Acute Myeloblastic Leukemia Patients Research Articles

Nitric Oxide and Hypochlorite Assessment and Screening of some β-Lactamase Encoding Genes among Gram Negative Bacteria in Patients with Acute Leukemia

Objective: The serologic levels of both nitric oxide (NO) and hypochlorite (ClO-) were assessed in this study for Iraqi acute leukemic patients infected with Gram negative bacteria (GNB). Alongside, the phylogenetic analysis for both blaSHV and blaTEM genes of GNB was performed. Methods: The clinical samples were recovered from acute leukemic patients at hematology center in Baghdad from January 2021 to December 2021. The identification of bacterial isolates and susceptibility test were performed using Vitek 2 Compact System. Serum levels of NO and ClO- were assayed by Enzyme Linked Immunosorbent assay and colorimetric method, respectively. Gene screening was done utilizing polymerase chain reaction. DNA sequencing, GenBank accession numbers submission, and phylogenetic analysis were also conducted. Results: From 260 patients with acute leukemia, 485 clinical samples were collected from different sites. Of this total, 70 (15%) isolates of GNB were obtained, distributing as Klebsiella pneumoniae 23 (33%), Escherichia coli 21 (30%), Pseudomonas aeruginosa 18 (26%), and Acinetobacter baumannii 8 (11%). These isolates were mainly collected from urine 39 (55.71%), followed by blood 23 (32.85%), and the least from swabs 8 (11.42%). The infections of GNB were higher among acute myeloblastic leukemia (AML) patients 40 (57.14%) than these with acute lymphoblastic leukemia (ALL) 30 (42.85%). The levels of NO were higher among groups of patients than control groups. Additionally, ClO-levels were observed to be slightly increased in patients above these of controls. Most isolates of K. pneumoniae and E. coli showed high resistance rates for penicillins (ampicillin and ticarcillin), cephalosporins (ceftazidime, cefotaxime, ceftriaxone, and cefepime), followed by gentamicin and ciprofloxacin. Cefoxitin, aztreonam, imipenem and meropenem were nearly more effective against GNB isolates. On the other hand, about half isolates P. aeruginosa and all A. baumannii were resistant to the tested antibiotics. Extended Spectrum β- Lactamases (ESBLs) were released in 49 (70%) of the tested isolates of GNB. The multidrug resistant (MDR) pattern was noticed among 58 (82.85%) of GNB. Interestingly, ESBLs producing MDR isolates were determined in 34 (58.62%) of the studied GNB. Genotypic screening revealed that blaSHV and blaTEM genes were characterized in 20 (28.57%) and 32 (45.71%) of GNB isolates, respectively, while all GNB were negative for blaCTX-M. DNA sequencing of blaSHV amplicons revealed the occurrence of one point mutation (111T>A) in six isolates of K. pneumoniae with a missense effect (p.31Q>L) on the encoded protein. Forty-five GenBank accession numbers were recorded in NCBI to represent the studied variants of ESBLs. Phylogenetic analysis of both blaSHV and blaTEM displayed the possible epidemiologic routes of the tested GNB by detecting the origin host, source of collection, and geographic spread as compared with the reference sequences. Conclusion: high serum levels of both NO and ClO- indicated the potential role of these microbicidal agents to predict the serious infections of GNB in acute leukemia population. Phylogenetic analysis represented an important tool, possibly aiding in early control and prevention of pathogenic GNB.

EFFICACY OF CHLORINE DIOXIDE ON ORAL LESIONS IN ACUTE MYELOBLASTIC LEUKEMIA PATIENTS UNDERGOING CHEMOTHERAPY

Objective: The aim of this study was to describe the pharmacological management of oral lesions, particularly using chlorine dioxide agents in an acute myeloblastic leukemia (AML) patient undergoing chemotherapy.
 Methods: A 9 y old girl was referred from the Department of Paediatrics at Hasan Sadikin Hospital to the Department of Oral Medicine with complaints of swelling on the lips, difficulty while eating, swallowing, and even opening the mouth. Extra oral examination showed angioedema on the lips with a prominent serosanguinolent crust. Intraoral examination revealed white plaques that could not be scrapped off in all parts of the mouth. The patient was diagnosed with AML and had received chemotherapy until the second cycle.
 Results: Gauze soaked in chlorine dioxide oral rinse was applied to compress the patient's lips. Following this, 0.2% hyaluronic acid gel was also administered. In terms of intraoral treatment, chlorine dioxide was also applied by spraying it throughout the entire mouth. After one month, the lesion had completely healed, allowing the patient to open their mouth without any issues.
 Conclusion: Chlorine dioxide significantly improves the oral mucosal lesions in an AML patient undergoing chemotherapy.

Lncrna Famlf-2 Promotes Leukemogenesis Via the DHX9/c-Myc Axis in Acute Lymphoblastic Leukemia

