| Objective:Renal cell carcinoma(RCC)is one of the most common malignant tumors in the urinary system.The overall incidence of renal cancer is increasing at a rate of 3-5%per year.In general,30-40%of RCC patients have advanced metastatic renal cell carcinoma(m RCC)at initial diagnosis.With the deepening understanding of angiogenesis pathways in the pathogenesis of RCC,small molecule targeted drugs represented by tyrosine kinase inhibitors(TKIs)quickly replaced traditional cytokine therapy,and became the standard treatment for m RCC,significantly improving the prognosis of m RCC patients.Even so,the median progression-free survival(PFS)and overall survival(OS)time were less than 15 and 30 months.With the discovery of the immunogenicity of renal cell carcinoma,new immunotherapy aimed PD-1/PD-L1 has drawn attention.In the Check Mate025 clinical trial,nivolumab showed its efficacy as second-line treatment of m RCC patients.Since then,the immune checkpoint inhibitors(ICIs)represented by PD-1/PD-L1 inhibitors began to be used in the treatment of m RCC patients,especially combined with TKI or CTLA-4 inhibitors,and generally became a new standard for the treatment of m RCC.However,regardless of ICIs or TKIs,almost all patients would experience disease progression after 8-20 months of treatment.Therefore,rationalized sequencing treatment has become the key for improving the prognosis of patients.In the era of targeted therapy,there are a large number of population-based retrospective studies on the sequencing treatment of different small molecule targeted drugs;in the era of ICIs,we are also faced with second-line choices after the failure of first-line therapy.Honestly speaking,ICI-based therapy has limited efficacy whether it is first-line or second-line treatment,and its impressive results in the field of RCC treatment are more achieved by combination therapy.On the other hand,TKI treatment was still an important role in the standard first-line treatment of m RCC patients,especially in patients with 0-1 IMDC risk score.It is worth noting that in China,due to the adverse reactions,economic factors and other factors,there are still a large number of m RCC patients using TKIs as first-line treatment.After showing a satisfying efficacy in the first-line treatment,we are wondering whether ICI plus TKI treatment could showed its efficacy as second-line treatment in m RCC patients.In addition,how to make individual treatment approach is the key to improving survival outcomes of m RCC patients.Currently,some biomarkers(such as PD-L1 and TMB)for predicting the efficacy of immune checkpoint inhibitors cannot show their efficacy in RCC.On the other hand,the tumor immune microenvironment of m RCC should be paid attention.Therefore,this study aimed to verify the effectiveness of second-line immunotherapy for renal cancer patients after failed from first-line TKI treatment.In addition,by evaluating the characteristics of the tumor immune microenvironment of the m RCC patients,we aimed to explore its predictive effect on the prognosis of m RCC patients,thus guiding the usage of immune therapy in clinical practice.In the end,we hoped to realize the individualized and precise treatment of m RCC patients.Materials and methods:(1)Retrospectively collect clinicopathological and follow-up data of m RCC patients with second-line treatment in our center,and evaluate the effectiveness of second-line ICI+TKI combination treatment as scond-line treatment approach of m RCC patients.(2)Gene expression and clinicopathological data were download from TCGA database.The relationship between renal cancer immune infiltration score and immune-related gene expression,clinical pathological characteristics and prognosis were evaluated using statistic software.(3)Retrospectively collected the information of m RCC patients who treated in our center.Patients who received systematic treatment were enrolled.Baseline and follow-up data were collected,and pathological specimens of the patient’s primary tumor were also obtained.According to the results of the second part of the TCGA data analysis,we selected CD4,CD8,PD-L1,CD73,LAG-3,and NRI-1 as studied biomarkers.Using immunohistochemistry(IHC)to detect the expression levels of the six biomarkers in the tissues of patients.Statistical software were used to analyze the correlation of the expression level of biomarkers with patient’s clinicopathological characteristics and prognosis with the,and evaluate its predictive value for the efficacy of immune or targeted therapy.Results:I.Efficacy of immune checkpoint inhibitor plus tyrosine kinase inhibitor in patients with metastatic renal cell carcinoma after targeted therapy failure1.ICI plus TKI has satisfactory effectiveness and safety as second-line treatment:This part of study included a total of 120 m RCC patients who received second-line treatment.Among them,7 patients who