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The primary objective of this trial was to assess the feasibility, toxicity profile, and antitumor activity of gemtuzumab ozogamicin (GO) combined with a chemotherapy remission-induction regimen in adults with untreated high-risk myelodysplastic syndrome (HR-MDS) or secondary acute myeloid leukemia (sAML). In this phase II trial, 30 patients with median age of 58 years received 1 day of GO as a 1-h infusion at the dose level of 5 mg/m(2) on day 7 of the remission-induction course further consisting of a continuous infusion of cytarabine 100 mg/m(2)/day for 10 days and idarubicin 12 mg/m(2)/day on days 1, 3, and 5. A consolidation course, consisting of intermediate-dose cytarabine (A) and idarubicin (I) followed by hematopoietic stem cell transplantation (HSCT) was planned for patients in complete remission (CR). The primary endpoints were response rate (CR/CRi) and severe toxicity rate. The secondary endpoint(s) were survival and progression-free survival (PFS) from start of treatment. Thirteen patients (43 %) achieved CR (eight patients) or CR with incomplete hematopoietic recovery (CRi) (five patients). In patients who achieved CR or CRi, the median time to recovery of neutrophils to 0.5 × 10(9)/l and of platelets to >50 × 10(9)/l was 29 and 30 days, respectively. Grade 3 to 4 severe toxicities occurred in nine patients. The most prominent was liver toxicity, as shown by elevated bilirubin levels in 16 patients and one case of nonfatal veno-occlusive disease (VOD). All 13 patients with CR/CRi received consolidation therapy, which was followed by allogeneic HSCT in five patients and autologous HSCT in three patients. According to the statistical design of the study, the idarubicin and cytarabine in combination with gemtuzumab ozogamicin (IAGO) regimen did not show sufficient activity to warrant further exploration of this regimen in adult patients with HR-MDS or sAML. [ABSTRACT FROM AUTHOR]

High-risk myelodysplastic syndrome (MDS) patients have usually a less favorable outcome after intensive treatment compared with de novo acute myeloid leukemia (AML) patients. This may reflect different disease-related and patient-related factors. The purpose of this analysis is to identify disease-specific prognostic factors and to develop prognostic scores for both patient groups. A total of 692 patients in the EORTC/GIMEMA AML-10 study and 289 patients in the CRIANT study received identical remission-induction and consolidation treatment. Estimated 5-year survival rate was 34 % in the AML-10 versus 27 % in the CRIANT study, and estimated disease-free survival was 40 % versus 28 %, respectively. In multivariate analysis, cytogenetic characteristics, white blood count, and age appeared prognostic for survival in both studies. French-American-British (FAB) subtype and performance status were prognostic in the AML-10 study only, whereas number of cytopenias and duration of antecedent hematologic disorder >6 months were prognostic in the CRIANT study only. The prognostic scores distinguish three groups with a 5-year survival rate of 54, 38, and 19 % in the AML-10 study versus 69, 37, and 5 % in the CRIANT study. The prognostic value of these scores has been validated on two external series. The new scoring systems form a practical tool to predict the outcome of individual MDS and AML patients treated with intensive antileukemic therapy. [ABSTRACT FROM AUTHOR]

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients< 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p =0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival. [ABSTRACT FROM AUTHOR]

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis ( p = 0.024). TET2 and IDH1/ 2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival. [ABSTRACT FROM AUTHOR]

