Acute Leukemia Blast Cells Research Articles

Significance of OCT3/4 and SOX2 antigens expression by leukemic blast cells in adult acute leukemia

ObjectiveThis study aimed to address the prognostic impact of SOX2 and OCT3/4 expression on adult acute leukemia patients’ outcomes.MethodsSOX2 and OCT3/4 expression by blast cells were evaluated by flow cytometry in 80 acute leukemia patients and 8 healthy controls.ResultsBaseline SOX2 and OCT3/4 expression were significantly higher in both ALL (P = < 0.001, P = 0.005 respectively) and AML patients (P < 0.001, P = 0.003 respectively) as compared to control, and decline at complete remission (CR) and elevated again at relapse. High SOX2 and OCT3/4 levels were significantly correlated with the presence of adverse risk stratification parameters.ConclusionOur findings indicated that both SOX2 and OCT3/4 could serve as biomarkers that could improve risk stratification of acute leukemia patients. Also, both SOX2 and OCT3/4 might be a therapeutic target, especially in resistant acute leukemia.

RISK FACTORS AND EARLY SIGNS OF CRITICAL CONDITIONS IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA ADMITTED TO THE INTENSIVE CARE UNIT

Relevance: Acute lymphoblastic leukemia (ALL) is the most common cancer among children, accounting for nearly a quarter of all childhood cancers.&#x0D; The study aimed to determine the risk factors and signs of critical conditions in children with acute lymphoblastic leukemia admitted to an intensive care unit (ICU).&#x0D; Methods: The approach used was a systematic review. Data was collected from sources published in 2019-2023. Four cohort studies, four retrospective analyses, two literature reviews, one case-control study, and one case study were included in this systematic review.&#x0D; Results: The prognosis in pediatric ALL depends on the initial number of blast cells in the peripheral blood. Patients with B-cell precursor acute lymphoblastic leukemia (BCP ALL) and low blast cell numbers survived better than patients with T-cell acute lymphoblastic leukemia (T-ALL) and low cell count. IL1B and NLRP1 genetic polymorphisms enhanced ALL risk and reduced infectious comorbidity. However, these gene polymorphisms must be confirmed in juvenile leukemia. KRAS, FLT3, NRAS, PTPN11, KMT2D, PTEN, and NOTCH1 gene mutations affected pediatric ALL patient features and treatment results. These mutations demonstrate the relevance of genetic profiling in risk classification and tailored management. Gene variations and availability of effective medication contributed. Pediatric BCP-ALL patients with the PAX5P80R mutation had worse 5-year overall survival, higher white blood cell counts, male preponderance, and more genetic abnormalities. Pediatric BCP-ALL focused on genetic analysis and risk stratification. Children of African American and European American ancestry showed varied incidence, recurrence, and outcome rates for ALL. African American children exhibited lower incidence but greater recurrence rates and poorer prognosis than European American children.&#x0D; Conclusion: Risk factors for these patients’ admission to ICU include comorbidities, infectious diseases, hypoxia, and hemodynamic instability, as well as age and baseline white blood cell count at diagnosis.

Targeting Proliferation Signals and the Cell Cycle Machinery in Acute Leukemias: Novel Molecules on the Horizon.

Uncontrolled proliferative signals and cell cycle dysregulation due to genomic or functional alterations are important drivers of the expansion of undifferentiated blast cells in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells. Therefore, they are largely studied as potential therapeutic targets in the field. We here present the most recent advancements in the evaluation of novel compounds targeting cell cycle proteins or oncogenic mechanisms, including those showing an antiproliferative effect in acute leukemia, independently of the identification of a specific target. Several new kinase inhibitors have been synthesized that showed effectiveness in a nanomolar to micromolar concentration range as inhibitors of FLT3 and its mutant forms, a highly attractive therapeutic target due to its driver role in a significant fraction of AML cases. Moreover, we introduce novel molecules functioning as microtubule-depolymerizing or P53-restoring agents, G-quadruplex-stabilizing molecules and CDK2, CHK1, PI3Kδ, STAT5, BRD4 and BRPF1 inhibitors. We here discuss their mechanisms of action, including the downstream intracellular changes induced by in vitro treatment, hematopoietic toxicity, in vivo bio-availability and efficacy in murine xenograft models. The promising activity profile demonstrated by some of these candidates deserves further development towards clinical investigation.

