| The Central Nervous System(CNS)is a common metastatic site of advanced lung cancer,including Brain Metastases(BM)and Leptomeningeal Metastases(LM).About 10-20% of non-small cell lung cancer(NSCLC)patients will develop brain metastases,and about 50% of lung cancer is diagnosed concurrently with brain metastases.The median Overall Survival(OS)of untreated BM patients was only 1-3 months.For The survival of BM patients can be extended to 4-11 months treated with surgery,radiotherapy,and chemotherapy.LM refers to the tumor cells infiltrate into the leptomeninges,meninges,and cerebrospinal fluid.Leptomeningeal Metastases occur in 9-25% of patients with lung cancer.Cancer cells reach the leptomeninges through a variety of pathways,such as diffusion through arterial or venous circulation,intrarenal or perineural diffusion,and brain surgery is also a risk factor for promoting cancer cells into the ventricular system.LM is traditionally associated with very poor outcomes,with median survival from diagnosis of only 4–6 weeks.In the era of immunotherapy,more and more evidence supports that Immune Checkpoint Inhibitors(ICIs)can reduce the tumor volume and prolong the survival time in the treatment of lung cancer patients.However,in the course of clinical application,single-degree application of immune checkpoint inhibitors has not achieved satisfactory clinical efficacy.Considering that both anti-angiogenesis agents and immune checkpoint inhibitors target tumor microenvironments(TME),this combination therapy has the potential to become a promising clinical strategy.However,most clinical trials use strict selection criteria,and patients with CNS metastases are often excluded.Therefore,the efficacy of immune checkpoint inhibitors combined with anti-angiogenesis therapy in central nervous system metastases of lung cancer is still unclear.The aim of this study is to further understand the central nervous system metastasis of lung cancer by retrospectively analyzing the general characteristics and survival outcomes of patients with CNS metastases of lung cancer.Then the cerebrospinal fluid of lung cancer patients with CNS metastases was analyzed by singel cell RNA sequence(sc RNA-seq)sequencing to understand the composition of tumor microenvironment and exhausted CD8+ T cells,and lay a theoretical foundation for the application of immune checkpoint inhibitors.We retrospectively analyzed the clinical efficacy and related adverse reactions in lung cancer patients with CNS metastases treatment with immune checkpoint inhibitors combined with anti-angiogenesis drugs.Finally,we studied the effect of immune checkpoint inhibitors combined with anti-angiogenesis drugs on brain metastases of lung cancer in mice and the possible mechanism,which provided ideas for the treatment of clinical lung cancer patients with CNS metastases.Part One Clinical characteristics and prognostic factors of lung cancer patients with central nervous system metastasesObjective: Central nervous system metastases is a common complication of advanced lung cancer,and its treatment is limited and the prognosis is poor.We mainly evaluated the OS and prognostic factors of lung cancer patients with CNS metastases.Methods: We retrospectively analyzed lung cancer patients with CNS metao October 2022 as the experimental group.At the same time,stage IV Non-Small Cell Lung Cancer(NSCLC)without CNS symptom and non-neoplastic diseases of the CNS in the Second Hospital of Hebei Medical University were selected as controls group.The clinical characteristics and treatment of the patients were recorded.Enzyme Linked Immunosorbent Assay(ELISA)was used to detect Vascular Endothelial Growth Factor(VEGF)levels in serum and CSF of the experimental group and the control group.SPSS 26.0 software was used for data analysis.Survival analysis was performed according to the Kaplan-Meier method,and the significance of survival rate between the two groups was compared by log-rank test.Cox proportional hazards regression model was used to study the effect of multivariate factors on survival time.Results: 1.General information: a total of 133 lung cancer patients with central nervous system metastases were included in this study.The median age was 60(35-85)years.There were 69 females and 64 males.Among the patients with CNS metastases,129 cases were adenocarcinoma,3 cases were non-adenocarcinoma and 1 case was unknown.Among