AML-M2 Research Articles

Differentiating leukemia subtypes based on metabolic signatures using hyperpolarized 13C NMR.

Leukemia is a group of blood cancers that are classified in four major classes. Within these four classes, many different subtypes exists with similar origin, genetic mutations, and level of maturity, which can make them difficult to distinguish. Despite their similarities, they might respond differently to treatment, and therefore distinguishing between them is of crucial importance. A deranged metabolic phenotype (Warburg effect) is often seen in cancer cells, leukemia cells included, and is increasingly a target for improved diagnosis and treatment. In this study, hyperpolarized 13C NMR spectroscopy was used to characterize the metabolic signatures of the six leukemia cell lines ML-1, CCRF-CEM, THP-1, MOLT-4, HL-60, and K562. This was done using [1-13C]pyruvate and [1-13C]alanine as bioprobes for downstream metabolite quantification and kinetic analysis on cultured cells with and without 2-deoxy-D-glucose treatment. The metabolic signatures of similar leukemia subtypes could be readily distinguished. This includes ML-1 and THP-1, which are of the similar M4 and M5 AML subtypes, CCRF-CEM and MOLT-4, which are of the similar T-ALL lineage at different maturation states, and HL-60 and K562, which are of the closely related M1 and M2 AML subtypes. The data presented here demonstrate the potential of hyperpolarized 13C NMR spectroscopy as a method to differentiate between leukemia subtypes of similar origin. Combining this method with bioreactor setups could potentially allow for better leukemia disease management as metabolic signatures could be acquired from a single biopsy through repeated experimentation and intervention.

Unpredicted transformation of acute myeloid leukemia with translocation (16;16) (p13; q22): a case report and review of the literature

IntroductionThe transformation of acute myeloid leukemia with translocation (16;16) (p13; q22) from AML M2 to acute monocytic leukemia (AML M5) during therapy is a rare clinical occurrence, and this is the first time it has been reported.Clinical complainA 19-year-old male patient was admitted for severe fatigue with anemic manifestation and weight loss, for more than 1 month, with exacerbation of the condition in the last 2 days.DiagnosisA primary diagnosis was made for AML M2 with t (16;16) (p13; q22) established on bone marrow (BM) morphology. A consequential detection of FLT-3 ITD mutation was done. At day 28 follow-up after induction and maintenance therapy, the diagnosis of AML M2 was maintained with a high bone marrow (BM) blast count, prompting the initiation of a more aggressive treatment protocol. After 1 month of implementing the recent protocol, the patient remains morphologically resistant with a notable transformation of bone marrow infiltration by an abnormal monocytic population (monoblasts and promonocytes). The final diagnosis of transforming FLT3-mutated AML with t (16;16) (p13; q22) was established.InterventionAfter the initial diagnosis of AML M2 with t (16;16) (p13; q22), the patient received the 3 + 7 induction protocol. The 2nd induction protocol initiated after the second evaluation and morphological resistance was the FLAG Adrian protocol. The 3rd protocol after transformation to AML M5 was 1 cycle of the MEC protocol. Anti-FLT3 treatment was considered.OutcomesThe patient was maintained on the 3rd protocol of chemotherapy. Unfortunately, he was admitted to the ICU unit complaining of neutropenic fever and severe sepsis where he died before final re-evaluation and the anti-FLT3 treatment initiation.ConclusionAML with t (16;16) (p13; q22) characterized by favorable outcome. However, identifying additional chromosome abnormality or genetic aberration, especially FLT3 gene mutation, is recognized as an important factor influencing final disease outcome. Therefore, early detection of FLT3 mutations will allow comprehensive disease course prediction and targeted therapy that might achieve longer and more durable remissions.

A case report of congenital leukemia – AML M0

Congenital leukemia (CL) refers to leukemia diagnosed at birth or within the 1st month of life. Incidence is reported to be 1 in 5 million. We report the case of a 22-day-old neonate, who presented to us with features of sepsis, with predominant blasts in the peripheral smear, and was subsequently detected to have CL. Although the etiology is unknown, the presence of leukemia at birth suggests possible intrauterine exposure to drugs or other toxins or the presence of genetic abnormalities. The course of CL is one of the rapid deterioration and death from hemorrhage and infection unless treated appropriately, with adequate chemo and radiotherapy. These neonates pose a huge challenge to treat due to their high mortality, owing to both the high risk of treatment-related mortality and disease relapse. Early diagnosis and prompt initiation of treatment are essential for a better prognosis.

TIM-3/Gal-9 interaction affects glucose and lipid metabolism in acute myeloid leukemia cell lines.

