Acute Megakaryocytic Leukemia Research Articles

Acute megakaryocytic leukemia with acquired trisomy 21 and structural chromosomal rearrangements in a young child

Acute megakaryocytic leukemia with acquired trisomy 21 and structural chromosomal rearrangements in a young child

Clinical Analysis of Pediatric Acute Megakaryocytic Leukemia With CBFA2T3-GLIS2 Fusion Gene.

CBFA2T3-GLIS2 is the most frequent chimeric oncogene identified to date in non-Down syndrome acute megakaryocytic leukemia (AMKL), which is associated with extremely poor clinical outcome. The presence of this fusion gene is associated with resistance to high-intensity chemotherapy, including hematopoietic stem cell transplantation (HSCT), and a high cumulative incidence of relapse frequency. The clinical features and clinical effects of China Children's Leukemia Group-acute myeloid leukemia (AML) 2015/2019 regimens and haploidentical HSCT (haplo-HSCT) for treatment of 6 children harboring the CBFA2T3-GLIS2 fusion gene between January 2019 and December 2021 were retrospectively analyzed. The 6 patients included 4 boys and 2 girls with a median disease-onset age of 19.5 months (range: 6-67mo) who were diagnosed with AMKL. Flow cytometry demonstrated CD41a, CD42b, and CD56 expression and lack of HLA-DR expression in all 6 patients. All the children were negative for common leukemia fusion genes by reverse transcription polymerase chain reaction, but positive for the CBFA2T3-GLIS2 fusion gene by next-generation sequencing and RNA sequencing. All patients received chemotherapy according to China Children's Leukemia Group-AML 2015/2019 regimens, and 4 achieved complete remission. Four children underwent haplo-HSCT with posttransplant cyclophosphamide-based conditioning; 3 had minimal residual disease negative (minimal residual disease <0.1%) confirmed by flow cytometry at the end of the follow-up, with the remaining patient experiencing relapse at 12 months after transplantation. Transcriptome RNA sequencing is required for the detection of the CBFA2T3-GLIS2 fusion gene and for proper risk-based allocation of pediatric patients with AML in future clinical strategies. Haplo-HSCT with posttransplant cyclophosphamide-based conditioning may improve survival in children with AMKL harboring the fusion gene.

Fasudil promotes polyploidization of megakaryoblasts in an acute megakaryocyte leukemia model.

Acute megakaryocytic leukemia (AMKL) is a rare neoplasm caused by abnormal megakaryoblasts. Megakaryoblasts keep dividing and avoid undergoing polyploidization to escape maturation. Small-molecule probes inducing polyploidization of megakaryocytic leukemia cells accelerate the differentiation of megakaryocytes. This study aims to determine that Rho kinase (ROCK) inhibition on megakaryoblasts enhances polyploidization and the inhibition of ROCK1 by fasudil benefits AMKL mice. The study investigated fasudil on the megakaryoblast cells in vitro and in vivo. With the differentiation and apoptosis induction, fasudil was used to treat 6133/MPLW515L mice, and the differentiation level was evaluated. Fasudil could reduce proliferation and promote the polyploidization of megakaryoblasts. Meanwhile, fasudil reduced the disease burden of 6133/MPLW515L AMKL mice at a dose that is safe for healthy mice. Combination therapy of ROCK1 inhibitor fasudil and reported clinical AURKA inhibitor MLN8237 achieved a better antileukemia effect in vivo, which alleviated hepatosplenomegaly and promoted the differentiation of megakaryoblast cells. ROCK1 inhibitor fasudil is a good proliferation inhibitor and polyploidization inducer of megakaryoblast cells and might be a novel rationale for clinical AMKL treatment.

Heat shock protein 90 inhibitors induce cell differentiation via the ubiquitin-dependent aurora kinase A degradation in a MPLW515L mouse model of primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by immature megakaryocytic hyperplasia, splenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Our preclinical study had demonstrated that aurora kinase A (AURKA) inhibitor MLN8237 reduced the mutation burden of PMF by inducing differentiation of immature megakaryocytes. However, it only slightly alleviated splenomegaly, reduced tissue fibrosis, and normalized megakaryocytes in PMF patients of the preliminary clinical study. So enhancing therapeutic efficacy of PMF is needed. In this study, we found that AURKA directly interacted with heat shock protein 90(HSP90) and HSP90 inhibitors promoted the ubiquitin-dependent AURKAdegradation. We demonstrated that HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), normalized peripheral blood counts, improved splenomegaly, attenuated extramedullary hematopoiesis, decreased tissue fibrosis and reduced mutant burden in a MPLW515L mouse model of PMF. Importantly, both 17-AAG and 17-DMAG treatment at effective doses in vivo did not influence on hematopoiesis in healthy mice. Collectively, the study demonstrates that HSP90 inhibitors induce cell differentiation via the ubiquitin-dependent AURKA and also are safe and effective for the treatment of a MPLW515L mouse model of PMF, which may provide a new strategy for PMF therapy. Further, we demonstrate that combined therapy shows superior activity in acute megakaryocytic leukemia mouse model than single therapy.

Clinical Significance of Flow Cytometry Findings in Brazilian Patients with De Novo Acute Myeloid Leukemia

Introduction: Immunophenotyping by flow cytometry (FC) is an essential method for diagnosis and classification of acute myeloid leukemias (AML), and its extensive use could identify blast cell subpopulations with phenotypes rarely seen in normal myelopoiesis, correlating with clinical, morphological and prognostic characteristics. Methods: In this study we analyzed 143 cases of AML, examining them for leukemia-associated immunophenotype (LAIP) by FC immunophenotyping in leukemic cells using a panel of monoclonal antibodies (MoAb) for diagnosis and classification of acute leukemia (AL). At the same time, clinical, demographic and hematological data of these patients were also investigated. Most patients were male adults and splenomegaly and hepatomegaly were present in most cases. Results: Immunophenotyping showed a characteristic profile of AML with expression of pan-myeloid antigens CD13, CD33 and myeloperoxidase (MPO), combined with CD34 and CD117 in most cases. Expression of CD14 and CD64 were observed in AML with monocytic component (AML-M4/M5), CD235a, CD36 and CD71 in cases of erythroleukemia (AML-M6) and platelet glycoproteins CD41, CD42b and CD61 in acute megakaryocytic leukemia (AML-M7). Regarding the aberrant phenotype, higher levels of expression of CD4, CD7 and CD56 were observed, corresponding to 24.5%, 22.4%, and 16.1% of the cases, respectively. Conclusion: We conclude that LAIP, as they are described here, were present in the vast majority of cases of investigated AML, with a relevant association with prognostic factors, clinical data and cytomorphological classification.