received second-line m TOR based therapy(10 cases in total)underwent treatment cessation due to intolerable adverse events,thus this part of patients was not included in further survival analysis.Further analysis showed that the objective response rate of patients receiving ICI plus TKI therapy were significantly higher than those of patients receiving TKI alone(36.5%vs.12.1%,P=0.002).In terms of safety,ICI plus TKI therapy had comparable safety profile with TKI alone.2.Second-line ICI plus TKI therapy could significantly improve the prognosis of m RCC patients:Survival analysis suggested that,compared patients treated with TKI alone,those who received ICI plus TKI treatment experienced longer PFS2(15.0 vs.9.0 months,P=0.009)and OS time(not reaching vs.16.0 months,P=0.018).3.Clinicopathological characteristics that indicate superior survival benefits of ICI plus TKI over alternative TKI alone:Cox-regression analysis suggested that patients with cc RCC feature,single metastatic site,PFS1≥9 months,and second-line IMDC favorable-/intermediate-risk groups could independently predict much more favorable survival benefits from ICI plus TKI therapy over alternative TKI alone.4.Clinicopathological factors related to the efficacy of second-line ICI plus TKI treatment:(1)Second-line IMDC risk group could stratify the prognosis of patients with second-line ICI plus TKI treatment.Survival analysis suggested that only second-line IMDC risk criteria could fairly stratify the prognosis of patients with second-line ICI plus TKI treatment.(2)The prognosis of patients in the IMDC intermediate-risk group is heterogeneous:In the IMDC intermediate-risk group,patients with1 risk factor experienced significantly longer PFS2(NR vs.10.0 months,p=0.013)and OS time(NR vs.15.0,p=0.007)time from ICI plus TKI treatment than those with 2 risk factors.Thus,we classified patients into two groups according to numbers of IMDC risk factors:group 1(0-1 risk factor)and group 2(≥2 risk factors),and found significantly favorable survival outcomes in group 1 over group 2.III.Characteristics of tumor immune microenvironment had predictive value for the prognosis of m RCC patients1.Immune score is closely related to the clinicopathological characteristics and prognosis of RCC patients:Data of 537 RCC patients were download form TCGA database.(1)Immune score is closely related to the clinicopathological characteristics of RCC patients:With the median of the immune/stroma score as the cut-off point,patients were divided into high and low immune/stroma score groups.High immune score is associated with higher T stage(P<0.01)and ISUP nuclear stage(P<0.01);on the other hand,stroma score did not show a correlation with the patient’s clinicopathological characteristics.(2)Tumor immune score can predict the prognosis of RCC patients:Survival analysis showed that compared with patients with low immune score,those with high immune score experienced shorter OS time(P=0.014);tumor stroma score did not show a correlation with patients’OS time.2.Differential expression genes(DEGs)between the high and low tumor immune/stromal score groups:(1)There is a difference in gene expression between the high and low immune/stromal score groups:Compared with the low tumor immune score group,the expression of 1433genes in the high tumor immune score group were increased and 230 genes were decreased.In terms of stroma score,compared with the low stroma score group,a total of 1910 genes in the high stroma score group were increased expression and 235 gene expression were decreased.There were722 DEGs with increased expression and 77 DEGs with decreased expression in both high immune and stroma score groups;(2)DEGs between the high and low immune score groups were related to the OS time of RCC patients:Among the 1433 up-regulated DEGs,a total of 890genes are related to the patient’s prognosis.Among them,855 genes are related to shorter OS time,and only 35 genes are related to prolonged OS time.3.The infiltration of different types of immune infiltrating cells in the high and low tumor immune score groups:Among the 22 different immune infiltrating cells,there are significant differences in the infiltration of 16 types of cells between the high and low tumor immune score groups.In the high immune score group,the infiltration level of CD8~+T lymphocytes was significantly increased,while the infiltration of CD4~+memory T cells and mast cells were decreased.In addition,the expressions of neutrophils,M1 macrophages,NK cells,regulatory T cells,etc.were also differentially infiltrated in different immune score groups.4.Correlation between different types of immune infiltrating cells and patients’prognosis:Survival analysis showed that the high infiltration levels of CD8+T cells,activated CD4+memory T lymphocytes,Treg cells,and memory B cells were related to poor prognosis of patients.On the other hand,high