HMG20B stabilizes association of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block

Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.

Systematic Review of Pharmacogenetics of ABC and SLC Transporter Genes in Acute Myeloid Leukemia.

Antineoplastic uptake by blast cells in acute myeloid leukemia (AML) could be influenced by influx and efflux transporters, especially solute carriers (SLCs) and ATP-binding cassette family (ABC) pumps. Genetic variability in SLC and ABC could produce interindividual differences in clinical outcomes. A systematic review was performed to evaluate the influence of SLC and ABC polymorphisms and their combinations on efficacy and safety in AML cohorts. Anthracycline intake was especially influenced by SLCO1B1 polymorphisms, associated with lower hepatic uptake, showing higher survival rates and toxicity in AML studies. The variant alleles of ABCB1 were related to anthracycline intracellular accumulation, increasing complete remission, survival and toxicity. Similar findings have been suggested with ABCC1 and ABCG2 polymorphisms. Polymorphisms of SLC29A1, responsible for cytarabine uptake, demonstrated significant associations with survival and response in Asian populations. Promising results were observed with SLC and ABC combinations regarding anthracycline toxicities. Knowledge of the role of transporter pharmacogenetics could explain the differences observed in drug disposition in the blast. Further studies including novel targeted therapies should be performed to determine the influence of genetic variability to individualize chemotherapy schemes.

Central Nervous System Involvement in Childhood Acute Lymphoblastic Leukemia: An Analysis of Day-One Versus Day-Eight Lumbar Punctures in Remission Induction Therapy.

IntroductionTraumatic lumbar puncture (TLP+) can lead to the iatrogenic infiltration of the central nervous system (CNS) by circulating leukemic blast cells in childhood acute lymphoblastic leukemia (ALL). The risk of TLP+ is increased by a number of factors at the time of presentation of the disease, such as a high white cell count (WCC), T-ALL phenotype, and unstable clinical condition of the patient. For this reason, the first lumbar puncture (LP) was deferred until Day Eight of prednisolone prophase during remission induction therapy in one set of patients. The objective was to compare the historical cohort of Day-One LP with Day-Eight LP with respect to the incidence of TLP+ and de novo CNS leukemia.MethodsA retrospective comparative data analysis of 1,185 childhood ALL patients aged 1-16 years was conducted based on the electronic medical records of the pediatric hematology-oncology department of The Indus Hospital (TIH), Karachi, from January 2010 to August 2018. A total of 600 patients whose LP was done on Day One (January 2010-May 2015) were placed in cohort A, whereas 585 patients whose LP was performed on Day Eight (June 2015-August 2018) were placed in cohort B. After the examination of the cerebrospinal fluid (CSF), the status of CNS infiltration was classified as CNS-1, CNS-2, CNS-3, and TLP+.ResultsA total of 1,185 patients were included in the study, of whom 600 patients were in cohort A and 585 patients in cohort B. The incidence of TLP+ was found to be lower in cohort B (1.7%) as compared with the incidence in cohort A (4.3%) (p-value=0.009). However, there was an increase in the incidence of CNS-3 cases in cohort B (8%) as compared to cohort A (3%) (p-value: <0.001). When the CNS status of both the cohorts was compared with that of the internationally published data, a low incidence of TLP+ cases was noted in patients with LP on Day Eight.ConclusionThe modified approach of performing the first LP on Day Eight significantly reduced the incidence of TLP+ cases. However, an unusual finding of a significant increase in the CNS-3 leukemia was noted. More prospective studies are needed to investigate this significant increase in CNS-3 cases.

P38 MAPK/AKT signalling is involved in IL-33-mediated anti-apoptosis in childhood acute lymphoblastic leukaemia blast cells