Clinical Feature and Effect of Demethylation Therapy in Pediatric Patients with Acute Myeloid Leukemia with Myelodysplasia-Related Changes

Objective:To better understanding of the clinical and biological features of childhood AML-MRC, in this study we retrospectively investigated 70 children with AML-MRC admitted to our hospital and compared with AML-NOS. We also analyzed the effect of demethylation drugs in introduction therapy among these patients. Study design: This retrospective cohort study was conducted at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (IHCAMS). Newly diagnosed AML patients at our hospital between January 2009 and December 2021 with complete data regarding baseline characteristics and treatment outcomes were reevaluated as having AML-NOS or AML-MRC according to the 2016 WHO classification of myeloid neoplasms and acute leukemia, strictly excluding cases of therapy-related myeloid neoplasms and AML with recurrent genetic abnormalities. Patients with acute megakaryocytic leukemia were also excluded. Clinical and laboratory data were searched in electronic medical records. Follow-up information was obtained from electronic records or by contacting family members and was initialized from the day of diagnosis to May 31, 2022, or the day of death. Results: Among the 779 newly diagnosed AML patients in our center, 70 patients met the diagnostic criteria of AML-MRC and 73 patients were AML-NOS. Patients with AML-MRC had lower white blood cell count (9.1 (IQR 3.0~30.0) ×109/L vs.17.6 (IQR 5.9~86.8) ×109/L, p=0.002), lower hemoglobin level (74.5 (IQR 63.5~ 86.0) g/L vs. 80.0 (71.0, 94.0) g/L, p=0.025) and lower bone marrow proliferation level (p=0.001) compared with patients with AML-NOS. More FLT3-ITD positive patients were found in AML-NOS patients than AML-MRC patients (33.8% vs. 7.1%, p<0.001). Among the 70 AML-MRC patients, 41(58.6%) were diagnosed by myelodysplasia-related cytogenetics, 10(14.2%) from MDS history, and 19 (27.1%) from a multilineage dysplasia. Monosomy 7 or deletion 7q(-7/7q-) is the most frequent cytogenetic abnormality (21.6%) followed by complex karyotype (18.8%) in AML-MRC patients. Totally 56 AML-MRC patients and 71 AML-NOS patients accepted chemotherapy in out center. Induction treatment for 69 AML-NOS patients were idarubicin/ cytarabine/ etoposide (IAE) or mitoxantrone/cytarabine/ etoposide (MAE) therapy. For AML-MRC patients, demethylation drugs alone or demethylation drugs combined above regime were also used (15 case) besides IAE/MEA therapy (35 case). Six AML-MRC patients and 2 AML-NOS patients accepted homoharringtonine/cytarabine/ granulocyte colony-stimulating factor (HAG) and 3 patients received hematopoietic stem cell transplantation (HSCT) without induction chemotherapy. Overall complete remission (CR) rate of 1 course were 51.0% in AML-MRC and 68.2% in AML-NOS patients. In IAE/MAE group, higher CR rate were seen in AML-NOS patients (70.3%) than AML-MRC patients (48.4%) p= 0.06). AML-MRC patients treated with demethylation drugs seemed to have higher CR rate than introduced by IAE/MAE (64.2 % vs. 48.3%, p= 0.39). HSCT in CR1 were conducted in 29 AML-MRC patients and 19 AML-NOS patients. Median follow-up periods were 2.17 in AML-MRC group and 2.43 years in AML-NOS. Three-year overall survival (OS) and event-free survival (EFS) were 54.4% (95%CI 42.4%~69.8%) and 75.7% (95%CI 62.8%~91.1%) for AML-MRC, 42.3% (95%CI 30.2%~59.2%) and 45.8% (95%CI 32.8%~63.9%) for AML-NOS patients. Cox analysis revealed that HSCT were associated with higher OS (HR 0.21,95%CI 0.1~0.41, p< 0.001). In AML-MRC patients, 3-year OS in HSCT group were significantly higher than that in non-HSCT group [83.4% (95%CI,69.6%~99.8%) vs. 28.0% (95%CI,14.6%~53.9%), p<0.001]. AML-MRC patients with myelodysplasia-related cytogenetics exhibited lower 3-year OS (41.9% ,95%CI,27.5%~63.8%) compared with MDS history group (64.8% (95%CI,39.3%~100.0%) and multilineage dysplasia (77.1% (95%CI,57.3%~100.0%). complex karyotype (41.9% ,95%CI,27.5%~63.8%) and -7(7q-) (40.0% ,95%CI,21.5%~74.3%) AML-MRC patients had lower 3-year OS than normal karyotype (42.3%, 95%CI,21.8%~82.0%) and the other abnormal karyotype (63.5% ,95%CI,37.3%~100.0%) but without statistical difference. Conclusions: Childhood AML-MRC exhibited a comparable adverse outcome with AML-NOS which can be significantly improved by HSCT therapy. Demethylation may help improve the CR rate of AML-MRC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Poor Outcomes Among Pediatric Non-Down Syndrome Acute Megakaryocytic Leukemia Patients Irrespective of Receipt of Hematopoietic Stem Cell Transplant

Poor Outcomes Among Pediatric Non-Down Syndrome Acute Megakaryocytic Leukemia Patients Irrespective of Receipt of Hematopoietic Stem Cell Transplant

