Development and application of a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain of acute myeloid leukemia / 中国生物制品学杂志

@#ObjectiveTo develop a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain(FLT3-TKD)of acute myeloid leukemia(AML)and apply to the monitor of minimal residual disease(MRD).MethodsRecombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed respectively and mixed at certain ratios.The obtained standard plasmids with mutation rates of 50%,1%,0.1% and 0% respectively were determined by restriction fragment length polymorphism(RFLP)in combination with Sanger method.The plasmid DNA standards and blood DNA standards,at various FLT3-D835Y mutation rates,were determined by the developed method to verify the sensitivity.The genomic DNA samples of patients with AML before and after treatment were determined by the developed method to monitor the MRD.ResultsSequencing proved that both the recombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed correctly.The sensitivity of developed method increased to 0.1% through Sanger method combined with digestion with EcoR Ⅴ/Xho Ⅰ and recovery of mutant fragments in determination of purified plasmid DNA and collected blood DNA samples.MRD was detected in the peripheral blood sample of a patients with AML in complete remission period by the developed method but not by Sanger method.ConclusionA highly sensitive method for detection of FLT3-TKD mutation was developed,which was of an important clinical significance in guiding the treatment of AML and monitoring the MRD in complete remission period.

A combination of lenalidomide, bortezomib, and dexamethasone (RVD) provides deep-er responses in patients with newly diagnosed multiple myeloma / 中国肿瘤临床

Objective: This study investigated the efficacy and safety of a combination of lenalidomide, bortezomib, and dexametha-sone (RVD) in patients with newly diagnosed multiple myeloma (NDMM). Methods: The clinical features and responses of 48 patients with NDMM who were treated with RVD from January 2015 to May 2019 in Beijing Chaoyang Hospital were retrospectively analyzed. Results: The median age of the 48 patients was 59 years (range: 34-79). Among these, 44 patients were Durie-Salmon stageⅢ, 15 were ISS stageⅡ, 19 were ISS stageⅢ, and 12 had plasmacytoma; 32.5% of all patients were cytogenetic high-risk. All patients re-ceived a median of four cycles (range: 1-9) of the RVD regimen as induction treatment. The overall response rate was 97.9%, with 35.4% of patients achieving complete response (CR) or better. The rate of very good partial remission (VGPR) or better was increased from 64.1% (after two cycles) to 84.6% (after four cycles). The mean collection of CD34+cells was 4.2 (± 2.6)×106/kg. Negative minimal residual disease (MRD), as indicated by next-generation flow (NGF), was achieved in 20.6% of patients after induction. Two patients with positive MRD after induction became MRD negative after transplantation. Two patients developed grade 3 or 4 hematologic toxic-ity. No nonhematologic toxicity of grade 3 or 4 was observed. Conclusions: In patients with NDMM, RVD treatment resulted in signifi-cantly improved response rates and exhibited an acceptable risk-benefit profile, with no adverse impact on stem cell collection. RVD combined with transplantation significantly improved the negative rate of MRD, as indicated by NGF.

Expression of RUNX1 isoformsand its clinical significance in acute leukemia / 中国肿瘤生物治疗杂志

@# Objective: : To investigate the relationship between the expression of RUNX1 isoforms and the clinical curative effect and the prognosis of acute leukemia (AL), in order to provide valuable experimental data for the individualized treatment, MRD (minimal residual disease) monitoring and prognosis prediction of AL. Methods: AL patients with primary treatment (PT, n=88) and recrudescence (RC, n=10) that treated at the Department of Hematology of Affiliated Hospital of Guangdong Medical University from April, 2012 to April, 2013 were included in this study. Real-time PCR was used to examine the mRNA expression of RUNX1 isoforms (RUNX1a and RUNX1b/c) in PT patients, RC patients and controls (non-malignant hematological disease patients).The changes in mRNA expression of RUNX1a and RUNX1b/c in patients before and after the chemotherapy were also observed. Results： ： (1)The expression levels of RUNX1a mRNAinAML andALL PT group andAML RC group was significantly higher than those of control group (P<0.05); The expression level of RUNX1a mRNAinAMLPT group was increased compared withALLPT group (P<0.05). (2) The expression levels of RUNX1a and RUNX1b/c mRNAinAML andALL patients at initial treatment were significantly higher than those after complete remission (CR) (P<0.05). (3) By comparing the expression levels of RUNX1a and RUNX1b/c mRNA at initial diagnosis, there was no significant difference between 6-month death group and survival group, CR group and NCR (non-complete remission) group after first cycle of chemotherapy, or the high leukocyte group and non-high leukocyte group (all P>0.05).The expression level of RUNX1a mRNA in AML-ETO positive group was higher than that of negative group (P<0.05). (4) The expression levels of RUNX1a and RUNX1b/c mRNA in patients with acute leukemia decreased with the increasing chemotherapy cycle, and significantly increased when had a relapse, which may even succeed the initial level.Conclusion: RUNX1a isoforms participate in the pathogenesis of acute leukemia, and isrelated to the relapse ofAML. The expression levels of RUNX1a and RUNX1b/c mRNAare related to the clinical efficacy that can be used as an indicator of curative effect, but have no significant correlation with the prognosis of the disease.Dynamic monitor of theexpression levels of RUNX1a and RUNX1b/c isomers can be used as an effective indicator of MRD monitoring after chemotherapy, which can be used to evaluate the efficacy and identify the risk of recurrence at early stage.

Efficacy of autologous hematopoietic stem cell transplantation on the treatment of patients with acute myeloid leukemia / 器官移植

Objective To observe the efficacy of autologous hematopoietic stem cell transplantation on the treatment of patients with acute myeloid leukemia (AML) in complete remission stage. Methods Clinical data of 14 AML patients underwent autologous hematopoietic stem cell transplantation were analyzed retrospectively, including 7 low-risk patients, 6 moderate-risk patients and 1 high-risk patient. After pretreatment, pre-cryopreserved autologous peripheral blood stem cells were retransfused. And component blood transfusion, increasing white blood cell (WBC) count and preventing from infection, etc. were given. Hematopoietic reconstitution of autologous stem cells in the patients was observed, and incidence of transplantation related complications was obtained. Furthermore, survival curves were drawn, and postoperative 1- and 3-year overall survival rates and disease-free survival (DFS) rates were calculated. Results Hematopoietic reconstitution was achieved in all 14 patients. The median time of WBC implantation was 12(9-28) d, and that of platelet implantation was 29(8-158) d. Two patients suffered from E. coli septicemia during neutropenia stage, 1 from proteus vulgaris septicemia, 1 from cytomegalovirus viremia within 29 d after transplantation and the remaining from infection or gastrointestinal reaction after pretreated. All patients were cured by anti-infection and other symptomatic relief and supportive treatment. All patients were followed up for 29.8(5.3-61.5) months. In 14 patients, 5 cases recurred. 11 patients survived and 3 died of recurrence. The postoperative 1- and 3-year overall survival rates were 86％ and 79％, and the postoperative 1- and 3-year DFS rates were 64％ and 57％. Conclusions Autologous hematopoietic stem cell transplantation is effective in the treatment of majority patients with low- or moderate-risk AML.

Minimal residual disease detection in Tunisian B-acute lymphoblastic leukemia based on immunoglobulin gene rearrangements

IGH gene rearrangement and IGK-Kde gene deletion can be used as molecular markers for the assessment of B lineage acute lymphoblastic leukemia (B-ALL). Minimal residual disease detected based on those markers is currently the most reliable prognosis factor in B-ALL. The aim of this study was to use clonal IGH/IGK-Kde gene rearrangements to confirm B-ALL diagnosis and to evaluate the treatment outcome of Tunisian leukemic patients by monitoring the minimal residual disease (MRD) after induction chemotherapy. Seventeen consecutive newly diagnosed B-ALL patients were investigated by multiplex PCR assay and real time quantitative PCR according to BIOMED 2 conditions. The vast majority of clonal VH-JH rearrangements included VH3 gene. For IGK deletion, clonal VK1f/6-Kde recombinations were mainly identified. These rearrangements were quantified to follow-up seven B-ALL after induction using patient-specific ASO. Four patients had an undetectable level of MRD with a sensitivity of up to 10-5. This molecular approach allowed identification of prognosis risk group and adequate therapeutic decision. The IGK-Kde and IGH gene rearrangements might be used for diagnosis and MRD monitoring of B-ALL, introduced for the first time in Tunisian laboratories.

Minimal Residual Disease Status in Childhood Acute Lymphoblastic Leukaemias by Flow Cytometry and Their Clinical and Haematological Features

Residual disease in patients with acute leukaemia indicates unfavorable prognosis. The evaluation of remission using flow cytometry allows a better estimation of minimal residual disease (MRD) after induction chemotherapy in childhood acute lymphoblastic leukaemia (ALL) cases. Patients in morphological marrow remission with presence of blast cells of less than 5%, may still have up to 1010 leukaemic cells. However with flow cytometric analysis, lower levels of the residual leukaemic cells (1 in 104 cells) can be detected and it can be used as a tool to predict relapse. This study compared the presenting clinical and haematological features of children with ALL and their residual disease status determined by flow cytometry. Analysis of their MRD status following remission-induction chemotherapy were done at day-28, week-12 and week-20. The cases were also followed up to five years, to determine their survival status. Their residual disease status by flow cytometric immunophenotyping was also compared with their bone marrow findings morphologically. Thirty-eight cases of precursor B-ALL in pediatric patients from UKM Medical Centre (UKMMC) were analyzed. There was no significant correlation between demographic, clinical and haematological features with MRD status at day-28. However, there was a significant correlation between MRD status by flow cytometry and by morphological marrow examination at week-12. Three cases showed persistent MRD findings until week-20 where two of the cases relapsed and died subsequently. Twenty four patients were still alive after five years of follow up.

Evaluation of the detection of PML-RARα fusion gene in acute promyelocytic leukemia to monitor minimal residual disease / 白血病·淋巴瘤

Objective To investigate the kinetics of PML-RARα fusion gene in acute promyelocytic leukemia(APL)to monitor minimal residual disease(MRD). Methods In induction therapy,consolidation and maintenance therapy courses, PML-RARα fusion gene was performed by RT-PCR. Results The long-term follow-up of 18 cases achieved complete remission (CR),two cases experienced molecular relapse. One case relapsed at 4 months after CR1 and achieved CR2 after induction therapy. However, molecular and hematology relapsed again at 2 months after CR2 and re-achieved CR3. The other case relapsed at 74 months after CR1 and achieved CR2 after induction treatment, who had survived for 106 months until the end of follow-up. Conclusion RT-PCR assay for detection of PML-RARα should be performed regularly during CR period so as to find molecular relapse eady. Hematological relapse could potentially be averted through treatment modification according to molecular monitoring results of PML-RARα.