Single-Cell Analysis Reveals Biological Characteristics Of Clinical Efficacy In Relapsed Or Refractory Autoimmune Hemolytic Anemia Cohort

Background and objectives:Autoimmune hemolytic anemia(autoimmune hemolytic anemia,AIHA)is a relatively rare and highly heterogeneous disease due to accelerated destruction of autologous erythrocytes by autoantibodies with or without complement activation.Other pathogenic mechanisms involve excessive activation of cellular immune effectors,dysregulation of cytokines and decompensation of bone marrow hematopoiesis.Whereas the biological characteristics of relapsed or refractory AIHA at single-cell level is completely unknown.Recently,approximately 70-80%of patients have an initial response to glucocorticoid monotherapy or combined with rituximab(RTX)as the first and second-line therapy,nevertheless more than 25%of patients will relapse again.Due to less supporting evidence and a considerable risk of serious adverse effects on therapies of splenectomy and other immunosuppressants,which cannot be widely applied in clinical.Therefore,it is an urgent clinical problem to establish a salvage treatment strategy for relapsed or refractory AIHA and to find new therapeutic drugs with greater efficacy and lower toxicity.In addition,using single cell transcriptomes to investigate the biological characteristics of relapsed or refractory AIHA patients is helpful to explore new targeted therapy,improve the response rate of therapy,design reasonable strategies of targeted therapy and reduce the risk of recurrence.Methods:Firstly,we retrospectively analyzed the clinical characteristics and efficacy of 33 AIHA patients who received treatment of RTX in our center from August 2019 to January 2022.Secondly,we designed a phase Ⅱ clinical trial(NCT04398459)to evaluate the efficacy and safety of ibrutinib in the treatment of recurrent or refractory AIHA and completed the efficacy and safety evaluation of 12 patients currently enrolled.Finally,a total of 77829 mononuclear cells from peripheral blood samples of 4 patients in the ibrutinib treatment cohort and 3 healthy controls were analyzed by single cell transcriptome and TCR sequencing.All data were analyzed by R package(3.6.3 version),Graphpad Prism(8.0.2 version)and SPSS(23.0 version)software.The continuous variables were in accordance with normal distribution using mean,standard deviation,ttest,skewness distribution using median,quartile,rank sum test,chi-square test or Fisher exact test,and Kaplan-Meier method was used to draw relapse free survival(RFS)curve.All tests were two-side,and P<0.05 was of statistical significance.Results:1.Analysis of efficacy in RTX treatment cohort:Among the 33 patients treated with RTX,the median age was 32(6-69)years old.Before treatment,the hemoglobin(Hb)value was 81(35-141)g/L and the overall response rate(ORR)was 81.8%.The CR and CRi rates were 60.6%(20/33 cases).The median response time was 4(1-14)weeks,the median follow-up time was 9.5(2-30)months,and the median RFS was 11.5(6.7-NA)months.The baseline of Hb in AIHA group(27 cases,Hb 72g/L)was significantly lower than that in Evans group(6 cases,Hb 104g/L),and the RFS of AIHA group was significantly longer than that of Evans group(12.3 months versus 5.8 months,HR=2.68,P=0.041).There was no significant difference of RFS between the low and high dose of RTX groups.The safety profile of RTX was acceptable.The incidence of infection was 27.3%,of which 24.2%were grade 3-4 infection,and the median time of infection was 11(1-14)weeks.2.Analysis of efficacy in ibrutinib treatment cohort:Among the 13 patients treated with ibrutinib,there were 8 cases of warm antibody type,2 cases of mix antibody type,2 cases of Coombs negative type and 1 case of Evans syndrome.The median age of the patients was 30(6-64)years old,and the median Hb was 67(24-88)g/L at baseline level.12 were evaluable for efficacy,the ORR was 66.6%and the CR and CRi rates were 58.3%(7/12 cases).The median response time was 5.3(1-10)weeks.After 12 weeks of using ibrutinib,they showed elevated levels of Hb and reduced level of lactate dehydrogenase(LDH)respectively(all P<0.05).Compared with the patients who response to ibrutinib,the level of C3 in the no response group was relatively higher at baseline level(NR group 0.855g/L vs Res group 0.695g/L,P=0.008),and the area under the ROC curve of predictive potency test was 1.Finally,the main adverse events of grade 3-4 were neutropenia(n=2),infection(n=2),variant vasculitis(n=1)and rash(n=2).3.Analysis of single cell sequencing:After clustering and annotating the single cell sequencing data of AIHA before treatment of ibrutinib and healthy controls,we identified 20 clusters of immune cells.It is