| Background and purpose: Gastric cancer(GC)is an important global health problem,especially in China’s mainland,where the morbidity and mortality are much higher than other countries,accounting for nearly 50% of the global morbidity.Metastasis has always been considered to be one of the main factors of postoperative recurrence and death of GC patients,and accurate GC staging is of great significance for guiding clinical treatment and prognosis.Therefore,the American Joint Committee on Cancer(AJCC)proposed a tumor-node-metastasis(TNM)clinical staging and prognostic stratification system,which is also the most commonly used prognostic stratification method in clinical practice.Because the current N staging of AJCC is only based on the number of lymph nodes,the technique of the surgeon,the experience of the pathologist,and other unavoidable circumstances affect the number of lymph nodes examined.As a result,10-25% of GC patients are classified incorrectly.Lymph node ratio(LNR)is the ratio of the number of metastatic regional lymph nodes to the number of lymph nodes examined.LNR has recently been reported to be associated with the prognosis of many tumors including GC.The LNR-based GC staging system has been proven to be better at predicting survival than the seventh version of the AJCC staging system.However,whether LNR can provide more prognostic prediction information for the eighth edition of the AJCC staging system has not yet been clarified.In recent years,with the emergence of tumor immunotherapy and checkpoint inhibitors,the field of oncology has undergone revolutionary changes.Tumor infiltrating lymphocytes(TILs)are monitoring indicators of tumor continuous immunity.Patients with higher levels of TILs usually have a better prognosis.Tumor immunotherapy has always been based on this knowledge to adjust T cell strategies,especially the so-called immune checkpoint blockade and adoptive T cell therapy.At present,the method of using monoclonal antibodies to block immune regulation pathways to enhance tumor-specific immune response has been successfully applied to tumor treatment.Studies have found that about one-third of GC patients have chromosomal instability and abnormal activation of human epithelial growth factor receptor-2(HER-2).At present,anti-HER-2 antibody trastuzumab combined with cytotoxic drug chemotherapy has become the first-line treatment recommended by the guidelines in GC patients with HER-2 amplification or HER-2 protein overexpression.Although the use of trastuzumab prolongs the survival of patients with HER-2 positive GC,the five-year survival rate is still low.Therefore,it is necessary to continue to seek better treatments in order to prolong the survival period of GC patients.It has recently been discovered that in HER-2 positive patients with advanced GC,the combined use of anti-PD-1 antibody pembrolizumab,anti-HER-2 trastuzumab and chemotherapy is more effective.However,the synergistic mechanism of anti-HER-2 therapy and immunotherapy is not yet clear.This article intends to explore the characteristics of the immune microenvironment and immune escape mechanism of HER-2 positive GC,and then provide a theoretical basis for the further development and optimization of anti-HER-2 therapy and immunotherapy combined treatment strategies.T cell co-suppressive molecules belonging to the B7 family in the tumor microenvironment have been considered as the main immunosuppressive mechanism of many solid tumors.Antibodies that inhibit the PD-1/PD-L1 pathway have produced long-lasting clinical responses in various solid malignancies,but it seems to be only beneficial to some patients with GC.Studies have found that most of the immune checkpoints are down-regulated in HER-2 positive GC,which may be related to the down-regulation of immune infiltration in HER-2 positive GC,while the immune checkpoint VTCN1,which mediates inhibitory signals,is up-regulated,suggesting that VTCN1 may be Involved in mediating the phenotype of cold tumors with HER-2 positive GC.VTCN1 belongs to the B7 family and is also a T cell co-suppressor molecule.Its overexpression in immune cells may reduce the activity or number of TILs through various mechanisms to block the body’s immune response to tumors.So far,studies have shown that the expression of VTCN1 is related to many clinicopathological parameters,such as tumor size,primary tumor classification,TNM