T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in acute myeloid leukemia and master cell survival and proliferation. In this study, we aimed to explore the effect of TIM-3 interaction with its ligand galectin-9 (Gal-9) on glucose and lipid metabolism in AML cell lines. HL-60 and THP-1 cell lines, representing M3 and M5 AML subtypes, respectively, were cultured under appropriate conditions. The expression of TIM-3 on the cell surface was ascertained by flow cytometric assay. We used real-time PCR to examine the mRNA expression of GLUT-1, HK-2, PFKFB-3, G6PD, ACC-1, ATGL, and CPT-1A; colorimetric assays to measure the concentration of glucose, lactate, GSH, and the enzymatic activity of G6PD; MTT assay to determine cellular proliferation; and gas chromatography-mass spectrometry (GC-MS) to designate FFAs. We observed the significant upregulated expression of GLUT-1, HK-2, PFKFB-3, ACC-1, CPT-1A, and G6PD and the enzymatic activity of G6PD in a time-dependent manner in the presence of Gal-9 compared to the PMA and control groups in both HL-60 and THP-1 cell lines (p > 0.05). Moreover, the elevation of extracellular free fatty acids, glucose consumption, lactate release, the concentration of cellular glutathione (GSH) and cell proliferation were significantly higher in the presence of Gal-9 compared to the PMA and control groups in both cell lines (p < 0.05). TIM-3/Gal-9 ligation on AML cell lines results in aerobic glycolysis and altered lipid metabolism and also protects cells from oxidative stress, all in favor of leukemic cell survival and proliferation.

Combination of Liposomal Mitoxantrone, Venetoclax, Homoharringtonine, and Olverembatinib (HQP1351) (MVHO) in Pediatric Patients with Refractory or Relapsed Acute Myeloid Leukemia (AML): Case Series

Introduction: AML accounts for up to 25% of all pediatric acute leukemias. Although overall survival (OS) in pediatric AML has increased to 70% in recent years because of enhanced risk stratified intensive chemotherapy and hematopoietic stem cell transplantation. Unfortunately, more than 30% of patients experience relapse. Further increase intensity of chemotherapy was proven unfeasible for the toxicity and treatment related mortality. Hence, specific targeted therapy has been being explored to treat pediatric AML in recent decade. Because of the heterogeneity of AML and multiple pathways involved in leukemogenesis, single agent targeting one pathway was effective in limited cases. In this study, we designed a regimen to block multiple pathways with combination of Liposomal Mitoxantrone, Venetoclax, Homoharringtonine, and Olverembatinib (MVHO) to investigate its efficacy and safety in treatment of refractory or relapsed AML. Methods: We enrolled patients with relapsed or newly diagnosed AML with poor prognosis with adverse molecular abnormalities, such as NUP98 rearrangements, FUS::ERG, CBFA2T3::GLIS2, and del7/7q, who did not achieve complete remission (CR) after first-line chemotherapy. Patients were administered the MVHO regimen, which included 1 dose of liposome mitoxantrone 8mg/m 2; venetoclax, 300 to 350mg/m 2 once daily, from day1 to day 7, (dose escalation for up to 3days from 50mg/m 2 if high tumor burden occur); homoharringtonine 2mg/m 2 once daily, from day1 to day 7; and Olverembatinib (HQP1351) 20-30mg/m 2 every other day, for 4 doses from day 1 to day 7. Prophylactic oral levofloxacin and posaconazole were administered from day 8 through whole myelosuppression period of every cycles. One cycle (28-35 days, depending on hematopoietic recovery) response, total response, cumulative incidence of relapse (CIR), event-free survival (EFS), hematologic toxicity, and infection during this study were evaluated. Results: A total of 18 patients were enrolled (9 boys and 9 girls), with a median (range) age of 7.3 (4 months-13 years) years, and underwent a total of 27 cycles of MVHO therapy. Half of the patients underwent 1 cycle and the other half, 2 cycles. Per French-American-British (FAB) classification criteria: patients had myelodysplastic syndrome (n = 2); mixed-phenotype acute leukemia (n = 2); and AML subtypes M7 (n = 4), M2 (n = 4), M5 (n = 4), M4 (n = 1), and M0 (n = 1). The median (range) follow-up time was 131 (26-256) days. After cycle one, the objective response rate (ORR, CR plus CR with incomplete hematological recovery [Cri] and partial response [PR]) was 94.4% (17/18), and the remission rate (CR + Cri) was 72.2% (13/18). The total ORR and remission rate before transplantation were 83.3% (15/18) and 66.7% (12/18), respectively. 8 patients among them reached minimum residual disease (MRD) negative remission. For 6 patients with relapsed AML, the ORR and one cycle remission rate was 100% and 66.7%, respectively. A total of 3 patients discontinued because of no response or disease progression, and 12 patients underwent hematopoietic stem cell transplantation (HSCT), after which 1 patient experienced relapse. Remaining 2 patients are being underwent further MVHO cycles. The CIR was 6.7% (1/15). The 8-month mean (± SD) EFS was 60.1% (±19%) and the OS, 100%. Among 26 cycles of MVHO treatment, there were no associated fatal infections or bleeding events. There were 21 episodes of breakthrough infection occurred in 26 cycles of MVHO. There was 1 case of septic shock, and the incidence of ≥grade 3 infection was 80.7%, which included pneumonia, bloodstream infection. Common grade 4 treatment-related adverse events were neutropenia, which was experienced by 100% of patients, and grade 4 thrombocytopenia, which was experienced by 46.1%. Most notably, platelets were stably normal in nearly 20% (5/26) MVHO cycles. Conclusions: MVHO therapy was effective and reasonably well tolerated in pediatric patients with refractory or relapsed AML, suggesting that it may comprise a suitable first-line treatment option for pediatric AML patients.

