A new form of familial platelet disorder caused by germline mutations in RUNX1 in a pedigree / 中华内科杂志

Objective: To investigate the clinical and biological characteristics of familial platelet disorder (FPD) with germline Runt-related transcription factor (RUNX) 1 mutations. Methods: Patients diagnosed with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with RUNX1 mutations from February 2016 to December 2021 in Wuhan No.1 Hospital underwent pedigree analysis and were screened for gene mutations (somatic and germline). Patients diagnosed with FPD with germline RUNX1 mutations were enrolled and evaluated in terms of clinical characteristics and biological evolution. Bioinformatics analysis was used to assess the pathogenicity of mutations and to analyze the effect of mutated genes on the function of the corresponding protein. Results: Germline RUNX1 mutations were detected in three out of 34 patients suffering from MDS/AML who had RUNX1 mutations. A pedigree of FPD with RUNX1 (RUNX1-FPD) c.562A>C and RUNX1 c.1415T>C mutations was diagnosed, and the mutations were of patrilineal origin. Bioinformatics analysis indicated that the locus at positions 188 and 472 in the AML-1G type of RUNX1 was highly conserved across different species, and that variations might influence functions of the proteins. The mutations were evaluated to be highly pathogenic. Of the nine cases with germline RUNX1 mutations: two patients died due AML progression; one case with AML survived without leukemia after transplantation of hemopoietic stem cells; four patients showed mild-to-moderate thrombocytopenia; two cases had no thrombocytopenia. During the disease course of the proband and her son, mutations in RUNX1, NRAS and/or CEBPA and KIT appeared in succession, and expression of cluster of differentiation-7 on tumor cells was enhanced gradually. None of the gene mutations correlated with the tumor were detected in the four cases not suffering from MDS/AML, and they survived until the end of follow-up. Conclusions: RUNX1-FPD was rare. The mutations c.562A>C and c.1415T>C of RUNX1 could be the disease-causing genes for the family with RUNX1-FPD, and these mutations could promote malignant transformation. Biological monitoring should be carried out regularly to aid early intervention for family members with RUNX1-FPD.

Analysis of 7 cases of pediatric acute myeloid leukemia with DEK-NUP214 fusion gene / 中华儿科杂志

Objective: To investigate the clinical features, treatment regime, and outcome of pediatric acute myeloid leukemia (AML) with DEK-NUP214 fusion gene. Methods: The clinical data, genetic and molecular results, treatment process and survival status of 7 cases of DEK-NUP214 fusion gene positive AML children admitted to the Pediatric Blood Diseases Center of Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from May 2015 to February 2022 were analyzed retrospectively. Results: DEK-NUP214 fusion gene positive AML accounted for 1.02% (7/683) of pediatric AML diagnosed in the same period, with 4 males and 3 females. The age of disease onset was 8.2 (7.5, 9.5) years. The blast percentage in bone marrow was 0.275 (0.225, 0.480), and 6 cases were M5 by FAB classification. Pathological hematopoiesis was observed in all cases except for one whose bone marrow morphology was unknown. Three cases carried FLT3-ITD mutations, 4 cases carried NRAS mutations, and 2 cases carried KRAS mutations. After diagnosis, 4 cases received IAE induction regimen (idarubicin, cytarabine and etoposide), 1 case received MAE induction regimen (mitoxantrone, cytarabine and etoposide), 1 case received DAH induction regimen (daunorubicin, cytarabine and homoharringtonine) and 1 case received DAE induction regimen (daunorubicin, cytarabine and etoposide). Complete remission was achieved in 3 cases after one course of induction. Four cases who did not achieved complete remission received CAG (aclarubicin, cytarabine and granulocyte colony-stimulating factor), IAH (idarubicin, cytarabine and homoharringtonine), CAG combined with cladribine, and HAG (homoharringtonine, cytarabine and granulocyte colony-stimulating factor) combined with cladribine reinduction therapy, respectively, all 4 cases reached complete remission. Six patients received hematopoietic stem cell transplantation (HSCT) after 1-2 sessions of intensive consolidation treatment, except that one case was lost to follow-up after complete remission. The time from diagnosis to HSCT was 143 (121, 174) days. Before HSCT, one case was positive for flow cytometry minimal residual disease and 3 cases were positive for DEK-NUP214 fusion gene. Three cases accepted haploid donors, 2 cases accepted unrelated cord blood donors, and 1 case accepted matched sibling donor. The follow-up time was 20.4 (12.9, 53.1) months, the overall survival and event free survival rates were all 100%. Conclusions: Pediatric AML with DEK-NUP214 fusion gene is a unique and rare subtype, often diagnosed in relatively older children. The disease is characterized with a low blast percentage in bone marrow, significant pathological hematopoiesis and a high mutation rate in FLT3-ITD and RAS genes. Low remission rate by chemotherapy only and very high recurrence rate indicate its high malignancy and poor prognosis. Early HSCT after the first complete remission can improve its prognosis.

