Variant Of Acute Myeloid Leukemia Research Articles

Monocytic Differentiation in Acute Myeloid Leukemia Cells: Diagnostic Criteria, Biological Heterogeneity, Mitochondrial Metabolism, Resistance to and Induction by Targeted Therapies.

We review the importance of monocytic differentiation and differentiation induction in non-APL (acute promyelocytic leukemia) variants of acute myeloid leukemia (AML), a malignancy characterized by proliferation of immature myeloid cells. Even though the cellular differentiation block is a fundamental characteristic, the AML cells can show limited signs of differentiation. According to the French-American-British (FAB-M4/M5 subset) and the World Health Organization (WHO) 2016 classifications, monocytic differentiation is characterized by morphological signs and the expression of specific molecular markers involved in cellular communication and adhesion. Furthermore, monocytic FAB-M4/M5 patients are heterogeneous with regards to cytogenetic and molecular genetic abnormalities, and monocytic differentiation does not have any major prognostic impact for these patients when receiving conventional intensive cytotoxic therapy. In contrast, FAB-M4/M5 patients have decreased susceptibility to the Bcl-2 inhibitor venetoclax, and this seems to be due to common molecular characteristics involving mitochondrial regulation of the cellular metabolism and survival, including decreased dependency on Bcl-2 compared to other AML patients. Thus, the susceptibility to Bcl-2 inhibition does not only depend on general resistance/susceptibility mechanisms known from conventional AML therapy but also specific mechanisms involving the molecular target itself or the molecular context of the target. AML cell differentiation status is also associated with susceptibility to other targeted therapies (e.g., CDK2/4/6 and bromodomain inhibition), and differentiation induction seems to be a part of the antileukemic effect for several targeted anti-AML therapies. Differentiation-associated molecular mechanisms may thus become important in the future implementation of targeted therapies in human AML.

Clinical analysis of 82 cases of acute promyelocytic leukemia with PML-RARα short isoform in children and adults.

Acute promyelocytic leukemia (APL) with PML/RARα fusion gene is a distinct variant of acute myeloid leukemia. According to the different break sites of the PML gene, there are three transcripts: Long (bcr1), Variant (bcr2) and Short (bcr3). We retrospectively analyzed 82 APL cases with PML-RARα short isoform. A total of 384 patients with APL were seen, of which 85(22.14%) had PML/RARα short isoform (bcr3) and 82 met the inclusion criteria. The median age was 33.5 years (range, 2-72 years). The incidences of hemorrhage in the intermediate- and high-risk group were higher, but only the incidence between medium and low risk differed statistically (P=0.006), and the incidences of fever, fatigue, splenomegaly, and lymph node enlargement and differentiation syndrome (DS) in those groups were not statistically significant (P>0.05). FLT3 gene mutation rate and the mortality rate of the high-risk group were significantly higher than that of other groups (P=0.040 and P=0.004, P=0.041 and P=0.037, respectively). The mortality rate was lowest (4.26%) in the group treated with ATRA combined with arsenic and anthracycline. The 3-year OS and the 3-year DFS of the low and intermediate-risk group were better (P=0.019 and P=0.017, respectively). ATRA combined with arsenic and anthracycline had significant impact on outcomes in APL with PML-RARα short isoform.

Cis-Acting Splicing-Associated Variants Can Redefine the Molecular Signature of Genes Commonly Mutated in Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a blood malignancy that occurs as a result of genomic alterations acquired in hematopoietic stem cells (HSCs). Several studies have recognized the importance of these alterations, including chromosomal rearrangements and single nucleotide variations (SNVs), for the classification and risk stratification of patients. However, none of these studies have thoroughly explored the functional impact of these lesions, potentially leading to the oversight or misinterpretation of certain driver mutations. In this study, we focused on the splicing process, as we aim to comprehensively characterize cis-acting splicing-associated variants (SAVs) in a large cohort of AML patients. Methods: We obtained recurrent driver gene variants (n= 3,847) from Table S5 reported by Papaemmanuil et al. and selected unique SNVs (n= 915) for our analysis. Among them, 628 (69%) were missense variants, 232 (25%) were nonsense variants, and 55 (6%) were located within splice sites. To assess their potential impact on splicing, we employed three splicing predictor tools (MaxEntScan, regSNP-splicing, and SpliceAI) and considered variants with two favorable predictions for further functional studies. Firstly, we attempted to locate the SNVs within the exomes of the TCGA-LAML (n= 149) and the BeatAML (n=342) datasets available in the Genomic Data Commons repository. If an SNV was identified, we obtained the RNA sequencing bam file from the corresponding patient's sample and utilized the rest of the cohort as a control for statistical analysis (p<0.05 for significance). In cases where the SNV was not found in these datasets, we employed the pSPL3-based splicing reporter minigene assay to study the variant. Results: The predictors unanimously identified all 55 splice-site affecting variants as true positives and reclassified 20 missense and nonsense variants as likely SAVs. Functional validation experiments confirmed that 11 of these variants (55%) indeed had an impact on splicing. Analysis of RNA sequencing data revealed interesting findings in commonly observed AML hotspots. For instance, in cases of U2AF1 c.470A>G and IDH1 c.394C>A, novel donor splice sites were created at distances of one (p=0.04522) and 34 nucleotides from the mutations (p=0.01878), respectively. Whereas, the TP53 c.395A>G change was found to activate a cryptic acceptor splice site located XX nucleotides away (p=0). Among private AML variants, minigene assays showed that eight variants resulted in aberrant splicing compared to the wild-type. Three variants led to exon skipping due to the loss of canonical donor splice sites ( EZH2 c.363G>A, FLT3 c.2523C>A, and TET2 c.3500G>A), four variants caused the activation of a novel donor splice site ( PTPN11 c.1508G>T, WT1 c.1157C>A, STAG2 c.545T>G, and KDM6A c.1516C>T), and one variant resulted in both exon skipping and the creation of a donor splice site ( RAD21 c.688G>A). Consequently, the predicted impact on the protein varied, with FLT3 and TET2 variants potentially leading to an in-frame deletion of the ATP-binding site and TET2-oxygenase domain, respectively, with the EZH2 variant likely resulting in an expression switch from the large to small isoform. The remaining SNVs were anticipated to trigger Nonsense-Mediated Decay or shorten proteins. Conclusions: This study revealed that approximately 7% of SNVs found in a large group of AML patients have the potential to impact splicing. Notably, 1% of these SNVs had been classified as missense or nonsense changes. These findings provide support for the significant role of mis-splicing in the development of leukemia and emphasize the importance of devising novel approaches to effectively characterize driver mutations.