Background Acute lymphoblastic leukaemia (ALL) is often characterized by broad clinical and biological heterogeneity, as well as recurrent genetic aberrations. Despite remarkable improvements in the treatment outcome in paediatric ALL over the past several decades, it remains a leading cause of morbidity and mortality among children. Cytokines have been extensively studied in haematologic diseases; however, the mechanisms by which cytokines contribute to ALL pathogenesis remain poorly understood. Methods IL-33 levels were measured by enzyme-linked immunosorbent assay (ELISA). IL1RL1 expression on ALL cell surface was accessed by flow cytometry. Expression of phosphorylated p38 MAPK, p38, pAKT, AKT and GAPDH were quantified by western blot. Cell survival signals were evaluated by apoptosis using flow cytometry. Results BM samples from ALL patients at diagnosis upregulated their cell surface expression of IL1RL1, and a higher interleukin (IL)-33 level in the serum was observed as compared to the healthy individuals. Moreover, exogenous IL-33 treatment significantly inhibited apoptosis by activating p38 mitogen-activated protein kinase (MAPK) and AKT pathway, while the inhibitor for p38 MAPK, SB203580, counteracted IL-33-induced anti-apoptosis via inactivation of p38 MAPK and AKT. Furthermore, IL-33 negatively regulates cyclin B1 protein level while increasing the expression of CDK1, with SB203580 inhibiting the effect. Conclusion Our study reveals an important role for IL-33/IL1RL1 axis in supporting ALL which may represent a novel treatment for paediatric patients. KEY MESSAGES Both IL-33 and IL1RL1 levels are upregulated in primary ALL samples. IL-33 increased both p38 MAPK and AKT activation in ALL. IL-33 promotes survival and cell cycle progression of ALL cells via activating p38 MAPK.

Gilteritinib: An FMS-like tyrosine kinase 3/AXL tyrosine kinase inhibitor for the treatment of relapsed or refractory acute myeloid leukemia patients.

Acute myeloid leukemia has recently undergone a significant transition into identifying and successfully inhibiting driver mutations leading to disease. One of the most common mutations in acute myeloid leukemia involves the protein FMS-like tyrosine kinase 3 (FLT3), which leads to ligand-independent activation of intracellular signaling cascades leading to the survival and proliferation of the acute leukemia blast cell. Preclinical studies have demonstrated the presence of two dominant types of mutations of this protein: internal tandem duplication and tyrosine kinase domain mutations. Successful inhibition of this protein has proven to be challenging. While FLT3 has been shown to be successfully inhibited and shown to improve overall survival in the frontline therapy of acute myeloid leukemia in combination with cytarabine and anthracycline, relapsed and refractory (R/R) patients have not been shown to be a successful population until recently. A phase III trial (ADMIRAL trial) demonstrated significant overall survival benefit in patients receiving gilteritinib compared to patients receiving salvage chemotherapy. This review will provide an overview of the preclinical, clinical, and practical use of gilteritinib in the treatment of patients with relapsed and refractory acute myeloid leukemia with FLT3 mutation.

PML/RARa Interferes with NRF2 Transcriptional Activity Increasing the Sensitivity to Ascorbate of Acute Promyelocytic Leukemia Cells.

NRF2 (NF-E2 p45-related factor 2) orchestrates cellular adaptive responses to stress. Its quantity and subcellular location is controlled through a complex network and its activity increases during redox perturbation, inflammation, growth factor stimulation, and energy fluxes. Even before all-trans retinoic acid (ATRA) treatment era it was a common experience that acute promyelocytic leukemia (APL) cells are highly sensitive to first line chemotherapy. Since we demonstrated how high doses of ascorbate (ASC) preferentially kill leukemic blast cells from APL patients, we aimed to define the underlying mechanism and found that promyelocytic leukemia/retinoic acid receptor α (PML/RARa) inhibits NRF2 function, impedes its transfer to the nucleus and enhances its degradation in the cytoplasm. Such loss of NRF2 function alters cell metabolism, demarcating APL tissue from both normal promyelocytes and other acute myeloide leukemia (AML) blast cells. Resistance to ATRA/arsenic trioxide (ATO) treatment is rare but grave and the metabolically-oriented treatment with high doses of ASC, which is highly effective on APL cells and harmless on normal hematopoietic stem cells (HSCs), could be of use in preventing clonal evolution and in rescuing APL-resistant patients.

Clinical-biological characteristics and treatment outcomes of pediatric pro-B ALL patients enrolled in BCH-2003 and CCLG-2008 protocol: a study of 121 Chinese children