A rare complication of leukostasis with AML M4 patient: Microcirculatory dysfunction of upper extremities

BackgroundIn children with acute myeloid leukemia, the incidence of hyperleukocytosis is 5–33%. Patients with AML and hyperleukocytosis have a higher early mortality rate than patients with nonhyperleukocytic AML because of the increased risk of severe pulmonary and neurologic complications. Leukapheresis provides rapid cytoreduction and reduces early mortality rates. Case presentationIn this report, we present a case with microcirculatory failure of upper extremities as a rare symptom of hyperleukocytic AML M4 at initial presentation. ConclusionsEarly diagnosis and treatment of patients with AML admitted to emergency services with these symptoms is too important to prevent from loss of extremities. Most of the complications of hyperleukocytosis can be reversible with early treatment.

Evaluation of Analysis Strategy for Cytoplasmic Lineage-Associated Markers in Mixed-Phenotype Acute Leukemia (MPAL) As Devised By Who Classification 2022

Background: Conventional rating of flow cytometrically determined expression of cytoplasmic lineage-associated markers MPO, CD3 and CD79a has defined positivity by ≥10% leukemic cells being positive compared to internal negative controls. WHO classification 2022 has devised a new approach defining positivity by part of leukemic population exceeding 50% of expression level of normal counterpart populations used as positive controls. Aim: Evaluate interpretation of cytoplasmic expression of MPO, CD3 and CD79a in MPAL and other acute leukemias. Methods: 277 acute leukemia cases were analyzed (MPAL-BM, n=18; MPAL-TM, 20; AML M0, 42; AML M1, 40; AML M2, 39; APL, 39; BCP-ALL, 40; T-ALL, 39). Following WHO 2022 positivity for cytoplasmic MPO, CD3 and CD79a was determined setting a cutoff at 50% of mean fluorescence intensity (MFI) of granulocytes, mature T-cells and mature B-cells, respectively. An alternative refined approach was evaluated calculating the MFI ratio between leukemic populations and positive controls. Results of MFI ratios are given as log differences, i.e. 0 representing identical MFIs, -1 meaning 1 log lower expression in leukemic population compared to controls. Log difference of -1.5 was set as cutoff. Results: Applying WHO 2022 majority of cases were rated positive for expected markers, i.e. AML, MPAL-BM and MPAL-TM for MPO, T-ALL and MPAL-TM for CD3 and BCP-ALL and MPAL-BM for CD79a (figure, A). However, there were cases rated positive in other groups although in general only few leukemic cells were causing positive rating, indicating limited specificity of this approach. In detail, MPO was positive in 69% of M0, 95% of M1, 92% of M2, 92% of APL, 61% of MPAL-BM and 70% of MPAL-TM, but also in other groups (BCP-ALL, 35%; T-ALL, 51%).Applying WHO 2022, diagnosis would have changed to BCP-ALL in 39% of MPAL-BM and to T-ALL in 30% of MPAL-TM cases. CD3 was positive in 92% T-ALL and 95% MPAL-TM cases, but also in other groups (M0, 29%; M1, 25%; M2, 15%, APL, 49%; BCP-ALL, 13%; MPAL-BM, 56%). Applying WHO 2022, diagnosis would have changed to AML in 5% of MPAL-TM cases. CD79a was positive in 100% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 55%; M1, 43%; M2, 51%, APL, 49%; T-ALL, 33%; MPAL-TM, 75%). Applying WHO 2022, diagnosis would have changed to AML in 6% of MPAL-BM cases. In order to refine the WHO 2022 approach and to improve specificity MFI ratio between leukemic cells and positive controls was applied. Similarly to the WHO 2022 approach the MFI ratio approach resulted in expected differences but also significant overlaps (figure, B), although to a lower degree for MPO. In detail, MPO was positive in 31% of M0, 75% of M1, 77% of M2, 85% of APL, 72% of MPAL-BM and 50% of MPAL-TM, but also in other groups (BCP-ALL, 13%; T-ALL, 23%). Applying the MFI ratio approach, diagnosis would have changed to BCP-ALL in 28% of MPAL-BM and to T-ALL in 50% of MPAL-TM cases. CD3 was positive in 92% of T-ALL and 100% of MPAL-TM cases, but also in other groups (M0, 14%; M1, 23%; M2, 18%, APL, 85%; BCP-ALL, 13%; MPAL-BM, 72%). Applying the MFI ratio approach there would have been no changes in MPAL-TM diagnosis. CD79a was positive in 98% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 50%; M1, 65%; M2, 46%, APL, 87%; T-ALL, 56%; MPAL-TM, 85%). Applying the MFI ratio approach, diagnosis would have changed to AML in 6% of MPAL-BM. In 6 and 7 cases analyzed by WHO 2022 and MFI ratio, respectively, changes in diagnosis from MPAL to ALL were not supported by NGS yielding mutations known to be associated with myeloid neoplasms while changes were supported by NGS in a minority of cases. Discussion: The WHO 2022 definition, as compared to conventional analysis, changed diagnosis in a significant number of MPAL cases, in particular due to rating MPO negative. At the same time, application of WHO 2022 approach to cases with AML, BCP-ALL and T-ALL suggests limited specificity with significant rates of positivity outside the lineage determined by conventional approach. The MFI ratio approach also changed diagnosis in a significant number of MPAL cases in comparison to the conventional approach, however, data suggests a better specificity for MPO. Further, MFI ratio considers the whole leukemic population and thus may result in higher reproducibility. Overall this data argues in favor of considering multiple lineage-associated markers instead of single markers. In addition, classification based on genetic markers should be maximized also in MPAL.