MALIGNANCY MASQUERADING AS STEROID RESISTANT HYPEREOSINOPHILIA SYNDROME

<h3>Introduction</h3> Hypereosinophilia syndrome (HES) is defined as 1500 eosinophils/mL or above with evidence of end-organ damage due to the eosinophils at least 4 weeks apart. <h3>Case Description</h3> A 3-year-old male was admitted for newly found anterior mediastinal mass and left pleural effusion. About 6 weeks prior to admission, he was seen in his primary care office for difficulty breathing. Despite the appropriate treatment for his symptoms, the patient continued to have significant respiratory symptoms and a chest x-ray showed a large left pleural effusion. He was immediately transferred to a tertiary care center and a CT chest showed large left pleural effusion, possible abscess, small pericardial effusion, and concern for an anterior mediastinal mass due to mediastinal shift. He underwent a thoracentesis, which showed eosinophilia within the pleural fluid. While his peripheral eosinophils had fluctuated at the beginning of his hospitalization, the patient's absolute eosinophil count suddenly increased from 600 to 25,048 and remained elevated above 20,000. He developed recurrent eosinophilic pericardial effusions requiring multiple pericardiocentesis. Despite high dose steroids and inpatient use of mepolizumab, his hypereosinophilia persisted. After two inconclusive fine-needle aspirations of his mediastinal mass, the patient's family agreed to a thoracotomy with removal of the mediastinal mass. Pathology eventually revealed myeloid sarcoma, a variant of acute myeloid leukemia. <h3>Discussion</h3> Steroid resistant HES should raise concern for underlying malignancy especially when eosinophils are above 20,000.

Intracranial hypertension associated with treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA): a case report

Case presentation&#x0D; Female, 18 years-old, without relevant family history, referred to Neurology by Hematology for headache. 3 years ago, she was diagnosed with Acute Promyelocytic Leukemia (APL) currently on maintenance therapy with All-Trans Retinoic Acid (ATRA). The patient reports that in all previous cycles presented headache with the following characters: one-side temporal location, pulsating quality, moderate intensity, with nausea and vomiting, photophobia and phonophobia, uncertain duration, with simple analgesics response. In the current cycle, she relates continuous pain, severe intensity and unresponsive to medication. On examination, there were no focal neurological findings and on funduscopic examination there was no papilledema. MRI and laboratorial tests were normal. Cerebrospinal fluid (CSF) opening pressure was 35 cmH2O, no other alterations. 15 milliliters of CSF were removed, with a closing pressure of 25 cmH2O. A hypothesis of Intracranial Hypertension associated with use of ATRA was made. Afterwards, there was important improvement of the headache, with residual pain of mild intensity. Therefore, Acetazolamide was started at a dose of 250 milligrams every 12 hours with complete resolution of symptoms.&#x0D; &#x0D; Discussion&#x0D; Acute myeloid leukemia (AML) comprises a heterogeneous group of aggressive blood cell cancers that arise from clonal expansion of malignant hematopoietic precursor cells in the bone marrow. Acute promyelocytic leukemia (APL) is a biologically and clinically distinct variant of AML. APL is classified as acute promyelocytic leukemia with PML-RARA. The cytogenetic hallmark of APL is a translocation involving RARA, the retinoic acid receptor alpha locus on chromosome 17. Without treatment, APL is the most malignant form of AML, with a median survival of less than one month.&#x0D; (To see the complete abstract, please, check out the PDF).

P1612: ACUTE MYELOID LEUKEMIA IN ADULT PATIENTS: FEATURES OF TREATMENT IN CONTEXT OF THE COVID-19 PANDEMIC.