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including leukemia. Our previous study identified Familial acute myelogenous leukemia related factor ( FAMLF) in an acute myeloblastic leukemia (AML) pedigree. Two splice variants of FAMLF, FAMLF-1 and FAMLF-2 were detected. FAMLF-1 is upgraded in AML patients and associated with poor prognosis. However, the full cDNA sequence of FAMLF-2 is not verified and its role in leukemogenesis remain unclear. Herein, we aim to investigate the functional and mechanistic roles of FAMLF-2 in leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of FAMLF-2 was at higher levels in Acute Lymphoblastic Leukemia(ALL) cell lines when compared to AML cell lines. Bone marrow cells were obtained from de novo ALL patients. qRT-PCR showed FAMLF-2 was up-regulated in ALL patients (N=115) compared to healthy controls (N=72). The FAMLF-2 expression was positively correlated with the poor prognosis in ALL patients by Kaplan-Meier analysis. These data demonstrated that FAMLF-2 is overexpressed in ALL patients and is associated with poor prognosis. To verify the full cDNA sequence of FAMLF-2, 5‘ and 3‘ rapid amplification of the cDNA ends (RACE) was performed, and we identified the 2393 nt of FAMLF-2. FAMLF-2 is predicted to not have protein-coding potential. Further subcellular analysis showed FAMLF-2 is mainly localized in the nucleus of Jurkat cells by lncRNA FISH and subcellular fractionation assay. To further verify the role of FAMLF-2 in ALL leukemogenesis, a xenograft model was established in NCG mice by subcutaneously or intravenously injection of engineered Jurkat cells (knockdown group: shNC and sh FAMLF-2, overexpression group: pCDH and FAMLF-2). We found that the size and weight of tumors in FAMLF-2 knockdown group were significantly decreased compared with the empty vector in subcutaneous ALL xenograft tumor model. and the mice in the sh FAMLF-2 groups survived longer than those in the shNC groups in introvenous ALL xenograft tumor model (sh FAMLF-2 vs. shNC: 55 days vs. 43 days, P<0.01). These data suggested that downregulation of FAMLF-2 inhibits leukemia development. Although no significant difference was found in the overall survival of mice overexpressed FAMLF-2. We observed the accelerated central nervous system(CNS) infiltration of leukemia cells when FAMLF-2 was overexpressed (clinical score at 32th day from Vector vs. FAMLF-2: 2.7 scores vs. 1.2 scores, p < 0.01 ). In vitro study revealed knockdown of FAMLF-2 inhibited cell proliferation and promoted cell apoptosis in ALL cells, and vice versa. Taken together, our clinical, mouse model, and AML cell data demonstrated that FAMLF-2 promotes leukemogenesis. RNA sequencing was performed to study how FAMLF-2 promotes ALL development. Gene Set Enrichment Analysis (GSEA) of genes downregulated by FAMLF-2 showed “Myc targets” with the highest normalised enrichment score (NES) in Jurkat cells. C-Myc is a well-recognized transcription factor that involves in leukemogenesis. To investigate whether FAMLF-2 performed direct interaction with c-Myc, we performed RNA pull-down followed by mass spectrometry analysis. Instead of c-Myc, DExH-Box Helicase 9 (DHX9) has the highest abundance. The Western blot and RNA immunoprecipitation (RIP) assay were further performed to validated the direct interaction between FAMLF-2 and DHX9. DHX9 is an RNA helicase that participates in c-Myc mRNA stability. Inhibition of FAMLF-2 suppressed DHX9 and c-Myc expression. Reduced interaction between DHX9 protein and c-Myc mRNA were observed when FAMLF-2 were downregulated. Furthermore, RNA stability assays suggested that knockdown of FAMLF-2 leads to a declined half-life of c-Myc mRNA. Collectively, these results demonstrated that FAMLF-2 regulates the stability of c-Myc mRNA through direct binding of DHX9, thus involved in the ALL development. In summary, our study revealed the previous undefined cDNA sequence of FAMLF-2, and provides new insights of FAMLF-2 in leukemogenesis via the DHX9/c-Myc axis, which might act as a prospective prognostic biological marker in ALL.

Repeated small-volume exchange transfusion for hyperleukocytosis in pediatric acute leukemia: A retrospective analysis.

Leukapheresis reduces hyperleukocytosis in children with acute leukemia. Although the usefulness of this procedure is under debate, a repeated small-volume exchange transfusion along with leukapheresis yielded satisfactory results. Forty-seven patients with acute leukemia [32 acute lymphocytic leukemia (ALL) and 15 acute myeloblastic leukemia (AML)] were enrolled between January 2017 and June 2022 and underwent repeated small-volume exchange transfusion. The following were measured: demographic and clinical characteristics, time of the procedure, PWBC (peripheral white blood cell) count, hemoglobin, platelet count, blood biochemistry, electrolytes, coagulation, leukostasis, TLS (tumor lysis syndrome), DIC (disseminated intravascular coagulopathy), adverse events (AEs), and serious AEs (SAEs). The demographic and clinical characteristics were not significantly different between ALL and AML patients, but differences were observed in PWBC counts (424.2 ± 135.6 vs. 223.8 ± 58.0 × 109/L). The procedures needed 3-8 processes, and the average procedure time was not significantly different between ALL and AML. The PWBC count gradually reduced to <100 × 109/L; hemoglobin, platelet count, K+, Na+, and Ca2+ were unchanged. Alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, troponin-I, creatine kinase-MB, prothrombin time, and activated partial thromboplastin time maintained normal or recovered from abnormal ranges. The manifestations of leukostasis, TLS, and DIC improved or disappeared. No AEs and SAEs occurred. The required total blood volume was based on initial PWBC count, manifestations of leukostasis, and age. Our finding suggests that repeated small-volume exchange transfusion is effective and safe for treating hyperleukocytosis in children with acute leukemia.

Factors influencing access to specialised haematology units during acute myeloblastic leukaemia patient care: A population-based study in France.

