The Research Of Mismatch Repair System And Microsatellite Status In Sporadic Colorectal Cancer And Long-term Prognosis

Colorectal cancer is a malignant tumor with high morbidity and mortaity.According to the "World Cancer Report" published by WHO in 2014,the incidence of colorectal cancer was the third place in all cancers,and the death rate was fourth.Colorectal cancer is divided into hereditary and sporadic characteristics according to the hereditary characteristics,with sporadic 85%of all colorectal cancers.There are three main types of gene changes:up-regulated expression or abnormal activation of tumor genes,up-regulated expression or loss of function of tumor suppressor genes,and structural abnormality or dysfunction of DNA repair genes.DNA repair system includes mismatch repair(MMR),base excision repair(BER)and nucleotide excision repair(NER),in which MMR is the main way to repair.The mismatch repair gene system is made up of highly conserved genome and its expressed protein products.It has the function of correcting the base pairing errors in DNA replication,recombination and gene damage repair.For example,if the base pairing error of oncogene occurs,it may lead to gene activation and over-expression,resulting in cell uncontrolled proliferation.If the mutation of tumor suppressor gene occurs,it may lead to the loss of inhibition of oncogene.The above situation can lead to the occurrence of malignant tumor.Previous studies have confirmed that the mutation of the mismatch repair gene system is both significantly correlated with the genetic basis of Hereditary non-polyposis colorectal cancer(HNPCC)and many other kinds of malignancies,such as endometrial cancer,breast cancer,esophageal cancer,gastric cancer,pancreatic cancer,lung cancer,prostate cancer and so on.About 15%of the patients with sporadic colorectal cancer have the same pathogenesis as HNPCC,and these patients have clinical pathological features and prognostic trends similar to lynch syndrome,which makes it important to screen out this part of patients with sporadic colorectal cancer and make individual diagnosis and treatment.In this study,the expression level of mismatch repair genes in sporadic colorectal cancer was detected by immunohistochemistry(IHC),and the correlation with prognosis was analyzed by long-term individualized follow-up.At the same time,this study also studied the conformance of mismatch repair gene protein expression and microsatellite status in sporadic colorectal cancer.Microsatellite sequences are abundant throughout the genome;they are polymorphic among individuals but are unique and uniform in length in every tissue in each person.Due to the lack of mismatch repair gene function,the base mismatch in DNA complex process can not be found and corrected,resulting in the change of microsatellite sequence length.This state is called microsatellite instability(MSI).Microsatellite instability exists widely in hereditary colorectal cancer,and it is also found in 10-15%patients with sporadic colorectal cancer.Microsatellite instability is associated with genetic defects in the mismatch repair,but some studies have shown that the two results of tests are not exactly the same.This study for mismatch repair system for sporadic colorectal cancer patients and microsatellite stability consistency ratio,aimed to study the feasibility of sequential screening,so as to improve the accuracy of diagnosis,reduce medical costs,prompt clinical prognosis.Part 1:Analysis of The Expression Level and Prognosis of Mismatch Repair Genes in Sporadic Colorectal CancerPurpose:Objective to detect the expression level of mismatch repair genes(MMR)including hMLH1,hMSH2,hMSH6 and hPMS2 in sporadic colorectal cancer,and to explore the correlation with prognosis.Methods and Materials:1.Research Object:Patients archives from the Department of Gastrointestinal Surgery of Weihai Municipal Hospital were retrospectively collected between January 2011 and January 2012,who underwent radical surgical treatment of colorectal cancer.Of the 221 consecutive patients identified,205 patients who met the criterion were deemed eligible for inclusion.The 16 cases of excluded patients included 1 cases of familial polyposis excluded,4 cases of hereditary nonpolyposis colorectal cancer,1 case died in postoperative cardiopulmonary cneoplasm omplications,1 cases of recurrent colorectal cancer,1 case of neuroendocrine confirmed by postoperative pathology,8 cases treated with neoadjuvant chemotherapy.There were 13 cases refusing to cooperate for personal reasons.So 192 cases were effectively enrolled in the study group.2.Demographic Characteristics of Sample Population:the age distribution 33-89 years old;the average age 63.2 + 10.8 years old;the ratio of male to female about 1.4:1;124 cases(64.6%)? 