Research Progress of m6A Demethylase FTO and Its Inhibitors in Acute Myeloid Leukemia --Review / 中国实验血液学杂志

Obesity-associated protein (FTO) is an important m6A demethylase that regulates RNA methylation modification and can promote the proliferation of acute myeloid leukemia(AML) cells. FTO regulates the methylation level of AML through multiple cellular signaling pathways such as FTO/RARA/ASB2, FTO/m6A/CEBPA, and PDGFRB/ERK, and participates in the occurrence, development, treatment and prognosis of AML. At present, studies have found that a variety of inhibitors targeting FTO have shown good anti-leukemia effects, and the study of FTO will provide new ideas for the treatment of AML. This review focus on the mechanism of action of FTO in AML and the research progress of FTO inhibitors in AML.

Efficacy and Safety of Decitabine Combined with Modified CAG Regimen in Patients Aged ≥ 70 Years with Newly Diagnosed Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To evaluate the clinical efficacy and safety of decitabine combined with modified CAG regimen (D-CAG regimen) in patients aged ≥70 years with newly diagnosed acute myeloid leukemia (AML).@*METHODS@#The clinical data of 59 AML patients (≥70 years old) who were newly diagnosed and treated in the Hematology Department of the First Affiliated Hospital of Nanjing Medical University from November 2010 to June 2021 were retrospectively analyzed.@*RESULTS@#Among the 59 AML patients, 28 were males and 31 were females, with a median age of 74 (70-86) years. The complete remission (CR) rate was 69.4% (34/49), and the median duration of CR was 10.7 (0.6-125.4) months after 2 courses of D-CAG treatment. According to the British Medical Research Council (MRC) classification, there was only one patient in the favorable-risk group, and the CR rate was 71.8% (28/39) in the intermediate-risk group, and 55.6% (5/9) in the adverse-risk group, respectively. There was no statistical difference in the CR rate between the intermediate-risk and adverse-risk group. Referring to ELN 2017 genetic risk classification, CR rate was 88.2% (15/17) in the favorable-risk group, 45.5% (5/11) in the intermediate-risk group, and 66.7% (14/21) in the adverse-risk group. There was no significant difference in CR rate between the favorable-risk and adverse-risk categories, but both were significantly higher than that in the intermediate-risk group (P <0.05). Next-generation sequencing (NGS) analysis showed that 11 gene mutations with a frequency of more than 10%, including TET2 mutation (35.6%), ASXL1 mutation (30.5%), NPM1 mutation (28.8%), FLT3-ITD mutation (27.1%), DNMT3A mutation (22.0%), IDH1 mutation (15.3%), CEBPA single mutation (13.6%), TP53 mutation (13.6%), IDH2 mutation (11.9%), RUNX1 mutation (11.9%), and NRAS mutation (10.2%). There were no statistical differences in mutation frequency of these 11 genes between CR group and non-CR group. Compared with normal karyotypes, patients with complex karyotypes were more likely to develop TP53 mutations (P <0.001), while FLT3-ITD and DNMT3A mutations were more likely to occur in patients with normal karyotypes (P =0.04, P =0.047). The median follow-up, overall survival (OS), and event-free survival (EFS) of all the patients was 11.7 (1.5-128.2) months, 12.3 (1.5-128.2) months, and 8.5 (1.5-128.2) months, respectively. The median OS and EFS of CR patients were 19.8 and 13.3 months, respectively, which were significantly longer than 6.4 and 5.7 months in patients experiencing treatment failure (P < 0.001, P =0.009). In regard to genes with mutation frequency >10%, there were no statistical differences in CR rate, median OS, and median EFS between mutated and wild-type patients by Chi-square test and survival analysis. Univariate analysis showed that age, hemoglobin, lactate dehydrogenase, cytogenetics and CR were factors affecting prognosis, while multivariate analysis showed that only CR failure was an independent adverse prognostic factor for OS. The major adverse reactions to D-CAG regimen were grade 3-4 myelosuppression, pulmonary infection, and fever (infection focus was not identified).@*CONCLUSION@#D-CAG regimen is safe and effective in the treatment of AML patients ≥70 years old, and can partially improve the prognosis of elderly and high-risk patients.