infiltration of dormant CD4~+memory T lymphocytes were related to prolonged OS time.The overall CD4+T lymphocytes,B cells,naive B cells,myeloid suppressor cells and mast cell infiltration levels did not show a correlation with the patient’s prognosis.5.Correlation between the level of immune cell infiltration and the expression of immune-related genes:The expression levels of CD274,HAVCR2,NT5E,LAG3,NRP1 and TIGIT were closely related to immune cell infiltration.Among them,the expression levels of CD274,HAVCR2,NT5E,LAG3,and TIGIT were positively correlated with the infiltration of CD8~+T,while NT5E and NRP1 were positively correlated with the infiltration of CD4+T cells.It is worth noting that the expression levels of LAG3 and TIGIT were positively correlated with Treg cell infiltration,and the expression levels of CD73 and NRP1 are negatively correlated with Treg cell infiltration.III.The predictive value of immune-related biomarkers on the prognosis of m RCC patients1.Clinicopathological characteristics of the included patients:This part of the study included a total of 135 patients diagnosed with metastatic renal cell carcinoma,with an average age of 53.4(±14.4)years old.The majority of patients were diagnosed with renal clear cell carcinoma(111,82.2%).In terms of treatment,a total of 102(75.6%)patients received TKI therapy,and 33(24.4%)patients received TKI+ICI combined therapy.The median PFS and OS time of all patients were 12.0 and 35.0 months,separately.2.The expression profile of biomarkers:(1)CD4:CD4 is mainly expressed on the cell membrane surface of tumor infiltrating lymphocytes(TIL).According to CD4~+T cell infiltration,it can be divided into high infiltration(43 cases,31.9%)and low infiltration(92 cases,68.1%)groups.(2)CD8:CD8 is mainly expressed in the cell membrane of TILs.According to CD8~+T cell infiltration,patients were divided into two groups:high infiltration(87 cases,64.4%)and low infiltration(48 cases,35.6%)groups.(3)PD-L1:PD-L1 is mainly expressed in the cell membrane of tumor cells and lymphocytes.According to its expression,it can be divided into PD-L1positive(45 cases,33.3%)and negative(90 cases,66.7%)groups.(4)CD73:CD73 is mainly expressed in the cell membrane of tumor cells.According to its expression,patients could be divided into two groups:high expression of CD73(63 cases,46.7%)and low expression(72 cases,53.3%)groups.(5)LAG-3:LAG-3 is mainly expressed on the surface of lymphocyte membrane.Among all patients,there were 55(40.7%)cases with high LAG-3expression and 80(59.3%)cases with low LAG-3 expression.(6)NRP-1:NRP-1 is mainly expressed on the cell membrane of tumor cells.Among all included patients,there were 99(73.3%)cases with NRP-1 high expression and 36(36.3%)cases with NRP-1 low expression.3.The correlation between the expression of biomarkers:Correlation analysis showed that the infiltration level of CD4+T lymphocytes was positively related to the expression of PD-L1(r=0.292,P=0.002)and LAG-3(r=0.177,P=0.040)on tumor cell.On the other hand,CD8+T lymphocyte infiltration was positively associated with the expression of PD-L1(r=0.263,P=0.002),CD73(r=0.265,P=0.002)and LAG-3(r=0.427,P<0.001).In addition,the expression levels of PD-L1,CD73 and LAG-3 also showed a correlation.The expression level of PD-L1 is positively related to LAG-3.The positive rate of LAG-3 expression in the PD-L1 positive group is as high as 49%,which is much higher than the expression level of LAG-3 in the PD-L1 negative group(22%)(P=0.001).The expression level of LAG-3 in CD73-positive group was much higher than that in CD73-negative group(64%vs.35%,P=0.001).4.The relationship between the expression of biomarkers and the clinicopathological characteristics of patients:CD4+T cell infiltration did not show the relationship with the clinicopathological characteristics of patients.The level of CD8+T cell infiltration was related to tumor necrosis.The ratio of tumor necrosis in the CD8+T cell high infiltration group was significantly higher than that in the CD8+T cell low infiltration group(35.6%vs.18.8%,P=0.035).In addition,PD-L1,CD73,LAG-3,and NRP-1all showed a correlation with the IMDC risk group.Among them,the high expression of PD-L1,CD73,LAG-3 were related to the poor IMDC groups.In the IMDC favorable-,intermediate-,and poor-risk groups,the positive expression of PD-L1 were 22.0%,38.2%,47.1%(P=0.025);the positive expression of CD73 were 30.0%,58.8%,47.1%(P=0.006);the positive rates of LAG-3 expression were 24.0%,48.5%,and 58.8(P=0.001).On the other hand,the high expression of NRP-1 is related to the lower IMDC prognosis group of patients.The positive rates of NRP-1 in the IMDC favorable-,intermediate-,and poor-risk groups were 90.0%,73.5%,and 52.9%(P=0.004).5.Correlation between the expression of biomarkers and the overall prognosis of patients:Survival analysis showed that except for CD4~+T cell infiltration,CD8,PD-L1,CD73,LAG-3,and