The excess mortality observed in Acute Myeloblastic Leukaemia (AML) patients, partly attributed to unequal access to curative treatments, could be linked to care pathways. We included 1039 AML incident cases diagnosed between 2012-2016 from the 3 French blood cancer registries (3,625,400 inhabitants). We describe patients according to age, the medical entry unit and access to the specialised haematology unit (SHU) during follow-up. Multivariate logistic regression model was done to determine the association between covariables and access to SHU. A total of 713 patients (69%) had access to SHU during care. The most common care pathway concerned referral from the general practitioner to SHU, n=459(44%). The univariate analysis observed a downward trend for the most deprived patients. Patients who consulted in SHU were younger (66 years vs. 83, p < 0.001), and 92% had access to cytogenetic analysis (vs. 54%, p < 0.001). They also had less poor prognosis AML-subtypes (AML-MRC, t-AML/MDS and AML-NOS) (38% vs. 69%); 77% with de novo AML (vs. 67%, p < 0.003)], more favourable cytogenetic prognostic status (23% vs. 6%, p < 0.001), less comorbidities (no comorbidity=55% vs. 34%, p < 0.001) and treatments proposed were curative 68% (vs. 5.3%, p < 0.001). Factors limiting access to SHU were age over 80 years (OR, 0.14; 95% CI, 0.04-0.38), severe comorbidities (OR, 0.39; 95% CI, 0.21-0.69), emergency unit referral (OR, 0.28; 95% CI, 0.18-0.44) and non-SHU referral (OR, 0.12; 95% CI, 0.07-0.18). Consultation in an academic hospital increased access to SHU by 8.87 times (95% CI, 5.64-14.2). The high proportion of access to cytogenetic testing and curative treatment among patients admitted to SHU, and the importance of early treatment in AML underlines the importance of access to SHU for both diagnosis and treatment.

The prognostic role of C-KIT, TET1 and TET2 gene expression in Acute Myeloid Leukemia

Aimwas to assess the role of C-KIT, TET1 and TET2 expression in the diagnosis and prognosis of acute myeloblastic leukemia (AML).MethodsThe expression levels of C-KIT, TET1 and TET2 were assessed in the bone marrow (BM) aspirate of 152 AML patients compared to 20 healthy control using quantitative real-time polymerase chain reaction (qRT-PCR). Data were correlated with the clinico-pathological features of the patients, response to treatment, disease-free survival (DFS), and overall survival (OS) rates.ResultsC-KIT, TET1 and TET2 were significantly upregulated in AML patients [0.25 (0–11.6), 0.0113 (0–3.301), and 0.07 (0–4); respectively], compared to the control group [0.013 (0.005–0.250), P < 0.001, 0.001 (0–0.006), P < 0.001, and 0.02 (0.008–0.055), P = 0.019; respectively]. The sensitivity, specificity, and area under curve of of C-KIT were (48.7%, 100%, 0.855; respectively, P = 0.001), and that of TET1 were (63.4%, 100%, 0.897; respectively, P = 0.001), while that of TET2 were (56.8%, 100%, 0.766; respectively, P = 0.019). When combining the three markers, the sensitivity was 77.5%, however it reached the highest sensitivity (78.6%) and specificity (100%) when combining both c-KIT + TET1 together for the diagnosis of AML. C-KIT overexpression associated with shorter DFS (P = 0.05) and increased incidence of relapse (P = 0.019). Lymph nodes involvement [HR = 2.200, P = 0.005] is an independent risk factor for shorter OS rate of AML patients. Increased BM blast % [HR = 7.768, P = 0.002], and FLT3-ITD mutation [HR = 2.989, P = 0.032] are independent risk factors for shorter DSF rate of the patients.ConclusionC-KIT, TET1, and TET2 could be used as possible useful biomarkers for the diagnosis of AML.

Plasminogen Activator Inhibitor-1 in Acute Myeloid Leukemia: Is It Useful in Predicting Response to Induction Remission Therapy?

BACKGROUND: Coagulation and fibrinolytic abnormalities are common in patients with acute myeloblastic leukemia (AML). Plasminogen activator inhibitor (PAI) activity is believed to be elevated during the initial diagnosis and relapse of AML patients. AIM: This study aimed to evaluate the levels of plasma PAI-1 activity in AML patients before and after remission induction. METHODS: Thirty AML patients and 20 healthy volunteers were included in this study. The patients were classified according to AML-FAB subtypes. All patients received 7+3 induction chemotherapy. They were evaluated for complete remission after induction chemotherapy and followed up for 6 months. RESULTS: PAI-1 activity was measured by ELISA immunoassay. PAI-1 activity was significantly higher in AML patients than the control group (P=0.016), whereas there was insignificant difference among patients in various AML subgroups (P&gt;0.05). Before and after treatment, there was a significant difference in PAI-1 activity between patients with active disease and those at remission (P= 0.023 and &lt;0.0001, respectively). CONCLUSION: High PAI-1 activity in AML patients is found to be associated with poor response to treatment.