C-Mpl-del, a c-Mpl alternative splicing isoform, promotes AMKL progression and chemoresistance

Acute megakaryocytic leukemia (AMKL) is a clinically heterogeneous subtype of acute myeloid leukemia characterized by unrestricted megakaryoblast proliferation and poor prognosis. Thrombopoietin receptor c-Mpl is a primary regulator of megakaryopoeisis and a potent mitogenic receptor. Aberrant c-Mpl signaling has been implicated in a myriad of myeloid proliferative disorders, some of which can lead to AMKL, however, the role of c-Mpl in AMKL progression remains largely unexplored. Here, we identified increased expression of a c-Mpl alternative splicing isoform, c-Mpl-del, in AMKL patients. We found that c-Mpl-del expression was associated with enhanced AMKL cell proliferation and chemoresistance, and decreased survival in xenografted mice, while c-Mpl-del knockdown attenuated proliferation and restored apoptosis. Interestingly, we observed that c-Mpl-del exhibits preferential utilization of phosphorylated c-Mpl-del C-terminus Y607 and biased activation of PI3K/AKT pathway, which culminated in upregulation of GATA1 and downregulation of DDIT3-related apoptotic responses conducive to AMKL chemoresistance and proliferation. Thus, this study elucidates the critical roles of c-Mpl alternative splicing in AMKL progression and drug resistance, which may have important diagnostic and therapeutic implications for leukemia accelerated by c-Mpl-del overexpression.

Diagnostic challenge in mixed phenotype acute leukemia with T/megakaryocyte or T/myeloid lineages accompanied by t(3;3)

BackgroundThe diagnosis of mixed phenotype acute leukemia (MPAL) with T/megakaryocyte or T/myeloid lineages accompanied by t(3;3) is always a challenge. Therefore, multiple experimental methods are usually required to avoid misdiagnosis. In this report, we presented a rare case of MPAL with T/myeloid lineages accompanied by t(3;3) and discussed the experience of differential diagnosis and our appreciation of the MPAL with T/megakaryocyte and T/myeloid lineages accompanied by t(3;3).Case presentation:A 31-year-old woman was admitted to our hospital due to recurrent fever for 20 days. Two distinct blast populations were detected by flow cytometry analysis: one population fulfills the immunophenotypic criteria for T-lymphoblastic leukemia, while the other population is highly suggestive of megakaryoblasts. These immunophenotypic features support the diagnosis of MPAL (T/megakaryocyte), which is rarely reported​. Interestingly, a complex karyotype was detected afterward by cytogenetics with t(3;3)(q21;q26.2), indicating a diagnosis of AML with t(3;3), a subset of which is also characterized by megakaryocytic markers such as CD41 and CD61. It seems that the second blast population detected by flow cytometry could not be classified into either diagnosis based on the morphology, immunophenotyping, and even cytogenetic findings, posing a real diagnostic problem because of the lack of clear-cut cytogenetic morphological defined criteria to distinguish between acute megakaryocytic leukemia and AML with t(3;3). Combining all of the examination data, this case was ultimately diagnosed as MPAL (T + My)-NOS with t(3;3) through differential diagnosis. Before the cytogenetic results were available, the patient received an acute lymphoblastic leukemia (ALL) regimen for MPAL treatment, but the effect was unsatisfactory. After the diagnosis was clear, she received an AML-like regimen with azacitidine for 7 days and venetoclax for 14 days, and achieved complete morphological remission.ConclusionMPAL with either T/megakaryocyte or T/myeloid lineages accompanied by t(3;3) is rare, and it is difficult to make a clear diagnosis. Thus, comprehensive examinations, including bone marrow cell morphology, flow cytometry analysis, cytogenetics, and molecular analysis are recommended to avoid misdiagnosis. AML-like regimen including azacitidine and venetoclax may be effective for treating MPAL (T + My)-NOS with t(3;3).

Pediatric non-Down's syndrome acute megakaryoblastic leukemia patients in China: A single center's real-world analysis.

Non-Down’s syndrome acute megakaryocytic leukemia (non-DS-AMKL) is a subtype of childhood acute myeloid leukemia (AML), whose prognosis, prognostic factors and treatment recommendations have not yet to be defined in children. We conducted a retrospective study with 65 newly diagnosed non-DS-AMKL children from August 2003 to June 2020 to investigate the clinical impact of factors and clinical outcome. Among all 65 patients, 47 of them were treated at our center who received three different regimens due to time point of admission (CAMS-another, CAMS-2009 and CAMS-2016 protocol), and the efficacy were compared. Patients with newly diagnosed non-DS-AMKL accounted for 7.4% of pediatric AML cases. The median age of the patients was 18 months at diagnosis, and over 90% of them were under three-years-old. The overall survival (OS) rates were 33.3% ± 1.7%, 66.7% ± 24.4% and 74.2% ± 4.0% for three groups (CAMS-another, CAMS-2009 and CAMS-2016 regimen), respectively. In CAMS-2016 group, the complete remission (CR) rate after induction was 67.7% (21/31), while the total CR rate after all phases of chemotherapy was 80.6% (25/31). The 2-year survival probability did not significantly improve in patients underwent HSCT when compared with non-HSCT group (75.0% ± 4.7% vs. 73.9% ± 4.6%, p=0.680). Those who had a “dry tap” during BM aspiration at admission had significantly worse OS than those without “dry tap” (33.3% ± 8.6% vs. 84.0% ± 3.6%, p=0.006). Moreover, the results also revealed that patients with CD34+ had significantly lower OS (50.0% ± 6.7% vs. 89.5% ± 3.5%, p=0.021), whereas patients with CD36+ had significantly higher OS than those who were negative (85.0% ± 4.0% vs. 54.5% ± 6.6%, p=0.048). In conclusion, intensive chemotherapy resulted in improved prognosis of non-DS-AMKL children and subclassification may base on “dry tap” and immunophenotypic. Although some progress has been made, outcomes of non-DS-AMKL children remain unsatisfactory, especially in HSCT group, when compared with other AML types.