Frequency of Fab Subtype and Linicohaematological Manifestation in Elderly Acute Myeloid Leukemia Patients in Tertiary Care Hospitals Peshawar

Aim And Objectives: AML is the most common acute leukemia affecting adults, its incidence increases with increasing age; the study aims to find out the clinical and haematological parameters of adult AML patients and the frequency of AML FAB subtype among these patients. Methods: This descriptive, cross sectional study was conducted in IRNUM; KTH and HMC tertiary care hospitals of Peshawar, 30 diagnosed AML patients of age ≥ 50 years were included in the study. The demographic details were recorded in a questionnaire, previous clinical record was obtained and the hematological findings at the diagnosis were recorded. Data was recorded in Microsoft Excel® (version 2013) data sheet, for further analysis the data was imported into Statistical Package for Social Sciences® (SPSS) version 22. Results: Overall 30 diagnosed denovo AML patients of age ≥50 years of age, included 19 male (63.3%) and 11 females (36.7%) patients were included in the study. The mean age of our study patients was 57.83 ± 5.26 years whereas the median was 59.5 years. The most common AML-FAB subtype found among study patients was AML-M2 found in 12 (40%) of patients followed by AML M4 9(30%), AML M1 6(20%) and AML M5 3(10%) in respective order. The mean hemoglobin level among the study patients was 72.10 g/L. Anaemia severity was significantly higher in AML-M2 subtype (z-score 2.0). The AML-subtypes have statistically similar pattern of WBC count (Fischer Exact P-value 0.681). Among the presenting clinical features in the study group fever, anaemia, bone pain and weakness is the most frequent presenting features in all the AML FAB subtypes. However gingivitis and hepatomegaly is the most common presenting feature in AML-M5 in the study group. Conclusion: AML M2 was the most frequent AML FAB subtype found in study patients with the male predominance M: F is (1.7:1). Anaemia, fever, weakness and bone pain was the most frequent presenting feature among the study group. Weight loss is most common ......

Clinicopathological study of acute myeloid leukemia in a tertiary care hospital

Background- The diagnosis of acute myeloid leukemia is based on peripheral blood smear and bone marrow examination. Immunophenotyping characteristics and cytogenetics have clinical relevance besides morphological features in these cases. Present study aims at clinicohematological evaluation of cases of acute myeloid leukemia diagnosed at hematology unit of our tertiary care hospital. Objectives –Present study aimed to diagnose and classify cases of acute myeloid leukemia with clinicohematological correlation. Material and methods- newly diagnosed cases of acute myeloid leukemia within a period of 1 year from May 2020 to May 2021 were included in this cross sectional and prospective study. In addition to hematological work up, flow cytometry, cytogenetics and molecular studies were taken into consideration for clinicohematological evaluation.Result- The study included 14 cases of acute myeloid leukemia which were classified as per FAB classification as AML M1- 7 cases, M3 – 3 cases and M4 and M5 – 2 cases each.

Aberrant Phenotype in Acute Myeloid Leukemia: Hematology Laboratory Experiment of the University Hospital of Casablanca, Morocco.

Aberrant phenotypes in acute myeloid leukemia have variable frequencies and their prognostic value with adverse hematological and other biological prognostic factors is still controversial, despite several reports of clinical significance. To date, no study has been reported evaluating the incidence of these phenotypic aberrations in the Moroccan population. The aim is to evaluate the incidence of aberrant phenotype expressions in acute myeloid leukemia and correlate their presence with the different AML subtypes, and clinical and biological characteristics in Moroccan patients. Fifty-four AML patients were diagnosed according to WHO classification 2016 criteria. Immunopheno-typing by flow cytometric analysis was performed to evaluate aberrant phenotypes on myeloblasts. The occurrence of lymphoid antigens in AML was higher (51.8%), which was closer to that reported in the literature. CD7 has been revealed to be the most commonly expressed lymphoid antigen. Besides, CD19 was expressed in all 3.7% of M2 AML subtype. However, we could find no statistically significant differences between these aberrant phenotypes regarding FAB subtypes or clinical and biological outcomes. The great majority of AML cases showed asynchronous expression (57%) with significant differences regarding FAB subtypes. Aberrant phenotypes might be associated with different leukemia subtypes that should be studied for a better understanding of their biological significance and adding important information for prognosis and, at the same time, could be of help when looking for minimal residual disease during morphologic remission.

Prognostic Impact of Poliovirus Receptor Expression (PVR) (CD155) in Context to FLT3-ITD and NPM1 Mutation Status at Egyptian Acute Myeloid Leukemia

Background: Acute myeloid leukemia is a heterogeneous hematological malignancy with diversity in molecular, phenotypic and genetic alteration. Accurate assessment of prognosis is mandatory to reduce relapse, changing treatment related mortality, decision making for appropriate therapy protocols. Aim: A case control observational analytical study to assess the prognostic value of poliovirus (CD155) in acute myeloid leukemia and its significant in-patient stratification. Subject & Methods: 100 newly diagnosed de novo AML selected from oncology center, Mansoura University. Measurement of PVR (CD155) by multiparameter flow cytometry in addition to cytogenetic and molecular stratification. Results: Statistical stratification according to median cut off CD155 > 5.35. Higher expression was associated with an increased risk of death and relapse, LDH. While lower expression is associated with longer OS & DFS. Cox regression with univariate analysis revealed that LDH, molecular stratification, CD155 were significant risk factors for shorter OS, while positive expression of CD16, CD56 AML M3 were significantly associated with longer survival. Multivariate analysis revealed that CD155 was a significant risk factor for shorter OS. Conclusion: Variability in expression of CD155 in AML patients due to dual costimulatory and inhibitory function, high expression associated with tumor bulk and progression, short OS, increased relapse rate. Cox regression analysis shows its independent risk factor. It could be implemented in routine laboratory for risk stratification into low, high risk that needs more therapeutic modalities with new immunotherapy. In future could be targeted therapy such as PDL, CTLA-4 and open more therapeutic hope for those patients.