BackgroundAlthough leukemic blast cells of Pro-B cell acute lymphoblastic leukemia (ALL) are arrested at the same stage of B cell differentiation, the immature B cell subtype is still biologically heterogeneous and is associated with diverse outcomes. This study aimed to explore the clinical-biological characteristics of pediatric pro-B ALL and factors associated with outcomes.MethodsThis study enrolled 121 pediatric patients aged 6 months to 14 years with newly diagnosed CD19+CD10− pro-B cell acute lymphoblastic leukemia (pro-B ALL) treated at Beijing Children’s Hospital from March 2003 to October 2018. Genetic abnormalities, immunophenotypic markers, minimal residual disease (MRD) at early treatment stage and long-term outcomes of children treated on two consecutive protocols were analyzed.ResultsKMT2A rearrangements were the most frequent abnormalities (incidence rate 33.06%), and were associated with lower frequency of CD13, CD33, CD22 and CD34 expression and higher frequency of CD7 and NG2 expression. Higher frequency of CD15 and CD133 expression was found in KMT2A-AFF1+ patients, exclusively. Presence of CD15 and absence of CD34 at diagnosis correlated with the high burden of MRD at the early stage of treatment. Outcomes were more favorable in patients older than 1 year, with absence of CD20 expression and KMT2A rearrangements, and with MRD lower than 1% at the end of induction and 0.1% before consolidation. Increased intensity of chemotherapy based on MRD analysis did not improve outcomes significantly (5-year EFS 73.9 ± 6.5% for BCH-2003 and 76.1 ± 5.3% for CCLG-2008, P = 0.975). Independent adverse prognostic factors were MRD ≥ 0.1% before consolidation and presence of KMT2A gene rearrangements (odds ratios [ORs] 9.424 [95% confidence interval (CI) 3.210, 27.662; P < 0.001]; 4.142 [1.535, 11.715, P = 0.005]; respectively).ConclusionsPediatric pro-B ALL is a heterogeneous disease. Genetic analysis and MRD evaluation can predict patients with dismal prognosis; however, intensive chemotherapy alone does not improve outcomes of these patients and targeted therapy or hematopoietic stem cell transplantation may be required.

Human-level recognition of blast cells in acute myeloid leukaemia with convolutional neural networks

Reliable recognition of malignant white blood cells is a key step in the diagnosis of haematologic malignancies such as acute myeloid leukaemia. Microscopic morphological examination of blood cells is usually performed by trained human examiners, making the process tedious, time-consuming and hard to standardize. Here, we compile an annotated image dataset of over 18,000 white blood cells, use it to train a convolutional neural network for leukocyte classification and evaluate the network’s performance by comparing to inter- and intra-expert variability. The network classifies the most important cell types with high accuracy. It also allows us to decide two clinically relevant questions with human-level performance: (1) if a given cell has blast character and (2) if it belongs to the cell types normally present in non-pathological blood smears. Our approach holds the potential to be used as a classification aid for examining much larger numbers of cells in a smear than can usually be done by a human expert. This will allow clinicians to recognize malignant cell populations with lower prevalence at an earlier stage of the disease. Deep learning is currently transforming digital pathology, helping to make more reliable and faster clinical diagnoses. A promising application is in the recognition of malignant white blood cells—an essential step for detecting acute myeloid leukaemia that is challenging even for trained human examiners. An annotated image dataset of over 18,000 white blood cells is compiled and used to train a convolutional neural network for leukocyte classification. The network classifies the most important cell types with high accuracy and can answer clinically relevant binary questions with human-level performance.

Development and Validation of a Sensitive UHPLC-MS/MS-Based Method for the Analysis of Folylpolyglutamate Synthetase Enzymatic Activity in Peripheral Blood Mononuclear Cells: Application in Rheumatoid Arthritis and Leukemia Patients.

Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs such as methotrexate (MTX), which is commonly used for the treatment of (pediatric) leukemia and the anchor drug in rheumatoid arthritis (RA) treatment. To date, assessment of FPGS catalytic activity relies on assays using radioactive substrates that are labor-intensive and require relatively large numbers of cells. Here, we describe a nonradioactive, ultra-high-performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS)-based method allowing for sensitive and accurate measurements of FPGS activity in low cell numbers (ie, 1-2 × 10) of biological specimens, including leukemic blast cells of acute lymphoblastic leukemia patients and peripheral blood mononuclear cells of patients with RA. The UHPLC-MS/MS assay was validated with 2 CCRF-CEM human leukemia cells, one proficient and one deficient in FPGS activity. Linearity of time and protein input were tested by measuring FPGS activity at 30-180 minutes of incubation time and 10-300 mcg protein extract. In addition, FPGS enzyme kinetic parameters were assessed. The FPGS enzymatic assay showed a linear relation between FPGS activity and protein input (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed for evaluation of FPGS enzyme kinetic parameters revealing Km values for the substrates MTX and L-glutamic acid of 64 µmol/L and 2.2 mmol/L, respectively. The mean FPGS activity of acute lymphoblastic leukemia blast cells (n = 4) was 3-fold higher than that of CCRF-CEM cells and 44-fold and 88-fold higher than that of peripheral blood mononuclear cells from MTX-naive (n = 9) and MTX-treated RA patients (n = 6), respectively. Collectively, given its sensitivity with low cell numbers and avoidance of radioactive substrates, UHPLC-MS/MS-based analysis of FPGS activity may be eligible for routine therapeutic drug monitoring of MTX in RA and leukemia for therapy (non)response evaluations.