Background: Acute myeloid leukemia (AML) is a disease with a poor prognosis and a high mortality rate, while patients with acute myeloid leukemia are at increased risk of complications from COVID-19 infection. Aims: To investigate the treatment effectiveness and survival of patients with AML who had the COVID-19 diagnosis confirmed, as well as evaluating predictors of hospital mortality among this group of patients. Methods: From April 2020 to November 2021, Moscow City Clinical Hospital 52 treated 149 patients with AML who had the COVID-19 diagnosis confirmed, with a median age of 61 years (range 18-91), while 56% of patients were over 60 years old. An equal ratio of men and women was performed, 70 and 79 respectively. 13% were diagnosed with APL, while the remaining 87% had other variants of AML. At the time of hospitalization the most of the patients admitted had first- or second-degree pulmonary lesions (28 and 35%, respectively), while 31% of patients performed more severe lesions and the other 6% had no pulmonary damage detected, but they had a positive PCR. At the time of hospitalization, 10 out of 149 patients (7%) achieved complete remissions (CR) and were withdrawn from therapy; 41 patients (28%) were diagnosed with AML for the first time in our clinic; and 98 patients (65%) were already in process of AML treatment in other clinics. The effectiveness of treatment was assessed by the frequency of CR, as well as by the amount of cases of lethality and in-hospital survival rates. The analysis of treatment results was performed as at 01 November 2021. Results: 60 patients (38%) with AML (excluding APL) and confirmed COVID19 received anti-tumor treatment. Palliative care was received by 23 patients (38%), these were predominantly elderly patients with a median age of 71 years (range 45-91 years), with severe COVID-19. 19 patients (32%) with a median age of 38 years (range 21-61 years) received the 7 + 3 treatment protocol. While 18 elderly patients (30%) with a median age of 70 years (range 60-85 years) received the low intensity therapy (low doses of cytarabine, hypomethylation therapy and venetoclax containing regimens). As a result of COVID-19, 79 patients (54%) died. The main factor determining the high mortality rate in our study was the absence of AML CR. The Survival of patients in CR was 96.3±3.6%, which means almost all patients have recovered from COVID-19 and were discharged to continue their treatment in other medical institutions. For patients out of remission and primary patients with AML the survival rate was 38.0±6.5% and 33.7±8.7%, respectively. For the purposes of analyzes of predictors of survival, we divided our patients into 2 groups, due to a great variety of prognosis. Remission status essentially outweighs all other factors, including presence of significant comorbidities, AML variants, isolated cytopenias, severity of COVID-19 (CT, CRP, D-dimer, etc.), nature of AML therapy and other factors. In the group of patients out of AML remission the negative predictive value for hospital survival was shown by age over 60 years, as well as by agranulocytosis, CT 3-4, the use of GCS and transfer to the ICU. Summary/Conclusion: The diagnosis of COVID-19 in patients with AML greatly worsens their life prognosis. The leading predictor of mortality becomes the lack of remission of AML. Thus, antitumor treatment allows achieving CR, which in turn reduces the risk of developing a severe course of coronavirus infection.

ACUTE MEGACARYOBLASTIC LEUKEMIA IN CHILDREN: DIAGNOSTICS AND MRD MONITORING

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) associated with poor prognosis for all patients except children with t(1;22) or Down syndrome. The frequency of complete remission in case of AMKL is comparable to the frequency of it in other variants of AML but the median survival is much lower. This determines the necessity of more thorough evaluation of treatment effect using flow cytometry accessment of minimal residual disease (MRD). The clinical and immunological profile of 8 girls and 9 boys with de novo AMKL between the ages of 3 months–11 years old was analyzed. The primary leucocytosis median was 10,25; only one patient had hyperleukocytosis (53х109/l) at presentation.The measurement of MRD was performed in 6 patients using multiparameter flow cytometry. The measurement of MRD performed after induction therapy on the basis of megakaryocytic markers, weak CD45 expression using the initial iimmunophenotype patterns. Adequate measurement of the level of MRD had required extensive diagnostic immunophenotyping in order to determine the aberration of megakaryoblasts. CD9(83,3%), CD33(75%), CD34(60%), CD13(50%) apart from megakaryocyte markers (100%) were most common for blast cells in case of AMKL. The expression of CD7 antigen was as frequent as of CD117-40%. The MRD level ranged from completely negative (0%; 0.006%) to evident (1.05%). The detection of residual tumor megakaryoblasts in AML M7 using flow cytometry is a promising method for assessing the effect of therapy. Adequate measurement of MRD requires detailed immunophenotyping in the diagnosis to determine the aberrations of megacaryoblasts immunophenotype.

Acute myeloid leukemia mmicking&#x0D; an Ewing’s sarcoma family tumor:&#x0D; A clinical case

Relevance: M6 variant of acute myeloid leukemia is extremely rare in pediatric practice. The diverse clinical manifestations&#x0D; of erythroid leukemia complicate the timely diagnosis of this group of diseases.&#x0D; Purpose: to describe a clinical case of congenital erythroid leukemia with multiple lesions of soft tissues and the skeletal&#x0D; system with complications in the form of the convulsive disorder, presented as a tumor of the Ewing’s sarcoma family, diagnosed at the Research Center of Pediatrics and Pediatric Surgery (Almaty, the Republic of Kazakhstan).&#x0D; Results: The presented clinical case demonstrated challenges in diagnosing and treating young patients with multiple&#x0D; life-threatening tumor lesions. The need to develop molecular genetic studies and expand diagnostic capabilities is an integral&#x0D; part of treating oncohematological diseases.&#x0D; Conclusion: The presented clinical case is of great interest due to its rareness. This case confirms the need to conduct all&#x0D; diagnostic manipulations to assess the process prevalence and choose and conduct molecular genetic studies on all examination and treatment stages.

Efficacy and Tolerability of First Line Arsenic Trioxide in Combination With All-Trans Retinoic Acid in Patients With Acute Promyelocytic Leukemia: Real Life Experience.