Research Progress of Next Generation Sequencing in Acute Leukemia --Review / 中国实验血液学杂志

With the advent of precision medicine, next-generation sequencing (NGS) is playing an increasingly important role in clinical oncology diagnosis and treatment with its advantages of high sensitivity, high accuracy, high efficiency and operability. NGS reveals the genetic characteristics of acute leukemia(AL) patients by screening for specific disease-causing genes to identify occult as well as complex genetic mutations in patients with AL, leading to early diagnosis and targeted drug therapy for AL patients, as well as to predict disease recurrence by detecting mnimal residual disease (MRD) and analyzing mutated genes to determine patient prognosis. NGS plays an increasingly important role in the diagnosis, treatment and prognosis assessment in AL, providing a direction for the pursuit of precision medicine. This paper reviews the research progress of NGS in AL.

The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML.@*METHODS@#Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05).@*CONCLUSION@#Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

Molecular features of 109 patients with chronic myelomonocytic leukemia in a single center / 中华血液学杂志

Objective: To explore the molecular features of chronic myelomonocytic leukemia (CMML) . Methods: According to 2022 World Health Organization (WHO 2022) classification, 113 CMML patients and 840 myelodysplastic syndrome (MDS) patients from March 2016 to October 2021 were reclassified, and the clinical and molecular features of CMML patients were analyzed. Results: Among 113 CMML patients, 23 (20.4%) were re-diagnosed as acute myeloid leukemia (AML), including 18 AML with NPM1 mutation, 3 AML with KMT2A rearrangement, and 2 AML with MECOM rearrangement. The remaining 90 patients met the WHO 2022 CMML criteria. In addition, 19 of 840 (2.3%) MDS patients met the WHO 2022 CMML criteria. At least one gene mutation was detected in 99% of CMML patients, and the median number of mutations was 4. The genes with mutation frequency ≥ 10% were: ASXL1 (48%), NRAS (34%), RUNX1 (33%), TET2 (28%), U2AF1 (23%), SRSF2 (21.1%), SETBP1 (20%), KRAS (17%), CBL (15.6%) and DNMT3A (11%). Paired analysis showed that SRSF2 was frequently co-mutated with ASXL1 (OR=4.129, 95% CI 1.481-11.510, Q=0.007) and TET2 (OR=5.276, 95% CI 1.979-14.065, Q=0.001). SRSF2 and TET2 frequently occurred in elderly (≥60 years) patients with myeloproliferative CMML (MP-CMML). U2AF1 mutations were often mutually exclusive with TET2 (OR=0.174, 95% CI 0.038-0.791, Q=0.024), and were common in younger (<60 years) patients with myelodysplastic CMML (MD-CMML). Compared with patients with absolute monocyte count (AMoC) ≥1×10(9)/L and <1×10(9)/L, the former had a higher median age of onset (60 years old vs 47 years old, P<0.001), white blood cell count (15.9×10(9)/L vs 4.4×10(9)/L, P<0.001), proportion of monocytes (21.5% vs 15%, P=0.001), and hemoglobin level (86 g/L vs 74 g/L, P=0.014). TET2 mutations (P=0.021) and SRSF2 mutations (P=0.011) were more common in patients with AMoC≥1×10(9)/L, whereas U2AF1 mutations (P<0.001) were more common in patients with AMoC<1×10(9)/L. There was no significant difference in the frequency of other gene mutations between the two groups. Conclusion: According to WHO 2022 classification, nearly 20% of CMML patients had AMoC<1×10(9)/L at the time of diagnosis, and MD-CMML and MP-CMML had different molecular features.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

