| Objective:1.212 newly diagnosed acute promyelocytic leukemia(APL)patients who are hospitalized in the Department of Hematology and Pediatrics of the First Affiliated Hospital of Nanchang University from May 2005 to December 2019 are selected to analyze the relationship between clinical laboratory characteristics and prognosis of APL patients.2.The possible fusion genes in variant APL with negative PML-RARA fusion gene is analyzed,then explore the clinical laboratory characteristics and prognosis of patients of variant APL.3.The cytology and animal experiments are used to study the leukemic mechanism of CPSF6-RARG fusion gene and provide a model for research drug screening.Methods:1.212 newly diagnosed acute promyelocytic leukemia(APL)patients who were hospitalized in the Department of Hematology and Pediatrics of the First Affiliated Hospital of Nanchang University from May 2005 to December 2019 were selected,the clinical data and laboratory indicators of the patients were collected to analyze the relationship between clinical features such as immunophenotype characteristics,fluorescence in situ hybridization(FISH)fluorescence signal pattern characteristics,chromosomal karyotype characteristics,and FLT3-ITD mutations of APL patients and clinical prognosis.According to immunophenotype,it was divided into cross-line expression group and non-cross-line expression group.According to FISH fluorescence signal pattern,it was divided into typical fusion signal pattern group and complex signal pattern group.According to the characteristics of chromosome karyotype,it was divided into separate t(15;17)group and atypical chromosomal karyotype groups(including atypical translocations,other chromosomal abnormalities,and complex chromosomal translocations),according to the FLT3-ITD mutations,they were divided into mutation-positive and mutation-negative groups.Graph Padprism 5 statistical software was used for statistics analysis,unpaired t-test(normal distribution)or Mann-Whitney U test(non-normal distribution)were used to explore the relationship between each group and the laboratory indicators of APL patients.The chi-square test was adopted to analyze the relationship between each group,P<0.05 indicated that the difference was statistically significant.2.Three variant APL patients who were negative for the PML-RARA fusion gene found in the analysis were taken as the research objects,and 16 myeloid leukemia fusion genes were screened.Bone marrow mononuclear cells of the patients with negative fusion genes were further screened by high-throughput gene sequencing technology undergoing transcriptome sequencing.Defuse software was used to analyze the gene fusion sequence in the transcriptome data,reverse transcription-polymerase chain reaction(RT-PCR)and Sanger sequencing were adopted to verify the fusion gene with clear pathological significance,and to summarize the clinical laboratory characteristics and prognosis of variant APL patients.3.APL cell line NB4 cells were infected with CPSF6-RARG lentivirus to verify the effect of CPSF6-RARG on the differentiation of NB4 cells induced by ATRA.CPSF6-RARG was expressed in mouse precursor cell 32Dcl-3 cells,and the effects of CPSF6-RARG on the proliferation,apoptosis and differentiation of 32Dcl-3 cells were analyzed;Overexpressed CPSF6-RARG in P53 loss mouse bone marrow stem cells,and then transplanted for mice of the same strain,the pathogenic effect of CPSF6-RARG was analyzed by bone marrow cell morphology analysis,flow cytometry analysis,PCR technology and WB technology after the mice developed disease.Results:1.The relationship between clinical laboratory characteristics and prognosis of212 APL patientsAmong the 212 newly diagnosed APL patients,111 were males,101 were females.The ratio of male to female was 1.10:1,and the median age was 43 years(3-87 years).There were 191 patients in the immunophenotyping group,and 29 cases in the cross-line expression group,accounting for 15.18%,of which 16 were CD2 positive patients,accounting for 55.17% of the cross-line expression group and8.38% of the immunophenotyping group.White blood cell counts(WBC,P<0.0001),prothrombin time(PT,P=0.03),and the proportion of patients in the NCCN prognosis stratified into the high-risk group(P=0.0009)of the cross-lineage expression group were significantly higher and were significantly correlated to FLT3-ITD mutation(P=0.004).The age,hemoglobin(Hb),platelet count(PLT),activated partial thromboplastin time(APTT),fibrinogen(Fbg),D-dimer,albumin(Alb),and lactate dehydrogenase(LDH)of the two groups of patients had no significant differences,and they were not related to FISH fluorescence signal patterns and chromosome karyotype characteristics.There were 179 patients in the FISH fluorescence signal pattern group,159 patients in the typical fusion signal pattern group,accounting for88.83%,and 20 patients in the complex signal pattern group,accounting for 11.17%.Patients in the complex signal pattern group were significantly related to their karyotype characteristics(P=0.02).There was no significant difference in age,WBC,Hb,PLT,PT,APTT,Fbg,D-Dimer,Alb,LDH between the two groups of patients,and they were not related to the immunophenotypic characteristics,FLT3-ITD mutations,and NCCN prognosis stratification.There were 113 patients in the karyotype analysis group,of which 80 patients were in separate t(15;17)group,accounting