malignant tumor score and overall survival rate.VTCN1 is also directly related to the increase of tumor burden,the formation of new blood vessels and the poor prognosis of patients.VTCN1 plays an important role as a negative regulator of immune response in patients with malignant diseases.However,few reports reveal the relationship between the expression of VTCN1 in primary tumors and the nature of tumors in GC patients.Recently,studies have reported that cancer therapy with anti-PD-L1 antibodies can prolong the lifespan of certain types of cancer,and not all cancer patients will respond to these drugs.PD-L1 is only expressed in certain human cancers,while VTCN1 is expressed in a wider range of tumors.Therefore,considering that VTCN1 is more widely expressed in human cancer than PD-L1 and the mechanism by which VTCN1 regulates tumorigenesis and development,we suspect that VTCN1 may be used as a potential cancer treatment target.In this study,we used the Surveillance,Epidemiology,and End Results(SEER)database and the GC population cohort data of the Lanzhou University Second Hospital to evaluate the value of LNR for the eighth edition of GC AJCC staging.At the same time,we have established a new LNR-based AJCC(r AJCC)staging system.Then,we conducted a differential analysis of the GC cohort data in The Cancer Genome Atlas(TCGA)to explore the characteristics of the immune microenvironment and immune escape mechanisms of HER-2 positive GC,and further develop and optimize anti-HER-2 the combined treatment strategy of treatment and immunotherapy provides a theoretical basis.Finally,we evaluated the expression of immune checkpoint VTCN1 in GC,and explored the relationship between VTCN1 expression and clinicopathological characteristics and prognosis of GC patients.In addition,animal experiments and other experiments are conducted to explore the important mechanism of VTCN1 in the process of GC.It hopes to provide a theoretical basis for the immunotherapy of HER-2 positive GC and to find new potential cancer treatment targets.Materials and Methods: 1.We collected data from the SEER database of patients diagnosed with non-metastatic GC who underwent surgical resection from 2004 to 2013.At the same time,the data of patients who were diagnosed with non-metastatic GC and were performed D2 lymph node dissection from December 2012 to July 2014 in Lanzhou University Second Hospital were collected.The Kalpan-Meier method was used to compare the overall survival(OS)of patients according to the eighth edition of the AJCC staging system.Multivariate Cox regression was used to analyze the association between staging systems.After adjusting the baseline covariates including age,gender,race,diagnosis year,marital status,SEER area,tumor location,tumor size,and tumor grade,hazard ratios(HRs)were calculated.The recursive segmentation method was used to determine the optimal critical point of LNR,and the rN staging based on LNR was designed,and the corresponding rN staging replaced the eighth edition AJCC N staging.The patients included in the study were regrouped according to the improved staging system,and the consistency index was used to compare the two staging systems.The chi-square test was used to evaluate the homogeneity of the prognosis,and the stratified survival analysis was used to evaluate the effect of rN staging and each N staging of the eighth edition of AJCC on the prognosis.2.We used the transcriptome data of GC patients in the TCGA database to analyze the differential expression of HER-2 positive vs.negative groups to find differentially expressed genes.Furthermore,based on the results of differential expression analysis,differentially expressed genes were selected according to the fold difference of the expression of HER-2 positive vs.HER-2 negative group and the P value of differential expression analysis corrected by Benjamini-Hochberg method.According to the multiple differences in expression levels between the two groups,the genes were arranged from low to high,and then gene enrichment analysis(GSEA)was performed.Based on the P value of the differential expression analysis corrected by the Benjamini-Hochberg method,the selection was made The HER-2 positive group significantly up-regulated/down-regulated pathways.Then,we used the single-sample GSEA method based on the CD8+ T cell label identified by Bindea et al.to quantitatively compare the abundance of CD8+ T cells in the HER-2 positive vs.negative group on the transcriptome data of GC patients,and screened the genes for potential immunochemokines that affect the infiltration of CD8 T cells.Finally,we used the gene list of common immune checkpoints compiled by Thorsson et al.to perform an integrated analysis with the previously screened differential expression analysis results to screen for those that are significantly up-regulated in HER-2 positive GC and mediate inhibitory signals,and immune checkpoints that have the potential to serve as targets for intervention. 