PB1781: THERAPEUTIC POTENTIAL OF DNA DAMAGE RESPONSE IN ACUTE MYELOBLASTIC LEUKEMIA

Background: Genome integrity is of utmost importance for cell survival and homeostasis maintenance. However, DNA integrity is threatened by endogenous and exogenous sources. DNA lesions can block genome replication and transcription, and if unrepaired or repaired incorrectly can lead to mutations and genomic instability. Hence, cells have developed a variety of mechanisms to protect genome integrity and repair damaged DNA, collectively termed the DNA damage response (DDR). These mechanisms comprise DNA damage recognition, damage tolerance, signal transduction, transcriptional regulation, cell cycle control, and DNA repair. Key DDR elements include sensor proteins that detect damage (e.g. PARP1 and 2), mediators and signaling transducers (e.g. DNA-PK) that regulate downstream targets, and effector molecules (e.g. CHK1 and 2). Increased DNA damage and altered DDR are critical features of genetic instability presumably implicated in the pathogenesis of acute myeloblastic leukemia (AML). AML is a clonal malignant disease of hematopoietic stem and/or progenitor cells characterized by a block in myeloid differentiation and increased proliferation. This neoplasia is considered the most common acute leukemia in adults and is characterized by recurrent genetic abnormalities. Recently, specific recurrent chromosomal translocations and genetic mutations typical of AML have been associated with DNA repair defects. Aims: Our goal is to assess the potential of DDR as a therapeutic target in AML, using in vitro models, in order to identify new therapeutic approaches. Methods: Five AML cell lines of different molecular and cellular subtypes were used (HEL, HL-60, K-562, LAMA-84, and NB-4). Cells were incubated in the absence and presence of increasing concentrations of two DDR inhibitors, CCT245737 (CHK1 inhibitor) and Niraparib (PARP1/2 inhibitor). Cell density and viability were assessed, for 72 hours, by trypan blue assay. Afterward, resorting to flow cytometry, the cell cycle was assessed using propidium iodide/RNAse, and the cell death was determined using Annexin V/7-AAD double staining as well as by optic microscopy (May-Grünwald-Giemsa staining). Results were statistical analyzed, considering a significance level of 95%. Results: CCT245737 reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 54 µM. In contrast, LAMA-84 was the most resistant cell line with an IC50 at 48 hours of 203 µM, 3.7-fold higher than NB-4. PARP1/2 inhibition also reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. Once again NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 25 µM and LAMA-84 was the most resistant with an IC50 at 48 hours of 66 µM, 2.7-fold higher than NB-4. Both inhibitors induced cell death by apoptosis since an increase in the percentage of cells in late apoptosis was observed. This result is in agreement with the observed by optic microscopy. We also detected a cytostatic effect since a cell cycle arrest in the S phase was observed after treatment with both inhibitors. Summary/Conclusion: AML cell lines have different sensibilities to DDR inhibitors, particularly CCT245737 and niraparib, with the NB-4 cell line appearing to be the most sensitive to DDR inhibition (namely CHK1 and PARP1/2 inhibition), whereas LAMA-84 appears to be the most resistant to this inhibition. In conclusion, this work may help to identify new therapeutic approaches that could eventually improve AML patients’ outcomes.

Single Center Clinical Analysis of Bloodstream Infection Pathogens in Children with Acute Leukemia

To investigate the clinical features, distribution of pathogenic bacteria, and drug resistance of bloodstream infection in children with acute leukemia. Clinical data of 93 blood culture-positive children with acute leukemia from January 2015 to December 2019 in Department of Pediatrics, The Second Hospital of Anhui Medical University were analyzed retrospectively. In these 93 cases, 78 cases were in the period of neutrophil deficiency. There were 54 Gram-negative bacteria (G-) (58.1%) found through blood culture, and the top 4 strains were Escherichia coli (15.1%), Klebsiella pneumoniae (13.9%), Pseudomonas aeruginosa (6.5%), and Enterobacter cloacae (6.5%). There were 39 Gram-positive bacteria (G+) (41.9%) detected, and the top 4 strains were Staphylococcus epidermidis (10.8%), Streptococcus pneumoniae (6.5%), Staphylococcus hemolyticus (5.4%), and Staphylococcus human (5.4%). Among 74 strains of pathogenic bacteria from acute lymphoblastic leukemia (ALL) children, there were 29 strains of G+ bacteria (39.2%) and 45 strains of G- bacteria (60.8%). While in 19 strains from acute myeloblastic leukemia (AML) patients, G- bacteria accounted for 47.4% and G+ bacteria accounted for 52.6%. In 15 ALL children without neutropenia, G+ bacteria made up the majority of the strains (66.7%). In the 93 strains of pathogenic bacteria, 13 (13.9%) strains were multidrug-resistant. Among them, extended-spectrum β-lactamases accounted for 42.9%, carbapenemase-resistant enzyme Klebsiella pneumoniae 15.4%, and carbapenemase-resistant enzyme Enterobacter cloacae strains 33.3%, which were detected from G- bacteria. While, 13.3% of methicillin-resistant coagulase-negative Staphylococci accounted for 13.3% detected from G+ bacteria, but linezolid, vancomycin, teicoplanin Staphylococcus and Enterococcus resistant were not found. The average procalcitonin (PCT) value of G- bacteria infection was (11.02±20.282) ng/ml, while in G+ infection it was (1.81±4.911) ng/ml, the difference was statistically significant (P<0.05). The mean value of C-reactive protein (CRP) in G- infection was (76.33±69.946) mg/L, and that in G+ infection was (38.34±57.951) mg/L. The prognosis of active treatment was good, and only one case died of septic shock complicated with disseminated intravascular coagulation (DIC) and gastrointestinal bleeding caused by carbapenemase-resistant enzyme enterobacteriaceae. G- is the major bacteria in acute leukemia children with bloodstream infection, but the distribution of ALL and AML strains is different. G- bacteria dominates in ALL, while G+ bacteria and G- bacteria are equally distributed in AML. Non-agranulocytosis accompanied by bloodstream infections is dominant by G+ bacteria. The mean value of PCT and CRP are significantly higher in G- bacteria infection than in G+ bacteria.

