Acute Megakaryoblastic Leukemia Research Articles

Landscape of driver mutations and their clinical effects on Down syndrome–related myeloid neoplasms

AbstractTransient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Myeloid sarcoma with RBM15::MRTFA (MKL1) mimicking vascular neoplasm.

Extramedullary involvement of acute myeloid leukemia (AML), aka myeloid sarcoma, is a rare phenomenon in acute megakaryoblastic leukemia with RBM15:: MRTFA(MKL1) fusion, which might mimic non-hematologic malignancies. A 7-month-old infant presented with leukocytosis, hepatosplenomegaly, multiple lymphadenopathies, and a solid mass in the right thigh. Initially, the patient was diagnosed with a malignant vascular tumor regarding the expression of vascular markers from the biopsy of the right thigh lesion that was performed after the inconclusive bone marrow biopsy. The second bone marrow biopsy, which was performed due to the partial response to sarcoma treatment, showed hypercellular bone marrow with CD34 and CD61-positive spindle cell infiltration and > 20% basophilic blasts with cytoplasmic blebs. RNA sequencing of soft tissue biopsy revealed the presence of RBM15::MRTFA(MKL1) fusion. Based on these findings, myeloid sarcoma/AML with RBM15::MRTFA(MKL1) fusion diagnosis was made. AML with RBM15::MRTFA(MKL1) fusion can initially present as extramedullary lesions and might cause misdiagnosis of non-hematologic malignancies.

Acute panmylelosis with myelofibrosis - A rare case report with review of literature.

Acute panmyelosis with myelofibrosis (APMF) corresponds to <1% cases of acute myeloid leukemia, which could be an underestimation due to missed diagnosis. Due to its rapidly fatal course, it warrants a timely and correct diagnosis. We present a case of a 44-year male who came with a short history of fever, generalised weakness, revealed pancytopenia with occasional circulating blast in the peripheral blood smear. Bone marrow aspirate was dry tap,biopsy revealed panmyelosis with myelofibrosis with increased (22%) blasts. Flowcytometric immunophenotyping, cytogenetics and molecular tests were undertaken. Together with clinical details, immunophenotypic profile, cytogenetics and molecular studies, the diagnosis of Acute panmyelosis with myelofibrosis was made and managed accordingly. 32 The WHO 2017 describes APMF as an acute panmyeloid proliferation with increased blasts (≥20% in the bone marrow or peripheral blood) and accompanying marrow fibrosis. APMF is rare with poor prognosis thus, must be differentiated especially from Acute megakaryoblastic leukemia to arrive at the correct diagnosis which will help reduce/prevent the early mortality by providing timely chemotherapy followed by upfront hemopoietic stem cell transplantation.

Preclinical Evaluation of an Anti-CD41 CAR T Cell in Acute Megakaryoblastic Leukemia on in Vitro and In Vivo Models

Preclinical Evaluation of an Anti-CD41 CAR T Cell in Acute Megakaryoblastic Leukemia on in Vitro and In Vivo Models

A retrospective analysis of gene fusions and treatment outcomes in pediatric acute megakaryoblastic leukemia without Down syndrome.

Not available.

Survival Outcomes of Acute Megakaryoblastic Leukemia in the United States: A 10-Year SEER-Based Study

