Study On The Relationship Between MSI And KRAS Gene Mutations In Colorectal Cancer And The Effect Of Neoadjuvant Therapy On Tumor Immune Microenvironment

In recent years,the treatment of colorectal cancer has made great progress,especially the use of targeted drugs has significantly improved the survival of patients with advanced colorectal cancer,but even the use of targeted drugs in the process of drug resistance will also occur,How to further treat colorectal cancer patients who have received multiline therapy remains a challenge for medicine.Since 2015,when PD-1 inhibitors were reported to be effective in the treatment of advanced colorectal cancer patients with MSI,Immunotherapy for colorectal cancer and search for appropriate immune markers has become a hot issue.At present,the role of microsatellite instability in colorectal cancer and the relationship between microsatellite instability and related factors are more studied.It is believed that microsatellite instability pathway is an important pathway in the development of colorectal cancer,and that microsatellite instability plays an important role in the pathogenesis and development of colorectal cancer.Microsatellite instability can lead to a large number of gene mutations in tumor cells,and then form a large number of new antigens,increase the immune recognition and immune response of the body,so as to suppress the purpose of tumor.Tumor immune microenvironment plays an important role in anti-tumor,whether there is interaction between microsatellite instability and tumor immune microenvironment,and whether tumor immune microenvironment will change in the course of tumor treatment,and how to play a role.Wait,need us to carry on in-depth research.Part one Expression of MSI and KRAS gene mutations in colorectal cancer and their correlationObjective: To investigate the expression of MSI and KRAS gene mutations in colorectal cancer and explore the correlation between them.Methods: The clinical and pathological data of 731 patients with colorectal cancer operated in the fourth Hospital of Hebei Medical University from 2015.2 to 2016.12 were retrospectively analyzed.The clinical and pathological information of the patients were collected,the results of KRAS gene detection after operation,the expression of MMR protein(MLH1,MSH2,MSH6 and PMS2)were detected by immunohistochemical method,and the data were collected.The data were analyzed by SPSS 20 statistical software.Results:1.Status of MSI in sporadic CRC patientsThe expression of MMR protein was analyzed by immunohistochemistry in 731 cases.According to the results of MSI typing,the results showed that 68 cases were MSI-H,the incidence rate was 9.3%,and the incidence rate was 1.3%.MSS was found in 654 cases(89.4%).2.Mutation of KRAS gene in patients with CRCAmong 731 patients with KRAS gene detected by PCR,291 cases(39.2%)had KRAS gene mutation,223 cases(30.0%)had mutation in codon 12,and the most common mutation was p.G12 D mutation in codon 12(109cases),the mutation rate of codon 12 was 30.0%,and the mutation rate of codon 12 was 30.0%.The mutation rate was 14.7%,followed by p.G13 V with 12 codon.69 cases(9.3%)had mutation.3.There were significant differences between MSI and MSS in tumor location,age of onset,degree of histological differentiation,nerve invasion and clinical stage(P < 0.05).In the MSI group,the age of onset was lower,and the proportion of right colon was higher than that of the control group(P < 0.05),but there was no significant difference between the two groups(P < 0.05).Most of the tissues were poorly differentiated or mucinous adenocarcinoma with less lymph node metastasis.However,there was no significant difference in tumor invasion depth,vessel tumor thrombus,distant metastasis and sex.4.Correlation between KRAS gene and clinicopathological features of sporadic CRC The results showed that there were significant differences in sex and pathological type of tumor between KRAS gene mutation and wild type patients(P< 0.05).The expression of KRAS gene mutation was more common in women than in poorly differentiated and mucinous adenocarcinoma patients(P <0.05).There was no significant difference in age,primary location,degree of differentiation,depth of invasion,vessel tumor thrombus,nerve invasion,lymph node metastasis and distant metastasis between the two groups(P > 0.05).5.Analysis of association between 5 KRAS gene mutation and MSI typing in CRCKRAS gene mutation occurred in 40.1% of MSS patients and 30.8% of KRAS gene mutations in 21 of them.There was no significant difference between the two groups(P < 0.05).We analyzed the mutation of different codon in MSI and MSS groups,and found that the mutation ratio of 12,13 codon was different,the difference was statistically significant(P < 0.05).Summary:1.The incidence of MSI-H in sporadic CRC was closely related to the age of onset,tumor location,differentiation degree,TNM stage and lymph node metastasis.2.The mutation rate of KRAS in sporadic CRC was 39.2%.The most common mutation sites are codon 12 and 13.KRAs mutation is only related to sex and pathological type of tumor.3.There was no correlation between MSI status and KRAS gene mutations in colorectal cancer.Part two: Effects of MSI status on immune microenvironment of colorectal