60 years old;68 cases(35.4%)<60 years old;male 112 cases(58.3%),the average age 63.5 ± 10.8 years old;female 80 cases(41.7%),the average age 62.9 ± 11.0 years old.Tumour Location:40 cases(20.8%)of ascending colon cancer(including hepatic flexure carcinoma),44 cases(22.9%)of colon cancer(including splenic flexure and sigmoid colon cancer),108 cases of rectal cancer(56.3%),and 5 cases(2.6%)of colorectal cancer with liver metastasis.Degree of differentiation:higher differentiation and moderate differentiation 139 cases(72.4%),low differentiation 53 cases(27.6%).TNM staging according to the NCCN Guide:T1+T2 42cases(21.9%),T3+T4 150 cases(78.1%).Lymphatic metastasis:pN0 121 cases(63%),pN1 45 cases(23.4%),pN2 26 cases(13.6%).Positive forvascular tumor thrombus 63 cases(32.8%),negative 129 cases(67.2%).3.Immunohistochemistry(IHC):The tumor samples were collected and the expression of mismatch repair genes were detected by IHC,including MLH1,MSH2,MSH6 and PMS2.The samples with>10%of tumor cells stained for any MMR protein were considered to be MMR-positive.The criteria used for semi-quantification of immunohistochemical staining included the stainin-gintensity and the percentage of positively stained cells.A range of 0-3 was defined for classifying the intensity of staining:0,Absence of staining;1,weak staining;2,moderate staining;and 3,intense staining.Furthermore,extent of staining was scored as 0(? 10%),1(11-25%),2(26-50%),3(51-75%),and 4(76-100%)for evaluation.The final scores were calculated by multiplying the staining intensity by the extension.In the present study,all the final scores were stratified as negative MMR expression(0 score)or positive MMR expression(>0).The MMR-positive group included low expression(1-4score),moderate expression(5-8 score)and high expression(9-12 score).All pathological sections were analyzed and defined by at least two experienced pathologists in the department of pathology of Weihai Municipal Hospital.The four kinds of detected antibodies showed that the expression of the mismatch repair gene was proficient(pMMR).Deficient mismatch repair(dMMR)is a mismatch repair gene expression defect in any of the tumor cells with one or more than one antibody negative staining.4.Follow-up:All patients were followed up for a long time(at least 48 months of follow-up)and the correlation between the expression level and the prognosis was analyzed.The follow-up methods included telephone foll-ow-up,home visit follow-up,outpatient follow-up,and hospitalization.Followed by the end of January 31,2016,follow-up data including patient survival time,tumor recurrence or metastasis time after operation,whether undergoing chemotherapy,radiotherapy,biological therapy and/or immunotherapy adjuvant therapy,the reason of death including colorectal cancer directly leading to death or other diseases and accident events.The time of overall survival(OS)is defined as the time from operation to patient death.If when the follow-up ended,the patients still alive,the total survival time was calculated from the date of the operation to the last follow-up day.The time of free disease survival(DFS)is defined as the total time from operation day to the date of recurrence or metastasis of colorectal tumor or the last follow-up day.The determination of tumor recurrence and metastasis should be supported by clinical,imaging tests,histological or pathological examination.5.Statistical Analysis:All data are presented as the mean ± standard deviation(SD)or the median± SD by SPSS 18.0 software(SPSS Inc.,Chicago,IL,USA)was used for statistical analysis.Fisher's exact test and X2 test were used to evaluate clinicopathological significance of enrolled patients' characteristics in SCRC.The Kaplan-Meier method and log rank test were used to calculate survival data.The Cox regression tests were used for independent prognosis factor analysis.Two-sided P-values were calculated,and P<0.05 was considered to indicate a statistically significant difference.Result1.In the study,the total deletion rate of mismatch