Blockade of Tigit on AML-Derived M2 Macrophages Results in Reprograming into the M1 Phenotype and Enhances CD47-Mediated Phagocytosis

Blockade of Tigit on AML-Derived M2 Macrophages Results in Reprograming into the M1 Phenotype and Enhances CD47-Mediated Phagocytosis

IDH1R132, IDH2R140 and IDH2R172 in AML: Different Genetic Landscapes Correlate with Outcome and May Influence Targeted Treatment Strategies

Introduction: In AML, IDH mutations can be found at a frequency of 7% for IDH1 and 10% for IDH2 . Precision medicine aims at specifically targeting different gene mutations. Novel drugs which inhibit mutated IDH activity, such as AG-120 (for IDH1), AG-221 (for IDH2) or AG-881 (for IDH1 and 2), are currently tested in clinical trials to assess whether they confer better prognosis and overall survival to patients that carry these mutations.Aim: To comprehensively investigate the genetics and prognosis of de novo AML with IDH1 R132, IDH2 R140, and IDH2 R172 mutations.Patients and Methods: In a cohort of 306 de novo AML patients (pts) - all IDH1 or IDH2 mutated - cytomorphology and cytogenetics were available. The cohort comprised 141 females and 165 males, the median age was 65 yrs (range: 21-90 yrs). In all pts a 25-gene panel was investigated by next generation sequencing: ASXL1, BCOR, CALR, CBL, CEBPA, CSNK1A1, DNMT3A, ETV6, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, and ZRSR2 . FLT3 -ITD and KMT2A -PTD were analyzed by gene scan. All pts were treated with intensive standard chemotherapy. Overall survival (OS) analyses were performed in comparison to a FAB and MRC (Grimwade et al., Blood 2010;116(3):354-365) matched IDH wild type (wt) reference cohort (n=540).Results: In total,106 pts (35%) were mutated in IDH1 R132, 166 (54%) in IDH2 R140 and 34 (11%) in IDH2 R172. Regarding amino acidic substitutions, IDH1 R132 was dominated by the R132H substitution in 44 (41%) and the R132C in 39 cases (37%); IDH2 R140 was largely skewed towards the R140Q substitution which occurred in 92% of cases; IDH2 R172 showed R172K in 32 cases (94%) and R172W in 2 cases (6%).The majority of pts had mutations in two (n=148, 44%) or three (n=78, 23%) of the other 24 analyzed genes (range: 0-6), while only six pts (2%) showed no additional mutations. Among FAB classification, 144 pts (47%) were classified as M1 and 94 (31%) as M2; this rather immature phenotype was most prominent in IDH1 mutated pts with an even higher percentage of M1 (64%) and less of M2 (22%). AML M3 was not present in this cohort at all, indicating a specific occurrence of IDH mutations in distinct AML types. Cytogenetic analyses revealed that 227 pts (74%) had a normal karyotype, 79 (26%) showed an aberrant karyotype with only 6 pts (2%) having a complex karyotype. This is also reflected by a high number of pts in the intermediate MRC group (n=285), while only 6 and 15 pts were grouped to the favorable MRC or adverse MRC group, respectively.Most frequently IDH mutated pts were also mutated in NPM1 (49%), followed by DNMT3A (40%), SRSF2 (29%), FLT3 -ITD (21%), ASXL1 (16%), NRAS (15%), RUNX1 (15%), and KMT2A -PTD(13%). All other analyzed genes were mutated in <10% of cases. Of note, no IDH2 R172 mutated patient carried a NPM1 mutation, but 63% and 50% of pts mutated in IDH1 R132 and IDH2 R140 (p<0.001). This was also true for NRAS with 18% and 17% of pts mutated in IDH1 R132 and IDH2 R140 (p=0.001). FLT3 was mutated rarely in patients with IDH2 R172 (5%) compared to those with mutations in IDH1 R132 (28%) or IDH2 R140 (32%; p=0.001). In contrast BCOR was often mutated in IDH2 R172 mutated pts (32%) compared to 5% and 7% of pts mutated in IDH1 R132 and IDH2 R140 (p<0.001). SRSF2 was frequently mutated in IDH2 R140 mutated pts (43%; p<0.001) compared to pts mutated in IDH1 R132 (13%) and IDH2 R172 (8%). ASXL1 mutations were rare in IDH1 R132 mutated pts (8%; p=0.007) compared to 22% and 16% in IDH2 R140 and R172 mutated pts.Overall survival analysis did not show significant differences between IDH1 and IDH2 mutated and IDH wt pts. However, when separating IDH1 R132, IDH2 R140, and IDH2 R172 mutated pts, the latter group showed a better prognosis (median OS not reached) compared to all others (median OS: 28 months; p=0.040; Fig. 1). Other worse prognostic factors in IDH mutated pts were higher white blood cell count (WBC) and age, or ASXL1, DNMT3A, SRSF2, and TP53 mutations. NPM1 mutations showed a favorable impact. In multivariate analyses, only IDH R172 mutation, WBC, and age remained independent prognostic factors.Conclusions: AML pts with IDH1 R132, IDH2 R140, and IDH2 R172 mutations differ in their morphological and genetic patterns. This translates into a more favorable outcome for IDH2 R172 mutated pts. Therefore, IDH mutated AML need further molecular subclassification to define prognosis and select optimal targeted treatment strategies accordingly. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