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs

AbstractHuman lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+ B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid–restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid–restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.

Monocyte-Macrophage Differentiation Suppresses the Expression of Proapoptotic Receptors to Apo2L/TRAIL and Increases Resistance to TRAIL-Induced Apoptosis

Abstract—A comparative study of the expression of pro- and anti-apoptotic receptors to cytokine TRAIL (TNF-related apoptosis-inducing ligand) on immature blast cells of acute myeloid leukemia, on the cells of acute myeloid leukemia differentiated into monocyte-like and macrophage-like cells, and on normal monocytes and macrophages has been performed. It has been shown that a decrease in the expression of proapoptotic receptors to TRAIL and the acquisition of resistance to TRAIL-induced apoptosis occurs not only during the differentiation of acute myeloid leukemia cells into monocyte-like and macrophage-like cells, but also during the maturation of normal monocytes into macrophages. Thus, the differentiation of both acute myeloid leukemia cells and normal myeloid cells in the direction of monocyte-macrophages mediates a decrease in the expression of proapoptotic receptors to TRAIL, which in turn can determine the resistance of normal myeloid cells to TRAIL-induced apoptosis.

Global Transcriptome Profiling Identifies a Key Mir-185-PAK6 Axis That Promotes Survival of Leukemic Stem Cells and Drug-Insensitive Blasts in BCR-ABL+ Human Leukemia

Chronic myeloid leukemia (CML) stem/progenitor cells and BCR-ABL+ acute lymphoblastic leukemia (ALL) blast cells are insensitive to tyrosine kinase inhibitor (TKI) monotherapies. These cells rapidly generate therapy-resistant clones in vitro and in vivo and are often responsible for disease relapse. Therefore, identification of predictive biomarkers and novel treatments that target key molecular events active in leukemic stem cells (LSCs) are needed. MicroRNAs (miRNAs) are small molecules that regulate the gene expression network and are highly deregulated in many cancers. Through global transcriptome profiling, we have recently identified 66 differentially expressed miRNAs in pre-treatment CD34+ stem/progenitor cells from CML patients (n=6) compared to healthy bone marrow (NBM) controls (n=3, adjusted P<0.05); 26 differentially expressed miRNAs were identified between subsequent IM-nonresponders and IM-responders (P<0.05). 21 differentially expressed miRNAs were successfully validated in additional IM-responders (n=11), IM-nonresponders (n=11) and NBM (n=11). Interestingly, miR-185 was discovered to be one of the most highly deregulated miRNAs, with significant reduction in CD34+ cells from IM-nonresponders compared to IM-responders (p=0.0006). This significant change was further demonstrated in CD34+ cells from CML patients (n=60) before and after 3-month TKI nilotinib treatment in a clinical trial (p<0.05). We further demonstrated that miR-185 functions as a tumor suppressor; its restored expression by lentiviral transduction in CD34+ IM-nonresponder cells significantly impaired survival of these cells and sensitized them to TKI treatment in vitro. Restored miR-185 expression in BCR-ABL+ ALL blasts led to a profound decrease in leukemia burden and significantly enhanced survival compared to controls in vivo (median survival 65 vs. 47 days, P=0.0005). Strikingly, mice injected with miR-185-transduced cells and treated with dasatinib (DA) survived much longer than recipients of control cells treated with DA (median survival 83 vs. 60 days, P=0.0018). Moreover, restoration of miR-185 expression combined with DA treatment greatly reduced in vivo long-term regenerative activity of LSCs from IM-nonresponders as compared to control cells treated with DA in NRG mice (<0.2% vs. 5% GFP+ patient cells in the BM, 25 weeks post-transplantation). We observed not only a marked reduction in GFP+CD34+ cells, but also a near elimination of GFP+CD34+CD38- LSCs that were transduced with miR-185 and treated with DA compared to control cells treated with DA, indicating that restored miR-185 expression combined with DA preferentially prevents the growth of patient-derived long-term leukemia-initiating cells in vivo. Several miRNA target genes were further identified by integrating miRNA expression profiles with gene expression profiles from the same patient samples using strand-specific RNA-seq. Based on three out of six prediction algorithms (mirBase, TargetScan, miRanda, tarBase, mirTarget2, and PicTar), PAK6, a serine/threonine-protein kinase, was found to be highly expressed in CD34+ IM-nonresponder cells compared to IM-responders (p<0.003), which correlated with reduced expression of miR-185 in these cells (p=0.0002). PAK6 was confirmed as a target gene of miR-185 by a luciferase reporter assay. Western blot analysis showed that restored miR-185 expression caused a marked decrease in protein levels of PAK6 in miR-185-transduced cells and suppression of PAK6 reduced viability of these cells. These results indicate that PAK6 is a critical target of miR-185, and that loss of miR-185 expression in CML may lead to up-regulation of PAK6, which in turn contributes to disease progression and drug resistance. Indeed, the use of a pre-clinically validated pan PAK inhibitor (PF-3758309) significantly inhibited the growth of IM-resistant cells and CD34+ IM-nonresponder cells and these effects could be enhanced by TKIs (p<0.05). Mechanistically, we observed that p-ERK and p-AKT were significantly reduced in PAK6 knockdown or miR185-restored IM-resistant cells in response to IM treatment. Thus, we infer that downregulation of PAK6 may sensitize TKI-resistant cells to TKI therapy through inhibition of the RAS/MAPK pathway. Taken together, PAK6, a novel target of miR-185, emerges as an attractive druggable target for combination therapy of TKI-resistant patients. DisclosuresNo relevant conflicts of interest to declare.