Background: Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), commonly observed in children with Down Syndrome (DS) but also seen in adults. The cytogenetic abnormality t(1;22)(p13.3;q13.1), has been reported in children with non-DS AMKL, which involves the fusion of RNA-binding motif protein-15 and megakaryoblastic leukemia-1 resulting in abnormal proliferation of megakaryocyte progenitors. Population-level data on outcomes of AMKL is scarce. Methods: We conducted a retrospective analysis using the Surveillance Epidemiology and End Results (SEER) database, a U.S. population-based registry maintained by the National Cancer Institute. We included all cases with histologically confirmed diagnoses of AMKL and AML with t(1;22)(p13.3;q13.1) between the years 2010 and 2019. Survival analysis was performed using the Kaplan-Meier method, and differences in overall survival (OS) were assessed using the log-rank test. Results: The analysis included a total of 217 cases, consisting of 54 patients with AML with t(1;22)(p13.3;q13.1) and 163 patients with AMKL. Among the AML cases with the cytogenetic abnormality, the median age was 58 years (ranging from 0 to 89 years), and 61% were female. 74% (n=40) of these patients received chemotherapy. The 1-year survival rate was 54.0% for the pediatric group (age &amp;lt; 18 years, n=10) and 44.6% for the adult group (age &amp;gt; 18 years, n=44) (Median OS 20 vs 8 months, p=0.69). Patients who received chemotherapy had a significantly higher 1-year survival rate of 57.9% compared to 14.3% in those who did not receive chemotherapy (p=0.0005). No significant difference in survival was observed when patients were grouped based on the year of diagnosis, with 1-year survival rates of 50.8% for the period 2010-2014 and 41.3% for 2015-2019 (p=0.66). Patients with AMKL were divided into two age groups: pediatric AMKL (age &amp;lt; 18 years, n=96) and adult AMKL (age &amp;gt; 18 years, n=67). The median age in this group was 3 years (ranging from 0 to 86 years), and 55% were male. 81% (n=132) of these patients were treated with chemotherapy. The 1-year survival rate for the pediatric group was 79.4%, whereas it was 17.6% for the adult group (p&amp;lt;0.0001). Chemotherapy significantly improved overall survival (OS) for both pediatric and adult groups, with 1-year survival rates of 82.5% vs. 35.7% in the pediatric group and 27.6% vs. 0.00% in the adult group in patients who did and did not receive chemotherapy, respectively (p=0.041 for pediatric group and p&amp;lt;0.0001 for adults). No significant differences in survival were observed among the groups based on the year of diagnosis, both in adults and the pediatric group. Conclusion: AMKL is commonly seen in infants and children, but it can affect individuals of all age groups. The prognosis is more favorable in pediatric patients. In contrast, outcomes for adults remain poor with current chemotherapeutic agents. Interestingly, AML with t(1;22)(p13.3;q13.1), which has been predominantly reported in children in the literature, was found to be more common in adults in this study. Regardless of age group, current treatments show poor outcomes. This study emphasizes on the urgent need for investigational agents.

Genomic Landscape of Pediatric Non-Down's Syndrome Acute Megakaryoblastic Leukemia in China