Acute promyelocytic leukemia is a variant of acute myeloid leukemia characterized by t(15;17) and PML/RAR alfa fusion gene. The discovery of the molecular pathogenesis has led to entitle all-trans retinoic acid (ATRA) as the first targeted therapy for acute leukemia. It is usually associated to anthracycline-based chemotherapy with high response rates, but potential long-term sequelae including therapy-related malignancies have been observed. Arsenic trioxide (ATO) was added to obviate these complications and investigational trials aimed to a new strategy with the incorporation of arsenic trioxide (ATO) into initial therapy instead of chemotherapy in selected patients. ATRA plus ATO without chemotherapy was the first attempt to treat low and intermediate-risk patients with APL. Our study aims to describe a monocentric cohort of patients with newly diagnosed APL effectively treated with ATO plus ATRA underlying its efficacy together with the high grade of tolerability of this association. From January 2009 to December 2019 23 APL patients were diagnosed and treated with ATO plus ATRA regimen: 14 males and 9 females patients with a median age of 45 years (range 18-72), for the majority intermediate risk (15 patients, 65%). The treatment was well tolerated and all patients achieved molecular remission after a median time of 3 months (range 1-6 months). All patients proceeded to consolidation phase as outpatients, they maintained complete molecular response at a median time of 44 months (range 15-127) except for 1 patient. All but one patient are alive and in response at a median follow-up of 48 months (range 9-141) without late effects. ATO plus ATRA regimen shows advantages in comparison to chemotherapy; in fact it allowed to treat patients in which chemotherapy could even not be applicable and it did not show secondary hematological diseases. The association of ATO to ATRA as chemo-free regimen enabled to treat APL even without chemotherapy.

The Application of Minimal Residual Disease in Hemato-Oncology: A Review of Its Current Utility in Trials, Regulatory Decisions and Clinical Practice

The concept of minimal residual disease (MRD) has become common across multiple hematological malignancies. However, there is considerable variation in the application of this concept to research activities and clinical practice across different diseases. We sought to investigate the current status of MRD testing in research, its standing with regulators, and its application in clinical practice, and interpreted the differences identified in light of the differentiating characteristics of each disease. Seven malignancies were considered: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM).The technology used to detect MRD, or its near equivalents of major or deep molecular response (MMR) in CML, is under constant development; current techniques are based on polymerase chain reaction for all indications considered (including next-generation sequencing) or flow cytometry for four indications (ALL, AML, CML, and MM), with a role for imaging also proposed in MM.A review of the published literature (via PubMed) for each of the diseases identified multi-study meta-analyses describing the prognostic significance of MRD in ALL, CML, and MM; evidence of this relationship was based on analysis of individual studies for the other four diseases.In the context of the prognostic significance of MRD status, MMR is well established as an outcome in clinical trials in CML and is emerging as a trial outcome measure in other indications. A review of registered active Phase 2 and 3 clinical trials (via clinicaltrials.gov) found that MRD (or an equivalent such as molecular response) was listed as an outcome measure in 21% of trials in ALL, 17% in CLL, and 12% in MRD; this was more common in Phase 3 trials (15/40, 16/51, and 14/73 trials, respectively). MRD was listed as an outcome measure in 6% of trials in AML, 2% in DLBCL, and 6% in FL. Furthermore, the European Medicines Agency formally accepted MRD as an intermediate endpoint in CLL (effective from 2016), while MMR was recommended as a primary endpoint in CML in 2013; consultation is ongoing regarding acceptance of MRD in MM. The US Food and Drug Administration has led a number of workshops on the use of MRD in four indications (MM, CLL, AML, and ALL).Aside from academic interest and clinical development programs, the use of MRD testing to guide treatment is recommended in US and European clinical guidelines for ALL, CML, and the acute promyelocytic variant of AML, and in US guidelines for CLL; US guidelines for MM recommend assessment of MRD but do not suggest that the result should inform the course of treatment. For other malignancies, prognostic implications and utility in trial settings are acknowledged, but MRD testing is not recommended in practice.The role of MRD in a range of hematological malignancies appears to be evolving rapidly. MMR in CML has been important in trials and in clinical practice for a number of years, but has recently been followed by MRD in CLL, while MRD testing in MM is on the horizon. MRD evaluation is now an integral part of both adult and pediatric treatment protocols. The biological basis of each disease (targets for detection, location of disease), interacting with the available technology, treatments, and treatment intent, may have contributed to different speeds of uptake. Given the growing body of research on detection technologies and its role in other malignancies, we expect MRD testing to become increasingly widespread and its role to shift from prognosis to a key factor in determining treatment pathways. DisclosuresNo relevant conflicts of interest to declare.

CAR T Cells Targeting FLT3 on AML Confer Potent Anti-Leukemic Activity and Act Synergistically with the FLT3 Inhibitor Midostaurin

