The versatile nature of miR-9/9* in human cancer

miR-9 and miR-9* (miR-9/9*) were first shown to be expressed in the nervous system and to function as versatile regulators of neurogenesis. The variable expression levels of miR-9/9* in human cancer prompted researchers to investigate whether these small RNAs may also have an important role in the deregulation of physiological and biochemical networks in human disease. In this review, we present a comprehensive overview of the involvement of miR-9/9* in various human malignancies focusing on their opposing roles in supporting or suppressing tumor development and metastasis. Importantly, it is shown that the capacity of miR-9/9* to impact tumor formation is independent from their influence on the metastatic potential of tumor cells. Moreover, data suggest that miR-9/9* may increase malignancy of one cancer cell population at the expense of another. The functional versatility of miR-9/9* emphasizes the complexity of studying miRNA function and the importance to perform functional studies of both miRNA strands in a relevant cellular context. The possible application of miR-9/9* as targets for miRNA-based therapies is discussed, emphasizing the need to obtain a better understanding of the functional properties of these miRNAs and to develop safe delivery methods to target specific cell populations

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia

© The Authors.Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.The co-ordination of this study was supported by the EuroFlow Consortium. The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA)

Classification of pediatric acute myeloid leukemia based on miRNA expression profiles

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease with respect to biology as well as outcome. In this study, we investigated whether known biological subgroups of pediatric AML are reflected by a common microRNA (miRNA) expression pattern. We assayed 665 miRNAs on 165 pediatric AML samples. First, unsupervised clustering was performed to identify patient clusters with common miRNA expression profiles. Our analysis unraveled 14 clusters, seven of which had a known (cyto-)genetic denominator. Finally, a robust classifier was constructed to discriminate six molecular aberration groups: 11q23-rearrangements, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17) (q21;q22), NPM1 and CEBPA mutations. The classifier achieved accuracies of 89%, 95%, 95%, 98%, 91% and 96%, respectively. Although lower sensitivities were obtained for the NPM1 and CEBPA (32% and 66%), relatively high sensitivities (84%-94%) were attained for the rest. Specificity was high in all groups (87%-100%). Due to a robust double-loop cross validation procedure employed, the classifier only employed 47 miRNAs to achieve the aforementioned accuracies. To validate the 47 miRNA signatures, we applied them to a publicly available adult AML dataset. Albeit partial overlap of the array platforms and molecular differences between pediatric and adult AML, the signatures performed reasonably well. This corroborates our claim that the identified miRNA signatures are not dominated by sample size bias in the pediatric AML dataset. In conclusion, cytogenetic subtypes of pediatric AML have distinct miRNA expression patterns. Reproducibility of the miRNA signatures in adult dataset suggests that the respective aberrations have a similar biology both in pediatric and adult AML

Prognostic Value of FLT3-Internal Tandem Duplication Residual Disease in Acute Myeloid Leukemia

PURPOSE The applicability of FLT3-internal tandem duplications (FLT3-ITD) for assessing measurable residual disease (MRD) in acute myeloid leukemia (AML) in complete remission (CR) has been hampered by patient-specific duplications and potential instability of FLT3-ITD during relapse. Here, we comprehensively investigated the impact of next-generation sequencing (NGS)-based FLT3-ITD MRD detection on treatment outcome in a cohort of patients with newly diagnosed AML in relation to established prognostic factors at diagnosis and other MRD measurements, ie, mutant NPM1 and multiparameter flow cytometry. METHODS In 161 patients with de novo FLT3-ITD AML, NGS was performed at diagnosis and in CR after intensive remission induction treatment. FLT3-ITD MRD status was correlated with the cumulative incidence of relapse and overall survival (OS). RESULTS NGS-based FLT3-ITD MRD was present in 47 of 161 (29%) patients with AML. Presence of FLT3-ITD MRD was associated with increased risk of relapse (4-year cumulative incidence of relapse, 75% FLT3-ITD MRD v 33% no FLT3-ITD MRD; P < .001) and inferior OS (4-year OS, 31% FLT3-ITD MRD v 57% no FLT3-ITD MRD; P < .001). In multivariate analysis, detection of FLT3-ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 3.55; P < .001) and OS (hazard ratio 2.51; P = .002). Strikingly, FLT3-ITD MRD exceeds the prognostic value of most generally accepted clinical and molecular prognostic factors, including the FLT3-ITD allelic ratio at diagnosis and MRD assessment by NGS-based mutant NPM1 detection or multiparameter flow cytometry. CONCLUSION NGS-based detection of FLT3-ITD MRD in CR identifies patients with AML with profound risk of relapse and death that outcompetes the significance of most established prognostic factors at diagnosis and during therapy, and furnishes support for FLT3-ITD as a clinically relevant biomarker for dynamic disease risk assessment in AML