Acute megakaryoblastic leukemia (AMKL) accounts for 4% to 15% of newly diagnosed pediatric acute myeloid leukemia (AML), and different from Down's syndrome AMKL (DS-AMKL) and non-AMKL, clinical outcomes of non-DS-AMKL are characterized by an inferior prognosis. Although comprehensive genomic studies have revealed key genomic aberrations of non-DS-AMKL in Western cohort, there is still no reports about Chinese patients. Here we report a comprehensive transcriptomic study on 100 Chinese pediatric non-DS-AMKL cases through 2009 to 2022 in Shanghai Children's Medical Center. A total of 133 rearrangements were detected involving 78 patients, including previously reported recurrent fusion events which defining subgroups of AMKL, namely CBFA2T3-GLIS2 (n=20, 20%), NUP98-KDM5A (n=16,16%), rearrangements of HOX cluster genes (HOXr, n=11, 11%), RBM15-MKL1 (n=5, 5%) and MLLr (n=3, 3%). Two new recurrent gene translocations, XPO1-TRNC18 and STAG2 rearrangements (STAG2r) were detected in 5 (5%) and 11 (11%) cases, respectively. Further unsupervised clustering analysis was done with the most variant coding genes defined by standard deviation, the results showed that cases carrying STAG2r and XPO1-TNRC18 fusion exhibited distinguishable gene expression pattern from other subgroups and were clustered together respectively. It suggested these two rearrangements as potential driver genomic alterations in AMKL. By introducing XPO1-TNRC18 fusion and three STAG2 aberrations (STAG2 knock-out, STAG2-GNA12 and STAG2-OCRL) into 32D cell line, we proved these gene alterations promoted cell proliferation and colonies formation, suggesting potential function in tumor transformation. Based on these evidences, we defined XPO1-TNRC18 and STAG2r as two new subgroups of AMKL. We identified 222 nonsynonymous sequency mutations affecting 137 genes totally, including 202 single-nucleotide variants and 20 insertion/deletion, with a median of two mutations for per case (range 0-6). Among driver abnormalities, 7 aberrations occurred in more than 3% of cases in current cohort, including JAK2 (n=15, 15%), MPL (n=13, 13%), PTPN11 (n=7, 7%), NRAS (n=5, 5%), GATA1 (n=4, 4%), CTCF (n=4, 4%) and JAK3 (n=3, 3%). All the mutations could be clustered into 8 pathways, and the most frequently mutated pathways were JAK signaling (30%), transcription regulation (29%) and RAS signaling (14%). Mutation frequency varied across different subgroups. For example, JAK/STAT lesions were enriched in STAG2r subgroup (p&amp;lt;0.05). Distinct genomic aberration profile was revealed while comparing results of this non-DS-AMKL cohort to the mutation profile of non-AMKL cohort which we have published before. CBFA2T3-GLIS2, NUP98-KDM5A, STAG2r, XPO1-TNRC18 and RBM15-MKL1 subgroups were merely detected in AMKL, while driver aberrations like FLT3-ITD, CBFB-MYH1 and so on were not detected in AMKL. For driver mutations, JAK2 (15% vs 3.7%, p&amp;lt;0.0001), MPL (13% vs 0%, p&amp;lt;0.0001 ) and CTCF (4% vs 0%, p=0.007) were detected more frequently in non-DS-AML, while NRAS (5% vs 18.8%, p&amp;lt;0.001), KRAS (2% vs 10.6%, p=0.008), KIT (1% vs 21.6%, p&amp;lt;0.001), CSF3R (1% vs 6.1%, p=0.047), FLT3 (0% vs 22.5%, p&amp;lt;0.001), CEBPA (0% vs 9.8%, p&amp;lt;0.001), AXSL2 (0% vs 8.6%, p&amp;lt;0.001), GATA2 (0% vs 6.1%, p=0.007) and DHX15 (0% vs 4.5%, p=0.038) occurred less frequently in non-DS-AMKL cohort. Survival analysis revealed association between key aberrations and clinical outcomes. Carrying CBFA2T3-GLIS2 (p=0.018), NUP98-KDM5A (p&amp;lt;0.001) and FUS-ERG (p=0.018) was associated with unfavorable prognosis. Driver mutations in JAK2 (p=0.03) and RAS pathway (p=0.01) were associated with shorter overall survival. Among the two new subgroups, the new XPO1-TNRC18 subgroup demonstrated similar clinical outcomes to MLLr cases, with 5-year EFS and OS of 50%, and STAG2r was characterized by a favorable clinical outcome, with 5-year EFS and OS over 75%. As the first comprehensive genomic landscape of Chinese pediatric non-DS-AMKL, our results defined two new subgroups with distinct expression patterns and prognosis characteristics. We further revealed a distinct mutation profile between AMKL and non-AMKL and shed light on association between driver aberrations and clinical outcomes. These findings revealed non-DS-AMKL as a heterogenous disease, and suggested the importance of exploration of risk-stratification and targeted therapy in different mutations driven cases.