Background: FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein that is expressed on normal hematopoietic stem (HSC) and progenitor cells. FLT3 is also uniformly present on malignant blasts in acute myeloid leukemia (AML) providing a target for immunotherapy with chimeric antigen receptor (CAR)-modified T cells. FLT3 is a driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3-ITD) and other gain-of-function mutations in the intracellular tyrosine kinase domain. Accordingly, the FLT3 inhibitor midostaurin has recently been approved in combination with chemotherapy to treat FLT3-mutated AML. Here, we explored the antileukemia efficacy of FLT3 CAR-T cells against FLT3 wild-type (wt) and FLT3-ITD+ AML, alone and in combination with midostaurin to determine whether the two therapeutic modalities could be used synergistically, particularly in high-risk FLT3-ITD+ AML.Methods: A FLT3-CAR (BV10 scFv_IgG4 hinge_CD28_CD3ζ_EGFRt) was encoded in the lentiviral vector epHIV7 for gene-transfer into T cells of healthy donors and AML patients (n>6). CAR-T cell cytolytic activity was evaluated in FACS-/luminescence-based assays, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. Immunodeficient NSG mice were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR-modified or control T cells (CD4:CD8 ratio = 1:1). MOLM-13 cells were cultured in the presence of 10nM midostaurin and doses titrated up to 50nM to induce resistance (MOLM-13R).Results: We confirmed specific recognition and high-level cytolytic activity of CD8+ FLT3 CAR-T cells against a panel of AML cell lines that included THP-1 (FLT3 wt), MOLM-13 (FLT3-ITD+/-) and MV4;11 (FLT3-ITD+/+), as well as FLT3-ITD+ primary AML blasts obtained from multiple patients. Both CD8+ and CD4+ FLT3 CAR-T cells produced IFN-γ and IL-2, and underwent proliferation after stimulation with native AML cell lines and primary AML blasts.We then treated AML cell lines with midostaurin and detected a significant increase in FLT3 expression in MOLM-13 and MV4;11 by flow cytometry (delta MFI = 8986 and 1442, respectively, p<0.05), but not THP-1 cells, indicating that upregulation specifically occurred in FLT3-ITD+ AML. We sought to determine whether higher FLT3 expression on midostaurin-resistant AML target cells translated into superior antileukemia efficacy of FLT3 CAR-T cells. Indeed, we observed superior cytolytic activity, cytokine production and proliferation of FLT3 CAR-T cells after stimulation with MOLM-13R compared to native MOLM-13 cells (n=3 experiments; p<0.05). In contrast to FLT3, expression of CD33 and CD123 as alternative CAR target antigens was decreased on MOLM-13R.We confirmed upregulation of FLT3 occurred on native MOLM-13 AML cells in vivo during midostaurin treatment in NSG mice, and observed synergistic antileukemia efficacy of FLT3 CAR-T cells and midostaurin in combination therapy. Intriguingly, we detected higher frequencies of CAR-T cells in all mice that received the combination treatment, compared to mice that received FLT3 CAR-T cells alone. At the end of the observation period, all mice in the combination treatment group had achieved complete remission of AML from peripheral blood, bone marrow and spleen.Importantly, we also show that FLT3 CAR-T cells recognize normal HSCs and interfere with hematopoiesis in colony formation assays in vitro . However, midostaurin treatment did not lead to an increase of FLT3 on normal HSC consistent with their expression of wt FLT3.Summary: Collectively, our data demonstrate that FLT3 CAR-T cells mediate potent reactivity against FLT3 wt and FLT3-ITD+ AML cells in vitro and in vivo . We further demonstrate that FLT3 CAR-T cells and midostaurin act synergistically, i.e. midostaurin-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSC, the clinical use of FLT3 CAR-T cells is likely restricted to a defined therapeutic window prior to allogeneic HSC transplantation, followed by CAR-T cell depletion and hematopoietic reconstitution. Our data provide an operational model to augment the antileukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk for relapse with FLT3-negative AML variants that may otherwise develop under therapeutic pressure. DisclosuresNo relevant conflicts of interest to declare.

Detection and monitoring of minimal residual disease in acute megakaryoblastic leukemia in children

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), which is associated with poor prognosis for all patients except children with t(1;22) or Down syndrome. The frequency of complete remission in case of AMKL is comparable to the frequency of complete remission in other variants of AML, and the median survival is much lower. This determines the necessity to update criteria for assessment of the effect of treatment using flow cytometry definition of the level of minimal residual disease (MRD). Nowadays, there are no unified and standardized approaches for the measurement of MRD in case of myeloid leukemia, including AMKL, which prohibits adequate assessment of the therapy effect and in some cases determination of the indications for allogeneic hematopoietic stem cells transplantation. The article identifies diagnostic features and describes approaches for the measurement of the level of MRD in case of AMKL.&#x0D; Aim. The aim is to demonstrate the algorithms for diagnosing and measuring MRD in case of AML-M7 in children.&#x0D; Materials and methods. The article analyzes the clinical and immunological profile of 10 boys and 4 girls with the initial diagnosis of AMKL between the ages of 3 months 12 years old, 13 of them have received treatment in the FSBI N.N. Blokhin National Medical Research Center of Oncology and one in the GBUZ Morozovsky DGKB between 1995 and 2020, The measurement of MRD was carried out in 6 patients. The measurement of MRD was carried out using both morphocytochemical method and multiparameter flow cytometry with megakaryocyte markers (CD61, CD42, CD41) in combination with other myeloid markers (CD13, CD33), CD34, CD117 and aberrant markers (mainly CD7).&#x0D; Results. We showed that adequate measurement of the level of MRD had required detailed immunophenotyping during diagnosis to determine the aberration of megakaryoblasts. CD9 marker (100%), CD33 myeloid marker (69.2%), stem cell antigen CD34 (46.2%), CD13 (38.2%) in addition to megakaryocyte markers (100%) were most often expressed on blast cells in case of AMKL. The CD117 antigen was present on the blasts in 33.3% of cases. The expression of the T-cell-associated CD7 antigen (46.2%) was frequent. The measurement of MRD was carried out during the treatment (usually after an induction course) on the basis of the markers of megakaryocytic cell line (CD61, CD41, CD42a, CD42b), weak CD45 expression, as well as the immunophenotype characteristics during initial diagnosis. The level of MRD ranged from completely negative (0%; 0.006%) to evident (1.05%).&#x0D; Conclusion. The detection of residual tumor megakaryoblasts in case of AML-M7 using flow cytometry is a promising method to evaluate the effect of therapy. The adequate measurement of the level of MRD requires detailed immunophenotyping during the diagnosis to determine the aberration of megakaryoblasts.

Proteomic Studies of Primary Acute Myeloid Leukemia Cells Derived from Patients Before and during Disease-Stabilizing Treatment Based on All-Trans Retinoic Acid and Valproic Acid.