3.We collected the clinicopathological characteristics of 80 eligible patients with GC from January 2015 to January 2016 in the Third Ward of General Surgery of the Lanzhou University Second Hospital and their corresponding GC tissues and paracarcinoma paraffin specimens.We evaluated the expression of VTCN1 in GC tissues and adjacent tissues of cancer and its relationship with the clinicopathological characteristics of GC.We used quantitative real-time PCR(q RT-PCR)experiments to detect VTCN1 m RNA expression in GC cell lines(MKN-45,HGC-27,KATO-Ⅲ and MGC-803)and human gastric mucosal epithelial cell line GES-1.We prepared sh RNA targeting human VTCN1 gene(sh-VTCN1)and negative control sh RNA(sh-NC)and screened effective targets and transfected MGC-803 and KATO-Ⅲ cells with effective sh-VTCN1 or sh-NC.We first detected the effect of VTCN1 inhibition on the proliferation of MGC-803 and KATO-Ⅲ cells through CCK-8 cell proliferation experiments,cell clone formation experiments and Ed U cell proliferation experiments.Then the cell scratch test,Transwell migration and invasion test were used to detect the effect of VTCN1 inhibition on the migration of MGC-803 and KATO-Ⅲ cells.All experiments were repeated three times.Immunohistochemistry(IHC)was used to detect the expression of VTCN1 in tissues.We used Western Blot(WB)experiments to check the expression of related proteins in cells or tissues.In addition,a nude mouse subcutaneous xenograft model was established to evaluate the growth and metastasis ability of VTCN1 expression on GC cells in vivo.Finally,we also used WB experiments to explore the relationship between VTCN1 and Epithelial-Mesenchymal Transition(EMT)and the AKT/PI3K/m TOR signaling pathway involved.Results: 1.The 5-year OS of GC patients in the SEER database was 42.2%(95% CI=41.2-43.2%),and the median LNR value was 0.1(interquartile range 0-0.4).The LNR cut-off points identified by the recursive segmentation method are 0.03,0.08,0.3 and 0.7.According to the definition of the LNR cut-off point,5 rN stagings were obtained.Among them,the corresponding 5-year OS of rN0,rN1,rN2,rN3 a,and rN3 b were 64.4%,54.1%,37.9%,19.1%,and 5.9%,respectively(P<0.01).The prognosis of each rN stage is significantly different(P<0.001 for all pairwise comparisons).The rN staging and the r AJCC staging system based on this regrouping have better discriminative ability and prognostic homogeneity than the eighth edition AJCC staging.A total of 12,037 patients with stage ⅢA(8th edition AJCC)were enrolled,and were further divided into five subgroups: r IIA,r IIB,r ⅢA,r ⅢB,and r ⅢC according to the proposed r AJCC staging.There was a difference in 5-year OS between patients classified as r IIA stage and those classified as r ⅢC stage(66.7% vs 5.1%).The 5-year OS of GC patients in the Lanzhou University Second Hospital was 65.7%(95% CI = 63.1-73.5%),and the median LNR value was 0.2(interquartile range 0-0.5).According to the LNR cut-off point definition obtained by the SEER database,the rN staging rate was 71.9%,56.6%,52.3%,34.3%,and 33.1%(log-rank test P<0.01).Based on rN staging,the 5-year OS of patients in each stage of r AJCC was 84.6%(r IA stage),77.4%(r IB stage),43.8%(r IIA stage),71.1%(r IIB stage),37.4%(r ⅢA stage),56.3%(r ⅢB stage)and 27.4%(r ⅢC stage)(log-rank test P<0.001).The distinguishing ability and prognostic homogeneity of rN staging and r AJCC staging are better than those of the eighth edition AJCC staging.2.A total of 417 GC patients with transcriptome data were enrolled through TCGA,grouped according to their HER-2 status and analyzed for differential expression.The differential expression analysis of HER-2 positive vs.negative group screened 996 differentially expressed genes up-regulated in the HER-2 positive group and 1220 differentially