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping

Background: While it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies. Methods: CD123 expression was evaluated using standardized EuroFlow immunophenotyping in 139 pediatric AML, 316 adult AML, 193 pediatric BCP-ALL, 69 adult BCP-ALL, 101 pediatric T-A

Molecular characterization of mutant TP53 acute myeloid leukemia and high-risk myelodysplastic syndrome

Substantial heterogeneity within mutant TP53 acute myeloid leukemia (AML) and myelodysplastic syndrome with excess of blast (MDS-EB) precludes the exact assessment of prognostic impact for individual patients. We performed in-depth clinical and molecular analysis of mutant TP53 AML and MDS-EB to dissect the molecular characteristics in detail and determine its impact on survival. We performed next-generation sequencing on 2200 AML/MDS-EB specimens and assessed the TP53 mutant allelic status (mono- or bi-allelic), the number of TP53 mutations, mutant TP53 clone size, concurrent mutations, cytogenetics, and mutant TP53 molecular minimal residual disease and studied the associations of these characteristics with overall survival. TP53 mutations were detected in 230 (10.5%) patients with AML/MDS-EB with a median variant allele frequency of 47%. Bi-allelic mutant TP53 status was observed in 174 (76%) patients. Multiple TP53 mutations were found in 49 (21%) patients. Concurrent mutations were detected in 113 (49%) patients. No significant difference in any of the aforementioned molecular characteristics of mutant TP53 was detected between AML and MDS-EB. Patients with mutant TP53 have a poor outcome (2-year overall survival, 12.8%); however, no survival difference between AML and MDS-EB was observed. Importantly, none of the molecular characteristics were significantly associated with survival in mutant TP53 AML/MDS-EB. In most patients, TP53 mutations remained detectable in complete remission by deep sequencing (73%). Detection of residual mutant TP53 was not associated with survival. Mutant TP53 AML and MDS-EB do not differ with respect to molecular characteristics and survival. Therefore, mutant TP53 AML/MDS-EB should be considered a distinct molecular disease entity

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry

Ibrutinib added to 10-day decitabine for older patients with AML and higher risk MDS

The treatment of older, unfit patients with acute myeloid leukemia (AML) is challenging. Based on preclinical data of Bruton tyrosine kinase expression/phosphorylation and ibrutinib cytotoxicity in AML blasts, we conducted a randomized phase 2 multicenter study to assess the tolerability and efficacy of the addition of ibrutinib to 10-day decitabine in unfit (ie, Hematopoietic Cell Transplantation Comorbidity Index ≥3) AML patients and higher risk myelodysplasia patients (HOVON135/SAKK30/15 trial). In total, 144 eligible patients were randomly (1:1) assigned to either 10-day decitabine combined with ibrutinib (560 mg; sequentially given, starting the day after the last dose of decitabine) (n = 72) or to 10-day decitabine (n = 72). The addition of ibrutinib was well tolerated, and the number of adverse events was comparable for both arms. In the decitabine plus ibrutinib arm, 41% reached complete remission/complete remission with incomplete hematologic recovery (CR/CRi), the median overall survival (OS) was 11 months, and 2-year OS was 27%; these findings compared with 50% CR/CRi, median OS of 11.5 months, and 2-year OS of 21% for the decitabine group (not significant). Extensive molecular profiling at diagnosis revealed that patients with STAG2, IDH2, and ASXL1 mutations had significantly lower CR/CRi rates, whereas patients with mutations in TP53 had significantly higher CR/CRi rates. Furthermore, multicolor flow cytometry revealed that after 3 cycles of treatment, 28 (49%) of 57 patients with available bone marrow samples had no measurable residual disease. In this limited number of cases, measurable residual disease revealed no apparent impact on event-free survival and OS. In conclusion, the addition of ibrutinib does not improve the therapeutic efficacy of decitabine. This trial was registered at the Netherlands Trial Register (NL5751 [NTR6017]) and has EudraCT number 2015-002855-85