cancerObjective:To study the expression of CD4+T cells,CD8+T cells,CD34 and PD-L1 molecules in different colorectal cancer tissues,and to explore the relationship between MSI and immune cells.Methods: 65 patients with MSI-H and 65 patients with MSS were selected.Immunohistochemical method was used to detect the expression of CD4+T cells,CD8+T cells,CD34 and PD-L1 in tumor tissues.SPSS 20.0 statistical software was used for analysis.Results:1.The expression of CD8+T cells in 37 patients with CRC was 28.4% and 93 cases of low expression(71.6%).The expression of CD8+T cells in the patients with MSI was significantly higher than that in the MSS group(P <0.05)..2.The expression of CD4 T cells was high in 37 cases(54.6%)of CRC patients in MSI group and 34 cases(54.6%)in CRC patients in MSS group.There was no significant difference in the proportion of 52.3%.CD4 T cells expression between MSI group and MSS group(P> 0.05).3.The expression of CD34 was high in MSI group and MSS group,but there was no difference between the two groups(P > 0.05).4.There was no significant difference in the expression of PD-L1 between MSI group and MSS group(P> 0.05).Summary:1.The infiltration of CD4+T cells in MSI and MSS groups did not change,but the expression of CD8+T cells in MSI group increased significantly,suggesting that MSI may produce more antigen expression,and CD8+T cells are the main immune effector cells.2.CD34 was highly expressed in both MSI and MSS groups,but there was no difference between the two groups,suggesting a higher vascular density in tumor tissues.However,the tumor MSI has no effect on the proliferation of blood vessels.3.The low expression of PD-L1 in colorectal cancer cells suggests that other mechanisms are more important in immune escape mechanism of colorectal tumors.Part three Changes of immune microenvironment in tumor tissue before and after neoadjuvant radiotherapy and chemotherapy for rectal cancerObjective: To investigate the changes of CD4+T,CD8+T cell and the density of TIL in tumor tissue before and after neoadjuvant therapy for rectal cancer,and to find out the immune markers sensitive to neoadjuvant radiotherapy and chemotherapy before operation.Methods: 52 patients with eligible rectal cancer were screened out by database.The changes of CD4+T,CD8+T and CD4+T / CD8+T ratio before and after tumor tissue were evaluated by HE staining and immunohistochemistry.SPSS 20 statistical software was used to analyze the changes.Results:1.After NCRT,the staging rate of T3 and T4 in the patients with rectal cancer was 46.2%,and that of N+ to N0 was 40.4%.2.The infiltration of CD4+T cells: the number of CD4+T cells increased in 2 cases of TIL grade 1 colorectal cancer,1 case of TIL2 grade and 3 cases of TIL3 grade colorectal cancer after radiotherapy.There was no significant difference in TIL grade of CD4+T cells before and after radiotherapy.CD8+T cell infiltration: the number of CD8+T cells decreased in 10 cases of TIL grade 1 colorectal cancer after radiotherapy,increased in 6 cases of TIL2 grade and increased in 4 cases of TIL3 grade colorectal cancer after radiotherapy.The TIL grade of CD8+T cells after radiotherapy was significantly lower than that of CD8 T cells before radiotherapy.Difference(P < 0.05).3.After radiotherapy,the CD4+T / CD8+T cell ratio≤increased in 9 patients and decreased in 9 patients with ratio > 1.The ratio of CD4+T / CD+T cells in rectal cancer tissue after radiotherapy was significantly lower than that before radiotherapy P< 0.05).4.After radiotherapy,the CD4+/ CD8+T cell ratio≤1 increased in 9 patients and decreased in 9 patients with ratio > 1.The ratio of CD4+ / CD8 +T cells in rectal cancer tissue after radiotherapy was significantly lower than that before radiotherapy(P< 0.05).4.There was no significant difference between the density of CD4+T TIL in tumor tissue before radiotherapy and the local curative effect of tumor tissue(P > 0.05).Summary:1.Patients with rectal cancer were treated with pre-operative NCRT,and the rate of T staging was 46.2% in the MRI and 40.4% in the N stage,and it was suggested that the pre-operative NCRT treatment could reduce the clinical stage and improve the surgical treatment effect.2.The ratio of CD4+/ CD8+ T cells to CD4+/ CD8+T cells may be related to the decrease of CD4+/ CD8+T cell ratio after radiotherapy..3.The higher the TIL grade of CD8+T cells is,the higher the tumor retraction rate is.The TIL grade of CD8+T cells may be a predictor of radiotherapy sensitivity.Conclusion:1.There is no correlation between MSI and KRAS genes in colorectal cancer.They play an important role in the pathogenesis and development of colorectal cancer.2.The increase of CD+T cells in MSI colorectal cancer indicates that CD8+T cells infiltrate more in MSI colorectal cancer,and its anti-tumor immune effect is stronger.3.The low expression of PD-L1 in colorectal cancer suggests that other escape mechanisms may be more important in the immune escape mechanism of colorectal cancer.4.The more TIL in rectal cancer tissues,the better the effect of radiotherapy and chemotherapy,and the more infiltration of CD8+T cells after radiotherapy and chemotherapy.It is suggested that TIL may be a predictor of preoperative radiotherapy and chemotherapy,and CD8+T cells are the main effector cells.