A Rare Case of a Germ-Cell Tumour Associated with Acute Megakaryoblastic Leukaemia- An Autopsy Report with Review of Literature

His association of mediastinal germ-cell tumours (MGCTs) with haematologic neoplasms is a rare though well known circumstance, and few cases are found in the literature. Most of these are non-seminomatous tumours in young males. The diagnosis of the haematological condition is usually either synchronic or metachronic with that of the germ-cell tumour. The prognosis is poor and basically determined by the haematologic neoplasia. Less than 20 cases of PMGCT with evolution into acute megakaryocytic leukemia have been reported in worldwide literature. Hematologic neoplasias associated with extragonadal germ cell tumors represent one of the most intriguing and biologically distinctive aspects of male germ cell cancers. The case report we present is that of a young 33 year old male with an initial diagnosis of both conditions which were detected synchronously. Despite timely intervention he succumbed to multiorgan failure following chemotherapy and an autopsy was performed.

Blocking PAK1 kinase activity promotes the differentiation of acute megakaryocytic leukemia cells and induces their apoptosis

Objective: To investigate the effect of blocking P21 activated kinase 1 (PAK1) activity on the proliferation, differentiation, and apoptosis of acute megakaryocytic leukemia (AMKL) cell lines (CHRF and CMK) . Methods: Cell counts were used to detect the effects of PAK1 inhibitors (IPA-3 and G5555) on AMKL cell proliferation inhibition and colony formation, and flow cytometry was used to detect its effects on AMKL cell cycle. The effect of PAK1 inhibitor on the expression of cyclin D1 and apoptosis-related protein Cleaved caspase 3 was detected using Western blot, while interference with the protein expression level of PAK1 in AMKL cells was assessed using lentivirus-mediated shRNA transfection technology. Flow cytometry was used to detect the effects of knockdown of PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in AMKL cells. Results: PAK1 inhibitors inhibited the proliferation of AMKL cells in a dose-dependent manner and reduced the ability of cell colony formation, and the difference was statistically significant when compared with the control group (P<0.05) . Moreover, they also reduced the percentage of AMKL cells in S phase, and Western blot detection showed that the expression levels of phosphorylated PAK1 and cyclin D1 decreased significantly. Finally, PAK1 inhibitors induced AMKL cell apoptosis by up-regulating Cleaved caspase 3 and showed different abilities to increase the content of polyploid DNA in megakaryocytes. Only high concentrations of IPA-3 and low doses of G5555 increased the number of polyploid megakaryocytes, while knockdown of PAK1 kinase activity promoted AMKL cell differentiation and increased the apoptosis rate. Conclusion: PAK1 inhibitor significantly arrests AMKL cell growth and promotes cell apoptosis. Knocking down the expression of PAK1 promotes the formation of polyploid DNA and induces AMKL cell apoptosis. The above findings indicate that inhibiting the activity of PAK1 may control AMKL effectively.

Project Stella: Development and Preclinical Assessment of FOLR1-Directed Chimeric Antigen Receptor T Cells in CBF2AT3-GLIS2/RAM AML

Background: A rare but highly aggressive type of AML that is only seen in infants with a unique immunophenotype (RAM phenotype) is caused by cryptic CBFA2T3-GLIS2 (CBF/GLIS) fusion. This infant AML is highly refractory to conventional chemotherapy with near uniform fatality despite highly intensive and myeloablative therapy (PMID 23153540). Transcriptome profiling of CBF/GLIS AML has revealed new insights into the pathogenesis of the fusion and uncovered fusion-specific molecular biomarkers that could be used for risk stratification and to inform treatment (PMID 30592296). Studying the largest cohort of these high-risk infants, we demonstrated several alterations in gene expression and transcriptional networks in these CBF/GLIS-positive patient samples that have potential for therapeutic targeting (PMID 31719049). FOLR1, which encodes for folate receptor alpha, was highly and uniquely expressed in CBF/GLIS AML but was entirely absent in AML with other cytogenetics abnormalities and in normal hematopoietic cells. Furthermore, we recently demonstrated that forced expression of CBF/GLIS enhances the proliferation and alters differentiation in cord blood (CB) CD34+ early precursors towards megakaryocytic lineage that recapitulates acute megakaryocytic leukemia seen in infants (PMID 31719049). Of significance, we showed that FOLR1 surface expression is causally linked to CBF/GLIS-induced malignant transformation, thus making it an attractive antigen for targeted therapies against CBF/GLIS AML cells. Given that chimeric antigen receptor (CAR) T cells are extremely effective at eradicating relapsed/refractory B-ALL malignancies, we developed FOLR1-directed CAR T cells for pre-clinical evaluation in CBF/GLIS AML.Methods: We generated a FOLR1-directed CAR using anti-FOLR1 binder (Farletuzumab), IgG4 intermediate spacer and 41-BB/CD3zeta signaling domains. The pre-clinical efficacy of FOLR1 CAR T cells was evaluated against CBF/GLIS AML cell lines in vitro and in vivo. CBF/GLIS AML models include CB CD34+ cells transduced with CBF/GLIS expression construct (CBF/GLIS-CB) and WSU-AML cell line. We also engineered Kasumi-1 cell line to express FOLR1 (Kasumi-1 FOLR1+) to evaluate target specificity (Figure 1A).Results: We tested the target specificity of FOLR1-directed CAR T cells against FOLR1-positive (CBF/GLIS-CB, WSU-AML, Kasumi-1 FOLR1+) and FOLR1-negative (Kasumi-1) cells. CD8 FOLR1 CAR T cells demonstrated cytolytic activity against FOLR1 positive but not FOLR1 negative cells (Figure 1B). Furthermore, both CD8 and CD4 FOLR1 CAR T cells produced higher levels of IL-2, IFN-γ, and TNF-α and proliferated more robustly than did unmodified T cells when co-incubated with FOLR1 positive but not FOLR1 negative cells (Figure 1C). These results indicate highly specific reactivity of FOLR1 CAR T cells against AML cells expressing FOLR1. We next investigated the in vivo efficacy of FOLR1-directed CAR T cells. In CBF/GLIS-CB, WSU-AML, and Kasumi-1 FOLR1+ xenograft models, treatment with FOLR1 CAR T cells induced leukemia clearance, while disease progression occurred in all mice that received unmodified T cells (Figure 1D). Activity of FOLR1 CAR T cells in vivo was target specific, as they did not limit the leukemia progression nor extend the survival of Kasumi-1 xenografts (Figure 1D).To determine whether FOLR1 is expressed on normal HSPCs, we characterized FOLR1 expression in normal CB CD34+ samples. FOLR1 expression was entirely silent in HSPC subsets (Figure 1E). Consistent with lack of expression, no cytolytic activity was detected against HPSCs Moreover, FOLR1 CAR T cells did not affect the self-renewal and multilineage differentiation capacity of normal HSPCs as compared to unmodified control T cells (Figure 1F), whereas significant eradication of colonies were detected in the CBF/GLIS-CB cells (Figure 1G).Conclusion: In this study, we demonstrate that FOLR1 CAR T effectively eradicates CBF/GLIS AML cells without compromising normal HSPCs, providing a promising approach for the treatment of high-risk CBF/GLIS AML. Transition of this CAR T to clinical development for infant AML is underway. [Display omitted] DisclosuresHylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