CSF3R Mutation in Acute Leukemia and MDS Patients

Introduction: CSF3R somatic mutations has been included in the 2016 revision of the WHO classification of myeloid neoplasms and acute leukemia as a specific molecular diagnostic marker of CNL. Besides CNL, CSF3R somatic mutation have been reported in about 30% of Severe Congenital Neutropenia (SCN) and in a low frequency of other myeloid malignancies (about 4% of CMML, 1.7% of adult AML, 1.9% of children AML), but not in MDS, JMML and ET (Lavallée Et al., Blood 2016; Sano H et al., Br J Haematol 2015). Lavallée et al. reported for the first time that CSF3R mutation are associated with AML with biallelic mutated CEBPA (4/14, 29%) (Lavallée et al., Blood 2016).We aimed to assess the frequency of CSF3R mutation in MDS, AML and ALL patients and the association with other genetic abnormalities.Methods: A retrospective analysis of 974 patients with hematological malignancies, including 297 cases of de novo AML, 144 cases of relapsed AML, 268 cases of de novo ALL, 191 cases of relapsed ALL and 74 cases of MDS patients. 58 genes which common mutated in hematological malignancies were tested by amplicon-based NGS with IonTorrent platform (PGM, Life Technology) and the average sequencing depth was 1000x. 36 leukemia related fusion genes were screened by RT-PCR.Results: We identified CSF3R mutation in 19 patients, which was found in 3.0% (9/297) of de novo AML, 5.6% (8/144) of relapsed AML, 0.5% (1/191) of relapsed ALL and 1.4% (1/74) of MDS. No CSF3R mutation was detected in de novo ALL. All 19 cases had no history of MPN, MDS/MPN or SCN. A total of 8 mutations of CSF3R were detected in 19 patients, 11 cases with T618I mutation, 3 cases with T615A mutation, 1 case with Y779H, Q781*, P809Hfs*8, S810Qfs*6, S813Lfs*2 and D837* mutation respectively, among which 1 patient accompanied with CSF3R T618I and Y779H mutation.In AML cohort, a total of 17 cases with mutant CSF3R, the median age was 20 years (rang, 6-49) and the frequency of CSF3R mutation have no difference between children and adult. Cytomorphology revealed AML M2 (n=11), AML M4 (n=3), AML M5 (n=1), and 1 case have no data available. The median of CSF3R variant allele frequency (VAF) was 25% (3%-49%), 3 cases with VAF <10%, 10 cases with VAF comparable to the concomitant gene mutation with highest VAF. CSF3R mutation was more common as the predominant clone in relapsed AML (87.5% VS 33.3%, P=0.05). The median of concomitant gene mutations was 2 (range 0-3), double-mutated CEBPA (CEBPAdm) was the most frequently concomitant partner of CSF3R mutation (7/17, 41.2% VS 51/382, 13.4%, P <0.05). We also identified 9 cases with AML1-ETO fusion gene which were mutually exclusive with CEBPAdm. In totally, 16 cases of 17 (94.1%) CSF3R mutated AML patient concomitant with core-binding factor gene abnormalities (Fig.1).CSF3R mutation have not been reported in ALL, we identified CSF3R mutation in one patient with B-ALL who was 10 years old and relapsed at 43 months after diagnosed. Bone marrow morphology and immunophenotyping at relapse shows no abnormal neutrophils and myeloid lineage expression were found. No fusion genes were detected. CSF3R T618I and ASXL1 Q803* mutations were detected with VAF of 50% and 51%, respectively, which were confirmed of somatic mutation rather than germline origin. As lack of genetic data at diagnosis, we could not analyze the order of mutation acquisition and clone evolution comprehensively. At the time of reporting, the patients have received allo-HSCT for 4 months and acquired sustained remission. We also detected 1 case of MDS patients (MDS-U) carried U2AF1 S34F, CSF3R Q781* and CBL G413S mutations with VAF of 45%, 32% and 31%, respectively.Due to the limited number of CSF3R mutated cases, we did not perform survival analysis in this study.Conclusion: (1) CSF3R mutation was found in 3.9% of total AML and 3% of de novo AML cohort, rarely seen in MDS and ALL. (2) In AML, CSF3R mutation are prevalent in M2 and M4, and all concomitant with core-binding factor gene abnormalities (CEBPAdm and AML1-ETO fusion gene) and NPM1 mutation which were associated with favorable-risk prognosis. (3) The CSF3R mutation could appeared as predominant clone or subclone in AML and seemly more common as the predominant clone in relapsed AML. As CSF3R mutation also have implications for therapeutic that JAK inhibitors may be efficacious in treating CSF3R-mutant CNL. The implications of prognostic and target therapy (eg. JAK inhibitors) of CSF3R mutation in AML warranting further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequency and Clinical Significance of P-Glycoprotein and Multidrug Resistance-Associated Protein-1 Expression in Leukemic Cells from Acute Myeloid Leukemia