Background aims. Adoptive transfer of cytomegalovirus (CMV)-specific memory T cells can be used for treatment of CMV reactivation after allogeneic stem cell transplantation. As co-ordinated CD8+ and CD4+ T cells specific for a broad repertoire of CMV epitopes may be most effective for adoptive immunotherapy, the aim of this study was to isolate these cells from peripheral blood of CMV seropositive donors, irrespective of their HLA type. Methods. Activation of CMV-specific CD8+ and CD4+ T cells was compared after stimulation of donor peripheral blood with minimal epitope peptides, pools of overlapping 15-mer peptides or full-length protein. Furthermore, the kinetics of interferon (IFN)-γ production after stimulation was analyzed to determine the optimal time-point for IFN-γ-based isolation of CMV-specific T cells. The specificity, phenotype and functionality of generated T-cell lines were analyzed. Results. CMV protein-spanning 15-mer peptide pools induced simultaneous activation of both CD8+ and CD4+ CMV-specific T cells, while full-length CMV protein only efficiently activated CD4+ CMV-specific T cells. Isolation of IFN-γ-secreting cells at the peak of the IFN-γ response after 4-h stimulation with CMV pp65 and IE1 peptide pools resulted in efficient enrichment of CMV-specific T cells. The T-cell lines contained high frequencies of CD8+ and CD4+ T cells recognizing multiple CMV pp65 and IE1 epitopes, and produced IFN-γ and tumor necrosis factor (TNF)-α upon specific restimulation. Conclusions. This study provides a feasible strategy for the rapid generation of clinical-grade CD8+ and CD4+ T-cell lines with high specificity for multiple CMV pp65 and IE1 epitopes, which may be used for effective adoptive immunotherapy. [ABSTRACT FROM AUTHOR]

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival. [ABSTRACT FROM AUTHOR]

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4+ T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II β gene. A polyclonal CD4+ T cell response against LB-Pl4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4+ T cells recognized and lysed the CD344 CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-γ and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4+ T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8+ T cell immunity as helper cells. [ABSTRACT FROM AUTHOR]

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors. [ABSTRACT FROM AUTHOR]

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2/HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8[sup +] T cells in the blood of the recipients 5-7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignances relapsing after alloSCT. [ABSTRACT FROM AUTHOR]

Abstract: Resistance to cytarabine (AraC) is a major problem in treatment of patients with acute myeloid leukemia (AML). In contrast to in vitro AraC resistance, deoxycytidine kinase (dCK) mutations are rarely found in patients with refractory or relapsed AML. Previously we have demonstrated alternatively spliced dCK mRNA predominantly expressed in leukemic blasts from patients with resistant AML. In this study we investigated wild-type (wt) dCK expression and activity to elucidate the possible role of decreased dCK expression or activity in unresponsiveness to AraC in patients with AML. No alterations in dCK mRNA and protein expression or in dCK activity were detected between patients with clinically resistant vs. sensitive AML. In addition, wt dCK expression and activity were not reduced in leukemic blasts expressing alternatively spliced dCK forms as compared to blasts with only wt dCK. Also, no major differences in wt dCK expression and activity were observed between samples obtained from patients with AML and bone marrow or peripheral blood samples from healthy donors. These data implicate that in our patient group of refractory or relapsed AML cases, alterations in dCK expression and/or activity cannot explain unresponsiveness to chemotherapy including AraC. [ABSTRACT FROM AUTHOR]

Twenty years of published literature was reviewed for chemotherapy regimens used to treat patients with acute myeloid leukemia (AML) in first relapse. Thirty-one trials containing at least 20 patients in first relapse and information on patient age, duration of first complete remission (CRI), and rate of second complete remission (CR2) were analyzed. These trials included 10 retrospective studies with CR2 rates ranging from 30 to 64%, two phase II single-agent studies with CR2 rates of 8 and 25%, 15 phase II combination-agent studies with CR2 rates ranging from 14 to 87%, and four phase III randomized studies with CR2 rates ranging from 40 to 89%. When reported, median duration of CR2 was ≤ 14 months and overall median survival was ≤ 12 months. The probability of 3-year survival ranged from 8 to 29%. Combination therapies resulted in higher CR2 rates but were associated with longer duration of myelosuppression and greater incidence of mucositis. None of the reviewed regimens provided durable remissions for the majority of AML patients in first relapse. The CR2 rates were closely associated with age and duration of CR1. Therefore, considering the poor clinical outcomes of patients with AML in first relapse, improved therapies need to be developed. [ABSTRACT FROM AUTHOR]

Examines the role of neutrophils in the hematopoietic stem cell mobilization induced by interleukin-8 in mice. Infusion of mononuclear cells; Circulation of metalloproteinase gelatinase B; Adherence of progenitor cells to the bone marrow.

Summary. Accelerated telomere shortening has been proposed as a possible long-term risk of allogeneic bone marrow transplantation (allo-BMT). In this study we monitored telomere length in white blood cells (WBC), granulocytes, and naïve and memory CD4+ T lymphocytes in recipients of allo-BMT at long-term follow-up. Peripheral blood was collected from 10 allo-BMT recipients and donors at a median interval of 18 years after allo-BMT. Telomere length was determined using Southern blot analysis. Similar to results previously reported at short-term follow-up, a small difference in telomere length (0·1–0·3 kb) between recipients and donors was detected in WBC, granulocytes and naïve CD4+ T cells. Our data therefore provide no evidence for sustained telomere shortening in leucocytes, and render the possibility of long-term haematopoietic graft failure unlikely. In addition, we observed two phenomena that may be related to involution of the thymus. First, the number of naïve CD4+ T cells in the blood was significantly lower in recipients (0·4 × 109 /l) than in donors (0·7 × 109 /l) (P < 0·05). Second, telomeres in memory CD4+ T cells from recipients were on average 0·6 kb shorter than those from donors (P = 0·01). The latter may be related to the reported rapid peripheral expansion of memory T cells immediately after transplantation. [ABSTRACT FROM AUTHOR]

Analyzes the expression of adhesion and co-stimulatory molecules in 99 acute myeloid leukaemia (AML) patients using monoclonal antibodies and flow cytometry. Correlation of the expression with French-American-British classification and survival; Discussion on flow cytometry analysis; Expression levels of the co-stimulatory and adhesion molecules on AML blasts.

Examines the specificity of both antigens, HLA class I A2 molecule and HLA class II DR1 by alloreactive human T cell clone MBM15. Dual reactivity mediated through single clonal T cell on specific peptide binding; Role of coreceptor CD8 in dual recognition.

Examines the polycomb group (PcG) proteins involved in the stable transmittance of the repressive state of their gene targets. Identification of PcG protein complexes; Enhancement of EZHZ expression; Effect between EZH2 upregulation and increased proliferation in hematopoietic cells.

The assays for the detection of unlabeled 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside, Ara-C) incorporation into DNA was simplified. The procedure includes DNA isolation from leukemic cells, quantification of DNA concentrations, breakdown by enzymatic digestion of DNA to nucleosides and a radioimmunoassay (RIA) using an antibody against Ara-C. Different techniques for quantification of DNA concentrations are compared. A fluorimetric technique using Hoechst 33258 is preferred because it is the most specific method. Comparison of this RIA assay with measurement of [3H]-Ara-C/DNA formation under similar conditions in HL-60 cells showed a correlation of 0.99. Ara-C incorporation into DNA of leukemic cells was studied using two rat-leukemia cell lines, one of which is sensitive to Ara-C and the other is an Ara-C-resistant wild type: BNML-Cl/0 and BNML-Cl/Ara-C, respectively. The results showed that Ara-C is incorporated when the cells are incubated at concentrations equal to or higher than the Ara-C concentration that induces 50% growth inhibition after 48 h incubation (IC50). This implies that at lower Ara-C concentration, i.e. levels that do not induce cytotoxicity, Ara-C is not incorporated into DNA. Similar results were obtained with human HL-60 myeloid leukemia cells. The detection limit of this assay is 2 pmol/ml Ara-C; therefore, the assay is more sensitive than measurement of Ara-C triphosphate (Ara-CTP), the only metabolite that can be measured in leukemic cells from patients after in vivo Ara-C administration. On the basis of in vitro studies, the finding of detectable Ara-C/DNA levels in vivo is expected to correlate with cytotoxicity; whether or not the Ara-C/DNA level itself is informative remains to be evaluated. [ABSTRACT FROM AUTHOR]