CONGENITAL ACUTE MEGAKARYOCYTIC LEUKEMIA (AML-7)

Background: Congenital Leukemia (CL) is a disorder that is very rare in newborns, most often originate from the myeloid series M4 / M5. Peripheral blood showed mononuclear cells with cytoplasmic bleb which is sometimes very difficult to distinguish between myeloid and lymphoid series. Acute megakaryocytic leukemia is a rare type of AML with an incidence of 0.5 per million per year and may accompanied by Down Syndrome. We report an AML-M7 case in a nenonate with Down syndrome. Case Description: A 3-day-old neonate was referred from RSIA Amanah Probolinggo with a suspected diagnosis of congenital leukemia and Down syndrome and main complaint of vomiting. Physical examination: Blood pressure 72/31 mmHg, pulse 137x/min; breathing 40x/ minute; temperature 36.5°C, body weight 2,700g, height 46cm, head circumference 33cm, jaundiced sclera, chest retraction, papular macular rash at abdomen, simian crease. Clinical chemistry: sodium 118 mmol/L, total bilirubin 7.36, CRP 11.6. Duodenal BOF atresia and duodenal web. Hematology: Hb 17.6 g/dl, MCV 90.4 fL, MCH 32.4 pg, MCHC 35.8 g / dl, WBC 58.00 x 103µL, platelets 74 x 103 / µL, IPF 25.8%. Peripheral blood smear: Leukocytosis with 70% megakaryoblasts. Specific immunophenotyping against megakaryocytes CD 36 (+) and myeloid CD 33, CD 34, CD 117 series with CD7 aberration. The patient died at the age of 8 days. Discussion: Neonates with a clinical manifestasion of Down Syndrome, morphology of peripheral blood smear dominated by megakaryoblasts, increased IPF and immunophenotyping CD 36 (+) help diagnose Congenital Acute Megakaryocytic Leukemia. Conclusion: AML-M7 is a congenital leukemia which is established based on blood smears and immunophenotyping and is related to Down Syndrome.

Pediatric Chronic Myeloid Leukemia with Megakaryocytic Blast Crisis as Initial Presentation: Case Report and Review of Literature

Abstract: Background: Pediatric Chronic Myeloid Leukemia (CML) is a rare entity accounting for 2-3% of pediatric malignancies. CML rarely presents as Blast Crisis (BC) at the time of diagnosis, and megakaryocytic blast crisis is even rarer. Case Presentation: We herein, report a case of a young female, 10-year-old who presented with anemia, leukocytosis and massive splenomegaly. Clinical features, peripheral film and bone marrow findings were consistent with CML in megakaryocytic blast crisis. Bone marrow cytogenetic analysis revealed karyotype of 46, XX, t(9:22)(q34;q11.2) in 20 metaphases and BCR-ABL P210 by PCR was detected with transcript level of 83%, which further confirmed our diagnosis. Conclusion: De novo presentation of chronic myeloid leukemia with megakaryocytic blast crisis is rarely observed in pediatric population with very few cases published till now. We are presenting this case because of its rarity, likelihood of misdiagnosis as AML (M7) and poor prognosis, if not treated precisely. Keywords: Chronic Myeloid Leukemia (CML), Acute Myeloid Leukemia (AML), Blast Crisis (BC), Acute Megakaryocytic Leukemia (AMKL), Chronic Phase (CP), Accelerated Phase (AP), Tyrosine Kinase Inhibitor (TKI).

LncRNA MONC suppresses the malignant phenotype of Endometrial Cancer Stem Cells and Endometrial Carcinoma Cells by regulating the MiR-636/GLCE axis