Therapeutic Outcomes of Pediatric Acute Myeloblastic Leukemia Patients at a Tertiary Hospital in Bandung, Indonesia

Background: Acute myeloblastic leukemia (AML) is a subtype of leukemia characterized by myeloid infiltration into the bone marrow, blood, and other tissues. AML ranks 5th malignancy in children and the prognosis is poor. After chemotherapy, the outcomes vary. Therefore, this study aimed to provide further insight into the therapeutic outcomes of pediatric AML patients. Methods: This study was conducted with a cross-sectional descriptive method. The data were obtained from the medical records of children diagnosed with AML at the Department of Child Health of Dr. Hasan Sadikin General Hospital in 2017–2019 with the total sampling method. Data including age at determination, gender, laboratory values including hemoglobin, leukocyte, thrombocyte as well as blast cell count on peripheral blood smear were collected. Also, the bone marrow punctures gathered were clustered based on the French-American-British (FAB) classification. Data were presented in tables. Results: In total, 46 data of AML patients were retrieved, with the age category at first diagnosis was &gt;5–12 years (48%) and predominantly males (63%), children with moderate anemia (41%), leukocytosis (35%), severe thrombocytopenia (46%), and blast cell count ≥20% (83%). AML-M2 was the most common subtype (30.4%). The majority of patients (91%) underwent chemotherapy and most (45%) patients died during chemotherapy Conclusion: The outcome of AML therapy among children is mostly poor, and particularly die during chemotherapy. Early detection and follow-up of patients to continue therapy are important aspects to reduce the mortality rate of AML.

Prognostic impact of homeobox and PR domain containing protein 16 genes expressions in patients with acute myeloid leukemia

BackgroundThe functions of the HOXA 5, HOXA 9, and PRDM16 are concerned with embryonic growth and the regulation of adult hematopoietic cells differentiation. The HOXA5, HOXA9, and PRDM16 are expressed in the majority of solid cancers and several hematological malignancies, including myeloproliferative disease and acute leukemia. The function and prognostic values of HOXA5, HOXA9, and PRDM16 quantitative PCR expression in novel patients with acute myeloblastic leukemia (AML) were to be studied. MethodsThis prospective case-control study was performed on 60 newly diagnosed AML patients and 40 healthy subjects acting as a control group. The patients underwent CBC, BM analysis, HOXA5, HOXA9, and PRDM16 expression by qRT-PCR testing. ResultsHigh expressions of HOXA5, HOXA9, and PRDM16 were significantly prevalent in patients compared with the control. The highest incidence of expression was found in AML patients with minimal maturation and acute megakaryoblastic leukemia (M1/M7). Interestingly, high levels of HOXA5, HOXA9, and PRDM16 expressions were correlated with older age and hepatosplenomegaly associated with poor clinical outcome, shorter overall and disease-free survival than low levels of HOXA5, HOXA9, and PRDM16 expressions. ConclusionHigh HOXA and PRDM16 expressions predict bad outcomes and are associated with weak prognostic factors which can promote new therapeutic trends for AML.

Does RAD21 Co-Mutation Have a Role in DNMT3A Mutated AML? Results of Harmony Alliance AML Database

Does RAD21 Co-Mutation Have a Role in DNMT3A Mutated AML? Results of Harmony Alliance AML Database

High risk of invasive fungal disease in children undergoing hematopoietic cell transplantation or complex anticancer therapy: the adverse role of post-transplant CMV replication

Introduction: We analyzed the epidemiology and outcomes of treatment of invasive fungal disease (IFD) in children during anticancer therapy (PHO, pediatric hematology and oncology) or after hematopoietic cell transplantation (HCT) over a period of eight consecutive years in a single-center study. Material and methods: Overall, a total of 254 HCTs were performed, and 415 children were newly diagnosed for malignancy. Incidence, epidemiology and outcome of IFD were analyzed. Results: The cumulative incidence of any IFD was 32.6% in allo-HCT, 22.2% in PHO, and 6.0% in auto-HCT patients. The incidence of proven +probable IFD was 12.6%, 10.4%, and 6.0%, respectively. As many as 77.0% HCT and 67.4% PHO of fungal episodes occurred in acute leukemia patients: the highest incidence of any IFD was observed for acute lymphoblastic leukemia (29.3% in HCT; 40.5% in PHO) and for acute myeloblastic leukemia (51.1% in HCT; 65.0% in PHO) patients. There were no significant differences in the incidence of fungal infections in both allo-HCT and PHO patients between the 2-year periods. Factors contributing to an increased risk of IFD in allo-HCT patients were: CMV replication, and acute and chronic graft-versus-host disease (GvHD). Survival from IFD was 91.9% in PHO, and 78.1% in HCT patients. Fungal pneumonia in HCT patients resolved in 62.9%, while in PHO patients it resolved in 93.5%. Conclusions: The risk of IFD in allo-HCT patients is much higher than in auto-HSCT and PHO patients. The outcome of IFD is better in PHO and auto-HCT than in allo-HCT settings.

Antifungal Prophylaxis in AML Patients Receiving Intensive Induction Chemotherapy: A Prospective Observational Study From the Acute Leukaemia French Association (ALFA) Group

