Detection Of Microsatellite Instability Of Sporadic Colorectal Cancer By Using Fluorescence-labelled Primers Multiplex　PCR

Colorectal carcinoma(CRC) can be divided into chromosomal instability and microsatellite instability(MSI).MSI in tumors is diagnostic for inactive DNA mismatch repair(MMR),resulting in failure to repair the errors that normally occur during replication of repetitive DNA sequences.Particularly,microsatellite mutations play an important role in tumorigenesis of DNA mismatch repair deficient tumors of colorectum. MSI is a well-recognized phenomenon that is classically a feature of tumors in the hereditary non-polyposis colorectal syndrome.Originally,MSI was shown to correlate with germline/inherited defects in MMR genes in families with HNPCC,where greater than 90%of colon cancers from patients exhibit MSI.MSI is also a recognised pathway of colorectal carcinogenesis responsible for about 15%of all sporadic colorectal cancers. The detection of MSI CRCs has prognostic value and can help screen for HNPCC.Microsatellites are repeating sequences of 1- to 6-basepair subunits interspersed throughout the genome that are useful markers for genetic mapping,forensic analysis, and bone marrow engraftment monitoring.There are hundreds of thousands of microsatellite loci throughout the genome that could potentially be used for MSI analysis.MSI is defined as alterations in the lengths of microsatellites due to deletion or insertion of repeating units to produce novel length alleles in tumor DNA when compared with the normal/germline DNA from the same individual.MSI in tumors, also known as replication errors,is the contraction or expansion of microsatellite lengths in the tumor relative to germline lengths and is a functional marker of mismatch repair defects.MSI is present in 85%to 90%of HNPCC,a condition most commonly due to germline mutations in DNA mismatch repair genes.Inactivation of any of several MMR genes,including hMLH1,hMSH2,hMSH6,and hPMS2,can result in MSI. However,it is now recognized that MSI also occurs in the absence of germline MMR mutations as the result of epigenetic inactivation of hMLH1 expression by promoter methylation.Promoter methylation at hMLH1 has been reported to be an important mechanism in MSI-positive sporadic colorectal cancer.The mechanisms causing MSI in sporadic tumors have not been elucidated completely and,possibly,may involve genes or pathways different from those implicated in HNPCC.The mechanism of MSI may be tumor-type specific and have different clinical implications for HNPCC and sporadic cancers.MSI tumors can be divided into two distinct MSI phenotypes:MSI-high (MSI-H) and MSI-low(MSI-L).MSI-H cancers may develop either sporadically or in the context of the HNPCC syndrome that is caused by germline mutations of MMR genes.These sporadic MSI-H colorectal tumors most often arise from the epigenetic silencing of the mismatch repair gene MLH1.Unlike MSI-H tumors,MSI-L tumors appear to arise through the chromosomal instability carcinogenesis pathway,similar to MSS tumors.Rather,it has been suggested that MSI-L tumors differ quantitatively from MSS tumors but do not differ qualitatively.Future studies will need to evaluate the specific mutations in non-MSI-H tumors in an attempt to sub-classify MSI-L tumors with regard to MSS tumors so that subtle differences between these two sub-groups can be identified.Determining the MSI status of a CRC has clinical use for identifying patients with HNPCC.In addition,MSI status,regardless of whether the causative defect is inherited or sporadic,may have use for prognostic and therapeutic decision-making purposes.Knowledge about the biological impact of microsatellite mutations in these genes will potentially help to develop modified clinical concepts for diagnosis,prevention,and treatment of microsatellite unstable human cancers.MSI status may also predict cancer response/resistance to certain chemotherapies.The loss of heterozygosity(LOH) on tumor suppressor genes is believed to be one of the key steps to carcinogenesis of colorectal cancer.The loss of one allele at a specific locus is caused by a deletion mutation or loss of a chromosome from a chromosome pair.The LOH analysis became an effective way to find informative loci and then to find candidate tumor suppressor genes.In order to identify representative genetic alterations in CRC and useful markers for future detection,a consecutive series of 63 sporadic colorectal cancers samples and the corresponding normal tissue between 2007 and 2008,were studied to evaluate the frequency of MSI and LOH in CRC patients,and investigate their role in screening sporadic colorectal cancers.It has been shown that the detected frequencies of MSI in the same type of cancer is different by using different