A Multicenter Clinical Study with Reduced Intensive Chemotherapy for Induction Remission of Pediatric Acute Megakaryoblastic Leukemia

Bcakground: Pediatric acute megakaryoblastic leukemia (AMKL) without Down syndrome (DS) is a genetically heterogenous myeloid malignancy and with dismal prognosis. Refractory and relapse remains a major challenge. Given to the young age distribution of AMKL and high frequency of abandonment in middle-income countries, we conducted a multicenter clinical trial (ChiCTR1800015875) in aims to lower the intensity of induction while without compromise the remission rate and survival. Methods: From June 2018 to December 2022, there were consecutively 43 cases with de novo non-DS AMKL, which accounted for 7.6% cases of acute myeloid leukemia (AML) during the same period, enrolled in this trial. All the patients received at least one cycle of low-dose induction with HAG regimen (homoharringtonine 1 mg/m 2, intravenous daily, days 1-7; cytarabine 10 mg/m 2, subcutaneously every 12 hours, 20 doses, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) followed by 3 to 4 courses of intensive chemotherapy or HSCT as post-remission consolidation. Besides conventional molecule testing, RNA-sequencing and whole exon sequencing (WES) were performed as well to reveal underlying genetic landscape of this type of disease. Results: The median age of the 43 patients at diagnosis was 18.5 (range 4-114) months. The median while blood cell (WBC) counts and platelet at diagnosis were 17.2×10 9/L (range 1.8-105.7 ×10 9/L) and 36.5×10 9/L (range 4-472 ×10 9/L), respectively. Among them, 39.5% of cases exhibited hepatosplenomegaly. A diversity of genetic fusions and mutations were uncovered. The recurrent fusions in this cohort include CBFA2T3-GLIS2 (16.3%), MLLr (11.6%), NUP98-(9.3%), RBM15-MKL1 (7.0%), HOX fusions (7.0%) and PICALM-MLLT10 (4.7%). Other non-recurrent fusions accounted for 18.6% (Figure 1A). The common mutations detected in our pediatric AMKL were JAK2, NRAS, MPL, BCOR, CTCF, KRAS and PTPN11. Regarding treatment response, 20 of 43 cases (46.5%) attained complete remission (CR) or complete remission with incomplete blood cell recovery (CRi) after one cycle of induction. Among the 20 cases, 9 of them got minimal residual disease (MRD) negative by flow cytometry. Ten patients (23.2%) achieved partial remission (PR) while 13 patients (30.2%) showed no response (NR) to treatment. Thirty-one patients continued with another cycle of the same regimen and 21 of the 28 cases who were available for evaluation (75.0%) attained CR/CRi. Disease relapse was observed in 16/43 (37.2%) cases, with 7/43 (16.2%) patients relapsed after transplantation. The 3-year overall survival (OS) and event-free survival (EFS) were 49.6±8.2% and 29.5±7.1%, respectively (Figure 1B). In total, more than half of patients (22/43, 51.5%) underwent HSCT. The 3-year OS of transplant group and chemotherapy group were 57.9±11.7% versus 39.5±11.2%, P=0.036. As genetic characteristics often predict treatment response and prognosis, we wonder which subgroup could benefit from the reduced intensive induction. We further compared the features of patients who got CR/CRi after induction I with those achieved PR/NR. Interestingly, we found more patients with adverse fusions in PR/NR group, such as CBFA2T3-GLIS2 (26.0% vs. 5.0%), NUP98- (17.4% vs. 0%), MLLs (13.0% vs. 10.0%), and RBM15-MKL1 (8.7% vs. 5.0%); while other non-recurrent fusions were more common in CR/CRi group (4.3% vs. 35.0%). Two thirds of patients (67.4%) experienced refractory or relapse and almost half of them harbored adverse fusions or mutations such as CBFA2T3-GLIS2, MLLr, NUP98- fusions, NRAS and PTPN11. On the other hand, 14 patients without refractory or relapse had genetic features with more JAK2 and MPL mutations. No treatment-related mortality (TRM) occurred during induction. Only one patient with KRAS mutation died of infection after consolidation III. Conclusion: Insight into the genetic and molecular landscape improved our understanding of pediatric AMKL. Distinct molecular features can serve as a tool to tailor the therapy. Though treatment still lags behind and novel therapy is urgent for the adverse subgroups, our low-dose induction with HAG regimen could benefit for a subgroup with non-recurrent fusions, which showed efficiency while is tolerable for the young age group. Based on our results, we provide an induction option for these AMKL patients, especially for those in the middle-income countries.

Clinical Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation Using Busulfan/Cyclophosphamide and Melphalan or Thiotepa As Conditioning Regimen in 37 Patients with Acute Megakaryoblastic Leukemia