the patients with CNS metastases,91 patients had meningeal metastases alone,12 patients had brain metastases alone and 30 patients had both meningeal and brain metastases.There were 60 patients with extracranial metastases and 73 patients without extracranial metastases.The sites of extracranial metastases were mainly bone and lymph nodes.2.Laboratory examination: in our study,there were 121 patients examined for Cerebrospinal Fluid(CSF)cytology,including 92 positive patients and 29 negative patients.Among the 133 patients,80 patients had positive enhancement of head and/or spinal cord Magnetic Resonance Imaging(MRI),41 patients had negative,and 12 patients had unknown results.Positive MRI findings showed linear,strip,or nodular enhancement.Among them,111 patients with CNS metastases were sent for next-generation sequencing of CSF and/or blood,of which 66 patients with Epidermal Growth Factor Receptor(EGFR)mutations,Three cases of Kirsten ratsarcoma viral oncogene(KRAS)mutation,Mesenchymal-epithelial transition factor(MET)mutation in 3 cases,Anaplastic Lymphoma Kinase(ALK)rearrangement in 2 cases,There were 2 cases of ROS proto-oncogene receptor tyrosine kinase 1(ROS1)fusion,and no sensitive mutations were detected in the rest.3.Treatment: Local treatment mainly includes intrathecal chemotherapy,radiotherapy,and systemic treatment includes systemic chemotherapy,targeted therapy,anti-angiogenesis therapy,and ICIs therapy.Irrespective of general supportive treatment,88 patients received combinded treatments and 45 patients received singel treatment.Among the 133 patients,81 patients were treated with targeted drugs and 52 patients were not.4.VEGF comparison: The median level of VEGF in CSF was 185.4(179.6,189.9)pg/ml in lung cancer patients with CNS metastases and169.8(160.4,179.1)pg/ml in patients with non-neoplastic diseases of the central nervous system.There was a significant difference in the level of VEGF in CSF between the above two groups(P < 0.001).Serum VEGF levels were 21.2(10.9,31.9)pg/ml and 38.1(15.6,112.6)pg/ml in patients with CNS metastases and without CNS symptoms lung cancer,respectively,and there was no significant difference in serum VEGF between the two groups.The level of VEGF in CSF was significantly higher than that in serum in patients with CNS metastases of lung cancer(P < 0.001).5.Survival prognosis analysis: The median OS of 133 patients with CNS metastases from lung cancer was 11.2 months in this study and the95% confidence Interval(CI)was 7.9-14.5 months.In the univariate survival prognosis analysis,leptomeningeal metastases,targeted therapy and combined therapy were the factors affecting the prognosis.Multivariate COX proportional hazards regression analysis showed that the combination therapy and targeted therapy were prognostic factors for longer overall survival.The prognostic factor associated with shorter overall survival was leptomeningeal metastases.Conclusions: The majority of lung cancer patients with CNS metastases are lung adenocarcinoma,the clinical prognosis is poor,with a median OS of only 11.2 months and EGFR mutations account for a large proportion of patients.The level of VEGF in CSF of lung cancer CNS patients is higher than that in serum.The level of VEGF in CSF of lung cancer CNS metastases group is higher than that of central nervous system non-neoplastic disease group,which lays the foundation for our application of anti-angiogenic drugs.The factors associated with outcomes may be used to guide patient management and to inform the design of future clinical trials for this population.Part two A preliminary study of tumor microenvironment in the CSF of lung cancer patients with central nervous system metastasesObjective: Tumor microenvironment is a regulator element of intracranial tumors to ICIs,so the study of tumor microenvironment is particularly important.We study the composition of the tumor microenvironment and the classification of CD8+ T cells and hope to provide ideas for the application immunotherapy in CNS metastases of lung cancer.Methods: In this study,CSF samples from lung cancer patients with CNS metastases were prepared for single-cell suspension preparation and cell quality detection,and then single-cell transcriptome sequencing was performed.Unsupervised clustering method was used to cluster the cells in the CSF tumor microenvironment,and the results were visualized by Uniform