Simple SummaryAcute myeloid leukemia (AML) is an aggressive hematological malignancy, and the only possibility of cure is intensive antileukemic treatment. Such treatment is only possible for relatively young and fit patients, but there is a large subset of elderly/unfit patients above 65–70 years of age who cannot receive intensive treatment. The low-toxicity combination of valproic acid (VP) plus all-trans retinoic acid (ATRA) has an AML-stabilizing effect for a subset of patients, even those with chemoresistant relapse. This response may be an indirect effect due to the modulation of the metabolic environment, but in this article, we describe that the in vivo treatment with ATRA/VP also has direct effects on the fundamental functions of human AML cells. Furthermore, new and less intensive antileukemic therapies are now available (i.e., venetoclax, azacitidine, decitabine), and the combination of these new agents with ATRA/VP may represent a new therapeutic strategy in AML.All-trans retinoic acid (ATRA) and valproic acid (VP) have been tried in the treatment of non-promyelocytic variants of acute myeloid leukemia (AML). Non-randomized studies suggest that the two drugs can stabilize AML and improve normal peripheral blood cell counts. In this context, we used a proteomic/phosphoproteomic strategy to investigate the in vivo effects of ATRA/VP on human AML cells. Before starting the combined treatment, AML responders showed increased levels of several proteins, especially those involved in neutrophil degranulation/differentiation, M phase regulation and the interconversion of nucleotide di- and triphosphates (i.e., DNA synthesis and binding). Several among the differentially regulated phosphorylation sites reflected differences in the regulation of RNA metabolism and apoptotic events at the same time point. These effects were mainly caused by increased cyclin dependent kinase 1 and 2 (CDK1/2), LIM domain kinase 1 and 2 (LIMK1/2), mitogen-activated protein kinase 7 (MAPK7) and protein kinase C delta (PRKCD) activity in responder cells. An extensive effect of in vivo treatment with ATRA/VP was the altered level and phosphorylation of proteins involved in the regulation of transcription/translation/RNA metabolism, especially in non-responders, but the regulation of cell metabolism, immune system and cytoskeletal functions were also affected. Our analysis of serial samples during the first week of treatment suggest that proteomic and phosphoproteomic profiling can be used for the early identification of responders to ATRA/VP-based treatment.

Clinical features and outcomes of hypocellular acute myeloid leukemia in adults: A Korean AML registry data.

The hypocellular variant of acute myeloid leukemia (AML) is defined as bone marrow cellularity of <20% in a biopsy specimen at presentation. We performed a retrospective analysis of the clinical features and survival outcomes of hypocellular AML in a Korean population. We reviewed the medical records of all patients diagnosed with AML at nine hospitals participating in the Korean AML registry from 2006 to 2012. Overall survival (OS) and event-free survival (EFS) rates were calculated from the time of diagnosis until death or an event, respectively. In total, 2110 patients were enrolled and 102 (4.8%) were identified as having hypocellular AML. Patients with hypocellular AML were older than those with non-hypocellular AML (median age: 59 vs 49 years; P < .001) and presented with leukopenia more frequently (mean white blood cell count: 5810/μL vs 40549/μL; P < .001). There was no difference between patients with and without hypocellular AML in terms of the presence of antecedent hematologic disorders (5.9% vs 5.3%; P = .809). FLT3-ITD and NPM1 mutations were less common in hypocellular than non-hypocellular AML (FLT3-ITD mutations: 1.2% vs 14.3%, P < .001; NPM1 mutations: 0% vs 9.5%, P = .019). No differences were seen between the hypocellular and non-hypocellular AML groups in the complete remission rate (53.9% vs 61.3%, P = .139) or early death rate (defined as any death before 8 weeks; 14.7% vs 13.0%, P = .629). The OS and EFS did not differ between the hypocellular and non-hypocellular AML groups (median OS: 16 vs 23 months, P = .169; median EFS: 6 vs 9 months, P = .215). Hypocellular AML is more frequently observed in older-aged patients and have fewer FLT3-ITD and NPM1 mutation, but the clinical outcomes of hypocellular AML do not differ from those of non-hypocellular AML.

A clinical case of acute myeloid leukemia&#x0D; mimicking a tumor&#x0D; of the Ewing sarcoma family

Relevance: M6 variant of acute myeloid leukemia is extremely rare&#x0D; in pediatric practice. The diverse clinical manifestations of erythroblastic leukemia complicate the timely diagnosis of this group of diseases.&#x0D; Purpose: to describe a clinical case of congenital erythroblastic&#x0D; leukemia with multiple lesions of soft tissues and the skeletal system&#x0D; with complications in the form of the convulsive syndrome, presented&#x0D; as an Ewing’s sarcoma tumor, diagnosed at the Research Center of&#x0D; Pediatrics and Pediatric Surgery (Almaty, the Republic of Kazakhstan).&#x0D; Results: The presented clinical case demonstrated challenges in&#x0D; diagnosing and treating young patients with multiple life-threatening&#x0D; tumor lesions. The need to develop molecular genetic studies and expand diagnostic capabilities is an integral part of treating oncohematological diseases.&#x0D; Conclusion: The presented clinical case is of great interest due&#x0D; to its rareness. This case confirms the need to conduct all diagnostic manipulations to assess the process prevalence and to choose and conduct molecular genetic studies on all stages of examination and treatment.