Aberrant Expression of CD Markers in Cases with Acute Leukemia in Sohag University Hospital

Introduction: Acute leukemias are a group of malignancies with clinical, morphologic, immunologic, and molecular patterns and display patterns of surface antigen expression (CD antigens). Precursor cells from different blast cells in acute leukemia express different subdivisions of surface molecules many of which are now defined as cluster of differentiation (CD) antigens. Every leukemia type has specific set of CD markers, what constitute nowadays current classification of hematopoietic malignancies and the basis of diagnosis. The expression of CD markers on leukocytes can be determined by flowcytometry. Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification. Flow cytometry is a technique used to analyze multiple phenotypic and functional parameters simultaneously within a single cell or a population of cells. Aim of the work: Assess the prevalence of aberrant expression of CD markers in cases with acute leukemia in Sohag University Hospitals. Patients and Methods: This study was carried out in the Clinical Pathology Department, faculty of medicine, Sohag university hospital on fifty (50) newly diagnosed acute leukemia patients who were subjected to bone marrow aspiration, their ages ranged between 1.5 to 71 years, 29 (58%) of them were males and 21 (42%) were females with a male to female ratio of 1.4:1, during the period from November 2015 to October 2016. Results: B-cell acute lymphoblastic leukemia positive for the following CD markers CD10, CD19, TdT, HLA-DR and expressing CD33 and case of AML (M4-M5) expressing CD7 lymphoid marker. Conclusion: Aberrant expression of CD markers is seen in several cases of acute leukemia. The frequency of aberrant antigen expression in acute leukemia was comparable with most published international data. The presence of aberrancy helps to identify a neoplastic process.