BACKGROUND: Despite advances in the cure rate for acute myeloid leukemia, a considerable number of patients die from the disease due to multidrug resistance (MDR). Overexpression of the transporter proteins P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) provide resistance to the treatment of these leukemias. OBJECTIVE: To analyze Gpp and MRP1 expression in patients with AML using flow cytometry (FC) and determine the correlation between expression and demographics and clinical and laboratory variables. METHODS: Bone marrow and peripheral blood samples from 346 patients diagnosed with AML were assessed for Pgp and MRP1 expression by FC. RESULTS: Pgp and MRP1 expression was found in 111 (32.1%) and 133 (38.4%) patients, respectively, with greater prevalence in older patients and lower in adolescents, in addition to a high incidence in patients with refractory, recurrent and secondary disease in comparison with cases of de novo AML. With respect to laboratory findings, we observed a statistically significant higher correlation between Pgp and MRP1 expression in AML CD34+ and FAB AML M7, M5A and M2 and lower in the M3 subtype, with no statistically significant correlation between the phenotype MDR and other laboratory data such as hemoglobin, leukocyte count, platelet count, and aberrant lymphoid antigen expression (CD2, CD7 and CD19) and clinical signs related to the disease. CONCLUSIONS: The results showed that detecting MDR phenotype by flow cytometry may be a molecular marker for the independent prognosis of patients diagnosed with AML. DisclosuresNo relevant conflicts of interest to declare.

A Case Report, An Adult with Acute Megakaryocytic Leukemia, Immunophenotyping Profile

Background: Acute Megakaryocytic Leukemia (AML-M7) is a subtype with 1% incidence of all adult AML cases. Variation morphology and limitations of immunophenotyping often make AML-M7 difficult to diagnose. The diagnosis of AML-M7 requires morphology and immunophenotyping. This case will discuss about immunophenotyping profile in establishing AML-M7 in an adult patient. Case Description: A 32-year-old-male, presented with fever, dizziness, loss of appetite and weakness on extremities, signs of bleeding were not found. Physical examination showed conjunctival anemia and decrease of power extremities. Laboratory results showed Hb 5.3 g/dL, WBC 79,700/?L, PLT 9,000/?L, IPF 37.3%. Blood smear evaluation and bone marrow aspiration resulted in dry tap but obtained formation resembling abnormal micro megakaryocytes and lymphoid cells. Immunophenotyping results were positive on megakaryocytes specific antigen CD 61 and CD36, as well as myeloid markers CD33, CD13, and CD117. Negative results of lymphoid markers were CD3 and CD79a. Patient did not receive chemotherapy and refused treatment. Two days after the patient passed away. Conclusion : AML-M7 cases in adult patients are very rare. Morphologies are very varied. In this case, morphology of blasts was difficult to distinguish with lymphoid cells because cytoplasm bleps were found. Immunophenotyping results showed positive CD61 and CD36 and negative lymphoid markers (CD3, CD79a) according to AML M7. Myeloid markers were positive in this case. Thus, it is concluded that this case was AML-M7 .

Neutrophil recovery with or without G-CSF in non M3 AML patient with DA 3+7 protocol

Background: Induction of acute myeloid leukaemia is associated with a high incidence of treatment related mortality mostly due to neutropenia related infections. This study primarily analyses the duration of neutrophil recovery with or without G-CSF during induction with DA 3+7 in non M3 AML patients. This study also evaluates role of G-CSF in duration febrile neutropenia, hospital stay, total blood products transfusion, total number of used injectable antibiotics and remission status. Methods: It was a Quasi Experimental - Non-Randomized Controlled Trail. There were two groups a) Case = Patients receiving G-CSF b) Control= Patients not receiving G-CSF. Sampling was purposive sampling. Results: 50 patients in two group, G-CSF group (n=25) and Control group (No G-CSF) (n=25) participated in this study. Among them 54% was female and 46% was male. Median age of the participants was 28 years. Neutrophil recovery duration in G-CSF group Vs Control group showed 22.64 Vs 24.64 days (p value 0.003). Duration of febrile neutropenia in G-CSF group Vs Control group showed 11.24 Vs 13.56 days (p value 0.038). Duration of hospital stay in G-CSF group Vs Control group showed 23.64 Vs 25.76 days (p value 0.002). Total number of blood and blood product transfusion in G-CSF group Vs Control group showed 3.80 Vs 3.88 (p value 0.597). Total number of injectable antibiotics in G-CSF group Vs Control group showed 2.72 Vs 3.12 days (p value 0.169). In G-CSF group 22 (88%) and in Control group 23 (92%) were in complete remission (p value 0.637). Conclusions: G-CSF significantly reduces the duration of neutropenia (p value 0.003), febrile neutropenia (p value 0.038) and hospital stay (p value 0.002) after induction with DA 3+7. This may cause reduction in treatment cost and sepsis related mortality. G-CSF can’t significantly reduce use of blood products (p value 0.597) and injectable antibiotics (p value 0.169) after induction with DA 3+7. G-CSF does not affect morphological remission status (p value 0.637) after induction with DA 3+7.