expressed genes down-regulated in the HER-2 positive group.GSEA analysis found that immune-related pathways such as interferon alpha pathway,interferon gamma pathway,IL-2 STAT5 pathway,IL-6 JAK STAT3 pathway,TNF-alpha pathway,and inflammation pathway were significantly down-regulated in the HER-2 positive group.Through immune infiltration analysis,it was found that the abundance of CD8+ T cells decreased significantly in the HER-2 positive group.The correlation between the differential expression information of chemokines and the infiltration abundance of CD8+ T cells was further integrated and analyzed,and it was found that the expression levels of multiple immunochemokines such as CCL19,XCL2,CXCL13 and the abundance of CD8+ T cells were significant Positive correlation,and its expression level was significantly down-regulated in the HER-2 positive group.In addition,considering that HER-2 status has a significant correlation with the immune microenvironment characteristics of GC patients,we further explored the correlation between the expression levels of common immune checkpoints and HER-2 status.The analysis found that the expression of most immune checkpoints in HER-2 positive GC was down-regulated,while the expression of immune checkpoint VTCN1 with immunosuppressive function was differentially up-regulated in the HER-2 positive group. 3.The expression of VTCN1 protein in GC tissues was significantly stronger than that of adjacent control tissues,and its positive rate was 77.5%(62/80).The expression of VTCN1 was significantly correlated with T stage(P<0.001),lymph node metastasis(P<0.001),TNM stage(P=0.001)and distant metastasis(P=0.011)in GC patients.The 5-year survival rate of patients with high VTCN1 expression was significantly lower than that of patients with low VTCN1 expression(P=0.008).Cox multivariate analysis found lymph node metastasis(HR=1.74,95% CI=1.07-2.82,P=0.025),TNM staging(HR=2.91,95% CI=1.19-7.10,P=0.019)and VTCN1 expression(HR= 2.54,95% CI=1.04-6.21,P=0.041)is an independent risk factor for the prognosis of GC patients.Compared with GES-1 cells,the expression of VTCN1 in GC cell lines MKN-45,HGC-27,KATO-Ⅲ and MGC-803 was significantly up-regulated.MGC-803 and KATO-Ⅲ cells with high expression of VTCN1 were selected for further analysis,and MGC-803 and KATO-Ⅲ cells were transfected with sh-VTCN1 and non-targeted sh-NC.CCK-8 proliferation experiments,cell clone formation experiments and Ed U cell proliferation experiments all showed that the down-regulation of VTCN1 can significantly inhibit the proliferation of GC cells.Cell scratch experiment,Transwell migration and invasion experiments proved that the high expression of VTCN1 promoted the invasion and migration of GC cells.The results of in vivo experiments showed that VTCN1 knockout can significantly inhibit the growth of MGC-823 tumor cells in vivo,and significantly reduce the positive rate of Ki67 staining in tumors formed by MGC-803 cells.In addition,WB experiments showed that VTCN1 can regulate the AKT/PI3K/m TOR pathway in GC cells.In order to explore the effect of PI3 K signaling pathway on GC cell proliferation and metastasis,we treated MGC-803 and KATO-Ⅲ cells with the p-PI3 K inhibitor 3-MA,and founded that VTCN1 regulates GC cell EMT partially dependent on AKT/PI3K/m TOR signaling pathway.Conclusions: 1.Compared with the 8th edition of the AJCC staging system,the new r AJCC staging system has prognostic advantages in GC patients and may become an effective tool in clinical practice.2.HER-2 positive GC presents an inhibitory immune microenvironment.Anti-HER-2 therapy may enhance the anti-tumor effect of anti-PD-1 therapy by up-regulating the abundance of CD8+ T cells in the tumor microenvironment.The treatment strategy of immune checkpoint VTCN1 is worthy of further exploration in HER-2 positive GC.3.VTCN1 is up-regulated in GC tissues and cells,and its expression is significantly related to T staging,lymph node metastasis,TNM staging and distant metastasis.VTCN1 expression is an independent risk factor for the prognosis of GC patients.VTCN1 knockdown can inhibit the proliferation,invasion,migration and EMT of GC cells.In terms of mechanism,VTCN1 regulates EMT of GC cells partially depends on the AKT/PI3K/m TOR signaling pathway.VTCN1 may be used as a potential therapeutic target for GC. |
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