BackgroundEmerging evidence shows that abnormal expression of long non-coding RNA is involved in the occurrence and development of various tumors. LncRNA MONC is abnormally expressed in head and neck squamous cell carcinoma, lung cancer, colorectal cancer, and acute megakaryocytic leukemia, but the biological function and potential regulatory mechanism of MONC in endometrial cancer stem cells (ECSCs) and endometrial cancer cells (ECCs) have not been studied. In this study, we aimed to explore the tumor suppressive effect and mechanism of MONC in regulating ECSCs and ECCs.MethodsWe used qRT-PCR to detect the expression of MONC, miR-636 and GLCE in normal human endometrial tissues and endometrial carcinoma (EC) tissues. Luciferase assay was used to verify the binding sites between MONC and miR-636 and between miR-636 and GLCE. Double fluorescence in situ hybridization was used to locate MONC and miR-636 in cells. ECSCs were obtained by flow cytometry sorting assay. Sphere formation assay, CCK-8 assay, transwell invasion assay, cell cycle analysis and apoptosis assay were used to detect the effects of MONC/miR-636/GLCE axis on the malignant biological behavior of ECSCs and ECCs. The effect of MONC on the epithelial-to-mesenchymal transition (EMT) process was detected using western blot. Finally, we conducted in vivo verification through Tumor xenografts in BALB/C nude mice.ResultsIn this study, we found MONC is low expression in endometrial carcinoma (EC) and patients in the MONC high-expression group had a better prognosis. MONC and miR-636 are relatively co-localized in the cytoplasm. MONC directly inhibits the malignant biological behavior of ECSCs and ECCs by directly inhibiting miR-636. Simultaneously, miR-636 may indirectly reduce the expression of MONC. Down-regulation of miR-636 may promote GLCE expression by targeting the 3′-untranslated region (UTR) of the downstream gene GLCE, thereby inhibiting the progression of ECSCs. MONC combined with miR-636 inhibited tumor epithelial-to-mesenchymal transition (EMT) process. In addition, we verified the tumor suppressive effect of MONC in nude mice, miR-636 can rescue the tumor suppressive effect of overexpressing MONC.ConclusionsIn conclusion, this study showed that MONC inhibits the malignant phenotypes of ECSCs and ECCs by regulating the miR-636/GLCE axis. Thus the MONC/miR-636/GLCE axis may provide novel treatment avenues for human EC.

NUP98-KDM5A Fusion Gene Is Not Exclusive to Acute Megakaryoblastic Leukemia and Is an Independent Predictor of Poor Prognosis

NUP98 rearrangements are recurrent genetic events in pediatric acute myeloid leukemia (AML). We identified NPU98-NSD1 as a recurrent poor risk abnormality (Hollink et al, Blood 2011). Another recurrent fusion is NUP98-KDM5A, which was found in pediatric acute megakaryocytic leukemia (AMKL) and was associated with poor clinical outcome (de Rooij et al, Nature Genetics 2017). To date, it is unclear whether NUP98-KDM5A exclusively occurs in AMKL or also in other FAB-subtypes of childhood AML. The aim of this study was to determine the incidence of NUP98-KDM5A in large pediatric AML cohorts, and to define the biological and clinical characteristics of this subgroup.The study comprised a COG and a European cohort of patients, and included 997 patients from COG trials 03p1 and 0531 (Gamis et al, Blood 2013) and 354 European pediatric AML patients, previously described by Balgobind et al (Haematologica 2011), NUP98-KDM5A rearrangements were detected either by paired-end RNA sequencing or RT-PCR. Patients with FAB M3 AML were excluded from this study. For comparison of clinical characteristics between NUP98-KDM5A positive and negative patients, the χ-squared and the Fisher's exact test were used. Event-free survival (EFS) and overall survival (OS) were estimated using the Kaplan-Meier method; the Cox proportional-hazard model was used for multivariate analysis. For this analysis, age, white blood cell count (WBC), cytogenetic risk group, allogeneic hematopoietic stem cell transplant (HSCT) as time dependent variable and study group (COG vs European cohort) were used as co-variables.NUP98-KDM5A rearrangements were detected in 31 out of 1351 patients (2.3%). There was no significant difference in prevalence between the COG and European cohorts. 41% of the NUP98/KDM5A+ patients were male, compared to 53% of the NUP98-KDM5A- patients (p=0.24). The median age of NUP98-KDM5A patients was significantly lower as compared to patients without this fusion gene (5.8 vs 9.4 yrs; p=0.03). There was no difference in WBC between NUP98-KDM5A positive and negative patients (13.1*109/l vs.25.2*109/l, respectively, p=0.13). NUP98-KDM5A occurred in all FAB types, with FAB M7 being most frequent (33% vs 8% in NUP98-KDM5A - patients; p<0.001).The 5-year EFS of NUP98-KDM5A + patients was 36.8 ± 18.8%, compared to 48.1 ± 2.8% in the NUP98-KDM5A - patients (p=0.12). The 5-year OS of NUP98-KDM5A + patients was 41.1 ± 19.7%, a value significantly lower compared to the NUP98-KDM5A - patients (63.9 ± 2.8%; p=0.01), this finding suggesting that NUP98-KDM5A+ patients are difficult to rescue when they relapse. In multivariate analysis, NUP98-KDM5A was an independent risk factor for OS, but not for EFS, with hazard ratios of 1.76 (p=0.045) and 1.58 (p=0.083), respectively. Non-M7 (n=19) patients had lower survival rates with EFS of 26.4 ± 21.7% and OS of 30.0 ± 22.3% compared to M7 (n=9) patients (EFS 55.6 ± 33.1% and OS 64.8 ± 33.1%). These differences were not statistically significant.This study shows that NUP98-KDM5A is an aberration that occurs in all FAB types and not just in AMKL. NUP98-KDM5A is an independent risk factor for OS but not for EFS and identifies a poor prognostic subgroup. Although there was a big difference in survival between M7 and non-M7 NUP98-KDM5A rearranged patients, this was not statistically significant, probably because of the low number of patients in the two cohorts. Overall, these data suggest to screen for the presence of NUP98-KDM5A rearrangements all children with non-APL AML irrespectively of their AML FAB subtype, and to stratify these patients in the high risk group. DisclosuresNo relevant conflicts of interest to declare.