Monocytic leukeima masquerading as a cutaneous lesion

Cutaneous lesions preceding a leukaemia are extremely rare and are referred to as ‘aleukaemic leukaemia cutis’. We present a case of acute monocytic leukaemia where skin infiltration of leukemic cells preceded any blood or bone marrow evidence of leukaemia. A 67-year-old woman presented with multiple cutaneous nodules all over the body of 3 months duration. Cutaneous examination showed multiple erythematous papules and plaques which were present over the face, trunk, extremities and back. Patient was evaluated at a nearby facility and was found to have normal blood parameters (TLC- 7130/cmm, N-57% L-34% M-09%). A skin biopsy done revealed infiltration of the dermis by atypical cells suggestive of a hematolymphoid malignancy. The patient was then shifted to our institution for tertiary care management. The blood counts over a period of one month showed gradually increasing TLC from an initial normal blood count to one showing absolute monocytosis (TLC-25500/cmm, AMC-3825/cmm) and finally abnormally high TLC (TLC-154050/cmm) with 79% monoblasts and promonocytes suggestive of acute leukaemia. A repeat skin biopsy again showed infiltration of atypical cells in the dermis. IHC done showed the atypical cells to be positive for monocytic markers (CD14, CD64). The patient had now started exhibiting systemic findings corroborating with the cutaneous lesions and skin biopsy. Bone marrow aspirate was hypercellular and showed replacement by monoblasts (82%). Cellular morphology was suggestive of AML-M5. Bone marrow biopsy showed a diffuse replacement of marrow by immature cells/blasts. Flow cytometry reports were also positive for monocytic markers. The patient was hence diagnosed as AML M5 with cutaneous metastasis/leukemia cutis and was immediately started on chemotherapy (3+7). Post induction phase, the patient was in remission and her skin lesions subsided. She was subsequently discharged and advised regular OPD follow up for maintenance therapy. This case is reported for its rarity.

Detection of Acute Myeloid Leukemia based on White Blood Cell Morphological Imaging using Naïve Bayesian Algorithm

The process of diagnosing AML is based on the complete blood-count analysis of the patients. As such, it involves high energy consumption, long completion times, and is rather expensive compared to conventional medical practices. One of the methods for identifying tumor cells involves the utilization of image-processing techniques based on the morphology of white blood cells (WBCs). The principal objective of this study involves the identification of AML cells—especially of the AML M1 and AML M2 types—through morphological imaging of WBCs using the Naïve Bayes' Classifier. The Image-processing methods used in this study include YCbCr color space classification, image thresholding, morphological operations, chain code representation, and the use of bounding boxes. Regardless of the processing technique used, all identification procedures, performed in this study, were based on the Naïve Bayes' Classifier. The test process was performed on 30 images of each of the AML M1 and M2 cell types. The use of the cell identification method proposed in this study demonstrated an accuracy of 73.33%. While the accuracy of cell type identification is 54.92%. Based on the results obtained in this study, it is inferred that the Naïve Bayes' Classifier method can be employed in the process of identifying dominant AML cell types amongst AML M1 and AML M2 (myeloblast, promyelocyte, myelocyte, and metamyelocyte) based on the morphology of WBCs.

CTSB Knockdown Inhibits Proliferation and Tumorigenesis in HL-60 Cells.

Background: Cathepsin B (CTSB) was well documented in solid tumors, up-regulated of CTSB expression is linked with progression of tumors. However, the study of CTSB in adult leukemia has not been reported. Methods: Total RNA was isolated from PBMC (peripheral blood mononuclear cell) of AML patients and healthy donors. qRT-PCR was performed to detect the expression of CTSB. The association of CTSB expression with the patients' overall survival (OS) and disease-free survival (DFS) were analyzed. Stable HL-60 CTSB-shRNA cell lines were established by retrovirus infection and puromycin selection. Cell proliferation was detected by CCK-8 analysis. Tumorigenesis ability was analyzed by soft agar and xenograft nude mice model. Western blot was performed to detect the expression of CTSB and the proteins of cell signaling pathway. Results: The mRNA expression level of CTSB was up-regulated in AML patients compared to healthy control (p<0.001), and CTSB expression was significantly higher in M1, M2, M4 and M5 AML samples than healthy control. The CTSB expression in AML was associated with WBC count (p=0.037). Patients with high CTSB expression had a relatively poor OS (p=0.007) and a shorter DFS (p=0.018). Moreover, the expression level of CTSB may act as an independent prognostic factor for both OS (p=0.011) and DFS (p=0.004). Knockdown CTSB expression in HL-60 cells could inhibit the cells' proliferation and tumorigeneses in vitro and in vivo. Further study showed knockdown CTSB expression in HL-60 cells could inactive the AKT signaling pathway. Conclusions: CTSB mRNA was upregulated in AML patients. CTSB overexpression was correlated with poor prognosis and may serve as an independent prognostic factor for both OS and DFS in AML patients. Knockdown CTSB expression in HL-60 cells could inhibit the cells' proliferation and tumorigenesis. The underlying mechanism may be the inhibition of the AKT signaling pathway.