BackgroundAlthough recommended in patients with acute myeloblastic leukaemia (AML) after induction chemotherapy, real-life use of antifungal prophylaxis (AFP) is different among centres. Materials and MethodsThis is an ancillary study to a randomized trial on intensive induction chemotherapy in AML patients (ALFA-0702/NCT00932412), where AFP with posaconazole was recommended. IFIs were graded by investigators and by central reviewers according to the revised EORTC definitions. Experts conclusions were compared to the investigators’ ones. ResultsA total of 677 patients were included. Four AFP strategies were reported: Group-1: no AFP (n = 203, 30%), Group-2: posaconazole (n = 241, 36%), Group-3: posaconazole with other AFP (n = 142, 21%), Group-4: other AFP (n = 91, 13%). Experts graded more IFI than investigators: proven/probable IFI, 9.0% (n = 61) versus 6.2% (n = 42). The cumulative incidence at day60 of probable/proven IFI was 13.9% (Group-1); 7.9% (Group-2); 5.6% (Group-3); and 6.6% (Group-4). IFI onset was 26 (19-31) days after induction in Groups 2-3, versus 16 (9-25) days in Group 1 and 20 (12-24) days in Group 4 (P< .001). After a median follow-up of 27.5 months (0.4-73.4), the mortality rate was 38.3%, with 5.4% attributed to IFI. In multivariate analysis, IFI occurrence was an independent risk of death (HR5.63, 95%-CI 2.62-12.08, P< .001). EORTC recommendations were applied in only 57% of patients. In patients without IFI, the rate of AML complete remission was higher. ConclusionsIn AML patients, AFP delayed the onset of IFI in addition of decreasing their rate. The frequent misidentification of IFI impacts their appropriate management according to recommendations. hematological remission was more frequent in patients without IFI.

Unsupervised Flow Cytometry Analysis Allows for an Accurate Identification of Minimal Residual Disease Assessment in Acute Myeloid Leukemia.

Simple SummaryIn acute myeloid leukemia (AML), minimal/measurable residual disease (MRD) can be assessed based on molecular markers or immunophenotypic features evaluated at diagnosis, through multiparameter flow cytometry (MFC) for the latter. New artificial intelligence tools allow to perform unsupervised analysis of MFC data. The Flow-Self-Organizing-Maps (FlowSOM) tool was used here to concomitantly compare MFC features of normal bone marrow together with diagnosis and follow-up bone marrow samples from 40 AML patients for the evaluation of MRD. MFC results were compared to molecular MRD, showing high concordance. This opens the road for a new easy and objective way of assessing MRD even in AML patients without molecular markers.The assessment of minimal residual disease (MRD) is increasingly considered to monitor response to therapy in hematological malignancies. In acute myeloblastic leukemia (AML), molecular MRD (mMRD) is possible for about half the patients while multiparameter flow cytometry (MFC) is more broadly available. However, MFC analysis strategies are highly operator-dependent. Recently, new tools have been designed for unsupervised MFC analysis, segregating cell-clusters with the same immunophenotypic characteristics. Here, the Flow-Self-Organizing-Maps (FlowSOM) tool was applied to assess MFC-MRD in 96 bone marrow (BM) follow-up (FU) time-points from 40 AML patients with available mMRD. A reference FlowSOM display was built from 19 healthy/normal BM samples (NBM), then simultaneously compared to the patient’s diagnosis and FU samples at each time-point. MRD clusters were characterized individually in terms of cell numbers and immunophenotype. This strategy disclosed subclones with varying immunophenotype within single diagnosis and FU samples including populations absent from NBM. Detectable MRD was as low as 0.09% in MFC and 0.051% for mMRD. The concordance between mMRD and MFC-MRD was 80.2%. MFC yielded 85% specificity and 69% sensitivity compared to mMRD. Unsupervised MFC is shown here to allow for an easy and robust assessment of MRD, applicable also to AML patients without molecular markers.

Efficacy, Safety, and Tolerance of Gemtuzumab Ozogamicin Combined with FLAG/FLAG-Ida or Azacitidine in Relapsed/Refractory Acute Myeloblastic Leukemia

Aim. To assess the efficacy, safety, and tolerance of gemtuzumab ozogamicin (GO) combined with FLAG/FLAG-Ida chemotherapy or azacitidine in patients with relapsed/refractory acute myeloblastic leukemia (AML) in clinical practice. Materials &amp; Methods. The study included 32 patients (16 men and 16 women). The median age was 44 years (range 23-83 years). Among them there were 15 (46.8 %) patients with refractory and 17 (53.2 %) patients with relapsed AML. GO combined with FLAG/FLAG-Ida was administered to 15 (46.8 %) patients, whereas 17 (53.2 %) patients were treated with GO and azacitidine combination. Therapy safety was assessed according to CTCAE v. 5.0. Results. Overall response rate including complete remission (CR), CR MRD-, CR with incomplete hematologic recovery, and morphologic leukemia-free status was 59.4 % (19/32). Refractoriness was observed in 31.25 % (10/32) of patients. Early mortality was 9.4 % (3/32). Overall response was 64.7 % (11/17) in the azacitidine and 53.3 % (8/15) in the FLAG/FLAG-Ida groups. In 4 (80 %) out of 5 patients with prior to FLAG treatment refractoriness, the response was achieved after GO + azacitidine therapy. In 58.9 % (10/17) of patients who received GO + azacitidine therapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be performed. The incidence of GO infusion complications in the tested groups did not significantly differ (p = 0.72) and was 46.7 % (7/15) (40 % with grade 1/2 and 6.7 % with grade 3) in the GO + FLAG/FLAG-Ida group and 35.3 % (6/17) (29.4 % with grade 1/2 and 5.9 % with grade 4) in the GO + azacitidine group. In the GO + FLAG/FLAG-Ida group 5 (33.3 %) patients experienced serious adverse events (SAE) of sepsis. In the GO + azacitidine group SAEs were reported in 6 (35.3 %) patients: 4 (66.6 %) with sepsis, 1 (16.7 %) with acute cardiovascular failure, and 1 (16.7 %) with acute respiratory failure. The median (range) duration was 23 (10-39) days for neutropenia grade 4, 24 (11-38) days for neutropenia grade 3, 21 (11-41) days for thrombocytopenia grade 4, 26 (16-45) days for thrombocytopenia grade 3, and 25 (22-45) days for thrombocytopenia grade 1/2. Thrombocytopenia duration was longer in patients with GO + FLAG/FLAG-Ida therapy, however, no significant differences were identified. No cases of veno-occlusive liver disease were reported. Median overall survival (OS) for both groups (n = 32) was 31.4 months, median disease-free survival (n = 21) was 13.3 months. In the group of patients with effective treatment, the median OS was not reached. In non-responders, it was 18 months (р = 0.0442). Conclusion. GO combined with FLAG/FLAG-Ida chemotherapy or azacitidine proved effective in relapsed/refractory AML patients. Remission did not appear to be associated with ELN risk, gender, age, CD33 expression, number of prior therapy lines, or number of relapses. GO + azacitidine combination showed efficacy, safety, and good tolerance in patients with prior high-dose chemotherapy refractoriness as well as low ECOG performance status. That allowed for the subsequent allo-HSCT administration to these patients. There was no significant difference between the groups of patients in the incidence of hematologic, non-hematologic toxicity, and time to hematologic recovery. Thrombocytopenia duration was longer in patients with GO + FLAG/FLAG-Ida therapy which is consistent with literature data. GO-based effective treatment in relapsed/refractory AML considerably improves OS: during 36 months of follow-up the median was not reached.