MicroRNAs and exosomes: promising new biomarkers in acute myeloid leukemias?

ABSTRACT Despite advances in understanding of carcinogenesis and of treatment of acute myeloid leukemia, this neoplasm still has a lethality of at least 30%. The search for biomarkers that can predict the response to treatment in the early stages of the disease is still necessary. In recent years, a new form of cellular communication between tumor and non-neoplastic cells has been discovered: the exchange of information through extracellular vesicles. These are small vesicles released by membrane-coated cells that carry proteins, lipids, messenger RNAs, microRNA and DNA, which can be internalized and promote biological changes in target cells. Exosomes are qualified as a type of extracellular vesicle and, in tumors, carry immunoinhibitory signals that promote the escape of immune control. Recent studies have showed their involvement in communication with the cells of the tumor microenvironment and with chemoresistance in several tumors. To date, there is no information about immunoregulatory microRNAs transported by exosomes and their correlation with clinical evolution during chemotherapy for acute myeloid leukemia. Knowledge about immunomodulatory microRNAs obtained by leukemic cells and transported by exosomes can direct us towards the design of new diagnostic and treatment tools in this type of leukemia.

Fanconi anemia gene-associated germline predisposition in aplastic anemia and hematologic malignancies / 医学前沿

Whether Fanconi anemia (FA) heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is a crucial but unsettled issue in cancer prevention and family consulting. We retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 patients with aplastic anemia (AA) and hematologic malignancy. Sixty-eight variants were identified in 66 patients (8.38%). FANCA was the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20). Compared with that of the ExAC East Asian dataset, the overall frequency of rare PSVs was higher in our cohort (P = 0.016). BRCA2 PSVs showed higher frequency in acute lymphocytic leukemia (P = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (P = 0.020; P = 0.008). FA-PSV-positive MDS/AML patients had a higher tumor mutation burden, higher rate of cytogenetic abnormalities, less epigenetic regulation, and fewer spliceosome gene mutations than those of FA-PSV-negative MDS/AML patients (P = 0.024, P = 0.029, P = 0.024, and P = 0.013). The overall PSV enrichment in our cohort suggests that heterozygous mutations of FA genes contribute to hematopoietic failure and leukemogenesis.

IRF4 and IRF8 expression are associated with clinical phenotype and clinico-hematological response to hydroxyurea in essential thrombocythemia / 医学前沿