decreased in the proportion of DC2,macrophages and NK cells in AIHA samples,while the opposite among γ δ TRDV1 and XCL-TE cell clusters.Correlation analysis between the two groups showed that there were significant differences among the clusters of CD8-TE,CD8-EM,γδ TRDV2-TRDV9 and monocytes.We totally founded 617 differential genes based on analyses of above different cell clusters and the whole differential genes between two samples.DEGs are mainly enriched in T cell activation,INF-y response,protein phosphorylation,B cell activation,NK cell mediated cytotoxicity,NF-κB signal pathway,B cell receptor signal pathway,IL-17 signal pathway,TNF signal pathway,JAK-STAT and PI3K-ARK signal pathway and other autoimmune response related signal pathways.Whereas the pathways related to T cell activation and many autoimmune response pathways were significantly downregulated in the samples after ibrutinib applied.It was interesting to note that almost up-regulated DEGs between no response and response group were enriched in the signal pathways of T cell activation,antigen processing and presentation and multiple autoimmune response single pathways.Lastly,through the analyses of TCR sequencing data of different samples,it tended to increase of the number of oligo clones and CDR3 length and decrease of the clone diversity.TCR clone size and type have changed in patients with response to ibrutinib,otherwise TCR clone was not obvious in patients with no response.We also found that it was no TRAV7 gene fragment,and elevated frequency of TRAJ25,TRBV5-7,TRBV6-9 and TRBV7-4 gene fragments in AIHA samples.A significant change in frequency of TCR gene fragment occurred after using of ibrutinib.Conclusion:RTX are the effective therapy for newly diagnosed or recurrent patients.We should pay attention to prevent infection within 6 months of treatment.Ibrutinib had presented significant short-term efficacy in relapsed or refractory AIHA patients,and it is still necessary to detect the long-term effect by expanding the sample size and follow-up period.Single cell transcriptome and TCR sequencing showed that DEGs were enriched in T cell activation,protein autophosphorylation and multiple autoimmune-related pathways and decreased of TCR clone diversity in relapsed or refractory AIHA patients before ibrutinib used.Treatment with ibrutinib would partially limit activation of T cell and various autoimmune-related pathways and restore TCR clone diversity.However,ibrutinib exhibited limited efficacy on excessive activation of T cell,dysregulation of Th or Treg cells and reduction of TCR clones diversity in patients with no response to ibrutinib therapy.Therefore,this study not only brings new treatment methods for relapsed or refractory AIHA patients,but also provides novel insights for researchers to design new treatment strategies for patients who failed to targeted B cell therapy.Acute myeloid leukemia(AML)is a genetically heterogeneous hematological malignancy with poor prognosis.We explored the RNA sequence data and clinical information of AML patients from The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)database to search the core molecular for prognosis.The DC-STAMP expression was significantly higher in AML patients,which was linked to older age,unfavorable cytogenetic risk,and death events(all P<0.05).Furthermore,it was revealed that high DCSTAMP expression was an independent unfavorable factor for overall survival(OS)by univariate analysis(hazard ratio[HR]:2.683;95%confidence interval[CI]:1.723-4.178;P<0.001)and multivariate analysis(HR:1.733;95%CI:1.079-2.781;P=0.023).The concordance index(C-index 0.734,95%CI:0.706-0.762),calibration curves,and decision curve analysis curves showed the certain predictive accuracy of nomogram model based on multivariate analysis for OS.In addition,we found the differentially expressed genes(DEGs)enrichment pathways of high-and low-DC-STAMP expression groups enrichment pathways were focused on channel activity and platelet alpha granule by gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),while gene set enrichment analysis(GSEA)pathways mainly involved in mTORC1 signaling,TNF-αsignaling via NF-kB pathway.Moreover,Protein-Protein interaction(PPI)network demonstrated that DC-STAMP interacted with two hub genes(PPBP and PF4),which were highly regulated and associated with worse survival.Finally,high DC-STAMP expression showed significantly positive correlation with four immune cells(NK CD56(dim)cells,Macrophages,Cytotoxic cells and CD8(+)T cells)infiltration and high level of immune checkpoint genes(PDCD1,CD274,CTLA-4 and TIGIT).Therefore,our results suggest that high expression of DC-STAMP predicts adverse outcomes of AML patients.