Study On The Characteristics Of Immunoglobulin Gene Sequences Of Tumor Infiltrating B Lymphocytes In Colon Cancer

ObjectiveColon cancer is one of the common gastrointestinal tumors,which seriously threatens people's health.About 101,420 new colon cancer cases and 51,020 deaths from colon cancer,the third leading cause of cancer-related deaths in both men and women,are expected in the United States in 2019,according to new cancer data released by the American Cancer Society in 2019.This is consistent with the latest available statistics showing that colorectal cancer was the third leading cause of cancer death in 2016.Colon cancer is a highly treatable disease.It is often curable when it is confined to the gut.Surgery remains the primary treatment and cures about 50%of patients.Postoperative recurrence is an important problem and often the final cause of death.Colon cancer can be simply divided into two molecular subtypes according to microsatellite instability:high level microsatellite instability(MSI-H)and microsatellite stability(MSS).MSI-H phenotype is present in 15%of early colorectal cancer with a good prognosis.MSI-H colon cancer has obvious histological characteristics.And MSI-H colon cancer also contains an increased number of tumor-infiltrating lymphocytes(TILs).Existing research shows that the dense infiltration of tumor infiltrating B lymphocytes(TIL-B),one of the important components of TILs,is related to improving the prognosis of patients with colon cancer.This is consistent with previous studies on breast cancer,ovarian cancer and other cancers,but the specific immune mechanism of TIL-B in the generation and development of colon cancer is still unclear.The purpose of our study is to explore the gene sequence characteristics of TIL-B immunoglobulin in colon cancer,and further analyze the differences between the gene sequences of MSI-H colon cancer patients and MSS colon cancer patients,so as to provide a feasible theoretical basis for the research on new immunotherapy targets for colon cancer.MethodsFresh tumor tissue from patients undergoing radical resection of colon cancer in Qilu Hospital of Shandong University was collected from November 30,2017 to January 3,2018.Take them to join ribonuclease(RNase)preservation solution,and put in storage in a freezer-80 ?,for later use.After confirmed by pathological results and immunohistochemistry,four MSI-H patients and four MSS patients were selected from the above patients.Age,gender,pathological type and TNM stage of the two groups of patients were matched as far as possible.Quickly grind up the tumor tissue,the extract of ribonucleic acid(RNA),and reverse transcription into complementary deoxyribonucleic acid(cDNA).Amplify heavy chain genes by nest polymerase chain reaction(Nest PCR),using primers of specific sequences of immunoglobulin heavy chains which are designed in advance.Agarose gel electrophoresis was used to detect DNA bands and recover the amplified DNA products.It was linked to the vector and then transformed into DH-5 alpha susceptible Escherichia Coli for further amplification.Monoclonal colonies were screened by ampicillin.50 to 100 colonies were selected from each patient and cultured in LB broth to reproduce Escherichia Coli.Extract DNA and test sequences.Peripheral blood of 2 normal adults was collected at the same time,mononuclear cells were isolated,and RNA was extracted for the above operation as the control.The frequency of use of VDJ genes,the length of amino acids which were encoded by complementarity determining region 3(CDR3),the positive and negative charge numbers of amino acids,the polarity of amino acids and the frequency of somatic hypermutation were analyzed by IMGT and other bioinformatics software.GraphPad Prism 8 statistical mapping software was applied to make charts.Results1.Patient MSI-H1 was expressed as lack of MLH1 and PMS2,while Patients MSI-H2 and MSI-H3 were both expressed as lack of MSH2 and MSH6,and Patient MSI-H4 was only expressed as lack of MSH6.Patients MSI-H2 and MSI-H4 received postoperative adjuvant chemotherapy.Patient MSI-H4 were found to have multiple metastases at reexamination 5 months after surgery,and died 9 months after surgery.The patient MSS-2 was suspected to have metastatic lung adenocarcinoma after reexamination 5 months after surgery,and his condition was stable after chemotherapy.Now she is under regular reexamination.No recurrence or metastasis was found in other patients.2.The average percentage of positive B-cell gene sequences in normal people's peripheral blood is about 86.99%,that is to say,about 86.99%of the submitted samples showed the rearrangement of V,D and J sequences in the complementarity determining region(CDR)of the heavy chain coding genes of immunoglobulin in peripheral blood B cells.The average percentage of effective gene sequences in MSI-H group was about 90.62%,which was close to that of normal people.The MSS group was only 75.93%,significantly lower than the control group.This suggests that TIL-B in patients with MSI-H may be a mutation induced by antigen stimulation,while MSS may not be induced by antigen.Results of TIL-B immunoglobulin sequencing in 8 patients with colon cancer showed no repetitive clone sequence.3.The frequency of VDJ usage in MSI-H and MSS groups was similar to that of normal pernpheral blood B cells,and the frequency of IGHV3 usage has a clear advantage,higher than 50%,which is same in the three groups.IGHD3 was the most frequently used gene fragment in segment D,and IGHJ2 in the MSI-H group was more frequently used than the other two groups,11.11%vs.3.82%,5.40%.4.Our analysis of the length of amino acids encoded by the CDR3 genes of immunoglobulin heavy chain showed that the ratio of amino acids length?16 in the MSI-H group was higher than that in the other two groups.In particular,when the amino acid length was>20,the proportion in the MSI-H group was 13.82%,the MSS group was 3.95%,and the normal group was 6.52%.But the ratio of amino acids length?15 was higher in MSS group.Further analysis of the charge of amino acids showed that the proportion of positively charged amino acids in the MSI-H group was higher than that in the other two groups,9.42%vs.5.09%and 5.44%.The data of polarity and nonpolarity of amino acids showed no significant difference between the three groups.5.Analysis of mutation frequency of variable region genes:The mutation frequencies of FW1,FW2,and FW3 were similar between the MSI-H group,the MSS group and the normal group,that is,no significant difference was found between them,suggesting that the FW genes in the MSI-H group,MSS group and the normal group were relatively stable.The mutation frequencies of CDR1,CDR2,and CDR3 in the three groups were significantly different.The frequencies of silent mutation(S-mutation)and replacement mutation(R-mutation)in the MSI-H group were increased compared with the normal group,and the frequencies of R and S mutation in the MSS group were decreased compared with the normal group.The R/S mutation rate of the three gene fragments of CDR1,CDR2,and CDR3 was increased in the MSI-H group compared with the normal group.In the MSI-H group,the mutation frequency with the practical significance of changing protein structure was higher.This suggests that the mutation may have been antigen-induced rather than random.Compared with the normal group,the R/S mutation ratio of the three gene fragments of CDR1,CDR2,and CDR3 were all decreased in the MSS group.It cannot be speculated that the MSS gene mutation is induced by antigen.ConclusionOur results showed that compared with the MSS group and the normal group,the length of amino acids encoded by CDR 3 in the MSI-H group was longer and amino acids had more positive charges.The immunoglobulin encoded by the MSI-H group may have higher autoimmunity,and the anti-tumor immunity effect was stronger than that in the MSS group.Patients in the MSI-H group had higher somatic hypermutation loads,and the somatic hypermutation in the CDR region was significantly more than that in the FR region,and the R/S mutation rate was significantly higher than that in the other two groups,suggesting that TIL-B patients in the MSI-H group experienced antigen stimulation and positive selection.This suggests that specificity of b-cell may have important immune function in colon cancer,providing a new direction for tumor immunotherapy.