Background Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of childhood AML and about 1% of adult AML. Patients with non-Down's syndrome AMKL (non-DS-AMKL) are often associated with a poor prognosis with a 3-year overall survival (OS) rate of less than 40%. Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative therapy for AMKL. However, the long-term survival post-transplant needs to be improved. Here we conducted a single-center study to evaluate the long-term efficacy and safety of allo-HSCT in 37 non-DS-AMKL patients using an innovative conditioning regimen of Busulfan/Cyclophosphamide (Bu/Cy) and Melphalan (MEL) or Thiotepa (TT). Methods From August 2015 to July 2023, a total of 37 non-DS-AMKL patients (M/F 21:16) were treated consecutively in our hospital (Hebei Yanda Lu Daopei Hospital). The median age was 2 years (range 1-9 years). Chromosomal abnormalities were present in 24 cases, with fusion genes as follows CBFA2T3/GLIS2 (5 cases) 13.5%, NUP98-KDM5A (2 cases) 5.4%, MLL-AF10 (3 cases) 8.1%, MLL-AF9 (1 case) 2.7%. Gene mutations: WT1 (10 cases) 27%, JAK2 (7 cases) 18.9%, EVI1 (6 cases) 16.2%, TET2 (4 cases) 10.8%, ASXL1 (4 cases) 10.8%, PTPN11 (4 cases) 10.8%, NRAS (3 cases) 8.1%, GATA1 (2 cases) 5.4%. Before transplantation, in bone marrow (BM), 29 patients were in complete remission (CR), 3 were in partial remission (PR) and the rest 5 were in non-remission (NR). Thirty-three patients received the haploidentical transplantation (donors from parents n=31, siblings n=2) including 1 with the 3 rd transplant, and 4 received matched-unrelated donor transplantation. The stem cell sources were from BM and peripheral blood stem cells (PBSCs) among the 33 related donors, and PBSCs for the 4 unrelated donors. Patients received a median cell number of total mononuclear cells 20.15×10 8/kg (10.7-32.85), CD34+ cells 12×10 6/kg(4.05-27.14) and CD3+ cells 4.66×10 8/kg(1.81-19.38). The conditioning regimens administered were modified Bu/Cy (fludarabine 30 mg/m 2/d×5d; cytarabine 2g/m 2/d×5d; Bu 3.2 mg/kg/d×4d, Cy 1.8g/m 2/d×2d; etoposide 100mg/m 2/d×2d; antithymocyte globulin 1.5mg/kg/d×5d) in 2 patients, Bu/Cy+MEL (MEL 55mg/m 2/d×2d) in 31 patients, and Bu/Cy+TT (TT 2.5mg/kg/d×2d) in the rest 4 patients. Cyclosporine A (CSA)+ mycophenolate mofetil (MMF)+ short methotrexate (sMTX) were used as graft-versus-host disease (GVHD) prophylaxis. Results The median follow-up time was 24 months (2-84 months), and all 37 cases survived the graft, with a median leukocyte engraftment of +13 days (9-21 days), and a median platelet engraftment of +9 days (4-38 days). BM was assessed one month after transplantation, with all grafts fully donor-type. The 5-year OS was 54.1% (Figure 1). There was a significantly higher OS in the CR group (5-year OS 69%) than the PR or NR groups (P&amp;lt;0.001) (Figure 2). Among the CR patients, no significant difference was observed in the 3 different condition regimen groups with modified BU/CY group OS of 50%, BU/CY+MEL group OS of 69.6% and BU/CY+TT group OS of 75% (P=0.869) (Figure 3). After transplantation, 17 patients died including 11 from relapse, 4 from GVHD, 1 from gastrointestinal bleeding and 1from cerebral hemorrhage. The cumulative recurrence rate (CIR) was 29.7%, among which the CIR of the CR group was 17.2%, the PR group of 66.7% and the NR group of 80% (P&amp;lt;0.001). Post-transplant, the incidence of acute GVHD (aGVHD) was 35.13% (Grade II-IV), and severe aGVHD (Grade III-IV) incidence was 18.91%. And 51.35% of patients developed chronic GVHD. Other complication incidences were cystitis at 16.21%, cytomegalovirus at 5.94%, Epstein-Barr viremia at 13.5% and infections at 21.62%. Conclusions Our study indicates that the use of modified Bu/Cy +MEL or +TT conditioning regimens in allo-HSCT procedures presents a feasible and effective approach, demonstrating an improvement in overall survival for non-Down Syndrome AMKL patients. Crucially, it was observed that achieving CR prior to transplantation significantly contributes to better outcomes. Therefore, it is strongly recommended that allo-HSCT is performed in CR status. However, to corroborate these findings, it is essential to conduct long-term, large-scale clinical research across multiple centers.

Down Syndrome with Acute Myeloid Leukemia in an Adolescent Male Successfully Treated with Low Dose Ara-C in Combination with Hypomethylating Agent