Manifold Approximation and Projection(UMAP).After the clustering and annotation of large classes of cells,the gene expression matrix of CD8+ T cells was extracted,and CD8+ T cells were clustered and grouped again.The expression of different types of inhibitory immune checkpoint genes was marked.Results: 1.In the sc RNA-seq performed on the CSF of four lung cancer patients with CNS metastases,a total of 15872 cell transcriptome data were included in the subsequent analysis through quality control.We roughly classified cells into 19 clusters based on cell characteristics and cell expression correlations.The proportion of cells varies from different patients.The tumor microenvironment in CSF of lung cancer patients with CNS metastases mainly contain T cells,myeloid cells,B cells and tumor cells.2.We divided CD8+ T cells in CSF into four classes based on the expression of different genes.CD8+ T cells express inhibitory immune checkpoint genes,such as Hepatitis A virus cellular receptor 2(HAVCR2),Lymphocyte Activation Gene-3(LAG3),Programmed death-1(PDCD-1),T cell immunoglobulin and receptor tyrosine inhibitory domains(TIGIT)proteins.There are exhausted CD8+ T cells in CSF,which lays a theoretical foundation for the application of immune checkpoint inhibitors.3.Myeloid cells are another important component of TME in addition to lymphoid cells,among which tumor Associated macrophages(TAM)account for the majority of myeloid cells.There are two main functional states of TAM,including the traditional M1 type that promotes inflammatory response,inhibits tumor development,and M2 type that promotes tumor development and metastases.In this study,in addition to TAM,there are a small number of dendritic cells and monocytes in the myeloid cells.Conclusions: Single-cell transcriptome sequencing technology can be used to comprehensively understand the tumor microenvironment of CNS metastases of lung cancer,which shows great heterogeneity and complexity.The presence of exhausted CD8+ T cells in the CSF microenvironment provides a theoretical basis for the application of ICIs in the treatment of lung cancer with CNS metastases.In addition,there are myeloid cells in the CSF microenvironment,and their role in immunotherapy needs to be further studied.Part three The clinical efficacy and adverse reactions of immune checkpoint inhibitors combined with anti-angiogenesis therapy in the treatment of lung cancer patients with CNS metastasesObjective: The prognosis of lung cancer patients with CNS metastases is poor and the treatment options are limited.The aim of this study is to investigate the clinical efficacy and safety of ICIs combined with anti-angiogenesis drugs in the treatment of lung cancer patients with CNS metastases,and analyze the factors that may be related to prognosis.Methods: The clinical data of patients with lung cancer CNS metastases treated with ICIs combined with anti-angiogenic drugs in the Department of Neurology of the Second Hospital of Hebei Medical University from January 2020 to October 2022 were retrospectively analyzed.The overall survival time,Disease Control Rate(DCR),Objective Response Rate(ORR)and adverse reactions were observed.SPSS 26.0 software was used for data analysis.Kaplan-Meier method was used for survival analysis,and log-rank test was used to compare the significance of survival rates between the two groups.Results:1:General information: in this study,15 lung cancer patients with CNS metastases were treated with ICIs combined with anti-angiogenesis drugs.There were 11 males and 4 females with a median age of 61(34-74)years.Of the 15 patients,6 cases were LM,6cases were LM+BM,and 3 cases were BM.Six of the 15 patients had extracranial metastases,and the metastatic sites were bone and lymph nodes.Among the 15 patients,1 case was squamous cell carcinoma,1case was unknown,and the remaining 13 cases were adenocarcinoma.2.Laboratory examination: 14 patients underwent lumbar puncture examination before ICIs and CSF cytology was positive in 13 cases and negative in 1 case.13 patients undego CSF and/or blood circulating tumor DNA(ct DNA)next-generation sequencing,of which 8 patients had EGFR mutation,3 patients had KRAS mutation,and the remaining 2patients had no sensitive mutation.Tumor Mutation Burden(TMB)in CSF and/or blood was detected in 7 patients,and the TMB in blood was13.8(8.5-16.9)mutations /Mb,and the TMB in CSF was 18.2(0-34)mutations /Mb.3.Treatment:15 patients received ICIs combined with anti-angiogenesis therapy,which was not the first-line treatment.There were 9 patients received second-line treatment,5 patients received third-line treatment,and 1 patient received fourth-line treatment.Three patients had a PD-L1 level of less than 1% in lung tissue,and the remaining 12 patients were unknown.In the 15 patients,11 received camrelizumab(anti-PD-1 monoclonal antibody)and 4 received emvolizumab(anti-PD-L1 monoclonal antibody).4.Survival prognosis analysis: the median OS of 15 patients was 5.8months,95%CI(0-11.86 months).The efficacy of 15 patients was evaluated since 8 weeks after the first ICIs,including 4 cases of partial response(26.7%),5 cases of stable disease(33.3%),6 cases of progressive disease(40.0%),DCR was 60.0%,and ORR was 26.7%.As of January 31,2023,12 patients(80%)had died and 3 patients(20%)were alive.The median follow-up of surviving patients was 17.0 months(range: 13.8 to 36.3 months).Univariate Log-Rank test was performed on the 15 patients treated with combination drugs.Gender,age,KPS score,number of combined treatment lines,extracranial metastases,absolute lymphocyte count in blood,and leptomeningeal metastases were not factors affecting the prognosis of patients.5.Adverse reactions:adverse reactions were observed in 13 patients,the overall incidence of adverse reactions was 86.7%(13/15),the incidence of grade 1-2 adverse reactions was 86.7%(13/15),the incidence of grade 3 adverse reactions was 13.3%(2/15),and no grade 4or above adverse reactions were observed.Conclusions: The combined use of ICIs and anti-angiogenesis drugs in lung cancer patients with CNS is effective and the adverse reactions are controllable.Gender,age,KPS score,number of combined treatment lines,extracranial metastases,absolute lymphocyte count in blood,and leptomeningeal metastases are not the prognostic factors of combination therapy in lung cancer patients with CNS.and More prospective multicenter clinical studies are expected to further explore factors affecting the prognosis of patients should be conducted.Part Four The effect and mechanism of ICIs combined with endostar on brain metastases of lung cancer in animal modelObjective:ICIs combined with anti-angiogenic drugs have achieved some effect in lung cancer,but there are few studies on brain metastases of lung cancer.The aim of this study is to investigate the efficacy and mechanism of ICI combined with anti-angiogenic drugs in the treatment of brain metastases from lung cancer in mice.Methods: To establish a mouse model of brain metastases,we used mouse Lewis lung cancer luciferase(LLC-Luc)cell line.Brain metastases and tumor burden were analyzed by bioluminescence imaging.The levels of cytokines and m RNA in tumor tissues were detected by ELISA and real-time quantitative PCR(q RT-PCR).Immunohistochemical staining was used to detect the expression of CD31 in brain tumor tissues.Results:1.Tumor burden:we observed that the fluorescence intensity of the combined PD-1 inhibitor and Endo group was significantly reduced compared with the monotherapy group or the vehicle group at day 28(P <0.001).2.The survival time:the survival time of combined PD-1 inhibitor and Endo group was significantly longer than that of single PD-1inhibitor group(P < 0.05),Endo group(P < 0.001)and vehicle group(P <0.001),the difference was statistically significant.3.Tumor angiogenesis:compared with vehicle group and PD-1inhibitor group,PD-1 inhibitor combined with Endo group can reduce the levels of CD31 and VEGF in lung cancer brain metastases.4.Cytokine:compared with the control group and Endo group,the combination of PD-1 inhibitor and Endo group could reduce the levels of cytokine secretion and m RNA level in brain metastases.Conclusions:In this study,we found that the combination of PD-1inhibitor and endostar could inhibit the growth of metastatic tumors,prolong the survival time and improve the survival rate in mice with brain metastases from lung cancer.The possible mechanism of PD-1 inhibitor combined with endostar in the treatment of brain metastases of lung cancer is to affect angiogenesis and cytokine levels.This study provides a theoretical basis for the clinical application of ICIs combined with anti-angiogenesis drugs in the treatment of lung cancer patients with brain metastases. |
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