High Tim-3 expression on AML blasts could enhance chemotherapy sensitivity

T-cell immunoglobulin and mucin domain-containing molecule3 (Tim-3) represents a novel mechanism of T-cell dysfunction and exhaustion. Tim-3 has also been identified in various solid tumors. However, the role of Tim-3 expression on blast cells in acute myeloid leukemia (AML) is not well understood. In this study, we aimed to explore the role of Tim-3 in patients with de novo AML, and the correlation between Tim-3 and clinicopathological prognosis. The study cohort consisted of 76 patients with de novo non-M3 AML. These patients’ bone marrow samples were collected and then bone marrow mononuclear cells (BMCs) were isolated for flow cytometry to detect Tim-3 expression on blasts. According to FAB type, 76 diagnosed AML patients included in this study were: M0 (n=2), M1 (n=16), M2 (n=20), M4 (n=20), M5 (n=16), and M6 (n=2). A positive expression (>20%) of Tim-3 was found in 87% (66/76) of patients with AML. The average percentage of Tim-3(+) blasts in these AML patients was 58.26 ± 29.23%. Moreover, the frequency of Tim-3 high expression was higher in M4 patients than that in other AML patients according to FAB type (P=0.004). Tim-3 high expression was also closely associated with inv(16) (P=0.01) and C/EBPA mutation (P=0.03). The mutations of the following six genes, i.e., FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, were independent of the Tim-3 expression. Additionally, it is more likely to find higher levels of Tim-3 in the low-risk group than in the intermediate- and high-risk groups (P=0.02). The expression of Tim-3 was positively correlated with CD13 (r=0.36, P=0.001), CD34 (r=0.41, P=0.000), and CD7 (r=0.27, P=0.02) in AML patients. AML patients with high Tim-3 expression achieved significantly high complete remission (CR) rate (P=0.01), while their Tim-3 expression significantly decreased after CR (P=0.01). Blockade of Tim-3 expression on AML blasts significantly reduced the Idarubicin (IDA)-mediated suppression of cell growth and reduction of cell apoptosis in vitro. Collectively, our study suggests that high Tim-3 expression on AML blasts could enhances chemotherapy sensitivity.

A decision support system for Acute Leukaemia classification based on digital microscopic images

In the era of digital microscopic imaging, Image Processing, data analysis, classification, decision support systems have emerged as one of the most important tools for diagnostic research. Physicians can observe cellular internal structures abnormalities by visualizing and analyzing images. Leukemia is a malignant disease characterized by the uncontrolled accumulation of abnormal white blood cells. The recognition of acute leukemia blast cells in colored microscopic images is a challenging task. The first important step in the automatic recognition of this disease, image segmentation, is considered to be the most critical step. In this study, we present a decision support system that includes the panel selection, segmentation using K-means clustering to identify the leukemia cells and features extraction, and image refinement. After the decision support system successfully identifies the cells and its internal structure, the cells are classified according to their morphological features of this analysis the decision support system was tested using a public dataset designed to test segmentation techniques for identifying specific cells, and the results of this analysis were compared with those of other techniques, which were suggested by other researchers, applied to the same data. The algorithm was then applied to another dataset, extracted under the supervision by an expert pathologist, from a local hospital; the total dataset consisted of 757 images gathered from two datasets. The images of the datasets are labeled with three different labels, which represents three types of leukemia cells: blast, myelocyte, and segmented cells. The process of labeling of these images was revised by the expert pathologist. The algorithm testing using this dataset demonstrated an overall accuracy of 99.517%, the sensitivity of 99.348%, and specificity of 99.529%. Therefore, this algorithm yielded promising results and warrants further research.

Distinct TP73-DAPK2-ATG5 pathway involvement in ATO-mediated cell death versus ATRA-mediated autophagy responses in APL.

We have previously demonstrated that the death-associated protein kinase 2 (DAPK2) expression is significantly reduced in acute myeloid leukemia (AML), particularly in acute promyelocytic leukemia (APL) blast cells. In this study, we aimed at further understanding DAPK2 function and regulation during arsenic trioxide (ATO) cytotoxic or all-trans retinoic acid (ATRA) differentiation therapy in APL cells. We found that the p53 family member transactivation domain-p73 isoform (TAp73) binds to and activates the DAPK2 promoter, whereas the dominant-negative ΔNp73 isoform inhibits DAPK2 transcription. Furthermore, the knocking down of tumor protein p73 (TP73) in NB4 cells resulted in reduced DAPK2 expression associated with decreased cell death and autophagy upon ATO and ATRA treatment, respectively. Moreover, the silencing of DAPK2 revealed that DAPK2 is an important downstream effector of p73 in ATO-induced apoptosis but not autophagy responses of APL cells. In contrast, the p73-DAPK2 pathway is essential for ATRA-induced autophagy that is mediated by an interaction of DAPK2 with the key autophagy-related protein (ATG)5. Lastly, we show that DAPK2 binds and stabilizes the p73 protein; thus, we propose a novel mechanism by which ATO- or ATRA-induced therapy responses initiate a positive p73-DAPK2 feedback loop.

The use of oligonucleotide aptamers in cancer therapy

Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers - particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug.