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1.
Virchows Arch ; 481(4): 621-646, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35819517

ABSTRACT

The first section of the bone marrow workshop of the European Association of Haematopathology (EAHP) 2020 Virtual Meeting was dedicated to pediatric myeloid neoplasms. The section covered the whole spectrum of myeloid neoplasms, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). The workshop cases are hereby presented, preceded by an introduction on these overall rare diseases in this age group. Very rare entities such as primary myelofibrosis, pediatric MDS with fibrosis, and MDS/MPN with JMML-like features and t(4;17)(q12;q21); FIP1L1::RARA fusion, are described in more detail.


Subject(s)
Myelodysplastic Syndromes , Myelodysplastic-Myeloproliferative Diseases , Myeloproliferative Disorders , Neoplasms , Bone Marrow/pathology , Child , Humans , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Neoplasms/pathology
2.
Leukemia ; 35(7): 1894-1906, 2021 07.
Article in English | MEDLINE | ID: mdl-33318611

ABSTRACT

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.


Subject(s)
Neoplasm, Residual/drug therapy , Neoplasm, Residual/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/pathology , Adolescent , Adult , Child , Child, Preschool , Female , Flow Cytometry/methods , Humans , Immunophenotyping/methods , Infant , Male , Middle Aged , Recurrence , Young Adult
3.
Int J Lab Hematol ; 39 Suppl 1: 76-85, 2017 May.
Article in English | MEDLINE | ID: mdl-28447425

ABSTRACT

We have developed a lymphoproliferative disorder screening tube (LPD-ST) with the aim to provide comprehensive immunophenotyping of lymphocyte subsets with minimal need for additional testing. The LPD-ST consists of CD4/kappa FITC, CD8/lambda PE, CD3/CD14ECD, CD38PC5.5, CD20/CD56PC7, CD10APC, CD19APC-A700, CD5APC-A750, CD57/CD23PB and CD45KO. The LPD-ST was validated against previously used lymphocyte subset panels in Canada (n=60) and in Sweden (n=43) and against the OneFlow™ LST (n=60). The LPD-ST panel was then implemented in clinical practice using dried monoclonal antibody reagents (Duraclone® ) on 649 patient samples in Sweden. In 204 of 649 samples (31%), a monotypic B-cell population was found. Of these cases, a final diagnosis could be rendered in 106 cases (52%), and in the remainder, additional B-cell immunophenotyping was performed. In 20 (3%) samples, an aberrant T-cell population was confirmed by additional testing. Of 425 samples diagnosed as normal/reactive lymphoid tissue, 50 (12%) required additional immunophenotyping, mostly due to an abnormal CD4/CD8 ratio. The LPD-ST tube significantly minimizes the need for additional testing, improves the turn-around time, and reduces the cost of LPD immunophenotyping. It is also suitable for investigating paucicellular samples such as cerebrospinal fluid or fine needle aspirates.


Subject(s)
Antibodies/chemistry , Antigens, CD/blood , B-Lymphocyte Subsets/metabolism , Flow Cytometry/methods , Immunophenotyping/methods , Lymphoproliferative Disorders/blood , T-Lymphocyte Subsets/metabolism , CD4-CD8 Ratio/methods , Female , Humans , Male
5.
Int J Lab Hematol ; 37 Suppl 1: 133-43, 2015 May.
Article in English | MEDLINE | ID: mdl-25976971

ABSTRACT

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10 - <20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia.


Subject(s)
Flow Cytometry/methods , Immunophenotyping/methods , Myelodysplastic Syndromes/metabolism , Pancytopenia/metabolism , Antibodies/immunology , Antigens, Surface/immunology , Color , Flow Cytometry/instrumentation , Humans , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytes/pathology , Myelodysplastic Syndromes/immunology , Myelodysplastic Syndromes/pathology , Pancytopenia/immunology , Pancytopenia/pathology , Reproducibility of Results
6.
Int J Lab Hematol ; 37(4): 431-49, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25977137

ABSTRACT

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.


Subject(s)
Bone Marrow Examination/standards , Bone Marrow/pathology , Flow Cytometry/standards , Immunohistochemistry/standards , Immunophenotyping/standards , Biopsy/standards , Bone Marrow/surgery , Decalcification Technique/standards , Humans , International Cooperation , Laboratory Proficiency Testing , Paraffin Embedding/standards , Quality Control , Tissue Fixation/standards
7.
Leukemia ; 28(9): 1793-8, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24919805

ABSTRACT

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.


Subject(s)
Flow Cytometry/methods , Myelodysplastic Syndromes/classification , Europe , Guidelines as Topic , Humans , Myelodysplastic Syndromes/diagnosis , World Health Organization
9.
Blood Cancer J ; 4: e189, 2014 Mar 07.
Article in English | MEDLINE | ID: mdl-24608733

ABSTRACT

This prospective phase II study evaluated the efficacy of azacitidine (Aza)+erythropoietin (Epo) in transfusion-dependent patients with lower-risk myelodysplastic syndrome (MDS). Patients ineligible for or refractory to full-dose Epo+granulocyte colony stimulation factors for >8 weeks and a transfusion need of 4 units over 8 weeks were included. Aza 75 mg m(-2) d(-1), 5/28 days, was given for six cycles; non-responding patients received another three cycles combined with Epo 60 000 units per week. Primary end point was transfusion independence (TI). All patients underwent targeted mutational screen for 42 candidate genes. Thirty enrolled patients received one cycle of Aza. Ten patients discontinued the study early, 7 due to adverse events including 2 deaths. Thirty-eight serious adverse events were reported, the most common being infection. Five patients achieved TI after six cycles and one after Aza+Epo, giving a total response rate of 20%. Mutational screening revealed a high frequency of recurrent mutations. Although no single mutation predicted for response, SF3A1 (n=3) and DNMT3A (n=4) were only observed in non-responders. We conclude that Aza can induce TI in severely anemic MDS patients, but efficacy is limited, toxicity substantial and most responses of short duration. This treatment cannot be generally recommended in lower-risk MDS. Mutational screening revealed a high frequency of mutations.

10.
Int J Lab Hematol ; 35(3): 275-82, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23590655

ABSTRACT

Currently, clinical laboratories face increasing demand for flow cytometry testing combined with limited funding. Therefore, many laboratories search for panels that would provide sufficient immunophenotyping information and meet economical requirements. At the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada, we apply two 10-color tubes of surface markers for diagnosis of lymphoproliferative disorders (LPDs). These tubes contain most of the mandatory B- and T-cell markers according to European Leukemia Net (www.leukemia-net.org) recommendations. The B-cell-oriented panel includes the following antibodies: Kappa-FITC/lambda-PE/CD19-ECD/CD38-PC5.5/CD20-PC7/CD34-APC/CD23 APC-AF700/CD10 APC-AF750/CD5-PB/CD45-KO. A different combination is applied to detect cytoplasmic Ig light chain expression and aberrant immunophenotype of plasma cells. The T-cell panel allows enumeration of various T- and NK-cell subsets: CD57-FITC/CD11c-PE/CD8-ECD/CD3-PC5.5/CD2-PC7/CD56-APC/CD7-APC-AF700/CD4-APC-AF750/CD5-PB/CD45-KO. The reported overall incidence of B-cell chronic LPDs presenting with more than one aberrant population is approximately 5%. Multicolor analysis facilitates the detection of multiple aberrant populations in the same sample because expression of multiple antigens can be studied simultaneously in each defined population. Examples of LPDs with multiple aberrant populations are presented.


Subject(s)
Flow Cytometry/methods , Hematologic Diseases/immunology , Immunophenotyping/methods , Lymphoproliferative Disorders/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Biomarkers/metabolism , Hematologic Diseases/diagnosis , Hematologic Diseases/metabolism , Humans , Lymphoproliferative Disorders/diagnosis , Lymphoproliferative Disorders/metabolism , Reproducibility of Results , Sensitivity and Specificity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
11.
J Intern Med ; 272(5): 465-71, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22469005

ABSTRACT

BACKGROUND: An increased percentage of CD4+ T cells is usually observed in bronchoalveolar lavage fluid (BALF) from patients with sarcoidosis. In HLA-DRB1*03-positive patients, such T cells express the T-cell receptor (TCR) AV2S3+ gene segment. It is not known whether cells found in BALF reflect those in enlarged regional lymph nodes (LNs). Therefore, the aim of this study was to compare T-cell phenotypes in BALF, blood and mediastinal LNs. METHODS: Fifteen patients underwent clinical investigation including bronchoscopy with bronchoalveolar lavage. Blood samples were drawn, and endoscopic ultrasound-guided fine-needle aspiration of enlarged mediastinal LNs was performed via the oesophagus. T cells from all three compartments were analysed by flow cytometry for markers of activity, differentiation and T regulatory function. RESULTS: The CD4/CD8 ratio was significantly higher in BALF compared with regional LNs and was also significantly higher in LNs than in blood. The CD4+ T cells were recently activated and more differentiated in BALF than in blood and LNs. There was an accumulation of T regulatory cells (FOXP3+) in LNs and a correlation between high levels of FOXP3+ cells in BALF and in LNs. In HLA-DRB1*03-positive patients, TCR AV2S3+ CD4+ T cells were predominantly localized within BALF. CONCLUSIONS: The CD4+ T-cell phenotype in BALF indicates an active ongoing specific immune response primarily localized to the alveolar space.


Subject(s)
Bronchoalveolar Lavage Fluid/immunology , CD4-Positive T-Lymphocytes/immunology , Lymph Nodes/immunology , Receptors, Antigen, T-Cell/immunology , Sarcoidosis, Pulmonary/immunology , Adult , Aged , Antigens/genetics , Antigens/immunology , Bronchoscopy/methods , Case-Control Studies , Female , Humans , Immunophenotyping , Male , Middle Aged , Receptors, Antigen, T-Cell/genetics , Sarcoidosis, Pulmonary/genetics , Statistics as Topic
12.
Leukemia ; 26(7): 1730-41, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22307178

ABSTRACT

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.


Subject(s)
Biomarkers, Tumor/metabolism , Flow Cytometry/standards , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/metabolism , Practice Guidelines as Topic/standards , Bone Marrow/metabolism , Bone Marrow/pathology , Flow Cytometry/methods , Humans , Immunophenotyping , International Agencies , Myelodysplastic Syndromes/immunology , Prognosis , Reference Standards , Societies, Scientific
14.
Leukemia ; 25(4): 567-74, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21252983

ABSTRACT

The European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.


Subject(s)
Leukemia/diagnosis , Lymphoproliferative Disorders/diagnosis , Acute Disease , Humans , Immunophenotyping , Leukemia/immunology , Lymphoproliferative Disorders/immunology
15.
Blood Cancer J ; 1(7): e31, 2011 Jul.
Article in English | MEDLINE | ID: mdl-22829187

ABSTRACT

Malignant cells are known to have increased glucose uptake and accelerated glucose metabolism. Using liquid chromatography and mass spectrometry, we found that treatment of acute lymphoblastic leukemia (ALL) cells with the glucocorticoid (GC) dexamethasone (Dex) resulted in profound inhibition of glycolysis. We thus demonstrate that Dex reduced glucose consumption, glucose utilization and glucose uptake by leukemic cells. Furthermore, Dex treatment decreased the levels of the plasma membrane-associated glucose transporter GLUT1, thus revealing the mechanism for the inhibition of glucose uptake. Inhibition of glucose uptake correlated with induction of cell death in ALL cell lines and in leukemic blasts from ALL patients cultured ex vivo. Addition of di-methyl succinate could partially overcome cell death induced by Dex in RS4;11 cells, thereby further supporting the notion that inhibition of glycolysis contributes to the induction of apoptosis. Finally, Dex killed RS4;11 cells significantly more efficiently when cultured in lower glucose concentrations suggesting that modulation of glucose levels might influence the effectiveness of GC treatment in ALL. In summary, our data show that GC treatment blocks glucose uptake by leukemic cells leading to inhibition of glycolysis and that these effects play an important role in the induction of cell death by these drugs.

16.
Int J Lab Hematol ; 32(1 Pt 2): 122-6, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19016916

ABSTRACT

Clinical diagnosis of the myeloproliferative disorders (MPD) has previously been based on clinical data and bone marrow morphology due to lack of specific molecular markers. The discovery of JAK2 V617F mutation has shed light on understanding of the molecular pathways involved in the pathogenesis of the myeloproliferative disorders. The thrombopoietin receptor gene (MPL) is expressed in megakaryocytes and exhibits the gain of function point mutation in approximately 5% of MPDs. Several research groups have used real-time PCR to detect and quantify the presence of JAK2 V617F mutation. We report here a highly specific real-time assay based on the TaqMan((R)) technology to detect the MPL W515L mutation with high sensitivity from the patient's blood. This assay can be easily performed together with the JAK2 V617F mutation assay on the same real-time PCR reaction plate.


Subject(s)
Mutation/genetics , Myeloproliferative Disorders/genetics , Polymerase Chain Reaction/methods , Receptors, Thrombopoietin/genetics , Humans , Janus Kinase 2/genetics , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/physiopathology , Sensitivity and Specificity , Signal Transduction , Time Factors
17.
Br J Cancer ; 101(8): 1393-401, 2009 Oct 20.
Article in English | MEDLINE | ID: mdl-19773754

ABSTRACT

BACKGROUND: Classical Hodgkin's lymphoma (cHL), although a malignant disease, has many features in common with an inflammatory condition. The aim of this study was to establish the molecular characteristics of the two most common cHL subtypes, nodular sclerosis (NS) and mixed cellularity (MC), based on molecular profiling and immunohistochemistry, with special reference to the inflammatory microenvironment. METHODS: We analysed 44 gene expression profiles of cHL whole tumour tissues, 25 cases of NS and 19 cases of MC, using Affymetrix chip technology and immunohistochemistry. RESULTS: In the NS subtype, 152 genes showed a significantly higher expression, including genes involved in extracellular matrix (ECM) remodelling and ECM deposition similar to wound healing. Among these were SPARC, CTSK and COLI. Immunohistochemistry revealed that the NS-related genes were mainly expressed by macrophages and fibroblasts. Fifty-three genes had a higher expression in the MC subtype, including several inflammation-related genes, such as C1Qalpha, C1Qbeta and CXCL9. In MC tissues, the C1Q subunits were mainly expressed by infiltrating macrophages. CONCLUSIONS AND INTERPRETATIONS: We suggest that the identified subtype-specific genes could reflect different phases of wound healing. Our study underlines the potential function of infiltrating macrophages in shaping the cHL tumour microenvironment.


Subject(s)
Gene Expression Profiling , Hodgkin Disease/classification , Hodgkin Disease/pathology , Inflammation/pathology , Wound Healing , Adolescent , Adult , Aged , Biomarkers , Extracellular Matrix/metabolism , Female , Fibrosis , Hodgkin Disease/genetics , Humans , Immunohistochemistry , Male , Middle Aged
18.
J Clin Pathol ; 62(6): 547-51, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19474355

ABSTRACT

BACKGROUND AND AIMS: In diagnostic immunohistochemistry (IHC), daily quality control/quality assurance measures (QC/QA) and participation in external quality assurance programmes (EQA) are important in ensuring good laboratory practice and patient care. Bone marrow trephine biopsies (BMTB) have been generally excluded from EQA programmes for diagnostic IHC due to a lack of standards for tissue processing. The European Bone Marrow Working Group (EBMWG) has set up an EBMWG IHC Committee with the task of exploring the plausibility of an EQA programme for BMTB IHC in Europe. METHODS: 28 laboratories participated in a web-based anonymous survey; 19 laboratories submitted a total of 109 slides stained for CD34, CD117, CD20, CD3, Ki-67 and a megakaryocyte marker of choice. RESULTS: Eight different fixatives and nine different decalcification methods were used. While 93% of participants believed that they produced excellent results in BMTB IHC, only 4/19 (21%) laboratories did not have any poor results. CD117 and Ki-67, with 53% and 50% poor results, respectively, were the most problematic immunostains, while CD20 was the least problematic, with only 11% poor results. CONCLUSIONS: The EBMWG IHC Committee calls for a reduction in the tissue processing methods for BMTB and establishment of an EQA programme for BMTB IHC to help diagnostic IHC laboratories calibrate their tests according to expert recommendations. This is especially necessary in the light of recent introduction of predictive IHC tests in BMTB.


Subject(s)
Bone Marrow Examination/standards , Hematologic Diseases/pathology , Hematology/standards , Immunohistochemistry/standards , Laboratories/standards , Quality Control , Antigens, CD20/analysis , Antigens, CD34/analysis , Bone Marrow Examination/methods , CD3 Complex/analysis , Europe , Humans , Ki-67 Antigen/analysis , Megakaryocytes/pathology , Pilot Projects , Proto-Oncogene Proteins c-kit/analysis
19.
Cell Death Differ ; 16(7): 1018-29, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19390558

ABSTRACT

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Apoptosis , Autophagy , Dexamethasone/pharmacology , Leukemia, Lymphoid/metabolism , Adolescent , Aged , Aged, 80 and over , Cell Line, Tumor , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Female , Humans , Male , Microscopy, Electron, Transmission , Microtubule-Associated Proteins/agonists , Microtubule-Associated Proteins/metabolism , Middle Aged , Morpholines/pharmacology , Nuclear Proteins/agonists , Nuclear Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Promyelocytic Leukemia Protein , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/agonists , Transcription Factors/metabolism , Tumor Suppressor Proteins/agonists , Tumor Suppressor Proteins/metabolism
20.
Leukemia ; 23(5): 852-5, 2009 May.
Article in English | MEDLINE | ID: mdl-19194467

ABSTRACT

The thrombopoietin receptor gene (MPL) is expressed in megakaryocytes and exhibits the gain of function point mutation W515K/L in approximately 5% of patients with primary myelofibrosis/idiopathic myelofibrosis (PMF) representing one subtype of the chronic myeloproliferative disorders (myeloproliferative neoplasm). A series of primary and secondary acute myeloid leukaemias (AML) with megakaryoblastic phenotype and myelofibrosis unrelated to PMF (n=12) was analysed for the MPL(W515K/L) mutation by pyrosequencing. In three cases (25%), MPL(W515L) was found and in two of these a combination with trisomy 21 or the Philadelphia chromosome occurred. None of the secondary AML cases evolving from pre-existing PMF showed MPL(W515K/L) (n=4). We conclude that MPL(W515L) occurs in a considerable proportion of acute megakaryoblastic leukaemias with myelofibrosis unrelated to PMF.


Subject(s)
Leukemia, Megakaryoblastic, Acute/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mutation/genetics , Primary Myelofibrosis/genetics , Receptors, Thrombopoietin/genetics , Adult , Aged , Aged, 80 and over , Blast Crisis , Bone Marrow Cells , Child , Child, Preschool , Female , Humans , Janus Kinase 2/genetics , Lasers , Male , Microdissection , Middle Aged
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