Effect Of Immunotherapy Combined With Metronomic Chemotherapy On Advanced Breast Cancer And Its Influence On Immune Environment

BackgroundBreast cancer has surpassed lung cancer with the highest incidence in the world,among which advanced breast cancer is considered a disease that cannot be completely cured.With the update iteration of treatment drugs,the overall survival of patients with advanced breast cancer has been extended,but its prognosis is still poor.Despite the proliferation of endocrine and targeted therapies,chemotherapy remains the main treatment of advanced breast cancer.Traditional chemotherapy can be discontinued due to adverse events,which affects the efficacy of treatment.Metronomic chemotherapy is a relatively low-dose,high-frequency,continuous application of cytotoxic drugs,which can greatly reduce adverse events.Metronomic chemotherapy has the effect of modulating the immune system which maybe contribute to immunotherapy in advanced breast cancer.ObjectiveThis study aims to explore the efficacy of immunotherapy combined with metronomic chemotherapy in patients with advanced breast cancer and search for the most appropriate combination of drugs.We aim to study the differences in immune environment between patients who response and non-response from the levels of systemic immune environment(including circulating immune system and gut microbiota)and tumor immune microenvironment,and to find prediction biomarkers and prediction models related to efficacy.MethodIn this study,patients with HER2-negative advanced breast cancer were included in our clinical trial,who were divided into five treatment cohorts,namely vinorelbine(NVB)metronomic chemotherapy(NVB monotherapy group),PD-1 monoclonal antibody+NVB(NVB group),PD-1 monoclonal antibody+NVB+bevacizumab(BEV group),PD-1 monoclonal antibody+NVB+cisplatin(DDP group),PD-1 monoclonal antibody+NVB+cyclophosphamide+capecitabine(VEX group),to evaluate the clinical benefit rate(CBR),adverse events,progression-free survival(PFS)and overall survival(OS)of the five cohorts.At baseline,after 1 cycle,2 cycles of treatment,and disease progression,the patients’ blood samples and fecal samples were retained.Patients’ tumor puncture specimens were retained at baseline.The following tests were performed:(1)Peripheral blood mononuclear cells(PBMC)and plasma were isolated from blood samples.The proportion of immune cells was detected by PBMC mass spectrometry.The secretion of cytokines in plasma was tested.(2)Multiplex immunofluorescence of patient tumor tissue samples detected the proportion and distribution of tumor cells and immune cells in the tumor immune microenvironment.Transcriptome sequencing and database bioinformatics analysis were conducted to elucidate the factors affecting the efficacy and survival in tumor immune microenvironment.(3)Metagenomic sequencing was performed on fecal samples to explore the relationship between gut microbiota and patient efficacy and survival.The therapeutic effect prediction model was established.Outcome(1)Among the five treatment cohorts,the DDP group and VEX group showed the efficacy advantage of CBR,and the CBR of the two groups was 73.1%and 69.2%,respectively.The median PFS in the BEV group,DDP group and VEX group was longer than that in the NVB monotherapy group and NVB group.The VEX group extended PFS to the most,reaching 6.7 months(NVB monotherapy group:1.3 months).The VEX group reduced 80%of disease progression risk.(2)Patients with the reponse of partial response(PR)and stable disease(SD)were defined as response group,patients with the response of progression disease(PD)were defined as non-response group.In the overall patient population,CD56bright NK cells and regulatory T cells(Treg)in the nonresponse group had a higher ratio,and immunomarker CD38 and CD85j expression was also higher.In BEV group,the proportion of CD56bright NK and CD85j expression were higher in non-response group.In DDP group,the proportion of memory B cells in the response group was higher,and the expression of marker CD4 and CD45RO in T cells was higher.In VEX group,the proportion of NKT,Treg,and effector memory T cells(Tem)in the non-response group was higher,and CD85j and PD-1 expression were higher.(3)In the tumor immune microenvironment,there were three pathological parameters differences in the response and non-response group,and all of them were higher in the response group,namely the I/T ratio(immune cells/tumor cells ratio),immune density(immune cells/total cells ratio),and immune ratio product(CD8+T cells/CD4+T cells)×(immune cells/total cells),indicating that the response group had a higher degree of immune cell infiltration and a higher proportion of CD8+T cells in the tumor microenvironment.Among them,I/T ratio had good predictive efficacy for patient efficacy and survival.The area under curve(AUC)of ROC curve was 0.89(p=0.005).(3)Senescence of immune cells and tumor cells in the tumor microenvironment is one of the factors affecting the therapeutic effect of patients,the degree of immune cell infiltration in the tumor immune microenvironment is negatively correlated with the senescence phenotype.Patients with low senescence phenotype have high immune cell infiltration and good prognosis.(4)Among the gut microbiota,there were different flora in the response and non-response group,and the different flora had different molecular characteristics and biological behaviors.The prediction model of immunotherapy combined with metronomic chemotherapy was established based on 9 characteristic microbiota,and the AUC of the ROC curve was 0.82(p=0.003).The sensitivity and specificity was 81.6%and 84.2%.The predictive effectiveness of this model for patient survival was also high.ConclusionThis study explored the efficacy and safety of immunotherapy combined with metronomic chemotherapy in patients with advanced breast cancer.We found that the VEX group not only had high CBR,but also had the greatest degree of reducing disease progression risk and prolonging PFS.We used multi-omics detection methods to elaborate the factors related to the difference in treatment efficacy and patient prognosis from the levels of systemic immune environment and tumor immune microenvironment.(1)In systemic immune environment,NK cell subset CD56bright NK and T cell subsets NKT,Treg,and Tem were associated with poor efficacy in patients,Immunomarker CD85j was significantly associated with poor efficacy.(2)In the tumor immune microenvironment,the response group had a higher degree of immune cell infiltration and the proportion of CD8+T cells.The pathological parameter I/T ratio had a good predictive effect on patient efficacy and survival,which is expected to become a predictive marker for advanced breast cancer patients with immunotherapy combined with metronomic chemotherapy.(3)Senescence of immune and tumor cells in the tumor microenvironment is one of the factors affecting the therapeutic effect of patients.(4)Among gut microbiota,the model based on characteristic flora is expected to predict the efficacy and survival of patients treated with immunotherapy combined with metronomic chemotherapy.Our findings provide a new exploration direction and theoretical basis for further research on immunotherapy combined with metronomic chemotherapy strategies.