RNA Sequencing Illustrates the Genetic Backgrounds of Pediatric Acute Myeloid Leukemia

BackgroundThe cytogenetic heterogeneity of acute myeloid leukemia (AML) has been recognized for more than three decades, and some chromosomal aberrations and gene mutations have been adopted for the risk stratification. However, approximately 50% of patients have disease relapse. Thus, novel and accurate prognostic markers are needed to improve the risk stratification. We have performed some targetting sequencing and gene expression array, and identified that PR DM16 and MECOM -overexpressing patients had significantly worse overall survival (OS) and event-free survival (EFS). Thisoverexpression was especially helpful for stratifying patients with FLT3 -ITD positive and/or normal karyotype. To establish a more appropriate risk classification, we investigated the correlations among genetic aberrations, gene fusions, and gene expression, which are significant prognostic markers for pediatric AML, and proposed a new risk stratification. In addition, we performed the whole-transcriptome analysis to reveal the comprehensive understanding of 57 patients whose aberrant genetic backgrounds were unclear.Patients and MethodsThis study enrolled 369 patients with de novo AML who had been registered with the Japan Children's Cancer Group study, JPLSG, AML-05 trial between November 2006 and December 2010. Patients diagnosed with acute promyelocytic leukemia or Down syndrome-associated AML were excluded from this study. We investigated gene mutations in KIT, CEBPA, NPM1, FLT3 -ITD, and MLL- PTD, gene fusions of RUNX1-RUNX1T1, CBFB-MYH11, KMT2A (MLL)- rearrangements, NUP98-NSD1, CBFA2T3-GLIS2, NUP98-KDM5A, FUS-ERG, and DEK-NUP214, and gene expression analyses of PRDM16 and MECOM . Although most of these patients were classified into five risk-dependent subgroups based on their genetic aberrations, the genetic background of 57 patients (15%), particularly in patients with complex karyotypes, was unclear. Thus, we additionally performed the whole-transcriptome analysis for these patients to reveal their genetic backgrounds.ResultsWe could re-stratify 369 patients into five new prognostic subgroups based on the genetic aberrations. Because genetic backgrounds of patients with acute megakaryocytic leukemia (AMKL) were different from non-AMKL patients, we proposed another stratification scheme for 34 patients with AMKL. For 335 patients with AML, as the first step, super low and extreme high groups were created. The super low group (5-year OS, 94%; 5-year EFS, 84%) consisted of CBFB-MYH11, KMT2A-R with low MECOM expression, NPM1 mutation, and CEBPA biallelic mutations. Conversely, the extremely high group (5-year OS, 24%; 5-year EFS, 16%) consisted of FUS-ERG, MLL-AF6, DEK-NUP214, CBFA2T3-GLIS2, MLL -PTD, NUP98-NSD1, MLL -R with MECOM and/or PRDM16 high, and FLT3 -ITD with high PRDM16 gene expression. Then, we chose low and intermediate groups as the second step. The low group (5-year OS, 86%; 5-year EFS, 76%) consisted of RUNX1-RUNX1T1 without KIT, MLL -R with low MECOM and PRDM16, and the intermediate group (5-year OS, 85%; 5-year EFS, 58%) consisted of RUNX1-RUNX1T1 with KIT, FLT3 -ITD with low MECOM, and PRDM16, according to the retrospective EFS and OS. For the remaining 57 patients (15%) whose genetic background was unclear, non-complete remission (CR) patients were assigned to the extremely high group, and patients who achieved CR were assigned to the high risk group (5-year OS, 53%; 5-year EFS, 44%). We performed the whole-transcriptome analysis for these 57 patients and identified gene fusions of PICALM-MLLT10 in 3, RPN1-MECOM in 2, CREBBP-KAT2A in 3, RUNX1 -rearrangements in 2, CBFA2T3 -rearrangement in 2, RARB -rearrangement in 1, and ETV6 -rearrangements in 3. The whole-transcriptome analysis was used to reveal the genetic background of these patients, particularly in patients with a complex karyotype. For the remaining 34 patients with AMKL, patients with CBFA2T3-GLIS2 or NUP98-KDM5A were assigned to the extremely high group and others to the intermediate group.ConclusionWe propose a new risk stratification comprising five prognostic subgroups based on the genetic aberrations. The whole-transcriptome analysis facilitated further understanding of the whole genetic background of pediatric patients with AML whose genetic background was unclear and was, thus, expected to improve more accurate risk stratification. DisclosuresNo relevant conflicts of interest to declare.

Targeting PRMT1 As a Novel Curative Therapy for Acute Myeloid Leukemia

Protein arginine methyltransferase 1 (PRMT1) is dynamically regulated in normal hematopoiesis and constitutively high expression of PRMT1 is associated with low survival rate in acute myeloid leukemia (AML) (Zhang et al. 2015). PRMT1 has been demonstrated for maintaining cell proliferation of AML1-ETO, MLL fusion initiated AML. Therefore, targeting PRMT1 may be a novel curative therapy for AML. In this report, we will use leukemia mouse models to address how to target protein arginine methyltransferase 1 as a novel therapy for treating acute megakaryocytic leukemia (AMKL).Pediatric AMKL is a fatal disease with poor prognosis. So far chemotherapy is still the first line therapy for AMKL, yet gene-specific therapy is not available. Chromosomal translocation t(1;22)(p13;q13), which generates the fusion protein RBM15-MKL1 (aka OTT-MAL), is exclusively found in pediatric AMKL patients. Knock-in mice that express Rbm15-MKL1 from endogenous Rbm15 locus develops leukemia with low penetrance and long latency, suggesting that additional genetic or epigenetic changes are required for the rapid leukemia progression. Previous study by Mercher et al. showed that addition of mutant thrombopoietin receptor tyrosine kinase c-MPLW515L to Rbm15-MKL1 translocation leads to aggressive leukemia development in mice. We further demonstrated that forced PRMT1 expression bypassed the need for c-MPLW515L to allow Rbm15-MKL1 cells to proliferate in a cytokine-independent fashion in cell culture. Viability of PRMT1-expressing cells as well as the c-MPLW515L expressing cells is highly sensitive to the treatment of the PRMT1 inhibitor MS203, indicating that the survival of transformed cells is dependent on PRMT1. Co-expression of PRMT1 and Rbm15-MKL1 in cord blood cells can sustain the long term culture while RBM15-MKL1 expressing cord blood cells will die quickly. Based on these results, we rationalize that PRMT1 is a key factor that cooperates with Rbm15-MKL1 to mediate malignant transformation and aggressive leukemia development. Inhibition of PRMT1 activity would have therapeutic effect on OTT-MAL-initiated leukemia. In this vein, we administered daily i.p. injection of PRMT1 inhibitor to mice that were transferred either c-MPLW515L- or PRMT1-expressing Rbm15-MKL1 cells. Inhibitor-treated group has greatly improved survival time. Vehichle-treated mice suffered from fast weight loss, early disease onset and then died within 21-23 days post transplantation while most inhibitor-treated mice remained alive with stable body weight. PRMT1 inhibitor treatment significantly blocks leukemia-associated splenomegaly. Flow cytometry analysis showed that inhibitor-treated mice have notably less leukemia cells in bone marrow and in peripheral blood, compared to their control counterparts. More than half of control mice developed rear limb paralysis and visible blood clots were found on their spine upon dissection. Two and four weeks post the last injection we analyzed the peripheral blood of surviving inhibitor-treated mice to detect residual disease and found that the percentage of leukemic cells was very low, in some cases completely undetectable. Up to 3 months those mice remained healthy and no signs of relapse without further MS203 treatment. How the PRMT1 inhibitor MS203 blocks leukemia progression with PDX models of Down syndrome AMKL will be presented.At the molecular level, we performed metabolomics analysis with mass spectrometry and Seahorse analyzer, and discovered that PRMT1 reprogrammed metabolism in leukemia cells through upregulation of glycolysis pathway and downregulation of fatty acid oxidation pathway. We reported that PRMT1 controls RBM15 protein stability by triggering methylation-dependent ubiquitilation (Zhang et al. 2015). In this report, we further demonstrated that RBM15 is directly responsible for controlling the mRNA stability of key metabolic enzymes. Thus we reasoned that the PRMT1-RBM15 axis is a key axis in controlling metabolism during leukemia progression. Based on these findings, we further tested whether we can use weight-controlling drugs such as etomoxir for treating leukemia. In summary, copious expression of PRMT1 in AML makes PRMT1 an encouraging new target for curative therapy, The PRMT1 inhibitor MS203 which has few side effects in mice will be a strong candidate for future clinical trials. DisclosuresNo relevant conflicts of interest to declare.