The morbidity and mortality of myeloproliferative neoplasms (MPNs) are primarily caused by arterial and venous complications, progression to myelofibrosis, and transformation to acute leukemia. However, identifying molecular-based biomarkers for risk stratification of patients with MPNs remains a challenge. We have previously shown that interferon regulatory factor-8 (IRF8) and IRF4 serve as tumor suppressors in myeloid cells. In this study, we evaluated the expression of IRF4 and IRF8 and the JAK2V617F mutant allele burden in patients with MPNs. Patients with decreased IRF4 expression were correlated with a more developed MPN phenotype in myelofibrosis (MF) and secondary AML (sAML) transformed from MPNs versus essential thrombocythemia (ET). Negative correlations between the JAK2V617F allele burden and the expression of IRF8 (P < 0.05) and IRF4 (P < 0.001) and between white blood cell (WBC) count and IRF4 expression (P < 0.05) were found in ET patients. IRF8 expression was negatively correlated with the JAK2V617F allele burden (P < 0.05) in polycythemia vera patients. Complete response (CR), partial response (PR), and no response (NR) were observed in 67.5%,10%, and 22.5% of ET patients treated with hydroxyurea (HU), respectively, in 12 months. At 3 months, patients in the CR group showed high IRF4 and IRF8 expression compared with patients in the PR and NR groups. In the 12-month therapy period, low IRF4 and IRF8 expression were independently associated with the unfavorable response to HU and high WBC count. Our data indicate that the expression of IRF4 and IRF8 was associated with the MPN phenotype, which may serve as biomarkers for the response to HU in ET.

Analysis of Gene Mutation Characteristics and Prognosis of Elderly Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the characteristics of gene mutation in elderly patients with acute myeloid leukemia (AML) and its effect on prognosis.@*METHODS@#The clinical and laboratorial characteristics of 54 AML patients (≥60 years old) in Department of Hematology, Tangdu Hospital were analyzed retrospectively during April 2016 to October 2019. Thirty-four AML/myelodysplastic syndrome/myeloproliferative neoplasm related mutant genes were detected by second-generation sequencing technology, and their clinical characteristics, treatment effect, and influence on prognosis were analyzed.@*RESULTS@#All the patients received DAC+CAG induction treatment, after 1-2 couses of treatment, 36 cases (66.7%) achieved complete response, with a total effective rate of 75.9%, and the median survival time was 17 months. The most frequent mutant genes were TET2 (33.3%), CEBPA (31.5%), DNMT3A (18.5%), ASXL1 (16.7%), NRAS (14.8%), RUNX1 (14.8%), FLT3-ITD (12.9%), TP53 (12.9%), NPM1 (12.9%), and IDH2 (12.9%). Among 7 patients with TP53 mutation, 6 cases obtained complete response after 1-2 courses of induction treatment, but there was no statistically significant difference in the effect on prognosis. Patients with FLT3-ITD and NRAS mutations had shorter overall survival time compared with who had no mutation (P=0.47, P=0.48). Multivariate analysis showed that FLT3-ITD and NRAS mutations were poor prognostic factors.@*CONCLUSION@#The incidence of TET2 gene mutation is high in elderly AML patients. AML patients with TET2 and TP53 mutations may benefit from Decitabine-based chemotherapy. However, patients with FLT3-ITD and NRAS mutations have a short survival time, and may have a poor prognosis.

Establishment of Drug-Resistant Cell Lines of Acute Myeloid Leukemia and Correlation of Sirt1 and PGC-1α Expression Levels with Drug Resistance / 中国实验血液学杂志

OBJECTIVE@#To construct cytarabine-resistant acute myeloid leukemia (AML) cell lines, and explore the correlation between Sirt1, PGC-1α expression levels and drug resistance.@*METHODS@#Human acute promyelocytic leukemia Kasumi-1 cells were induced by the method of gradually increasing the concentration of Ara-C drug. The IC50 value of Kasumi-1 cells before and after drug addition was detected by CCK-8 method, so as to construct Ara-C resistant cell lines. The expression levels of Sirt1 and PGC-1α mRNA in Kasumi-1 drug-resistant cell lines and their parental cell lines were detected by real-time fluorescence quantitative PCR, and the expression levels of Sirt1 and PGC-1α protein in kasumi-1 drug-resistant cell lines and their parental cell lines were detected by Western blot.@*RESULTS@#The constructed Kasumi-1 cell line had common morphological characteristics of drug-resistant cell lines under microscope, and the drug resistance index was greater than 5, indicating that Kasumi-1 drug-resistant cells had good drug resistance after the construction. The RT-qPCR and Western blot assays showed that the expression levels of Sirt1 and PGC-1α mRNA and protein in the drug-resistant cell lines were higher than those of the parental cell lines (P<0.001).@*CONCLUSION@#AML cell lines resistant to Ara-C can be successfully induced by the method of gradually increasing the concentration, and the co-high expression of Sirt1 and PGC-1α may mediate the drug resistance of AML cells.