for 70.80%.There were 33 patients in the atypical karyotype group(including atypical translocations,other chromosomal abnormalities,and complex chromosomal translocations),accounting for 29.20%.Patients in the atypical karyotype group were significantly related to their complex signal patterns(P=0.02).The age,WBC,Hb,PLT,PT,APTT,Fbg,D-Dimer,Alb,and LDH were not significantly different,and were not related to immunophenotypic characteristics,FLT3-ITD mutations and NCCN prognostic stratification.There were 65 patients in the FLT3-ITD mutation group,of which 17 were mutation-positive,accounting for26.15%,and 48 were mutation-negative,accounting for 73.85%.FLT3-ITD mutation-positive patients had WBC counts(P=0.0004)and ratio of NCCN prognosis stratified into high-risk groups(P <0.0001),both significantly increased,and were significantly related to immunophenotypic characteristics(P=0.004).There were no significant differences in age,Hb,PLT,PT,APTT,Fbg,D-Dimer,Alb,and LDH between the two groups,and they were not related to FISH fluorescence signal patterns and chromosomal karyotype characteristics.2.All 3 variant APL patients were diagnosed as acute promyelocytic leukemia by clinical manifestations,bone marrow cell morphology,immunology and cytochemical staining,but fluorescence in situ hybridization detected PML-RARA fusion gene negative,RT-PCR detection of 16 myeloid fusion genes found that patient 1 was positive for STAT5b-RARA,the other fusion genes were negative,while patients 2 and 3 were negative for 16 myeloid fusion genes.Through futher transcriptome sequencing analysis,it was found that patient 2 and patient 3 habored the rare fusion genes CPSF6-RARG and NUP98-RARG with clear pathological significance,respectively.Patient 1 was treated with all-trans retinoic acid(ATRA)to induce differentiation but failed,and achieved complete remission after treatment with demethoxydaunorubicin and cytarabine(IA regimen),and then undergo hematopoietic stem cell transplantation,in a state of continuous remission.Patients 2and 3 were not responded to all-trans retinoic acid(ATRA)induction differentiation therapy and standard AML 3+7 chemotherapy regimens.Patient 2 was administrated to homoharringtonine combined with cytarabine(HHT+A-arc)chemotherapy and was in a state of continuous remission.Patient 3 died of cerebral hemorrhage after treatment failed.3.The CPSF6-RARG lentiviral expression plasmid was successfully constructed,and CPSF6-RARG was found to be mainly located in the nucleus by infecting NB4 cells.Further experiments found that overexpression of RARG and CPSF6-RARG in NB4 cells could inhibit ATRA-induced CD11 b expression.Overexpression of CPSF6-RARG and RARG in 32Dcl-3 cells could significantly inhibit the cell growth inhibition caused by IL3 deprivation,and could inhibit the apoptosis induced by IL3 deprivation,but it had no effect on the growth of 32Dcl-3 cells in the presence of IL3.In addition,overexpression of CPSF6-RARG and RARG could significantly inhibit the differentiation of 32Dcl-3 cells induced by G-CSF.Overexpression of CPSF6-RARG in P53-deficient bone marrow hematopoietic stem cells could lead to leukemia in mice.PCR could detect that the bone marrow cells of the diseased mouse express CPSF6-RARG,and there was also P53 loss,which could also be seen in mouse thymus,but there was no CPSF6-RARG gene.Western Blot(WB)showed the presence of CPSF6-RARG fusion protein in mouse bone marrow cells,but no expression of CPSF6-RARG fusion protein was seen in mouse thymus and control mice.Cell morphology analysis showed that the characteristics of mouse bone marrow leukemia cells were similar to myeloid precursor cells.Flow cytometry analysis found that these cells mainly expressed CD34,Sca-1,CD117,partly expressed Gr-1,and did not express B and T lymphocyte antigens CD3,CD19,B220.It showed that CPSF6-RARG could cause AML in mice in the absence of P53.Conclusion:1.The APL immunophenotype may express across lines and the cross-line expression CD2 was the most common.The cross-line expression was significantly correlated with WBC,PT,NCCN prognosis stratification and FLT3-ITD mutation.The karyotypes and FISH signaling patterns of APL patients were not significantly correlated with clinical and laboratory characteristics.FLT3-ITD mutation was significantly correlated with WBC,NCCN prognosis stratification and immunophenotypic characteristics,and was an indicator of poor prognosis in patients.2.The clinical phenotype,bone marrow cell morphology and immunological characteristics of PML-RARA fusion gene negative variant APL were highly similar to APL with PML/RARA,but its pathogenesis was different,and its sensitivity to ATRA and ATO treatment as well as the prognosis were also different.Transcriptome sequencing could accurately analyze rare fusion genes that may exist in patients,aiming to provide experimental support and theoretical basis for further typing and precise treatment of APL.3.Overexpression of RARG and CPSF6-RARG could inhibit the differentiation of NB4 cells induced by ATRA.The growth inhibition and apoptosis of 32Dcl-3 cells caused by IL3 deprivation could be inhibited,and the effect of G-CSF on the differentiation and maturation of 32Dcl-3 cells was also inhibited.Simultaneous expression of CPSF6-RARG combined with P53 deficiency could lead to AML phenotype in mice. |
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