Target-Informed Repurposing of Immunotherapies in AML - a Transcriptome Based Approach for Identifying Immediately Available Therapeutics

Acute Myeloid Leukemia (AML) is an aggressive hematopoietic malignancy with limited therapeutic options and a great need for innovative treatment. However, the identification of novel molecular biomarkers remains challenging, and significant lag time from target discovery to clinical development impedes timely implementation of novel therapies. We previously demonstrated aberrant expression of non-hematopoetic (NH) antigens on leukemic cells in AML; one such antigen, mesothelin (MSLN), has recently begun AML clinical development, providing evidence for the use of NH directed therapeutics in AML. Given this observation, we questioned whether we could build a computational platform to identify misregulated genes in AML that are targeted by currently available immunotherapies in other malignancies. Early phase trial data for these targets will provide vital safety and tolerability information, thereby supporting a rapid path to AML clinical development. To this end, we curated a knowledgebase of antibody-drug conjugate (ADC) and chimeric antigen receptor T-cell (CAR T) therapies by querying all cancer immunotherapy trials registered in the U.S. National Library of Medicine clinical trials database (https://clinicaltrials.gov) and the Journal of Antibody-Drug Conjugates (https://adcreview.com) in November of 2019. In total, our knowledgebase contained 893 clinical trials assessing 141 prospective gene targets. We interrogated transcriptome expression of these targets using ribodepleted RNA-seq data from the TARGET AML and TpAML initiatives (N = 1,394, age &amp;lt;0 to 30 years) and the TCGA LAML cohort (N = 173, age 18 - 88 years). We selected for therapeutic targets expressed in AML by employing a minimum transcript expression threshold of 5 TPM in &amp;gt;50% of our AML cases. This led to the identification of 35 targets of interest for further analysis (Figure 1A). A total of 131 immunotherapies (70 ADCs and 61 CAR Ts) directed against these targets are in various stages of clinical development (Figure 1B). These included 62 AML-directed therapies targeting a total of 13 antigens; among these, CD33, CD123, FLT3, CD117, and CLEC12A have been well described in the context of AML. The remaining 69 therapies (43 ADCs and 26 CAR-Ts) target 30 NH antigens. Nine (30%) of these NH antigens, including CD74, BSG, TFRC, CXCR4, and CD44, all possesed a median of &amp;gt;90 TPM in our AML cohort and were subjects of therapies in Phase I or later. Additionally, these genes were universally expressed across major fusion groups (Figure 1C), suggesting potential as therapeutic targets regardless of age or genetic abnormality. A table of NH targets with high expression in AML is provided in Figure 1D. Conversely, we also identified targets for which expression was uniquely restricted to rare, high-risk, and therapeutically challenging AML variants. Upregulated genes in these variants were identified via differential expression analysis of CBFA2T3-GLIS2, NUP98-NSD1, and NUP98-KDM5A fusions. Genes were filtered using a log fold-change (logFC) threshold of 1 and FDR-adjusted p &amp;lt; 0.05, then cross-referenced with the full knowledgebase to identify 37 rare-variant enriched targets. The top 5 most highly enriched targets (ranked by logFC) were FOLR1, NCAM1, MMP14, TNF, and DLK1. The association of NCAM1 with CBFA2T3-GLIS2 has been previously described. However, FOLR1, a member of the folate receptor family that is overexpressed in epithelial-derived tumors, is a novel target in the context of AML. Like NCAM1, FOLR1 expression in our AML population is almost entirely limited to the CBFA2T3-GLIS2 fusion: 36/43 FOLR1-expressing patients possessed a CBFA2T3-GLIS2 fusion, comprising 94.74% of all CBFA2T3-GLIS2 fusions in our cohort. We screened transcriptome data from a large AML cohort for expression of immunotherapeutic targets with potential utility in AML. This enabled the identification of both broadly expressed and subtype-restricted putative targets. Future expansion of this methodology into other cellular therapies (CAR NK or BiTE) and the examination of new data sources could reveal additional targets with robust expression in AML. Demonstration of cell surface expression of these targets, and appropriate preclinical evidence of efficacy, could lay the groundwork for quickly moving these therapies to clinical trials in AML. Figure Disclosures No relevant conflicts of interest to declare.

Acute Promeylocytic Leukemia Presenting as Fatal Intracranial Bleeding in a Rural Setting

Acute promyelocytic leukemia (APL) is a biologically and clinically distinct variant of acute myeloid leukemia (AML), and accounts for 5 to 20 percent of all AML cases. It is a highly curable hematological malignancy with targeted therapy. Patients usually present with symptoms of the complications, typically bleeding. Intracranial bleeding with APL is rarely seen at the presentation, but more common in relapsed cases. APL is a medical emergency with a high rate of early mortality, usually due to catastrophic bleeding if it is not treated timely. It is vital to start treatment once the diagnosis is suspected without waiting for definite diagnosis. We presented a 35-year-old foreign worker who presented with headache and blurring of vision for 5 days, associated with fever and multiple episodes of vomiting on the day of admission. Subsequently he developed status epilepticus and disseminated bleeding. He had raised leukocyte, low platelet, prolonged international normalized ratio and prolonged prothrombin time. His computed tomography (CT) brain revealed intracranial hemorrhage. He was intubated and was transfused one cycle of disseminated intravascular coagulation regime. He was referred to intensive care unit and neurosurgical team. Unfortunately, the patient passed away within 5 hours of hospitalization. His urgent peripheral blood film subsequently came back after he passed away and showed increased white blood cell with more than 90% of abnormal promyelocytes, Auer rods and Faggot cells, suggestive of APL. This case highlights the importance of high index of suspicion; the timely and urgent treatment, which is crucial and might prevent the catastrophic event.