NRP-1 showed a relationship with patient’s prognosis.As for PFS,CD8~+T cell high infiltration(9.0 vs.18.0 months,P=0.005),as well as high expression level of PD-L1(8.0 vs.18.0 months,P=0.010),CD73(10.0 vs.16.0 months,P=0.046),and LAG-3(8.0 vs.18.0 months,P<0.001)were related to the shortened PFS time of m RCC patients,while the high expression of NRP-1 was related to the prolonged PFS time of patients(15.0vs.8.0 months,P=0.002).In terms of OS,high infiltration level of CD8+T cells(30.0 vs.45.0 months,P=0.013),as well as high expression level of PD-L1(30.0 vs.48.0 months,P=0.022)and LAG-3(30.0 vs.54.0 months,P=0.003)were associated with shortened OS time,while high NRP-1 expression were associated with prolonged OS time(35.0 vs.30.0 months,P=0.048).CD73 expression level did not show a relationship with OS time(high expression vs.low expression:32.0 months vs.not reached,P=0.438).Multivariate analysis showed that the high expression levels of CD73 and LAG-3 were independent risk factors for the shortened of PFS time,while the high expression of NRP-1 was an independent protective factor for PFS time.For the patient’s OS time,only the level of CD8+T cell infiltration showed a correlation with the patient’s OS time.6.The expression of immune-related biomarkers could aid the selection of appropriate treatment options for m RCC patients:(1)The value of single biomarker for the selection of treatment options:COX regression analysis showed that in patients with high CD8+T cell infiltration and PD-L1 expression,ICI plus TKI combined therapy showed better efficacy over TKI alone.(2)Relationship between tumor microenvironment immune types(TMIT)and patient prognosis:According to the expression level of CD8+T lymphocyte infiltration level and PD-L1 expression level,patients can be divided into TMIT Group I-VI.Among them,patients with TMIT I had the shortest PFS(7.0 months)and OS(17.0 months)time.In TMIT type I patients,ICI combined with TKI therapy has shown better efficacy over TKI alone,which could significantly prolong PFS(not reaching vs.6.0 months,P=0.026)and OS(not reaching vs.14.0 months,P=0.036)time of patients.(3)Prediction of patient prognosis based on comprehensive evaluation of lymphocyte infiltration and biomarker expression:By comprehensively evaluating the expression of PD-L1,LAG-3,CD73 and NRP-1,patients could be divided into 3groups:51(37.8%)patients in the low-risk group(0-1 points),62(45.9%)patients in the intermediate-risk group(2-3 points),and 22(16.3%)patients in the high-risk group(4-5 points).There were significant differences in prognosis among the three groups of patients.It was worth noting that in the intermediate-and high-risk groups,compared with TKI treatment alone,ICI combined with TKI treatment could significantly prolong the PFS(11.0 vs.5.5 months,P=0.023)time of patients.Although a prolonged OS time was also noticed in patinets with ICI plus TKI treatment,the difference had no statistically significant(25.0 vs.15.0 months,P=0.216).Conclusions:1.Second-line ICI plus TKI therapy has better efficacy than TKI therapy,with reliable safety.Patients with pathological types of cc RCC,oligometastasis,and first-line PFS time≥9 months are more likely to be benefit from ICI plus TKI treatment;2.Based on the TCGA database,we found that the tumor immune microenvironment has a significant impact on the prognosis of RCC patients.Patients with high immune score were tended to have a poor prognosis.In addition,the gene expression profile was quite different between high and low immune scores groups.3.The infiltration of CD8~+T cells,Treg cells and other lymphocyte subtypes in tumor tissue are closely related to the prognosis of RCC patients.In addition,and the expression of CD274,HAVCR2,NT5E,LAG3,NRP1and TIGIT are closely related to the infiltration of immune cell;4.In m RCC patients,the high expressions of CD8,PD-L1,CD73,and LAG-3 in tumor tissue are associated with a poor prognosis,while the high expression of NRP-1 indicates a better prognosis.In addition,The expressions of PD-L1,CD73,LAG-3 and NRP-1 are all related to the efficacy of TKI treatment.It is worth noting that in patients treated with ICI combined with TKI,the high expression of LAG-3 indicates that the prognosis of patients is poor,and LAG-3 It may be one of the reasons why some m RCC patients are resistant from immunotherapy;5.The high expression of PD-L1,CD73,LAG-3 is related to the high infiltration of CD8+T cells in tumor tissue;in addition,the expression of PD-L1,CD73,LAG-3 were consistant in m RCC patients.6.The TMIT classification of RCC patients and the prognostic stratification based on the expression of CD8,PD-L1,CD73,LAG-3,and NRP-1 could fairly stratify the prognosis of m RCC patients,and can be used to help clinicians choose the appropriate treatment option for m RCC patients. |
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