A CHAF1B-Dependent Molecular Switch in Hematopoiesis and Leukemic Pathogenesis

Children with Down syndrome (DS) are at substantial risk of developing acute leukemia, which suggests that there are leukemia promoting genes on chromosome 21. Chromatin Assembly Factor 1B (CHAF1B), which resides in the DS critical region, is a member of the heterotrimeric CAF1 chromatin assembly complex that is responsible for depositing H3/H4 heterodimers at the replication fork during S-phase. We previously demonstrated that CHAF1B is overexpressed in acute megakaryocytic leukemia cells with trisomy 21 and have now discovered that it is also elevated in AML cell lines and primary tumor samples. This elevated expression is correlated with poor prognosis but the mechanism by which CHAF1B may promote leukemogenesis is unknown. In this study, we demonstrate that CHAF1B acts as regulator of transcription, maintaining an undifferentiated state by competing with CEBPA, FLI1, and RUNX2 for occupancy at the promoters and enhancers of myeloid differentiation genes.To study CHAF1Bin the context of normal hematopoiesis, we generated Chaf1b floxed mice and found that homozygous deletion was associated with rapid pancytopenia and bone marrow failure due to a cell cycle arrest and cell death. These effects were associated with downregulation of cell adhesion and metabolic pathways, as well as upregulation of genes related to sensitivity to stress and inflammatory stimuli. On the other hand, consistent with a pro-leukemic function, overexpression of CHAF1B promoted serial replating in vitro and was associated with the upregulation of genes involved in metabolism and proliferation. However, CHAF1B overexpression alone was not sufficient to promote leukemia in vivo .Since CHAF1B expression is elevated in cell lines with MLL rearrangements and primary AML samples, we next studied its requirements in the murine MLL-AF9 AML model. First, we found that overexpression of CHAF1B enhanced the formation of MLL-AF9 leukemic cells (LCs) in vitro and significantly accelerated disease in vivo . In contrast to the cell death and cell cycle arrest observed in HSPCs following Chaf1b deletion, there were no changes in proliferation or survival of LCs at 48 hours following Chaf1b deletion. Instead, we found Chaf1b deletion led to terminal myeloid differentiation of LCs, suggesting that CHAF1B has a major role in maintaining leukemic stem cells. In fact, heterozygous deletion of Chaf1b or overexpression of a dominant negative allele was sufficient to substantially reduce colony formation, as well as completely prevent leukemia in vivo .Transcriptome analysis of Chaf1b- deficient LCs revealed an increase in expression of over 1500 genes, including many of those associated with differentiation of myeloid leukemia cells. In comparison, we only identified 255 significantly downregulated genes, suggesting that CHAF1B primarily functions as a transcriptional repressor. By ChIP-seq, we determined that CHAF1B, but not the DNA replication factor PCNA, co-occupied the promoter and enhancer regions of many of these upregulated genes. Surprisingly, by ATAC-seq we found a widespread decrease in accessibility at CHAF1B binding sites following its knockdown, suggesting that other mechanisms are likely the key contributors to the differentiation phenotype. Motif analysis of CHAF1B-bound peaks in MLL-AF9 cells revealed specific enrichment of differentiation-driving transcription factor binding sites, including CEBPA, FLI1, and RUNX2. Therefore, we hypothesized that CHAF1B competes with transcription factors for occupancy at promoters of differentiation genes, preventing their expression and maintaining a leukemia stem cell state. We tested our hypothesis by comparing the occupancy of CEBA, FLI1, and RUNX2 at CHAF1B bound sites by ChIP-seq in cells replete or deficient for CHAF1B. We found that occupancy of these factors was significantly enhanced at CHAF1B-bound regions in LCs following Chaf1b deletion, and that this was associated with concomitant increases in expression of the differentiation target genes. We conclude that CHAF1B maintains the leukemic stem cell state by preventing differentiation through direct competition for chromatin occupancy with transcription factors at regulatory regions of genes that promote terminal maturation. DisclosuresNo relevant conflicts of interest to declare.