Effects of Paclitaxel and Quizartinib Alone and in Combination on AML Cell Line MV4-11 and Its STAT5 Signal Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of paclitaxel, quizartinib and their combination on proliferation, apoptosis and FLT3/STAT5 pathway of human leukemia cell line MV4-11 (FLT3-ITD+).@*METHODS@#MV4-11 cells were treated with paclitaxel and quizartinib at different concentrations for 24 h, 48 h and 72 h, respectively, and then the two drugs were combined at 48 h to compare the inhibition of proliferation, the apoptosis rate was detected by flow cytometry, the expression of FLT3 and STAT5 mRNA was determined by fluorescence quantitative PCR, and the protein expression of FLT3, p-FLT3, STAT5 and p-STAT5 was determined by Western blot.@*RESULTS@#Different combination groups of paclitaxel and quizartinib had synergistic inhibitory effect. The cell survival rate in the combination group was significantly lower than that in the single drug group (P<0.05). The cell apoptosis rate in the combination group was significantly higher than that in the single drug group (P<0.001). The expression of FLT3 mRNA in combination group was significantly higher than that in two single drugs (P<0.01). The expression of STAT5 mRNA in combination group was significantly higher than that in quizartinib group (P<0.001); increased compared with paclitaxel group, but there was no statistical significance. The expression level of p-FLT3、p-STAT5 protein in the combination group was significantly lower than that in the single drug group (P<0.05, P<0.05).@*CONCLUSION@#Paclitaxel combined with quizartinib can synergistically inhibit the proliferation of MV4-11 cell line and promote the apoptosis of MV4-11 cell line by inhibiting the activity of FLT3/STAT5 pathway.

Characteristics of N6-methyladenosine modification patterns in t(8;21) acute myeloid leukemia / 南方医科大学学报

OBJECTIVE@#To investigate the relationship between AML1-ETO (AE) fusion gene and intracellular N6-methyladenosine (m6A) modification pattern in t(8;21) acute myeloid leukemia (AML).@*METHODS@#RNA m6A sequencing was performed in SKNO-1 and AE knockdown SKNO-1 (SKNO-1 siAE) cells using RNA-protein co-immunoprecipitation and high-throughput sequencing (methylated RNA immunoprecipitation sequencing, MeRIP-Seq) to analyze the changes in m6A modification of the entire transcriptome. Transcriptome sequencing (RNA-seq) was performed using high-throughput sequencing. The differentially modified mRNAs were further functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The changes in m6A-related enzyme expressions were detected using real-time PCR.@*RESULTS@#A total of 26 441 genes were identified in AE knockdown AML cells and AE-expressing cells, containing 72 036 m6A peaks. AE knockdown caused a reduction of the number of intracellular m6A peaks from 37 042 to 34 994, among which 1278 m6A peaks were significantly elevated and 1225 were significantly decreased; 1316 genes with newly emerged m6A modification were detected and 1830 genes lost m6A modification after AE knockdown. The differential peaks were mainly enriched in pathways involving cancer and human T-lymphocytic leukemia virus I. RNA-seq results showed that 2483 genes were up-regulated and 3913 genes were down-regulated after AE knockdown. The combined analysis of MeRIP-Seq and RNA-Seq results revealed relatively high expression levels of m6A-modified genes as compared with the genes without m6A modification (SKNO-1: 0.6116±1.263 vs 2.010±1.655, P < 0.0001; SKNO-1 siAE: 0.5528±1.257 vs 2.067±1.686, P < 0.0001). The m6A modified genes located in the 3'UTR or 5 'UTR had significantly higher expression levels than those located in exonic regions (SKNO-1: 2.177± 1.633 vs 1.333 ± 1.470 vs 2.449 ± 1.651, P < 0.0001; SKNO-1 siAE: 2.304 ± 1.671 vs 1.336 ± 1.522 vs 2.394 ± 1.649, P < 0.05). Analysis of RNA-seq data identified 3 m6A-related enzymes that showed significantly elevated mRNA expression after AE knockdown, namely WTAP, METTL14, and ALKBH5 (P < 0.05), but the results of real-time PCR showed that the expressions of WTAP and ALKBH5 were significantly increased while the expression of METTL14 was lowered after AE knockdown (P < 0.05).@*CONCLUSION@#AE knockdown results in differential expressions of m6A-associated enzymes, suggesting that the AE fusion gene regulates the expression of one or more m6A-associated enzymes to control cellular methylation levels.