One Tube 24 Color Full Spectral Flow Cytometry and Multi-Dimensional Software to Study the Maturation Pattern and Antigen Expression of the Myeloid

Introduction Flow cytometry(FC) plays an important role in the diagnosis of hematologic diseases and the study of cell maturation. Spectral multicolor flow cytometry(SMFC) has shown an advantage over traditional FC that more fluorescent markers could be detected simultaneously, more antigen combinations could be made, and the expression of cells could be scrutinized. However, published studies focused on lymphocyte subsets and the differentiation between hematogones and B-acute lymphoblastic leukaemia/lymphoma(ALL/LBL) minimal residual disease(MRD) detection, and there are few studies on myeloid development and expression. Besides, more powerful and cutting-edge software are needed for complicated combinations from SMFC because traditional dot plots are unable to meet the demand of analysis. Here we design a one-tube 24-color panel combining with the multidimensional data analysis software to study the expression and maturation of normal and malignant myeloid cells including minus subgroups. We hope to improve the sensitivity of MRD by FC and explore more information about myeloid diseases, finally promote the development of artificial intelligence(AI) in clinical FC diagnosis. Methods: the one-tube 24-color panel was designed according to our experience and Euroflow recommendation. it is composed of backbones including CD45 and myeloblast markers CD34, CD117 and HLA-DR, adding myeloid markers CD33, CD13, CD371, CD15, CD64, CD11c,CD14, CD36 and CD11b, routine leukaemia associated immunophenotyping(LAIP) or different from normal(DFN) markers CD4, CD19, CD7, CD2, CD56,CD96,CD123, CD38, CD200, CD71 and CD9. The control database consisted of 20 normal bone marrow(BM) specimens, including 8 healthy donors and 12 patients with other diseases that were in complete remission(CR) after treatment. To verify the effectiveness of the panel, 4 BM samples from acute myeloblastic leukaemia(AML) patients with MRD positive or relapsed status were selected, with malignant myeloblasts of 0.23% (sample A1), 4.3%, 30.31% (and 6.47% abnormal mast cells, sample A3), 0.29% (16.49% basophils) (sample A4), respectively. The data was acquired by a 3 laser 38-color Cytek spectral FC, and analyzed by Kaluza and Flowjo software. The results were compared with those of conventional 3 - laser 8 - color Canto FC. Results when analyzing the common antibodies acquired by two kinds of FCs and software, the similarities and good correlations were shown about the percentage of myeloid subsets and expression pattern of antigens, no matter in normal or abnormal specimen. However, SMFC can analyze subsets of myeloid cells more clearly and detailedly, especially in minus subgroups like mast cells or basophils. Abnormal expression of tumor cells could be clearly observed in both FCs, and more abnormalities could be found with 24 color analysis, especially mast cell abnormalities. The t-SNE plug-in in Flowjo was used to conduct dimension-reduction analysis on 20 samples (see figure), with hundreds of dot plots being merged into one multi-dimensional picture. The normal samples were similar, but the abnormal samples were different from the normal ones. However, the t-SNE pictures were same only for the specimens that malignant myeloblasts were the only abnormality. The other two samples with abnormal mast cells or basophils showed different t-SNE figures. In the process of automatic clustering, CD117 and CD34 positive myeloblasts, basophils and mast cells were classified as three clusters according to the proportion of cells, surface antigen expression, and of fluorescence intensity of antigen expression. A more obvious aggregation was formed by malignant cells than normal cells, and was positively related to tumor burden. Since mast cells and basophils were low percentages in normal specimens, the abnormal clusters were more obvious when the proportion of these two types of cells increased. Conclusion Combined with cutting-edge software(BD FlowJO), SMFC can offer more cellular information that is unmatched by traditional FC. This study is only the preliminary attempt of our laboratory. With detailed data from more samples and longer-termed clinical trials, it would be a promising method to explore and study the myeloid maturation, improve the sensitivity of MRD, and promote the application of AI. Disclosures No relevant conflicts of interest to declare.