Introduction Patients with Down syndrome (DS) are prone to developing hematological malignancies, with risk of acute myeloid leukemia (AML) being 150 -fold higher compared to the general population. DS patients with leukemia are likely to experience significant chemotherapy related toxicities, hence require dose modifications. The myeloid leukemia of Down Syndrome (ML-DS) typically reflects acute megakaryoblastic leukemia (AMKL) a rare subtype of AML. ML-DS typically evolves from transient aberrant/abnormal myelopoiesis (TAM), aka transient myeloproliferative disorder (TMD), a pre-leukemic syndrome that almost always precedes myeloid leukemia in Down syndrome. GATA1, the hallmark driver mutation of TAM, is found in all TMD/TAM cases. AML in DS can occur de-novo which represents a different entity, appears immunophenotypically as (non-AMKL) AML, but differs morphologically from the classical ML-DS. The de-novo type AML in DS is reported to have worse prognosis. Poor outcomes are due to increased chemosensitivity and resulting chemotherapy-related toxicities when treated on standard high dose Ara-C based AML regimens. There is currently a lack of consensus on standard-of-care AML therapy applicable to this population. Methods Case report, retrospective electronic medical record review with institutional board review exemption Results We report a case of a 25-year-old male with trisomy 21, presenting with a one-month history of low-grade fevers, fatigue, and upper back pain. Initial laboratories showed a white cell count of 6.6 K/uL, hemoglobin 11.9 gm/dL, platelets 352 K/uL. CT chest showed a large right-sided pleural effusion, pleural thickening and multiple sclerotic bone lesions concerning for malignancy. Positron emission tomography (PET) CT revealed multiple hypermetabolic foci in the pelvis, spine, ribs, and cervical lymph nodes. Thoracocentesis was completed and pleural fluid pathology revealed atypical cells, flow cytometry showed 18% blasts positive for CD38, CD45, CD117 and negative for CD34 and HLA DR indicative of a myeloid neoplasm. Bone marrow aspirate and biopsy showed 10% CD117 positive immature myeloid cells. Morphology was not megakaryoblastic. Bone marrow flow cytometry results were consistent with that of pleural fluid with CD117 and myeloperoxidase (MPO) positive blasts. Fluorescent in-situ hybridization was negative for fusions/rearrangements. Next generation sequencing (NGS) detected NPM1 mutation and also a low level (variant allelic frequency &amp;lt;3%) FLT3 p.Y599C mutation (not internal tandem duplication or tyrosine kinase domain). GATA1 mutation was absent. A diagnosis of AML with NPM1 mutation and staging CNS1 was confirmed. Our patient was defined as DS and AML. Given innate fragility and higher chemo sensitivity in patients with DS, we elected to treat him off protocol with Cladribine and low dose Ara-C alternating with Decitabine. This regime was derived from an adult leukemia protocol. Cycle 1 was complicated by central line related occlusive thrombus requiring anticoagulation, slow count recovery, and a brief episode of febrile neutropenia managed with antibiotics. End of induction marrow evaluation was negative for minimal residual disease (MRD). Flow cytometry on pleural fluid was also negative for abnormal blasts that were previously detected. Repeat molecular testing with NGS was negative for NPM1, DNMT3A, and FLT3 mutations. The patient received a total of 8 cycles of chemotherapy. On subsequent bone marrow evaluations, blasts remained &amp;lt;5% and MRD was negative. Interval PET scan showed significant improvement. Patient is currently more than a year off therapy and remains in remission. Conclusion Patients with DS and AML have a higher mortality due to increased risk of cardiotoxicity and infection from chemotherapy. There is a risk of either undertreatment or less commonly overtreatment leading to early mortality. Our case describes an adolescent male with DS and AML with pulmonary comorbidity who was successfully treated with a low-dose Ara-C regimen adapted from an adult protocol and had great clinical outcome. AML treatment in DS remains a challenge and further research is warranted to develop tailored regimens effectively balancing curative intent and reduced toxicity. This regimen could be considered in pediatric patients with Down Syndrome and other comorbidities.

Single-Cell Transcriptomics Reveal Altered Hematopoietic Mechanisms Driven By T21 and GATA1s

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p &amp;lt;0.05 was considered statistically significant. Results Cluster annotation revealed HPC, early and committed erythroid and MK, and myeloid cells (Figure 1). D7 T21/G1S HPCs surprisingly showed an erythroid bias, with upregulation of genes such as GYPA and AHSP, while euploid/G1S HPCs showed a MK bias, with upregulation of genes including PF4V1 and PF4. With wtGATA1, neither euploid nor T21 D7 HPCs showed a significant lineage skew. On D9 and D11, the euploid/WT, T21/WT, and T21/G1S cells developed subpopulations committing to the erythroid lineage, while the euploid/G1S cells demonstrated minimal erythroid potential (Figure 1). Across all 3 timepoints, early and committed erythrocytes from T21 and euploid cells with G1S had a greater number of combined DEGs than their wtGATA1 counterparts (G1S vs. WT, T21: 692 vs. 378; euploid: 1235 vs. 599). On D9, T21 was associated with a greater erythroid skew (38.6% of T21/WT and 32.2% of T21/G1S cells vs. 27.8% of euploid/WT cells; p &amp;lt;0.00001). By D11, this erythroid drive was no longer apparent, and the T21/G1S erythrocytes were significantly less mature (Table 1; p &amp;lt;0.00001). These data suggest that T21 initially enhances erythropoietic drive, while G1S results in a near-complete erythroid block in euploid cells but an incomplete developmental block with T21. All 4 genotypes yielded MK subpopulations on D9 and D11 (Figure 1). With wtGATA1, euploid and T21 MKs were predominantly early MKs by D11 (Table 1). Interestingly, G1S was associated with enhanced MK commitment and maturation in the euploid context but arrest at the early stage with T21 (Table 1; p &amp;lt;0.00001). G1S was again associated with increased DEGs compared to wtGATA1 (G1S vs. WT, T21: 594 vs. 454; euploid: 1689 vs. 1310). These data suggest that G1S in a euploid background promotes MK differentiation, but T21 and G1S cooperate to generate immature, abnormal MKs. Conclusions Although the hematopoietic abnormalities and leukemic risk of T21 have been well-described, the underlying developmental events remain unclear. This scRNA-seq timecourse of HPCs differentiated from isogenic iPSCs reveals the individual and synergistic effects of T21 and G1S. Overall, T21 enhances erythropoietic drive while G1S alone suppresses erythropoiesis. G1S in euploid cells enhances megakaryopoiesis, but with T21, megakaryopoietic commitment and maturation are uncoupled with a strong bias to early MKs. Together, the competing effects of T21 and G1S appear to result in abnormal, immature erythrocytes and MKs with impaired lineage commitment, consistent with a pro-leukemic phenotype.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank &amp;lt; 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

Immunophenotypic markers for the evaluation of minimal/measurable residual disease in acute megakaryoblastic leukemia

Acute megakaryoblastic leukemia is characterized by heterogeneous biology and clinical behavior. Immunophenotypic characteristics include the expression of megakaryocytic differentiation markers (e.g. CD41, CD42a, CD42b, CD61) associated with immaturity markers (CD34, CD117, HLA-DR) and myeloid markers (e.g. CD13, CD33) and even with lymphoid cross-lineage markers (e.g. CD7, CD56). Although the diagnostic immunophenotype has already been well described, given the rarity of the disease, its immunophenotypic heterogeneity and post-therapeutic instability, there is no consensus on the combination of monoclonal markers to detect minimal/measurable residual disease (MRD).Currently, MRD is an important tool for assessing treatment efficacy and prognostic risk. In this study, we evaluated the immunophenotypic profile of MRD in a retrospective cohort of patients diagnosed with acute megakaryoblastic leukemia, to identify which markers, positive or negative, were more stable after treatment and which could be useful for MRD evaluation. The expression profile of each marker was evaluated in sequential MRD samples. In conclusion, the markers evaluated in this study can be combined in an MRD immunophenotypic panel to investigate for megakaryoblastic leukemia. Although this study is retrospective and some data are missing, the information obtained may contribute to prospective studies to validate more specific strategies in the detection of MRD in acute megakaryoblastic leukemia.

Second Hematologic Malignancies Associated With Primary Mediastinal Germ Cell Tumors: A Population-based Study.

Studies addressing second hematologic malignancies (SHMs) in patients with primary mediastinal germ cell tumors (PMGCTs) are scarce. To better describe this phenomenon, we analyzed a large case series from a population-based registry. The Surveillance, Epidemiology, and End Results database was used to report the clinical characteristics and incidence of SHMs in patients with PMGCT. Among 1297 PMGCTs, 27 cases (2.08%) of SHM were found, with a median latency period of 12 months (95% CI: 5-41). All SHM occurred in males, 20 of whom (74.1%) had a previous nonseminomatous tumor. Acute myeloid leukemia was the most frequent SHM, accounting for 13 cases, 4 of which were acute megakaryoblastic leukemia that occurred within 5 months of diagnosis. The median survival after the diagnosis of SHM was 6 months (95% CI: 2-41). The risk of SHM was significantly higher than expected for the reference population, with a standardized incidence ratio of 6.21 (95% CI: 3.31-10.62) and an absolute excess risk of 19.19 per 10,000 person-years. Patients with PMGCT are at a higher risk of developing SHMs than the general population, particularly acute myeloid leukemia. This risk ranges from synchronous diagnosis of acute megakaryoblastic leukemia to the later onset of other hematological disorders that might be related to PMGCT therapies. Our findings may help create follow-up schedules for patients with PMGCT and raise the level of suspicion surrounding this association.

CBFA2T3::GLIS2 pediatric acute megakaryoblastic leukemia is sensitive to BCL-XL inhibition by navitoclax and DT2216

AbstractAcute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood–derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia.

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.

Long-term follow-up of haploidentical haematopoietic stem cell transplantation in paediatric patients with high-risk acute myeloid leukaemia: Report from a single centre.