Interleukin-1 (IL-1) refers to a hormonelike polypeptide that mediates a broad spectrum of activities in host defence as well as a variety of disease processes. Originally described as a substance produced by activated macrophages, IL-1 is now recognized as a polypeptide produced by many other cell types. Two distinct genes have been identified that code for two structurally related forms of the molecule, termed IL-1 alpha and beta. IL-1 is the primary mediator of the acute phase response and is responsible for many of the changes associated with the onset of infection. It is involved in the immune response to antigenic challenge. IL-1 induces fever and has profound endocrinologic, neurologic, metabolic and hematologic effects. Both forms of IL-1 bind to a common receptor that has been identified on a variety of cell types including lymphocytes, hepatocytes, endothelial cells and fibroblasts. Many of the activities of IL-1 are mediated by the induction of other cytokines like IL-2, IL-6, interferons, tumor necrosis factor, and colony-stimulating factors. In animals IL-1 protects against the effects of radation, it enhances natural resistance of infection, and it stimulates bone marrow recovery after myelosuppression. These studies suggest that IL-1 may be used as a therapeutic agent. [ABSTRACT FROM AUTHOR]

Presents a study conducted which focuses on the development of an assay which measures the frequency of T lymphocyte percursor cells, with the capabilities of suppressing the leukemic progenitor cells, in an attempt to identify the effector and target cell populations responsible for the elimination of the leukemic cells. Description of target cells in the assay; Definition of a cytogenic remission; Properties of effector cells.

Presents an experimental study conducted to identify and purify subpopulations of hematopoietic progenito cells in murine cytikine-mobilized blood. Information on the study methodology and the materials used; Detailed information on the study results; Discussion on the results.

Intensive chemotherapy regimens with or without stem cell transplantation are the current standard treatment in patients with acute myeloid leukemia (AML). The identification of new mutations in AML may provide the basis for a more targeted therapeutic approach. In one or several genetic subsets, some new drugs, as single drug or in combination with standard antileukemic drugs, demonstrated promising results. Typical examples are tyrosine kinase inhibitors, such as dasatinib for f c-Kit mutated core binding factor positive AML or FLT-3 inhibitors, such as midostaurin or sorafenib, in cases with FLT-3 mutations. Clofarabine is a second generation hybrid purine nucleoside analogue: standard induction regimens containing clofarabine provided a high remission rates in patients with intermediate and high risk AML. In patients unfit for intensive chemotherapy, treatment with hypomethilating agents is widely used. Gemtuzumab ozogamicin, anti-CD33 monoclonal antibody conjugated to calicheamicin, has shown significant antileukemic activity in older patients with relapsed AML. [ABSTRACT FROM AUTHOR]

The human interleukin-6 receptor α chain (IL6Rα) gene was regionally mapped to chromosome 1 bridization probes, partially overlapping lambda clones encompassing 28 kb of the genomic region of the gene were used. These clones were isolated using a polymerase chain reaction (PCR)-generated fragment of the 3′ noncoding region of the gene. This localization confirms and extends the provisional assignment of the IL6Rα gene to chromosome 1, for which a panel of somatic cell hybrids was used. [ABSTRACT FROM AUTHOR]

A patient with chronic lymphocytic leukaemia (CLL) and severe persisting neutropenia due to marrow infiltration of his leukaemia, developed bilateral Legionella pneumophila pneumonia for which he was treated with erythromycin, rifampin and ciprofloxacin. To increase the number of circulating polymorphonuclear neutrophils, the patient was treated with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) at a dose of 2 micrograms protein/kg bodyweight s.c./12 h. GM-CSF therapy resulted in a sustained rise of the neutrophil count from the fifth day of treatment onwards, without showing an effect on the number of circulating leukemic cells. The patient completely recovered from his pneumonia. It is suggested that the rise of the neutrophil count, due to GM-CSF, contributed to the improvement of the infection of this patient. Our observation illustrates that GM-CSF can be given safely to CLL-patients and that it can be used effectively in CLL patients with severe bacterial infections to restore neutropenia. [ABSTRACT FROM AUTHOR]