repair genes in colorectal cancer patients was 14.58%(28/192),of which MSH6 0.52%(1/192);PMS2 4.17%(8/192);MSH2/MSH6 3.65%(7/192)and MLH1/PMS2 6.25%(12/192).2.Contrast the clinicopathological features of two groups of patients,dMMR group in the right-side colon incidence rate and low differentiation,lymph node metastasis rate were significantly higher than the pMMR group(P<0.05),while on the sex,age and vascular invasion of the two groups,there is no obvious difference(P>0.05).3.The disease-free survival rate(DFS)of sporadic colorectal cancer patients with mismatch repair gene deletion(dMMR)was 37.64 ± 11.98 months and the overall survival rate(OS)was 43.29 ±9.39 months.The DFS of mismatch repair gene deletion(pMMR)was 30.72 ± 14.11 months and OS was 37.74 ± 11.34 months.The difference was statistically significant.(P(OS)=0.017 and P(DFS)=0.027)Conclusion1.Mismatch repair gene deletion not only exists in the hereditary colorectal cancer,but has a certain proportion in the sporadic colorectal cancer.2.Deletion of mismatch repair genes is associated with clinicopathological features and biological behavior in patients with sporadic colorectal cancer,such as high incidence of poor differentiation,lymph node metastasis and occourence in right colon.3.In the group of sporadic colorectal cancer,the prognosis of the patients with dMMR is better than that of the patients with pMMR.Part 2:Analysis of The Conformance of Mismatch Repair Gene Expression and Microsatellite StatusPurpose:Through different detection methods for mismatch repair system and microsatellite sequences in sporadic colorectal cancer patients,aim to prove the consistency between the two,to evaluate the reasonableness of the IHC method for MMR to replace the PCR method for MSI.Metheods and Materials:1.Research Object and Demographic Characteristics of Sample Population are the same as Part One.2.PCR:According to the U.S.National Cancer Institute(NCI)recommendation,five standard sites of BAT25,BAT26,D2S123,D5S346 and D17S250 were detected,and in according to the degree of instability of detected markers,microsatellite instability tumor were divided into MSI-H(2 or more sites instability)and MSI-L(1 site instability),and no site is classified as microsatellite stable(MSS).3.Statistical Analysis and Follow-up are the same as Part 1.Result1.According to the measurement method and standard of the aforementioned microsatellite,the consistency between MSI-H and dMMR expression:31 cases were detected by MSI-H group,1 of which was corrected to MSI-L by dMMR,and 4 cases of pMMR corrected to MSI-H,with a specificity of 99.4%and sensitivity of 90.0%.2.Contrast the clinicopathological features of two groups of patients,MSI-H group in the right-side colon incidence rate and low differentiation,lymph node metastasis rate were significantly higher than the MSS and MSI-L group(P<0.05),while on the sex,age and vascular invasion of the two groups,there is no obvious difference(P>0.05).3.Survival Analysis:The disease-free survival time(DFS)of the MSI-H group was 37.18 ± 12.42 months,while the total survival time(OS)was 42.79 ± 9.63 months.The disease-free survival time(DFS)of MSS group was 31.82 ± 14.26 months,while the total survival time(OS)was 38.08 ± 11.60 months.The disease-free survival time(DFS)of the MSI-L group was 29.44 ± 13.88,while the total survival time(OS)was 38.08 ± 11.60 months.The disease-free survival time(DFS)of the MSI-L/MSS group was 30.54 ±4.06,while the total survival time(OS)was 37.65±11.37 months.The log-rank method was used to analyze the data of three groups of patients and found that both DFS and OS of MSI-H group were better than MSI-L group(P<0.05).Patients with MSI-H presented with longer OS times compared with those with MSS(P<0.05),but the difference of DFS between the two group was not significant(P>0.05).There was no significant difference between MSS group and MSI-L group in DFS and OS(P>0.05).The MSS group and MSI-L group were combined and then compared with the MSI-H group,both DFS and OS in msi-h group were better than the former(P<0.05).Conclusion1.The IHC of MMR and PCR of MSI are not identical in detection rate,but can be used as a consistency detection method in biological efficiency.2.In the clinical work,IHC which has the advantages of convenience and low price is can be used for the initial screening of MMR with MSI complementary to improve the efficiency and reduce the cost.