1. The Role Of CD19 Epitope Deletion In BiTE Therapy For Immune Escape. 2. The Role Of The Combined Effect Of NPM1 Mutation And IDH1 Mutation In The Development Of AML

Objective:One CD19+B-ALL patient did not achieve remission after BiTE treatment,and flow cytometry revealed that CD 19 antigen turned negative.Despite of the disease progression,this patient has achieved complete remission by sequential infusions of our own developed CD22 CAR-T and CD 19 CAR-T.The purpose of this study is to explore the expression of CD 19 isoforms and the changes in lineage markers before and after BiTE treatment in this patient,and to understand the response of CD 19 isoforms to BiTE treatment,and to further explore the mechanism and solutions to BiTE immunotherapy of resistance.Methods:Flow cytometry was performed for the determination of leukemia-associated immune phenotype(LAIPs)and B-ALL minimal residual disease(MRD).The proportion of leukemia cells and the expression levels of CD 19 antigen were determined.Total RNA from bone marrow mononuclear cells was extracted and reverse-transcribed into cDNA.Semi-quantitative RT-PCR(qRT-PCR)was used to detect the expression of CD 19 mRNA isoforms before and after BiTE treatment.CD 19 isoforms were analyzed by Sanger sequencing.RT-PCR was used to identify the level of CD 19 mRNA in bone marrow specimens,and primer pairs specific for different exon regions were designed for CD 19 expression.Specific primers spanning exons 1 and 3 were used to amplify exon 2 deleted CD 19 isoform.Transcriptome sequencing was performed to analyze the expression of bone marrow cell lineage-specific molecules,differentially expressed genes involved in alternative splicing,and the expression of different CD19 transcripts before and after BiTE treatment.Results:Flow cytometry before BiTE treatment revealed that abnormal cells occupied 60.51%of nuclear cells and CD19 was positive;on the 11th day after the first period of BiTE infusion,there was an increase in leukocytes and peripheral blood naive cells,and a morphological relapse in this patient.Flow cytometry showed that abnormal cells increased to 80.43%,and was CD79a,CD 10 positive,CD34 low,CD 19 negative,CD22,CD20,CD38,and CD45 dim,which was found to be abnormal B lymphoblasts.This patient did not achieve remission after BiTE treatment,and flow cytometry revealed that CD 19 antigen turned negative.Exon 1-5 of CD 19 cDNA were amplified by semiquantitative RT-PCR in bone marrow samples before and after treatment and were visualized by agarose gel electrophoresis.The lengths of the amplified bands were 800bp,533bp,and 670bp,respectively,consistent in size with the full length CD 19,partial deletion of exon 2 and the isoform lacking exon2;Sanger sequencing further verified the sequence of purified products of the three bands.CD19 isoform lacking exon 2 exist before BiTE treatment.Amplifying exon 4-8 in our samples resulted in a single 490bp band,which was confirmed by sanger sequencing.Skipping of exon 5 and 6 is not expressed at the time of diagnose.CD 19 mRNA levels were found to be downregulated about 2 to 3 fold depending on the choice of primers?Transcriptome sequencing showed that the expression level of two CD 19 protein encoding transcripts decreased approximately 1 to 2 times after treatment.CD 19 was not screened in the differentially spliced genes(FDR<0.05),and the expression of exon 2 deleted CD 19 isoform did not accumulate after treatment.Transcriptome sequencing was used to analyze the expression of lineage-specific molecules in bone marrow cells before and after treatment.The expression of CD 19 decreased after BiTE treatment,and we found no increase in the expression of myeloid line specific molecules CD33,CD123,and CD117.Conclusions:The expression of CD 19 isoform with exon 2 deletion was found at diagnosis before BiTE therapy.The patient did not achieve remission after BiTE treatment,and the expression of CD 19 antigen turned negative by flow cytometry detection.But the expression ratio of exon 2 deleted CD 19 was not increased,and the flow cytometry phenotype and transcriptome sequencing confirmed that no linage switching occurred,which suggested the expression of CD 19 isoform caused by exon alternative splicing and lineage switching was not the driving mechanism of CD 19 epitope loss in this patient.Immune escape could not be prevented by targeting alternative exons.This patient achieved complete remission by sequential infusions of our own developed CD22 CAR-T and CD 19 CAR-T after disease progression.Targeting another antigen or a combination of CAR approach may be a promising treatment option.Objective:NPM1 is one of the most common recurrently mutated genes in AML.NPM1 mutations often co-occur with IDH1 mutation.It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis.In this study,we investigate the potential role of IDH1 and NPM1 mutation in the pathogenesis of AML in vivo.Meanwhile we try to find the possible mechanisms in this co-existing mutation by determining its biological function of primary mouse c-kit cells.Methods:Construction of pMSCV-NPMcA-myc-GFP,pMSCV-IDH1R132H-flag-GFP and a combined mutation retroviral vector pMSCV-IDH1R1 32H-T2A-NPMcA-GFP.And transfect the 293T packaging cells with co-mutation plasmid to produce retroviruses,which were then used to transduce mouse bone marrow c-kit+cells.Western blot were used to identify the expression of mutant gene.C-kit+ cells from mice bone marrow were purified with anti-CD117 microbeads and then infected with retroviral supernatant.The proliferation ability was measured by colony formation assay.For differentiation assay in vitro,the GFP+c-kit+ cells were plated and characterized by analyzing the myeloid markers(CD11b and GR-1)and erythroid-specific antigen.The ratio of G0 phase was detected by flow cytometry after Ki67 and 7-AAD staining.For the in vivo experiment,1×105 GFP+c-kit+cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an AML mouse model.Numbers of GFP+cells in peripheral blood as well as general condition of mice were monitored regularly.Results:The pMSCV-NPMcA-myc-GFP,pMSCV-IDH1R132H-flag-GFP and pMSCV-IDH1R132H-T2A-NPMcA-GFP retroviral plasmid was successfully constructed.The expressing of mutant gene was confirmed at mRNA and protein level.Colony formation assay showed that compared with the control group,clonogenic potential of c-kit cells expressing combined mutant gene did not increase.What's more,the differentiation experiment indicated that co-mutation may had no significant effect on the differentiation of the first generation of colony cells.Although the mutation may to some extent lead to an increase in the proportion of c-kit cells in the G0 phase,leading to more c-kit cells in the stationary phase,the increase in the proportion is not significant.In vivo experiments showed that no GFP+cells were detected in the co-mutation group during the observation period,and development of leukemia was not observed in any of the mice in both experimental and control group.Conclusions:In vitro experiments,the combined mutation of NPM1 and IDH1 had no significant effect on colony-formation,differentiation ability,and cell cycle of mouse c-kit+,and it could not induce leukemia and any other hematological diseases in vivo.