Basophil-lineage commitment in acute promyelocytic leukemia predicts for severe bleeding after starting therapy

Severe hemorrhagic events occur in a significant fraction of acute promyelocytic leukemia patients, either at presentation and/or early after starting therapy, leading to treatment failure and early deaths. However, identification of independent predictors for high-risk of severe bleeding at diagnosis, remains a challenge. Here, we investigated the immunophenotype of bone marrow leukemic cells from 109 newly diagnosed acute promyelocytic leukemia patients, particularly focusing on the identification of basophil-related features, and their potential association with severe bleeding episodes and patient overall survival. From all phenotypes investigated on leukemic cells, expression of the CD203c and/or CD22 basophil-associated markers showed the strongest association with the occurrence and severity of bleeding (p ≤ 0.007); moreover, aberrant expression of CD7, coexpression of CD34+/CD7+ and lack of CD71 was also more frequently found among patients with (mild and severe) bleeding at baseline and/or after starting treatment (p ≤ 0.009). Multivariate analysis showed that CD203c expression (hazard ratio: 26.4; p = 0.003) and older age (hazard ratio: 5.4; p = 0.03) were the best independent predictors for cumulative incidence of severe bleeding after starting therapy. In addition, CD203c expression on leukemic cells (hazard ratio: 4.4; p = 0.01), low fibrinogen levels (hazard ratio: 8.8; p = 0.001), older age (hazard ratio: 9.0; p = 0.002), and high leukocyte count (hazard ratio: 5.6; p = 0.02) were the most informative independent predictors for overall survival. In summary, our results show that the presence of basophil-associated phenotypic characteristics on leukemic cells from acute promyelocytic leukemia patients at diagnosis is a powerful independent predictor for severe bleeding and overall survival, which might contribute in the future to (early) risk-adapted therapy decisions.This work was supported by the Fundación Científica de la Asociación Española Contra el Cáncer (AECC, Madrid, Spain) and the Fundación Rafael del Pino (Madrid, Spain) and both CIBERONC (CB16/12/00400, CB16/12/00233, CB16/12/00480) and grant PI16/00787 from Instituto de Salud Carlos III (Ministerio de Economía y Competitividad, Madrid, Spain)

Inferior outcome of addition of the aminopeptidase inhibitor tosedostat to standard intensive treatment for elderly patients with aml and high risk mds

Treatment results of AML in elderly patients are unsatisfactory. We hypothesized that addition of tosedostat, an aminopeptidase inhibitor, to intensive chemotherapy may improve outcome in this population. After establishing a safe dose in a run-in phase of the study in 22 patients, 231 eligible patients with AML above 65 years of age (median 70, range 66–81) were randomly assigned in this open label randomized Phase II study to receive standard chemotherapy (3+7) with or without tosedostat at the selected daily dose of 120 mg (n = 116), days 1–21. In the second cycle, patients received cytarabine 1000 mg/m2 twice daily on days 1-6 with or without tosedostat. CR/CRi rates in the 2 arms were not significantly different (69% (95% C.I. 60–77%) vs 64% (55–73%), respectively). At 24 months, event-free survival (EFS) was 20% for the standard arm versus 12% for the tosedostat arm (Cox-p = 0.01) and overall survival (OS) 33% vs 18% respectively (p = 0.006). Infectious complications accounted for an increased early death rate in the tosedostat arm. Atrial fibrillation w