markers or various amount of markers, most likely due to variable sensitivity of individual markers to detect MSI.In an attempt to standardize MSI analysis,a 1997 National Cancer Institute workshop recommended a"reference panel"of five microsatellite markers for the detection of MSI and established MSI classification guidelines based on the results.This fluorescent multiplex polymerase chain reaction(PCR) assay analyzes the five primary microsatellite loci: BAT25,BAT26,D2S123,D5S346,and D17S250.Amplicon detection is accomplished by capillary electrophoresis using the ABI 3130 Genetic Analyzer.We tested 63 tumours for MSI using the five microsatellite markers.The clinicopathological features of patients with MSI and MSS were investigated.Tumors showing instability in＞or = 2/5 loci were classified as MSI-H.The others without any alteration of microsatellite were classified as microsatellite stability(MSS).In this study,we analyzed LOH at 5 loci in sporadic colorectal cancer.We have set up a clinical molecular diagnostic assay to test for MSI at multiple loci. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis(CE) runs,and compared directly to identify novel length alleles in tumor tissue.The assay presented is superior to standard radioactive monoplex PCR,polyacrylamide gel electrophoretic analysis,primarily due to the multiplex PCR format.CE is a commonly used tool in the analysis of fluorescently labeled PCR amplification products.This assay was originally designed for forensic purposes;however,it is increasingly being used by clinical laboratories for patient samples secondary to its sensitivity and relatively rapid turnaround time.Therefore,the details of the manipulation will affect the success(or not) of MSI detection,leading to the mistaken results in analysis.Some high-frequency problems are discussed in the present report.Simultaneously we developed a procedure for DNA extraction from formalin-fixed tissue samples for conventional MSI analysis studies on archival samples with known clinical outcome.Older samples are particularly valuable because they are associated with longer clinical follow up.This provided suitable DNA for various types of molecular analyses,including polymerase chain reaction,fragment analysis,and direct sequencing.DNA extracted from formalin-fixed tissue is problematic due to chemical modifications and continued degradation over time.A formalin fixative solution is added to the tissue;usually the fixative removes lipids and denatures proteins, making the cell membrane remnants very fragile,which allows subsequent chromosome spreading and consequently exposes the DNA.We compared quantity and quality of DNA extracted by two different protocols from archived formalin-fixed CRC tissues. DNA quantity from the conventional protocol was similar to that extracted from the improved protocol,but quality and success rate were generally higher for the improved protocol group.DNA fragment size was assayed by agarose gel electrophoresis,testing DNA fragment sizes of 500～1000bp or=1000bp.Microsatellite analysis and detection of LOH was performed by using 5 markers selected for sensitive detection of sporadic colorectal cancer(SCRC).Of all the SCRC samples examined,22(34.92%) samples exhibited MSI,including 16(25.4%) cases with MSI-H,6(9.52%) cases with MSI-L,and 41(65.08%) cases with MSS.These results indicate that MSI might be an important event in these subset SCRC cases.The instability of D5S346 was found in 9 cases(14.29%),BAT-26 in 6(9.52%)cases, BAT-25 in 14(22.22%) cases,D17S250 in 15(23.81%) cases,and D2S123 in 16 cases (25.4%).MSI colorectal tumors have not been shown to differ in their clinicopathologic features from MSS tumors(P＞0.05).In addition,more than 9%of the tumor samples showed LOH in at least one of the 5 markers.LOH were observed in 1/41(2.44%) MSS CRCs and 5/22(22.73%) MSI CRCs.The differential rate of LOH between MSI and MSS colorectal cancer was statistically different(P=0.008).LOH were observed in 3/16(18.75%) MSI-H CRCs and 2/6(33.33%) MSI-L CRCs.In summary,the fluorescence multiplex PCR-CE assay presented has become a popular tool for MSI analysis due to its relatively low PCR product requirements(high detection sensitivity),highly accurate sizing capability(resolution of 1 base or less), relatively high throughput,analysis,storage,management of MSI data and an automated format that requires minimal user intervention.We have developed a clinical molecular diagnostic assay for MSI analysis studies from formalin-fixed tissue samples by a modified procedure for DNA extraction and multiplex assay.The accumulation of MSI may be an important molecular event in the development of sporadic colorectal cancer,which contributes probably to the multistep carcinogenesis of sporadic colorectal cancer.