Crucial Role of BAALC-Expressing Progenitor Cells in Emergence and Development of Post-Transplantation Relapses in Patients with Acute Myeloid Leukemia

This article presents data demonstrating frequent BAALC hyperexpression, also in combination with WT1 hyperexpression, in children and adults with acute myeloid leukemia (AML). Treatment included allogeneic hematopoietic stem cell transplantation. The analysis of serial measurements of BAALC and WT1 expression level in 50 AML patients (37 adults and 13 children) showed that the increased BAALC expression is more common in patients with M1, M2, M4, and M5 FAB variants of AML with equal frequency in adults and children. Furthermore, the increased BAALC expression was rather common in combination with the increased WT1 expression, which predicted poorer prognosis. Since BAALC expression level in AML patients is closely related to AML-producing progenitor cells of leukemia hematopoiesis, a serial study of this phenomenon offers insights into the role of these cells in emergence and development of post-transplantation relapses, which is of both theoretical and practical importance.

Outcomes in Adult Acute Promyelocytic Leukemia: A Decade Experience

BackgroundAcute promyelocytic leukemia (APL), a distinct variant of acute myeloid leukemia (AML), accounts for 10% of AML cases. Over the past decade, APL has emerged from a highly fatal disease to a highly curable one. The published data on outcomes of APL from India are scant. The present study was designed to analyze the clinicopathologic features and outcomes in adult APL. Materials and MethodsThe present report is a single institutional, observational, retrospective study, and data of patients diagnosed with APL from 2006 to 2018 were analyzed. All patients with APL, diagnosed by morphology and confirmed by reverse transcriptase polymerase chain reaction (promyelocytic leukemia/retinoic acid receptor alpha) and over 18 years of age, were included in this study. SPSS software v25.1 was used for statistical analysis. ResultsA total of 1396 patients with AML were seen between January 2005 and June 2018, of which 190 (13.6%) had APL. Of these, 111 patients met the inclusion criteria and were analyzed. The median age at presentation was 33 years (range, 19-60 years). The median duration of symptoms before presentation was 15 days (range, 3-180 days). High risk, intermediate risk, and low risk were seen in 43.3%, 41.4%, and 15.3% of patients, respectively. At the end of induction chemotherapy, 88 (79%) patients achieved complete hematologic remission, and 23 (21%) succumbed during induction. The median time to attain complete hematologic remission was 30 days (range, 19-47 days). At a median follow-up of 34 months, the event-free survival and overall survival were 69.3% and 74.7%, respectively. On univariate analysis, bcr3 variant and high-risk category at presentation had significant impact on event-free survival (P = .029 and P = .003, respectively) and overall survival (P ≤ .001 and P = .007, respectively). On multivariate analysis, only high risk at presentation was significant for event-free survival (P = .04) and overall survival (P = .02). ConclusionAPL constituted one-tenth of patients with AML. The majority of patients were high risk at presentation. Sanz high risk category and bcr3 variant at presentation had significant impact on outcomes in APL.

PS981 LONG TERM AML SURVIVORS HAVE INCREASED MORTALITY AND HIGH PREVALENCE OF CLONAL HEMATOPOIESIS

Background:Acute myeloid leukemia (AML) is defined by the accumulation of immature blasts in the bone marrow (BM) resulting, if left untreated, in acute BM failure. While AML presents in an acute form, it is clear today that most AML cases are preceded by a long phase of age related clonal hematopoiesis (ARCH) with almost no symptoms. The clinical and molecular trajectories of AML both before and after diagnosis are highly predictable. Recent studies identified the high risk individuals destined to evolve from ARCH to AML based on blood counts parameters (mainly red cell distribution width (RDW)) and molecular attributes such as clone size and the number of ARCH defining events. Most AML patients will achieve complete remission after induction chemotherapy, however most will relapse within a median time of 11 months despite post remission consolidation or bone marrow transplantation (BMT). A small fraction of AML patients will maintain at long term remission (LTR).Aims:The course of AML from ARCH to relapse is well characterized both clinically and molecularly, however, this information is missing for long term AML survivors, specifically for AML patients who did not receive BMT. The fact that AML has a long chronic history before its acute presentation might suggest that a recovery from the acute phase will not necessary result in resetting the hematopoietic system but rather to turn it back to its chronic phase before AML presentation.Methods:To answer the question whether preleukemic events are still present at LTR and what important clinical implications it might have, we studied morbidity, mortality and molecular structure among two unique LTR cohorts. One cohort is the electronic health records (HER) of 4.2 million individuals covering over 15 years of follow up and include 61 LTR survivors who did not undergo BMT (LTSnoBMT). The second cohort which contains molecular data, included analysis of genetic variation in 30 LTR cases at diagnosis and LTR, and was compared to a group of lymphoid malignancies patients at remission.Results:We found that the mortality among LTSnoBMT 10 years after diagnosis was 20%&gt;50% higher in comparison to controls. In addition, several lab results were different between the LTSnoBMT and controls including higher red cell distribution width (RDW) among older (&gt;55) LTSnoBMT. Recent studies found a correlation between high RDW and mortality among individuals carrying age related clonal hematopoiesis (ARCH) mutations. We also found that ARCH defining events were significantly more prevalent among LTSnoBMT in comparison to controls (63% vs 37% p = 0.039). Furthermore of the ARCH defining events among the LTSnoBMT 62% were recurrent AML variants as oppose to 21% among the controls (p = 0.002). Of the recurrent mutations IDH1/2 were the most common mutations and 2 out of 7 IDH1/2 cases experienced a late relapse (Figure 1).Summary/Conclusion:Altogether, even after the sustainable eradication of the malignant clone, the hematopoietic system/microenvironment does not normalize (abnormal blood counts, high prevalence of ARCH and higher mortality).imageThese results suggest that AML at LTR is a unique situation and that this group of patients are not fully recovered and should be further studied and treated.