Data from 200 children with high-risk acute myeloid leukaemia who underwent their first haploidentical haematopoietic stem cell transplantation (haplo-HSCT) between 2015 and 2021 at our institution were analysed. The 4-year overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR) were 71.9%, 62.3% and 32.4% respectively. The 100-day cumulative incidences of grade II-IV and III-IV acute graft-versus-host disease (aGVHD) were 41.1% and 9.5% respectively. The 4-year cumulative incidence of chronic GVHD (cGVHD) was 56.1%, and that of moderate-to-severe cGVHD was 27.3%. Minimal residual disease (MRD)-positive (MRD+) status pre-HSCT was significantly associated with lower survival and a higher risk of relapse. The 4-year OS, EFS and CIR differed significantly between patients with MRD+ pre-HSCT (n = 97; 63.4%, 51.4% and 41.0% respectively) and those with MRD-negative (MRD-) pre-HSCT (n = 103; 80.5%, 73.3% and 23.8% respectively). Multivariate analysis also revealed that acute megakaryoblastic leukaemia without Down syndrome (non-DS-AMKL) was associated with extremely poor outcomes (hazard ratios and 95% CIs for OS, EFS and CIR: 3.110 (1.430-6.763), 3.145 (1.628-6.074) and 3.250 (1.529-6.910) respectively; p-values were 0.004, 0.001 and 0.002 respectively). Thus, haplo-HSCT can be a therapy option for these patients, and MRD status pre-HSCT significantly affects the outcomes. As patients with non-DS-AMKL have extremely poor outcomes, even with haplo-HSCT, a combination of novel therapies is urgently needed.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both invitro and invivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Analysis 33 patients of non-DS-AMKL with or without acquired trisomy 21 from multiple centers and compared to 118 AML patients

ABSTRACTBackgroundAcute megakaryoblastic leukemia (AMKL) without Down syndrome (non-DS-AMKL) usually a worse outcome than DS-AMKL. Acquired trisomy 21(+21) was one of the most common cytogenetic abnormalities in non-DS-AMKL. Knowledge of the difference in the clinical characteristics and prognosis between non-DS-AMKL with +21 and those without +21 is limited.ObjectiveVerify the clinical characteristics and prognosis of non-DS-AMKL with +21.MethodWe retrospectively analyzed 33 non-DS-AMKL pediatric patients and 118 other types of AML, along with their clinical manifestations, laboratory data, and treatment response.ResultsCompared with AMKL without +21, AMKL with +21 has a lower platelet count (44.04 ± 5.01G/L) at onset (P > 0.05). Differences in remission rates between AMKL and other types of AML were not significant. Acquired trisomy 8 in AMKL was negatively correlated with the long-term OS rate (P < 0.05), while +21 may not be an impact factor. Compared with the other types of AML, AMKL has a younger onset age (P < 0.05), with a mean of 22.27 months. Anemia, hemorrhage, lymph node enlargement, lower white blood cell, and complex karyotype were more common in AMKL (P < 0.05). AMKL has a longer time interval between onset to diagnosis (53.61 ± 71.15 days) (P < 0.05), and patients with a diagnosis delay ≥3 months always presented as thrombocytopenia or pancytopenia initially.ConclusionsDue to high heterogeneity, high misdiagnosis rate, and myelofibrosis, parts of AMKL may take a long time to be diagnosed, requiring repeated bone marrow punctures. Complex karyotype was common in AMKL. +21 may not be a promising indicator of a poor prognosis.

Acute megakaryoblastic leukaemia shows high frequency of chromosome 1q aberrations and dismal outcome.

Acute megakaryoblastic leukaemia (AMKL) is associated with poor prognosis. Limited information is available on its cytogenetics, molecular genetics and clinical outcome. We performed genetic analyses, evaluated prognostic factors and the value of allogeneic haematopoietic stem cell transplantation (allo-HSCT) in a homogenous adult AMKL patient cohort. We retrospectively analysed 38 adult patients with AMKL (median age: 58 years, range: 21-80). Most received intensive treatment in AML Cooperative Group (AMLCG) trials between 2001 and 2016. Cytogenetic data showed an accumulation of adverse risk markers according to ELN 2017 and an unexpected high frequency of structural aberrations on chromosome arm 1q (33%). Most frequently, mutations occurred in TET2 (23%), TP53 (23%), JAK2 (19%), PTPN11 (19%) and RUNX1 (15%). Complete remission rate in 33 patients receiving intensive chemotherapy was 33% and median overall survival (OS) was 33 weeks (95% CI: 21-45). Patients undergoing allo-HSCT (n = 14) had a superior median OS (68 weeks; 95% CI: 11-126) and relapse-free survival (RFS) of 27 weeks (95% CI: 4-50), although cumulative incidence of relapse after allo-HSCT was high (62%). The prognosis of AMKL is determined by adverse genetic risk factors and therapy resistance. So far allo-HSCT is the only potentially curative treatment option in this dismal AML subgroup.