Unsupervised Flow Cytometry Analysis: Application to Minimal Residual Disease Detection in a Cohort of 40 Acute Myeloblastic Leukemia Patients with Molecular Markers

Minimal/measurable residual disease (MRD) assessment in the course of acute myeloblastic leukemia (AML) monitoring is a topic that currently reaches undisputable interest. Multiparameter flow cytometry (MFC) appears as a valuable, rapid tool to assess response to therapy. Within the European LeukemiaNet (ELN) a task force has promoted the use of a backbone panel of monoclonal antibodies likely to cover both the most common leukemia associated immunophenotyping patterns (LAIP) and different from normal (DfN) immunophenotypes (Schuurhuis et al., Blood 2018). Classical gating strategies of MRD assessment however rely on supervised/operator-dependent approaches. The development of mass-cytometry has led to the emergence of new unsupervised analysis software. Among them, the FlowSOM (Flow Self Organizing Maps) R-based solution has been recognized as the most efficient, applicable to both mass and classical flow cytometry. Combination of the R-based FlowSOM definition of self-organized minimal spanning trees (MST) and the Kaluza® classical flow cytometry analysis software allows for a combined exploration of a normal bone marrow (NBM) matrix (derived from 19 normal BM samples)together with individual diagnosis and follow-up (FU) samples. This strategy was applied to 40 AML patients treated with a conventional 3+7 regimen for whom at least post induction FU was available, together with targets allowing for molecular MRD assessment (NPM1, RUNX1, CBFB-MYHorBCR-ABL). According to previously published panels (GEIL, Cytometry part B, 2018), two tubes were tested in MFC and results were compared to molecular MRD results for 96 FU time points. All patients could be evaluated in MFC at all time points. Comparison of molecular and MFC data showed an overall good concordance (80.2%), especially for FU1 time points (87.5%) with a sensitivity of 0.89 and a specificity of 0.75. Yet, outcome after induction appeared to be more correlated with MFC-negative MRD. This work also allowed to demonstrate the particular ability of the FlowSOM algorithm to disclose subtle chemoresistant subclones during FU. Moreover, by systematic comparison of normal bone marrow and FU points, MRD could be asserted without ambiguity and regenerating BM identified as such. Of note, this work could be performed on retrospective data, based on the fact that instruments were harmonized as reported (Lacombe et al. Leukemia, 2016) and identical panels were used all along. This makes this strategy easily transferrable to a network of laboratories using consensus settings and panels, as proposed by the European LeukemiaNet (Schuurhuis et al., Blood 2018). All in all, this study shows that unsupervised MFC MRD assessment appears as an accurate tool to appreciate response to chemotherapy in AML, and the FlowSOM + Kaluza method, combined with a systematic diagnostic FU / NBM multicomparison approach, increases the resolving power in the monitoring of MRD compared to the classic LAIP/different from normal approaches as implemented to date. Disclosures Dumas: Abbvie:Honoraria;Jazz-pharmaceutical:Consultancy;Daiichi Sankyo:Consultancy, Research Funding;Astellas:Research Funding, Speakers Bureau.

Gene expression analysis of activating and inhibitory receptors of natural killer cells in patients with acute myeloblastic leukemia

PurposeNatural killer (NK) cells are cytotoxic lymphocytes, which have long been known to play an essential role in immune surveillance of tumor cells. The results of several clinical studies imply evidence of impaired activity of NK cells in acute myeloblastic leukemia (AML). The aim of this study was to investigate the gene expression of activating and inhibitory receptors of NK cells in patients with newly diagnosed AML before and after induction therapy using 7 + 3 regimen in comparison to healthy donors. Materials and methodsSixteen AML patients aged 16–64 years as well as 16 matched healthy individuals were studied. Peripheral blood samples from patients were obtained in two steps, namely, in newly diagnosed patients and 28 days after receiving induction therapy. Real-time PCR was performed to evaluate the expression levels of activating receptors, including DNAM-1 and NKp46 as well as inhibitory receptors of KIR2DL1 and NKG2A. ResultsOur results demonstrated that the newly diagnosed patients showed over 50% decrease in NKp46 expression and a 6-fold increase in KIR2DL1 expression compared to healthy controls. The mRNA expression analysis in patients after induction therapy suggested a significant decrease in mRNA expressions of KIR2DL1 and NKG2A in comparison to newly diagnosed patients. ConclusionHerewith, we show a statistical difference in mRNA expression levels of activating (NKp46) and inhibitory receptors from NK cells in newly diagnosed AML patients when compared with healthy controls or patients who received induction therapy, supporting the findings of researchers who reported the impaired NK cells cytotoxicity in AML patients.

A general view of CD33+ leukemic stem cells and CAR-T cells as interesting targets in acute myeloblatsic leukemia therapy.

Acute myeloblastic leukemia (AML) is the most frequent acute leukemia in adulthood with very poor overall survival rates. In the past few decades, significant progresses had led to the findings of new therapeutic approaches and the better understanding of the molecular complexity of this hematologic malignancy. Leukemic stem cells (LSCs) play a key role in the initiation, progression, regression, and drug resistance of different types of leukemia. The cellular and molecular characteristics of LSCs and their mechanism in the development of leukemia had not yet been specified. Therefore, determining their cellular and molecular characteristics and creating new approaches for targeted therapy of LSCs is crucial for the future of leukemia research. For this reason, the recognition of surface maker targets on the cell surface of LSCs has attracted much attention. CD33 has been detected on blasts in most AML patients, making them an interesting target for AML therapy. Genetic engineering of T cells with chimeric antigen receptor (CAR-T cell therapy) is a novel therapeutic strategy. It extends the range of antigens available for use in adoptive T-cell immunotherapy. This review will focus on CAR-T cell approaches as well as monoclonal antibody (mAB)-based therapy, the two antibody-based therapies utilized in AML treatment.