Research Advance of Gene Mutation and Targeted Drug Therapy in Childhood Acute Myeloid Leukemia --Review / 中国实验血液学杂志

The clinical therapeutic regimen for acute myeloid leukemia (AML) is not significantly different between adults and children, which is mostly based on IA (idarubicin and cytosine arabinoside) induction chemotherapy. With the rapid development of sequencing technique, people's understandings towards the molecular and biological abnormalities of AML are increasing, diverse AML gene mutation-based targeted drugs have been rapidly developed and applied. In this review, several commonly gene mutations in AML (such as FLT3, NPM1 and C/EBPA) was described, and the therapeutic effects and differences of targeted drugs that used in clinical treatment or had been reported (like tyrosine kinase inhibitor, IDH1 mutation inhibitor and epigenetic modification inhibitor) in child and adult AML patients were summrized.

Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with inv(16)/t(16;16)(p13.1;q22) and/or CBFβ-MYH11 / 中国实验血液学杂志

OBJECTIVE@#To summarize the clinical and laboratory characteristics of patients with acute myeloid leukemia (AML) with inv(16)/t(16;16) (p13.1;q22), and to analyze the risk factors affecting the prognosis of the patients.@*METHODS@#AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFβ-MYH11+ admitted to the Department of Hematology, The First Affiliated Hospital of Soochow University from January 1, 2008 to October 30, 2019 were retrospective analyzed, the clinical and laboratory indicators, as well as treatment plans and efficacy evaluations of the patients were all recorded. Furthermore, related factors affecting the overall survival (OS) and event-free survival (EFS) of the patients were analyzed.@*RESULTS@#Among 151 AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFβ-MYH11+, the percentage of additional chromosomal abnormalities was about 27.8%, and the most common additional chromosomal abnormality was +22 (33/151, 21.8%), followed by +8 (11/151, 7.3%). There were 112 patients with perfect NGS examination, and the result showed the most common accompanying gene mutations were KIT mutation (34/112, 30.4%) and FLT3 mutation (23/112, 20.5%). Univariate analysis showed that factors affecting EFS included: NE≤0.5×109/L (P=0.006) and combined K-RAS mutation (P=0.002); Factors affecting OS included: Age≥50 years old (P<0.001) and NE≤0.5×109/L (P=0.016). Multivariate analysis showed that NE≤0.5×109/L (P=0.019) was the risk factors affecting OS. The proportion of bone marrow eosinophilia (BME)≥10.00% (P=0.029) was the risk factors affecting EFS.@*CONCLUSION@#The prognosis for those newly diagnosed AML patients who were of advanced age, the high proportion of bone marrow eosinophils, K-RAS mutations, and agranulocytosis is poor. The treatment plans can be adjusted in the early stage to improve the prognosis of such patients.

Analysis of Coexisting Gene with NRAS in Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the coexisting mutations and clinical significance of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in acute myeloid leukemia (AML) patients.@*METHODS@#High-throughput DNA sequencing and Sanger sequencing were used to detect 51 gene mutations. The occurrence, clinical characteristics and treatment efficacy of coexisting genes with NRAS were investigated.@*RESULTS@#A total of 57 NRAS mutations (17.5%) were detected in 326 patients with AML. Compared with the patients in NRAS non-mutation group, patients in the mutant group were younger (P=0.018) and showed lower platelet count (P=0.033), but there was no significant difference in peripheral leukocyte count, hemoglobin, and sex. For FAB classification, NRAS mutation and M2 subtype showed mutually exclusive (P=0.038). Among 57 patients carried with NRAS mutation, 51 (89.5%) patients carried with other gene mutations, 25 (43.9%) carried with double gene mutations, 10 (17.5%) carried with 3 gene mutations, and 16 (28.1%) corried with ≥ 4 gene mutations. The most common coexisting gene mutation was KRAS (24.6%, 14/57), followed by FLT3-ITD (14.0%, 8/57), RUNX1 (12.3%, 7/57), NPM1 (10.5%, 6/57), PTPN11 (10.5%, 6/57), DNMT3A (10.5%, 6/57) and so on. The age (P=0.013, P=0.005) and peripheral platelet count (P=0.007, P=0.021) of patients with NPM1 or DNMT3A mutations were higher than those of the patients with wild type, but there was no significant difference in peripheral leukocyte count and hemoglobin. Also, there was no significant difference in age, peripheral leukocyte count, hemoglobin, and peripheral platelet count between the patients in KRAS, FLT3-ITD, RUNX1 or PTPN11 mutant group and the wild group. Patients with FLT3-ITD mutations showed a lower complete remission (CR) rate (P=0.044). However, there was no significant difference in CR rate between the patients with KRAS, NPM1, RUNX1, PTPN11 or DNMT3A mutations and the wild group. The CR rate of the patents with single gene mutation, double gene mutations, 3 gene mutations, and≥ 4 gene mutations were decreased gradually, and there was no significant difference in CR rate between pairwise comparisons.@*CONCLUSION@#The mutation rate of NRAS mutation is 17.5%, 89.5% of AML patients with NRAS mutation coexist with additional gene mutations. The type of coexisting mutations has a certain impact on clinical characteristics and CR rate of patients with AML.

Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML / 中国实验血液学杂志

OBJECTIVE@#Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.@*METHODS@#The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.@*RESULTS@#In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.@*CONCLUSION@#The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Establishment and Validation of Immune Risk Score for Predicting Survival of Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To establish an immune gene prognostic model of acute myeloid leukemia (AML) and explore its correlation with immune cells in bone marrow microenvironment.@*METHODS@#Gene expression profile and clinical data of TCGA-AML were downloaded from TCGA database. Immune genes were screened by LASSO analysis to construct prognosis prediction model, and prediction accuracy of the model was quantified by receiver operating characteristic curve and area under the curve. Survival analysis was performed by Log-rank test. Enriched pathways in the different immune risk subtypes were evaluated from train cohort. The relationship between immune prediction model and bone marrow immune microenvironment was verified by flow cytometry in the real world.@*RESULTS@#Patients with low-risk score of immune gene model had better prognosis than those with high-risk score. Multivariate analysis showed that the immune gene risk model was an independent prognostic factor. The risk ratio for AML patients in the training concentration was HR=24.594 (95%CI: 6.180-97.878), and the AUC for 1-year, 3-year, and 5-year overall survival rate was 0.811, 0.815, and 0.837, respectively. In addition, enrichment analysis of differential gene sets indicated activation of immune-related pathways such as cytokines and chemokines as well as autoimmune disease-related pathways. At the same time, real world data showed that patients with high immune risk had lower numbers of CD8+T cells and B lymphocytes compared with low immune risk patients.@*CONCLUSION@#We constructed a stable prognostic model for AML, which can not only predict the prognosis of AML, but also reveal the dysregulation of immune microenvironment.