alteration in p tumour suppressor gene, while  MSS tumours of sporadic tumours had genetic alterations in tumour suppressor gene p.
Our results showed that loss of heterozygosity was Rebishung et al. () concluded that RER+ phenotype was rather less present at rectal tumours with prior loss of heterozygosity.Mutation of APC locus had smaller frequency than mutation of p locus at colorectal tumours with the same phenotype.Loss of heterozygosity at p was presented at  samples and at APC in  cases.Tumours belonging to RER-phenotype which have no loss of heterozygosity can be developed as a result of carcinogenesis model.. HNPCC is an inherited disease characterized by the development of cancer at a predominance of proximal colon, excess of multiple cancers increased risk for selected extracolonic adenocarcinomas and better prognosis ().


Our analysis of genetic alteration and clinico-pathological characteristics showed that loss of heterozygosity in locus APC,RB and Nm gene were presented at females in ,and at males had allele loss in locus APC in ,.Age group below , loss of heterozygosity was presented in locus APC gene in  samples, but in age group over  this loss was found in locus APC in ,.At tumour localization, loss of heterozygosity was presented at left side tumours in locus APC with , , and at right side tumours allele loss in locus APC,DCC and DCC was .According to histological classifi cation, LOH APC was presented at adenocarcinomas in / or ,, but at mucinous adenocarcinomas in locus DCC and DCC with  (Table ).Germ-line mutations in the mismatch-repair genes MLH, MSH, MSH, and PMS lead to the development of the Lynch syndrome (hereditary nonpolyposis colorectal cancer), conferring a strong susceptibility to cancer (,) The disorder () has traditionally been recognized in kindreds with a clustering of related cancers in association with mutations in DNA mismatch repair genes.HNPCC is associated with a substantially increased risk for several forms of malignancy but particularly colorectal and endometrial cancer.
BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2009; 9 (1): 16-18 VESNA HADŽIAVDIĆ ET AL.: MOLECULAR ANALYSIS: MICROSATELLITY INSTABILITY AND LOSS OF HETEROZYGOSITY OF TUMOR SUPPRESSOR GENE IN HEREDITARY NONPOLYPOSIS COLORECTAL CANCERS HNPCC found at tumour suppressor gene APC in , (Figure ), then NM  tumour suppressor gene , (Figure ), DCC , tumour suppressor gene DCC, p and RB in , tumour tissues.Homozygosity of locus was presented at DCC tumour suppressor gene in , , at DCC in , .There is a statistic significant difference tween tumour with allele and without allele (p<, ).Indinnimeo et al. () concluded that genetic instability of tumour suppressor genes p and NM  as clinicopathological forms at rectal cancers were found out that NM  showed alteration at all RER+ tumours, while p tumour suppressor showed this occurrence only in one case.There is no statistic difference between clinical parameters and RER status.Loss of heterozygosity was confirmed on q chromosome in , samples and RER+ phenotype was found in , cases.()There is no significant difference between LOH of q chromosome and clinicopathological characteristics regardless of sex, tumour state and differentiation level.Allele loss () which was usually detected at adenocarcinomas in distal part of colon than at tumours in proximal part.Our results of loss of heterozygosity of tumour suppressor gene APC showed in tumours belonging RER+ phenotype in ,, and low frequency showed at p in ,, at RER-phenotype LOH APC in  samples, and alterations were not find at DCC (), but LOH DCC had it in ,.Watatani et al.() found that loss of heterozygosity of p chromosome in  was presented at right -side tumours and only in  cases at left-side tumours.RER+ phenotype had  of right-side tumours and   of left-side tumours belong to RER+ phenotype According to Ikenaga et al. () the occurrence of microsatellite instability at younger patients with colorectal cancer was ,  and it was significantly higher than at older patients ().At  patients without MSI, it was established that only one case belongs to HNPCC and two have familial history of illness.

TABLE 1
. Microsatellite instability of mononucleotide and dinucleotide markers Microsatellite instability Analysis of genetic alterations of tumour suppressor in HNPCC Genetic alteration of tumour suppressor genes APC appears in / (, ) samples, then NM  tumour suppressor gene in / (, ) (Figures  and ), DCC  in / (,), tumour suppressor gene DCC, p and RB in / (,) of tumour tissues The highest frequency of homozogosity in locus is present in DCC tumour suppressor gene with / (, ), in DCC  with / (,) (Table ).locus p, RB in / () and DCC in / () and marker DS which was appeared in tumours with loss of heterozygosity in locus DCC in / () samples.There is no significant difference (p>,) in appearance of microsatellite instability of dinucleotide markers in tumour tissues with heterozygosity loss.Loss of heterozygosity of tumour suppressor gene APC was found in tumours which belong to RER+ phenotype in / (, ), and low frequency was showed at p in / (, ), in RER+ phenotype LOH APC in / () samples.Alterations were not seen at DCC () in regard to LOH DCC in / (, ) (Table )./ (, ), in right side tumours allele loss in locus APC, DCC and DCC was / ().According to histopathological classification, LOH APC was found in adenocarcinomas in / (, ), and at mucinous adenocarcinomas in locus DCC and DCC with  (Table ).Th ere is no signifi cant diff erence (p>,) in appearance of genetic alterations in tumour tissues with heterozygosity loss and clinico-pathological characteristics.

TABLE 5 .
Relation between genetic alterations of tumour suppressor genes and clinicopathological characteristics VESNA HADŽIAVDIĆ ET AL.: MOLECULAR ANALYSIS: MICROSATELLITY INSTABILITY AND LOSS OF HETEROZYGOSITY OF TUMOR SUPPRESSOR GENE IN HEREDITARY NONPOLYPOSIS COLORECTAL CANCERS HNPCC VESNA HADŽIAVDIĆ ET AL.: MOLECULAR ANALYSIS: MICROSATELLITY INSTABILITY AND LOSS OF HETEROZYGOSITY OF TUMOR SUPPRESSOR GENE IN HEREDITARY NONPOLYPOSIS COLORECTAL CANCERS HNPCC Conclusion Th e results of the study show importance of usage of Amsterdam and Bethesda criterion in detection of HNPCC patients.Unless this is not done, all tumours should be treated as occasional.Th e analysis of microsatellite instability showed that mononucleotide marker Bat  was present in , of tumour samples.Bat  and Bat  were present in , of the samples.From dinucleotide markers, microsatellite instability was present in TP in , and in DS in  of tumour samples.Th e result show that the analysis of loss of heterozygosity was marked in tumour suppressor gene APC in ,, in NM  tumour suppressor gene at ,, DCC  at , tumour suppressor gene DCC, p and RB in , of tumour tissues.Th e study reveals that mononucleotide marker Bat  has signifi cant microsatellite instability and as such it is effi cient for the fast microsatellite screening with HNPCC patients.Tumour suppressor APC gene can be highly sensitive marker of mutations which are only related to HNPCC group of patients.